**Chromatin Remodelers and Their Way of Action**

Laura Manelyte and Gernot Längst

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55683

### **1. Introduction**

Chromatin is the packaged form of the eukaryotic genome in the cell nucleus, presenting the substrate for all DNA dependent processes. The basic packaging unit of chromatin is the nucleosome core, a nucleoprotein structure consisting of 8 histone proteins and 147 bp of DNA. Two of each H2A and H2B, H3 and H4, form an octameric, disc like particle on which 1.65 turns of DNA is wrapped [1]. Nucleosomal cores are separated by a linker DNA, with a varying length of 7 bp to 100 bp, with distinct lengths in different organisms and tissues. Even within one cell type the linker length can vary about 40 bp between the actively transcribed and repressed genes [2].

Binding of the DNA to the histone octamer and the bending of the molecule on the protein surface present a strong barrier to sequence specific recognition of the nucleosomal DNA molecule. That's why the packaging of DNA into nucleosomes and higher order structures is generally inhibitory to all kind of DNA dependent processes. To overcome DNA sequence accessibility problems, cells have developed mechanisms to open higher order structures of chromatin and to disrupt nucleosomes allowing the binding of sequence specific regulators. In general, two major mechanisms exist which regulate chromatin accessibility: First, histones can be posttranslationally modified and recruit specific effector proteins to chromatin [3]. Second, specific chromatin remodeling enzymes displace the histone octamers from DNA or translocate them on DNA, thereby exposing or protecting underlying DNA sequences to regulatory factors that control the DNA dependent processes [4].

The presence of 53 different chromatin remodeling enzymes in the human cell suggests specialized functions of these enzymes and the associated complexes. Chromatin remodelers are DNA translocases that apply an ATP-dependent torsional strain to DNA, providing the force to reposition nucleosomes; i.e. moving the histone octamer to a different site on the DNA [4,5]. Diverse remodeling enzymes and complexes have distinct nucleosome positioning

© 2013 Manelyte and Längst; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

activities. In other words, the remodelers interpret the DNA sequence/structure information in different ways, establishing target site-specific nucleosome positioning patterns. The exact nucleosome positions at a given site depends on both, the type of the ATPase motor protein and the composition of the multiprotein complex where it is integrated [6]. The specialized functions of remodeling enzymes may result from their different nucleosome positioning behavior and the distinct targeting to genomic sites.

There is plenty of data available on the remodeling mechanism *in vitro*, however not much is known about the targeting and regulation of the remodelers *in vivo.* It remains unclear whether these complexes form a dynamic chromatin environment or a rather static chromatin structure with defined nucleosome positions in the cell nucleus. Many chromatin remodelers are believed to bind DNA and nucleosomes in a sequence independent manner *in vitro*, however there is mounting evidence for specific chromatin signals that are recognized by chromatin remodelers. This is best demonstrated by the recognition of histone variants, modified histone tails, the preferential binding to nucleosome free regions of DNA and binding to specific DNA and RNA structures and sequences. In addition, interacting proteins and/or accessory domains of the remodeling complexes may serve as an additional layer of signal recognition and recruitment of remodelers to the right place at the right time.

### **2. Remodeler families**

The catalytic subunit of the remodeling enzymes consists of a conserved ATPase domain and unique flankingdomains,usedfor a simplifiedseparationintofourdistinctfamilies (Fig. 1).The ATPase domain consists of two tandem RecA-like folds (DExx and HELICc), containing seven conserved helicase-related sequence motifs that classify the enzymes as part of the Superfami‐ ly 2 grouping of helicase-like proteins [7,8]. Chromatin remodelers are lacking the ability to separate nucleic acid strands, so they are not bona fide helicases. However, they are DNA translocases that use the energy of ATP to create a necessary force to reposition nucleosomes.

suggesting a global role for Swi/Snf in gene expression. However, recent whole-genome expression studies have shown that Swi/Snf controls transcription of a small percentage of all *S. cerevisiae* genes [10]. The SWI/SNF family members are defined by the presence of an Nterminally located HSA (helicase-SANT) domain, which is known to recruit actin and actinrelated proteins, and a C-terminally located bromo domain, suggested to bind to the acetylated-lysines of histones. This family of remodeling enzymes was shown to slide and to evict nucleosomes from DNA, but lacking chromatin assembly activities. Remodelers belong‐ ing to this family are large, multi-subunit complexes containing 8 or more proteins. Most eukaryotes utilize two related SWI/SNF family remodelers, built around the two related catalytic subunits Swi2/Snf2 or Sth1 in yeast, and BRM or BRG1 in humans (Table 1). Although SWI/SNF is not essential for yeast growth, a genome-wide analysis demonstrated that ~3 to 6% of yeast genes are regulated by SWI/SNF, with functions that contribute to both gene activation and repression [10,11]. On the other hand, RSC complex containing the Sth1 ATPase is essential for growth and about 10-fold more abundant than the SWI/SNF complex. RSC function is required for normal cell cycle progression [12]. Human BAF and PBAF complexes share eight identical subunits and are distinguished by the presence of only several unique subunits: BAF180, BAF200 and BRD7 for PBAF and BAF250a for BAF [13]. Variant subunits are thought to contribute to targeting, assembly and regulation of lineage-specific functions of those complexes. For example only PBAF, but not BAF, is capable of facilitating liganddependent transcriptional activation by nuclear receptors *in vitro* and to mediate expression of an interferon-responsive genes [14,15]. Both appear to be associated with lung cancer, as 90% of non-small cell lung carcinomas stained positively for BRG1 and BRM [16]. BRG1

**Figure 1.** Classical organization of remodeler families defined by their catalytic domain. All remodeling enzymes con‐

Chromatin Remodelers and Their Way of Action

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sist of a shared ATPase domain and unique flanking domains.

In a qualitative and quantitative study, the Snf2 family members were further subdivided into 24 distinct subfamilies based on similarities within the Snf2-specific motifs. Increased genomic complexity is paralleled by an increasing number of subfamilies and members of a given subfamily: the *S.cerevisiae* genome encoding some 6000 genes has 17 Snf2 family members belonging to 13 subfamilies, and the human genome encoding some 21000 genes has 53 Snf2 family genes from 20 subfamilies [8].

#### **2.1. SWI/SNF family**

The SWI/SNF complex was first described in *Saccharomyces cerevisiae*. In 1984 genetic screens revealed that the mutations in sucrose non-fermenting (SNF) genes caused defects in expres‐ sion of the SUC2 gene, which is required for growth on sucrose and raffinose as a carbon sources [9]. Similarly, mutations in SWI genes were identified as defective for expression of the HO gene, which is required for mating type switching (the name Swi is derived from switching defective). Mutations in both SNF and SWI genes cause pleiotropic phenotypes,

activities. In other words, the remodelers interpret the DNA sequence/structure information in different ways, establishing target site-specific nucleosome positioning patterns. The exact nucleosome positions at a given site depends on both, the type of the ATPase motor protein and the composition of the multiprotein complex where it is integrated [6]. The specialized functions of remodeling enzymes may result from their different nucleosome positioning

There is plenty of data available on the remodeling mechanism *in vitro*, however not much is known about the targeting and regulation of the remodelers *in vivo.* It remains unclear whether these complexes form a dynamic chromatin environment or a rather static chromatin structure with defined nucleosome positions in the cell nucleus. Many chromatin remodelers are believed to bind DNA and nucleosomes in a sequence independent manner *in vitro*, however there is mounting evidence for specific chromatin signals that are recognized by chromatin remodelers. This is best demonstrated by the recognition of histone variants, modified histone tails, the preferential binding to nucleosome free regions of DNA and binding to specific DNA and RNA structures and sequences. In addition, interacting proteins and/or accessory domains of the remodeling complexes may serve as an additional layer of signal recognition and

The catalytic subunit of the remodeling enzymes consists of a conserved ATPase domain and unique flankingdomains,usedfor a simplifiedseparationintofourdistinctfamilies (Fig. 1).The ATPase domain consists of two tandem RecA-like folds (DExx and HELICc), containing seven conserved helicase-related sequence motifs that classify the enzymes as part of the Superfami‐ ly 2 grouping of helicase-like proteins [7,8]. Chromatin remodelers are lacking the ability to separate nucleic acid strands, so they are not bona fide helicases. However, they are DNA translocases that use the energy of ATP to create a necessary force to reposition nucleosomes. In a qualitative and quantitative study, the Snf2 family members were further subdivided into 24 distinct subfamilies based on similarities within the Snf2-specific motifs. Increased genomic complexity is paralleled by an increasing number of subfamilies and members of a given subfamily: the *S.cerevisiae* genome encoding some 6000 genes has 17 Snf2 family members belonging to 13 subfamilies, and the human genome encoding some 21000 genes has 53 Snf2

The SWI/SNF complex was first described in *Saccharomyces cerevisiae*. In 1984 genetic screens revealed that the mutations in sucrose non-fermenting (SNF) genes caused defects in expres‐ sion of the SUC2 gene, which is required for growth on sucrose and raffinose as a carbon sources [9]. Similarly, mutations in SWI genes were identified as defective for expression of the HO gene, which is required for mating type switching (the name Swi is derived from switching defective). Mutations in both SNF and SWI genes cause pleiotropic phenotypes,

behavior and the distinct targeting to genomic sites.

recruitment of remodelers to the right place at the right time.

**2. Remodeler families**

4 Chromatin Remodelling

family genes from 20 subfamilies [8].

**2.1. SWI/SNF family**

**Figure 1.** Classical organization of remodeler families defined by their catalytic domain. All remodeling enzymes con‐ sist of a shared ATPase domain and unique flanking domains.

suggesting a global role for Swi/Snf in gene expression. However, recent whole-genome expression studies have shown that Swi/Snf controls transcription of a small percentage of all *S. cerevisiae* genes [10]. The SWI/SNF family members are defined by the presence of an Nterminally located HSA (helicase-SANT) domain, which is known to recruit actin and actinrelated proteins, and a C-terminally located bromo domain, suggested to bind to the acetylated-lysines of histones. This family of remodeling enzymes was shown to slide and to evict nucleosomes from DNA, but lacking chromatin assembly activities. Remodelers belong‐ ing to this family are large, multi-subunit complexes containing 8 or more proteins. Most eukaryotes utilize two related SWI/SNF family remodelers, built around the two related catalytic subunits Swi2/Snf2 or Sth1 in yeast, and BRM or BRG1 in humans (Table 1). Although SWI/SNF is not essential for yeast growth, a genome-wide analysis demonstrated that ~3 to 6% of yeast genes are regulated by SWI/SNF, with functions that contribute to both gene activation and repression [10,11]. On the other hand, RSC complex containing the Sth1 ATPase is essential for growth and about 10-fold more abundant than the SWI/SNF complex. RSC function is required for normal cell cycle progression [12]. Human BAF and PBAF complexes share eight identical subunits and are distinguished by the presence of only several unique subunits: BAF180, BAF200 and BRD7 for PBAF and BAF250a for BAF [13]. Variant subunits are thought to contribute to targeting, assembly and regulation of lineage-specific functions of those complexes. For example only PBAF, but not BAF, is capable of facilitating liganddependent transcriptional activation by nuclear receptors *in vitro* and to mediate expression of an interferon-responsive genes [14,15]. Both appear to be associated with lung cancer, as 90% of non-small cell lung carcinomas stained positively for BRG1 and BRM [16]. BRG1 possesses tumor suppressor functions, whereas BRM loss is a contributing factor and potential marker of tumorigenesis in lung, prostate and gastric cancers [17].

**Complex Catalytic subunit Auxillary subunits Organism**

ACF ACF1 *Fly*

ISWI1b loc2, loc4 *Yeast*

The CHD (Chromodomain-Helicase-DNA binding) family is defined by the presence of two chromodomains, arranged as a tandem, N-terminal of the ATPase domain. Additional structural motifs are used to further divide the CHD family into the subfamilies CHD1, Mi-2

Members of the CHD1 subfamily contain a C-terminal DNA-binding domain that preferen‐ tially binds to AT-rich DNA *in vitro* (members are Chd1 and Chd2 proteins in higher eukar‐ yotes) [22,23]. Recently, the crystal structure of the DNA binding domain of Chd1, revealed a SANT-SLIDE like fold. This domain was shown to be required for the remodeling activity of

The Mi-2 subfamily members contain a pair of PHD domains (plant homeodomain) in their N-terminal part (human Chd3 and Chd4, also known as Mi-2α and Mi-2β in *Drosophila*,

The CHD7 subfamily members have additional C-terminal domains, like the SANT or BRK

The biological properties of CHD family members are highly heterogenous. Some exist as monomers *in vivo*; others are subunits of multiprotein complexes, many of which have not yet been fully characterized [26]. The best studied is the NURD (nucleosome remodeling and deacetalase) complex, containing Chd3/Chd4, histone deacetylases (HDAC1/2) and methyl CpG-binding domain (MBD) proteins. It was shown to be involved in transcriptional repres‐ sion of a specific set of genes during *C.elegans*, *D.melanogaster* and mammalian development [26]. Chd1 together with Isw1 are also termed nucleosome-spacing enzymes that are required

loc3

ACF1

NURF301, NURF55/p55, NURF38

*Human*

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ISWI

ISWI1

Snf2H

ISWI2 ISWI2 Itc1

NoRC Tip5 RSF Rsf1 WICH Wstf

respectively), implicated in nucleosome binding [25].

**Table 2.** Selected SWI/SNF family remodelers.

CHRAC ACF1, CHRAC 14, CHRAC 16

NURF Snf2L BPTF, RbAp46 or RbAP48

CHRAC ACF1, CHRAC17, CHRAC15

NURF

ISWI1a

ACF

**2.3. CHD family**

and CHD7 [8,21].

Chd1 *in vitro* and *in vivo* [24].

domains (Chd5 to Chd9 proteins).


**Table 1.** Selected SWI/SNF family remodelers from yeast and human.

#### **2.2. ISWI family**

The ISWI (imitation switch) family ATPases harbour a C-terminal SANT domain adjacent to a SLIDE domain (SANT-like ISWI), which together form a nucleosome recognition module that binds to DNA and unmodified H4 tails [4]. The ISWI remodeling enzyme in *Drosophila*, is known to be present in several chromatin remodeling complexes such as NURF, CHRAC and ACF. Snf2H and Snf2L are the mammalian homologues of ISWI, which can act on their own or in the presence of one or more auxilary subunits forming different remodeling complexes with different properties. For example, Snf2H is known to interact with Tip5, RSF1 and WSTF proteins to form NoRC, RSF and WICH complexes. Specialized accessory proteins contain many chromatin binding domains, including histone fold motifs (in CHRAC), plant homeodomain (in Tip5), bromodomains (in BPTF, ACF1, Tip5) and additional DNA-binding motifs (HMGI(Y) in NURF301; AT hooks in Tip5). Many ISWI family complexes (ACF, CHRAC, NoRC) catalyze nucleosome spacing, promote chromatin assembly and confer transcriptional repression. However, NURF escapes theses general rules by disturbing nucleosome spacing and assisting ecdysone dependent transcriptional activation, showing that functional diversity is determined by the additional subunits [4]. The steroid hormone ecdysone directly modulates germline stem cells maintenance, activates transcription and proliferation in a cooperation with the NURF remodeler [18]. In *Drosophila*, loss of ISWI causes global transcriptional defects and results in dramatic alterations of the higher-order structure of chromatin, especially on the male X chromosome [19]. NoRC action correlates with specific changes in nucleosome positioning at the rDNA promoter region, causing heterochromatin formation and gene silencing [20].


**Table 2.** Selected SWI/SNF family remodelers.

#### **2.3. CHD family**

possesses tumor suppressor functions, whereas BRM loss is a contributing factor and potential

**Complex Catalytic subunit Auxillary subunits Organism**

The ISWI (imitation switch) family ATPases harbour a C-terminal SANT domain adjacent to a SLIDE domain (SANT-like ISWI), which together form a nucleosome recognition module that binds to DNA and unmodified H4 tails [4]. The ISWI remodeling enzyme in *Drosophila*, is known to be present in several chromatin remodeling complexes such as NURF, CHRAC and ACF. Snf2H and Snf2L are the mammalian homologues of ISWI, which can act on their own or in the presence of one or more auxilary subunits forming different remodeling complexes with different properties. For example, Snf2H is known to interact with Tip5, RSF1 and WSTF proteins to form NoRC, RSF and WICH complexes. Specialized accessory proteins contain many chromatin binding domains, including histone fold motifs (in CHRAC), plant homeodomain (in Tip5), bromodomains (in BPTF, ACF1, Tip5) and additional DNA-binding motifs (HMGI(Y) in NURF301; AT hooks in Tip5). Many ISWI family complexes (ACF, CHRAC, NoRC) catalyze nucleosome spacing, promote chromatin assembly and confer transcriptional repression. However, NURF escapes theses general rules by disturbing nucleosome spacing and assisting ecdysone dependent transcriptional activation, showing that functional diversity is determined by the additional subunits [4]. The steroid hormone ecdysone directly modulates germline stem cells maintenance, activates transcription and proliferation in a cooperation with the NURF remodeler [18]. In *Drosophila*, loss of ISWI causes global transcriptional defects and results in dramatic alterations of the higher-order structure of chromatin, especially on the male X chromosome [19]. NoRC action correlates with specific changes in nucleosome positioning at the rDNA promoter region, causing heterochromatin

Swi1/Adr6, Swi3, Swp73, Snf5, Arp7, Arp9, Swp82, Snf11, Taf14, Snf6, Rtt102

Sth1, Rsc8/Swh3, Rsc6, SfhI, Arp7, Arp9, Rsc1,2 or 4, Rsc7, Rsc30, Rsc3, Rsc5, Rtt102, Rsc14/Ldb7, Rsc10, Rsc9

BAF250, BAF155, BAF170,BAF60(A,B or C), SNF5, BAF57, BAF53(A or B), β-actin, BAF45(A,B,C or D)

BAF180, BAF200, BRD7, BAF155, BAF45(A,B,C or D), BAF170,BAF60(A, B or C), SNF5, BAF57, BAF53(A or B), βactin

*Yeast*

*Human*

marker of tumorigenesis in lung, prostate and gastric cancers [17].

SWI/SNF Swi2/Snf2

RSC Sth1

BAF BRM or BRG1

PBAF BRG1

formation and gene silencing [20].

**2.2. ISWI family**

6 Chromatin Remodelling

**Table 1.** Selected SWI/SNF family remodelers from yeast and human.

The CHD (Chromodomain-Helicase-DNA binding) family is defined by the presence of two chromodomains, arranged as a tandem, N-terminal of the ATPase domain. Additional structural motifs are used to further divide the CHD family into the subfamilies CHD1, Mi-2 and CHD7 [8,21].

Members of the CHD1 subfamily contain a C-terminal DNA-binding domain that preferen‐ tially binds to AT-rich DNA *in vitro* (members are Chd1 and Chd2 proteins in higher eukar‐ yotes) [22,23]. Recently, the crystal structure of the DNA binding domain of Chd1, revealed a SANT-SLIDE like fold. This domain was shown to be required for the remodeling activity of Chd1 *in vitro* and *in vivo* [24].

The Mi-2 subfamily members contain a pair of PHD domains (plant homeodomain) in their N-terminal part (human Chd3 and Chd4, also known as Mi-2α and Mi-2β in *Drosophila*, respectively), implicated in nucleosome binding [25].

The CHD7 subfamily members have additional C-terminal domains, like the SANT or BRK domains (Chd5 to Chd9 proteins).

The biological properties of CHD family members are highly heterogenous. Some exist as monomers *in vivo*; others are subunits of multiprotein complexes, many of which have not yet been fully characterized [26]. The best studied is the NURD (nucleosome remodeling and deacetalase) complex, containing Chd3/Chd4, histone deacetylases (HDAC1/2) and methyl CpG-binding domain (MBD) proteins. It was shown to be involved in transcriptional repres‐ sion of a specific set of genes during *C.elegans*, *D.melanogaster* and mammalian development [26]. Chd1 together with Isw1 are also termed nucleosome-spacing enzymes that are required to maintain nucleosomal organization in yeast [27]. To date, Chd3, Chd4, Chd5 and Chd7 have been implicated in human disease processes. Chd3 and Chd4 have been identified as autoan‐ tigens in patients with dermatomyositis, a connective-tissue disease characterized by inflam‐ mation of both muscles and skin. Chd3 is associated with Hodgkin's lymphoma and Chd5 is associated with neuroblastoma, a malignant neoplasm of the peripheral sympathetic nervous system frequently affecting infants and children [28]. Haploinsufficiency of Chd7 in humans results in the CHARGE syndrome. Chd7 is essential for the develompment of multipotent migratory neural crest cells, which contribute to the formation of many tissues affected in CHARGE syndrome [29].

**Complex Catalytic subunit Auxillary subunits Organism**

Chromatin remodelers use the energy of ATP hydrolysis to assemble, reposition or evict histones from DNA. Nucleosome repositioning by remodelers can be described as a 3-step mechanism: 1) initiation step that requires the recognition and specific binding to the substrate, 2) several translocation steps with varying step-lengths and kinetics depending on the particular remodeling enzyme and on the properties of the underlying DNA sequence, 3) release step, which occurs at energetically favourable positions depending on the combination of remodeler and DNA sequence/structure at this site [6,38]. This chapter will focus on the

Proposed models for nucleosome remodeling suggest that only a minor fraction of the 358 direct and indirect histone-DNA interactions are disrupted at a given time of the reaction, as the energy of ATP hydrolysis would not be sufficient to fully disrupt the nucleoprotein structure [39,40]. One of the first mechanisms proposed, is the ''twist diffusion model'' describing moving of the DNA over the histone octamer surface in 1 bp intervals. Thus, a single base pair distortion is continuously propagated through the nucleosome, transiently storing one additional basepair in the realm of the nucleoprotein structure. This model is supported by nucleosomal crystal structures exhibiting such a single-basepair "twist defect" [39,41]. However, several studies could not confirm such a translocation model. Experiments using nicked or gapped DNA substrates that uncouple DNA rotation mediated processes still allowed SWI/SNF and ISWI dependent nucleosome remodeling, arguing against a sole twist-

Alternatively, it was suggested that nucleosomes are repositioned according to the ''loop recapture model'', proposing a detachment of a DNA segment from the histone octamer surface at the entry site of the nucleosome. The exposed octamer surface would interact with more distant regions of the DNA molecule, resulting in the formation of a DNA loop on the histone octamer surface. This DNA loop would translocate over the octamer surface in an energy-neutral process, by releasing and rebinding adjacent sequences on the protein surface. DNA loop propagation would change the translational position of the nucleosome, according to the size of the DNA loop [45]. This model is strengthened by biochemical and recent single molecule studies. ACF remodeling complex was shown to cause the unwrapping of DNA,

Nhp10

**3. Translocation mechanism of chromatin remodelers**

Rvb1, Rvb2, Arp5, Arp8, Arp4, Act1, Taf14, les1, Ies2, les3, les4, les5, Ies6,

Rvb1, Rvb2, Arp6, Arp4, Act1, Yaf9, Swc4/Eaf2, Swc2, Swc3, Swc4, Swc5, Swc6, Yaf9, Bdf1, Swc7, H2AZ, H2B

*Yeast*

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INO80 Ino80

SWR1 Swr1

**Table 4.** Selected INO80 family remodelers.

mechanisms of the translocation step.

diffusion mechanism [42-44].


**Table 3.** Selected CHD family remodelers.

#### **2.4. INO80 family**

The specific feature of the remodeling enzymes belonging to the INO80 (inositol requiring 80) family is the split ATPase domain. This unique module retains ATPase activity, and acts as a scaffold for the association with the RuvB-like proteins, Rvb1 and Rvb2. RuvB is a bacterial ATP-dependent helicase that forms a double hexamer around Holliday junctions to promote their migration during homologous recombination [30]. Unlike remodelers of other families, the INO80 complex exhibits DNA helicase activity and binds to specialized DNA structures *in vitro.* These DNA structures resemble Holliday junctions and replication forks consistent with the function of the complex in homologous recombination and DNA replication [31,32]. Yeast INO80 was shown to control the genome-wide distribution and dynamics of the histone variant H2A.Z. INO80 and SWR1 were shown to exhibit histone-exchange activity, being capable to replace nucleosomal H2A.Z/H2B with free H2A/H2B dimers [33,34]. Both remod‐ eling complexes can slide nucleosomes *in vitro* on a reconstituted chromatin template and evict histones from DNA [35-37]. In addition to the role of INO80 in recombination and DNA replication, it is suggested to regulate the transcription level of about 20% of the yeast genes and to participate in DNA double-strand break repair via the interaction with γ–H2AX and recruit the MRX and Mec1 complexes to the DNA damage site [33].


**Table 4.** Selected INO80 family remodelers.

to maintain nucleosomal organization in yeast [27]. To date, Chd3, Chd4, Chd5 and Chd7 have been implicated in human disease processes. Chd3 and Chd4 have been identified as autoan‐ tigens in patients with dermatomyositis, a connective-tissue disease characterized by inflam‐ mation of both muscles and skin. Chd3 is associated with Hodgkin's lymphoma and Chd5 is associated with neuroblastoma, a malignant neoplasm of the peripheral sympathetic nervous system frequently affecting infants and children [28]. Haploinsufficiency of Chd7 in humans results in the CHARGE syndrome. Chd7 is essential for the develompment of multipotent migratory neural crest cells, which contribute to the formation of many tissues affected in

**Complex Catalytic subunit Auxillary subunits Organism**

Chd2 Chd2 *Fly*

RbAp46/48, p66α/β, DOC-1?

*Human*

NuRD Mi-2 MBD2/3, MTA, RPD3, p55, p66/68

NuRD Chd3/Chd4 MBD3, MTA1/2/3, HDAC1/2,

recruit the MRX and Mec1 complexes to the DNA damage site [33].

Chd5 Unknown

Chd7 PARP1, PBAF complex

The specific feature of the remodeling enzymes belonging to the INO80 (inositol requiring 80) family is the split ATPase domain. This unique module retains ATPase activity, and acts as a scaffold for the association with the RuvB-like proteins, Rvb1 and Rvb2. RuvB is a bacterial ATP-dependent helicase that forms a double hexamer around Holliday junctions to promote their migration during homologous recombination [30]. Unlike remodelers of other families, the INO80 complex exhibits DNA helicase activity and binds to specialized DNA structures *in vitro.* These DNA structures resemble Holliday junctions and replication forks consistent with the function of the complex in homologous recombination and DNA replication [31,32]. Yeast INO80 was shown to control the genome-wide distribution and dynamics of the histone variant H2A.Z. INO80 and SWR1 were shown to exhibit histone-exchange activity, being capable to replace nucleosomal H2A.Z/H2B with free H2A/H2B dimers [33,34]. Both remod‐ eling complexes can slide nucleosomes *in vitro* on a reconstituted chromatin template and evict histones from DNA [35-37]. In addition to the role of INO80 in recombination and DNA replication, it is suggested to regulate the transcription level of about 20% of the yeast genes and to participate in DNA double-strand break repair via the interaction with γ–H2AX and

CHARGE syndrome [29].

8 Chromatin Remodelling

Chd1 Chd1

Chd1 Chd1

Chd2 Chd2

**Table 3.** Selected CHD family remodelers.

**2.4. INO80 family**

#### **3. Translocation mechanism of chromatin remodelers**

Chromatin remodelers use the energy of ATP hydrolysis to assemble, reposition or evict histones from DNA. Nucleosome repositioning by remodelers can be described as a 3-step mechanism: 1) initiation step that requires the recognition and specific binding to the substrate, 2) several translocation steps with varying step-lengths and kinetics depending on the particular remodeling enzyme and on the properties of the underlying DNA sequence, 3) release step, which occurs at energetically favourable positions depending on the combination of remodeler and DNA sequence/structure at this site [6,38]. This chapter will focus on the mechanisms of the translocation step.

Proposed models for nucleosome remodeling suggest that only a minor fraction of the 358 direct and indirect histone-DNA interactions are disrupted at a given time of the reaction, as the energy of ATP hydrolysis would not be sufficient to fully disrupt the nucleoprotein structure [39,40]. One of the first mechanisms proposed, is the ''twist diffusion model'' describing moving of the DNA over the histone octamer surface in 1 bp intervals. Thus, a single base pair distortion is continuously propagated through the nucleosome, transiently storing one additional basepair in the realm of the nucleoprotein structure. This model is supported by nucleosomal crystal structures exhibiting such a single-basepair "twist defect" [39,41]. However, several studies could not confirm such a translocation model. Experiments using nicked or gapped DNA substrates that uncouple DNA rotation mediated processes still allowed SWI/SNF and ISWI dependent nucleosome remodeling, arguing against a sole twistdiffusion mechanism [42-44].

Alternatively, it was suggested that nucleosomes are repositioned according to the ''loop recapture model'', proposing a detachment of a DNA segment from the histone octamer surface at the entry site of the nucleosome. The exposed octamer surface would interact with more distant regions of the DNA molecule, resulting in the formation of a DNA loop on the histone octamer surface. This DNA loop would translocate over the octamer surface in an energy-neutral process, by releasing and rebinding adjacent sequences on the protein surface. DNA loop propagation would change the translational position of the nucleosome, according to the size of the DNA loop [45]. This model is strengthened by biochemical and recent single molecule studies. ACF remodeling complex was shown to cause the unwrapping of DNA, roughly 20 and 40 bp, from the nucleosomal border [46]. ATP dependent translocation of SWI/ SNF and RSC on DNA and nucleosomal templates produces DNA loops and nucleosome remodeling by RSC was shown to produce a remodeled intermediate containing internal DNA loops [47].

Nucleosomal translocation and its step-size depend on the size of the DNA loop, a parame‐ ter that depends on the nature of the remodeling enzyme. Single molecule studies with the remodeling complex ACF suggested an initial step size of 7 bp and subsequent steps of 3-4 bp [48], whereas RSC was shown to exhibit a step size of 2 bp [49]. Within a strong nucleo‐ somal positioning sequence both recombinant *Drosophila* Mi-2 and native RSC from yeast repositioned the nucleosome at 10 bp intervals, which are intrinsic to the positioning sequence. Furthermore, RSC-catalysed nucleosome translocation was noticeably more efficient when beyond the influence of this sequence. Interestingly, under limiting ATP conditions RSC preferred to position the nucleosome with 20 bp intervals within the positioning sequence, suggesting that native RSC preferentially translocates nucleosomes with 15 to 25 bp DNA steps [38]. Lately, it was proposed that loops do not freely diffuse about the exterior of the nucleosome but rather feed through specific restriction points by threading past fixed constrictions [47].

#### **4. Targeting remodelers: Signals**

One of the enigmas is the cellular requirement for 53 types of remodeling enzymes in humans that are capable to form hundreds to thousands of different remodeling complexes [6]. Such high numbers already suggest specialized functions for individual complexes and that remodeling enzymes mobilize nucleosomes in a specific manner. Many chromatin remodelers bind to DNA and nucleosomes in a sequence independent manner *in vitro*, albeit they exhibit complex specific features in nucleosome positioning and many of the complex subunits recognize specific chromatin features, targeting the complexes to defined genomic regions *in vivo*. The redundancy of enzymes and remodeling complexes suggest that they establish local and context specific chromatin structures and thereby regulate the DNA dependent processes. This chapter addresses the known and potential targeting mechanisms via DNA binding factors, the recognition of local chromatin features via functional RNA molecules and the impact of sequence context on the local chromatin structures (Fig. 2).

different complexes with various non-catalytic subunits, changed the outcome of the remod‐ eling reaction (Fig. 3). Additionally, recent genome-wide studies compared 4 different remodeling complexes and similarly, it was observed that each remodeler exhibits a unique set of genomic targets correlating with distinct chromatin signatures [52]. Thus, these data suggest that the remodelers are capable to recognize the underlying DNA sequence/structure

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The remodeling complexes contain DNA-binding motifs that are present in the catalytic or/and in accessory subunits (Fig. 1). For example, catalytic subunit Snf2H contains a SANT-SLIDE domain and in addition the WAC and AT hook motifs in the Acf1 and Tip5 proteins [4, 53-57]. These modules allow the specific recognition of DNA sequences and determine the outcome of a remodeling reaction, as it was shown by exchanging such domains between remodeling enzymes [38,58-60]. Nucleosome positioning is most probably affected by the

and accordingly establish specific chromatin structures.

**Figure 2.** Targeting signals for chromatin remodeling complexes.

#### **4.1. Direct chromatin targets**

#### *4.1.1. DNA and RNA sequence/structure*

Mechanistical analysis of the nucleosome remodeling process revealed that binding of a remodeling complex to a mononucleosomal substrate results in a specific and ATP-dependent repositioning of the nucleosome on the DNA [50,51]. An *in vitro* study compared 7 different remodelers on different nucleosomal templates [6]. It appeared that each enzyme placed the nucleosomes at distinct positions and that even the same remodeling enzyme present in a

**Figure 2.** Targeting signals for chromatin remodeling complexes.

roughly 20 and 40 bp, from the nucleosomal border [46]. ATP dependent translocation of SWI/ SNF and RSC on DNA and nucleosomal templates produces DNA loops and nucleosome remodeling by RSC was shown to produce a remodeled intermediate containing internal DNA

Nucleosomal translocation and its step-size depend on the size of the DNA loop, a parame‐ ter that depends on the nature of the remodeling enzyme. Single molecule studies with the remodeling complex ACF suggested an initial step size of 7 bp and subsequent steps of 3-4 bp [48], whereas RSC was shown to exhibit a step size of 2 bp [49]. Within a strong nucleo‐ somal positioning sequence both recombinant *Drosophila* Mi-2 and native RSC from yeast repositioned the nucleosome at 10 bp intervals, which are intrinsic to the positioning sequence. Furthermore, RSC-catalysed nucleosome translocation was noticeably more efficient when beyond the influence of this sequence. Interestingly, under limiting ATP conditions RSC preferred to position the nucleosome with 20 bp intervals within the positioning sequence, suggesting that native RSC preferentially translocates nucleosomes with 15 to 25 bp DNA steps [38]. Lately, it was proposed that loops do not freely diffuse about the exterior of the nucleosome but rather feed through specific restriction points by threading past fixed

One of the enigmas is the cellular requirement for 53 types of remodeling enzymes in humans that are capable to form hundreds to thousands of different remodeling complexes [6]. Such high numbers already suggest specialized functions for individual complexes and that remodeling enzymes mobilize nucleosomes in a specific manner. Many chromatin remodelers bind to DNA and nucleosomes in a sequence independent manner *in vitro*, albeit they exhibit complex specific features in nucleosome positioning and many of the complex subunits recognize specific chromatin features, targeting the complexes to defined genomic regions *in vivo*. The redundancy of enzymes and remodeling complexes suggest that they establish local and context specific chromatin structures and thereby regulate the DNA dependent processes. This chapter addresses the known and potential targeting mechanisms via DNA binding factors, the recognition of local chromatin features via functional RNA molecules and the

Mechanistical analysis of the nucleosome remodeling process revealed that binding of a remodeling complex to a mononucleosomal substrate results in a specific and ATP-dependent repositioning of the nucleosome on the DNA [50,51]. An *in vitro* study compared 7 different remodelers on different nucleosomal templates [6]. It appeared that each enzyme placed the nucleosomes at distinct positions and that even the same remodeling enzyme present in a

impact of sequence context on the local chromatin structures (Fig. 2).

loops [47].

10 Chromatin Remodelling

constrictions [47].

**4. Targeting remodelers: Signals**

**4.1. Direct chromatin targets**

*4.1.1. DNA and RNA sequence/structure*

different complexes with various non-catalytic subunits, changed the outcome of the remod‐ eling reaction (Fig. 3). Additionally, recent genome-wide studies compared 4 different remodeling complexes and similarly, it was observed that each remodeler exhibits a unique set of genomic targets correlating with distinct chromatin signatures [52]. Thus, these data suggest that the remodelers are capable to recognize the underlying DNA sequence/structure and accordingly establish specific chromatin structures.

The remodeling complexes contain DNA-binding motifs that are present in the catalytic or/and in accessory subunits (Fig. 1). For example, catalytic subunit Snf2H contains a SANT-SLIDE domain and in addition the WAC and AT hook motifs in the Acf1 and Tip5 proteins [4, 53-57]. These modules allow the specific recognition of DNA sequences and determine the outcome of a remodeling reaction, as it was shown by exchanging such domains between remodeling enzymes [38,58-60]. Nucleosome positioning is most probably affected by the

methylated DNA, but binds to the pRNA (promoter RNA). The pRNA is folded into the hairpin-like structure which is bound by NoRC and participates in the recruitment NoRC to

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The histone code hypothesis suggests that individual covalent modifications of histones or combinations of these modifications are recognized by specific readers which determine downstream events [3]. Chromatin remodeling complexes contain histone code reader domains, allowing the targeting to specifically modified chromatin domains and thereby enabling the establishment of a remodeler dependent nucleosomal positioning landscape.

The SWI/SNF type of remodelers contain bromodomains, interacting specifically with acetylated lysines on the histone tails [69]. Acetylation of the histone H3 N-terminal tail facilitated the recruitment and nucleosome mobilization by SWI/SNF and RSC. Tetra-acety‐ lated H3 tails, but not tetra-acetylated H4 tails, increased the affinity of RSC and SWI/SNF for nucleosomes, which is dependent on the SWI/SNF bromodomain, but is not further enhanced by additional bromodomains present in RSC [70]. By contrast, the SANT domain of the ISWI type of remodelers is known to interact with unmodified histone tails. The H4 tail has been shown to play a decisive role in ISWI remodeling, in that both, the complete removal of the H4 tail [71,72] and its site-specific acetylation suppress the remodeling action of ISWI [73]. Human Chd1 protein interacts with H3K4me2/3 via its double chromodomains, which fold into a functional unit. On the other hand, nucleosomal H3K4 methylation reduces the affinity of the NuRD complex for H3 tail binding. It was shown that the second PHD finger of Chd4 preferentially interacts with unmodified H3K4 and H3K9me3 [74,75]. Full-length NURF301 the large subunit of the ISWI containing NURF complex contains a C-terminal bromodomain and a juxtaposed PHD finger that bind H3K4me3 and H4K16Ac, respectively. However, a NURF301 isoform lacking these C-terminal domains is also detected in cells, suggesting that alternative splicing can change targeting signals and localisation of the complexes within the genome. It was concluded, that the specific recognition of the posttranslational marks by NURF is important for the regulation of primary spermatocyte differentiation in *Drosophila* [76].

Non-canonical histone variants differ from the canonical histones at the level of their primary sequence,whichcanrangefromafewaminoacidchanges tolargedomains.Thesevariants show distinct regulatory mechanisms for their expression and deposition, resulting in the establish‐ ment of chromatin domains with specific properties. The exchange of canonical histones for the variant ones is an active process, requiring the activity of remodeling enzymes and the action of

Analyzing the dynamic changes in the composition of histone variants in nuclear-transferred embryos revealed that the donor cell-derived histone H3 variants H3.1, H3.2, and H3.3, as well as H2A and H2A.Z, were rapidly eliminated from the chromatin of nuclei transplanted into enucleated oocytes. In parallel to this removal, oocyte-stored histone H3 variants and H2A.X

RNA and DNA polymerases that actively displace the histones from DNA [77].

the rRNA gene promoter region [56,66-68].

*4.1.2. Histone modifications*

*4.1.3. Histone variants*

**Figure 3.** Bandshift assay showing that the chromatin remodelers position nucleosomes in a DNA sequence-specific manner. The 350 bp DNA, containing the hsp70 promoter sequence, was assembled into the nucleomes via salt dialy‐ sis. Five different single-nucleosomes were observed in the bandshift assay (mapped as N1, N2, N3, N4 and N4') and this was used as a substrate for seven recombinant chromatin remodelers (lane 1). Brg1, Chd1, ISWI, Snf2H, Mi-2, ACF and NURF in the presence of ATP repositioned nucleosomes in a remodeler-specific manner (lanes 2-8) [6].

different binding affinities of those motifs to the non-remodeled and remodeled substrates and the sequence dependent flexibility and stability of the particle, impacting the final outcome of the reaction. The role of specific DNA sequences in nucleosome positioning was shown for the ISWI-containing complex ACF, which positions a nucleosome relative to an intrinsically curved DNA sequence element [6].

Not only individual positions, but also internucleosomal distances depend on the DNA binding domains of the enzymes. ACF interacts with linker DNA and is capable to sense its length [61]. This structural element appears to play a key role in the positioning of nucleosomes in regular arrays, as the remodeler-induced mobility of the nucleosome is biased towards the longer flanking DNA [62]. Similarly, the Chd1 remodeler was described to sense the length of linker DNA [63].

Moreover, unusual DNA structures like quadruplexes could represent specific targeting signals. ATRX recognises G-rich repeat sequences, which are prevalent in telomeres [64]. These repeat sequences likely to form G-quadruplex (G4) structures, and ATRX preferentially binds to such a G4 structure *in vitro*. Such alternative DNA structures are believed to destabilize the genome and it is enticing to think that ATRX is responsible for stabilizing G-rich regions of the genome by remodeling G4 DNA and incorporating H3.3-containing nucleosomes [64].

Methylated CpG islands in the DNA were shown to be recognized by MBD (methyl-binding domain) domains, so it can serve as a targeting signal for particular remodelers. For example, MBD2 recruits the NuRD complex to methylated promoters [65]. The related TAM domain (MBD-like) in Tip5, the noncatalytic subunit of the NoRC complex, does not recognise methylated DNA, but binds to the pRNA (promoter RNA). The pRNA is folded into the hairpin-like structure which is bound by NoRC and participates in the recruitment NoRC to the rRNA gene promoter region [56,66-68].

#### *4.1.2. Histone modifications*

The histone code hypothesis suggests that individual covalent modifications of histones or combinations of these modifications are recognized by specific readers which determine downstream events [3]. Chromatin remodeling complexes contain histone code reader domains, allowing the targeting to specifically modified chromatin domains and thereby enabling the establishment of a remodeler dependent nucleosomal positioning landscape.

The SWI/SNF type of remodelers contain bromodomains, interacting specifically with acetylated lysines on the histone tails [69]. Acetylation of the histone H3 N-terminal tail facilitated the recruitment and nucleosome mobilization by SWI/SNF and RSC. Tetra-acety‐ lated H3 tails, but not tetra-acetylated H4 tails, increased the affinity of RSC and SWI/SNF for nucleosomes, which is dependent on the SWI/SNF bromodomain, but is not further enhanced by additional bromodomains present in RSC [70]. By contrast, the SANT domain of the ISWI type of remodelers is known to interact with unmodified histone tails. The H4 tail has been shown to play a decisive role in ISWI remodeling, in that both, the complete removal of the H4 tail [71,72] and its site-specific acetylation suppress the remodeling action of ISWI [73]. Human Chd1 protein interacts with H3K4me2/3 via its double chromodomains, which fold into a functional unit. On the other hand, nucleosomal H3K4 methylation reduces the affinity of the NuRD complex for H3 tail binding. It was shown that the second PHD finger of Chd4 preferentially interacts with unmodified H3K4 and H3K9me3 [74,75]. Full-length NURF301 the large subunit of the ISWI containing NURF complex contains a C-terminal bromodomain and a juxtaposed PHD finger that bind H3K4me3 and H4K16Ac, respectively. However, a NURF301 isoform lacking these C-terminal domains is also detected in cells, suggesting that alternative splicing can change targeting signals and localisation of the complexes within the genome. It was concluded, that the specific recognition of the posttranslational marks by NURF is important for the regulation of primary spermatocyte differentiation in *Drosophila* [76].

#### *4.1.3. Histone variants*

different binding affinities of those motifs to the non-remodeled and remodeled substrates and the sequence dependent flexibility and stability of the particle, impacting the final outcome of the reaction. The role of specific DNA sequences in nucleosome positioning was shown for the ISWI-containing complex ACF, which positions a nucleosome relative to an intrinsically

and NURF in the presence of ATP repositioned nucleosomes in a remodeler-specific manner (lanes 2-8) [6].

**Figure 3.** Bandshift assay showing that the chromatin remodelers position nucleosomes in a DNA sequence-specific manner. The 350 bp DNA, containing the hsp70 promoter sequence, was assembled into the nucleomes via salt dialy‐ sis. Five different single-nucleosomes were observed in the bandshift assay (mapped as N1, N2, N3, N4 and N4') and this was used as a substrate for seven recombinant chromatin remodelers (lane 1). Brg1, Chd1, ISWI, Snf2H, Mi-2, ACF

Not only individual positions, but also internucleosomal distances depend on the DNA binding domains of the enzymes. ACF interacts with linker DNA and is capable to sense its length [61]. This structural element appears to play a key role in the positioning of nucleosomes in regular arrays, as the remodeler-induced mobility of the nucleosome is biased towards the longer flanking DNA [62]. Similarly, the Chd1 remodeler was described to sense the length of

Moreover, unusual DNA structures like quadruplexes could represent specific targeting signals. ATRX recognises G-rich repeat sequences, which are prevalent in telomeres [64]. These repeat sequences likely to form G-quadruplex (G4) structures, and ATRX preferentially binds to such a G4 structure *in vitro*. Such alternative DNA structures are believed to destabilize the genome and it is enticing to think that ATRX is responsible for stabilizing G-rich regions of the genome by remodeling G4 DNA and incorporating H3.3-containing nucleosomes [64].

Methylated CpG islands in the DNA were shown to be recognized by MBD (methyl-binding domain) domains, so it can serve as a targeting signal for particular remodelers. For example, MBD2 recruits the NuRD complex to methylated promoters [65]. The related TAM domain (MBD-like) in Tip5, the noncatalytic subunit of the NoRC complex, does not recognise

curved DNA sequence element [6].

linker DNA [63].

12 Chromatin Remodelling

Non-canonical histone variants differ from the canonical histones at the level of their primary sequence,whichcanrangefromafewaminoacidchanges tolargedomains.Thesevariants show distinct regulatory mechanisms for their expression and deposition, resulting in the establish‐ ment of chromatin domains with specific properties. The exchange of canonical histones for the variant ones is an active process, requiring the activity of remodeling enzymes and the action of RNA and DNA polymerases that actively displace the histones from DNA [77].

Analyzing the dynamic changes in the composition of histone variants in nuclear-transferred embryos revealed that the donor cell-derived histone H3 variants H3.1, H3.2, and H3.3, as well as H2A and H2A.Z, were rapidly eliminated from the chromatin of nuclei transplanted into enucleated oocytes. In parallel to this removal, oocyte-stored histone H3 variants and H2A.X were incorporated into the transplanted nuclei, while the incorporation of H2A and H2A.Z was minimal or not detected. The incorporation of these variant histones was independent of DNA replication suggesting an active process depending on the remodeling complexes [78].

the nuclear receptor co-activator protein Flightless-I, which then directly binds to both, the ER and the BAF53 subunit of the SWI/SNF complex [86]. The ISWI subfamily containing remod‐ eling complex NoRC is directly recruited to the rRNA gene by the transcription factor TTF-I,

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Several studies demonstrated the targeting of Chd4 to sites of DNA double strand breaks in a PARP dependent manner [87]. The enzyme was shown to bind to the poly(ADP-ribose) polymer *in vitro*. Also ALC1 binds to PAR via its macrodomain and is recruited to sites of DNA

The human genome is packaged into some 30 millions of nuclesosomes that have to be organized into functional chromatin domains with specific local structures. In order to identify target sites or to detect nucleosomes that have to be repositioned, the remodeling complexes have to detect such sites in chromatin very quickly. Potential genome screening mechanisms

In the seventies, JJ Hopfield introduced the kinetic proofreading mechanism for reducing errors in biological systems. He used Michaelis Menten kinetics to explain how enzymes discriminate between different substrates [89]. A similar kinetic proofreading mechanism can be used to describe the action of remodelers, where "good" substrates are characterized by a high affinity of the remodeler for the nucleosome substrate (low value of Michaelis-Menten constant *K*M) and a high catalytic conversion rate *k*cat, efficiently moving the nucleosome to the end position of the translocation reaction. Thus, the *k*cat/*K*M ratio is high as expected for an efficient catalytic process. The opposite would be true for "bad" nucleosomal substrates, i. e. having a low *k*cat/*K*<sup>M</sup> ratio. According to this model, remodeler bind to "good" substrates and move them as long, as they are converted to "bad" substrates, exhibiting a lower affinity for the remodeler. The remodelers are released from the low affinity substrates, a mechanism termed "release model" (Fig. 4). In an alternative ''arrest model", all nucleosomal substrates are recognized with similar affinities, but remodeler has a slow translocation rate on a ''bad'' substrate. *In vitro* binding assays showed that the Chd1 and ACF complexes were bound with lower afiinity to the nucleosomes at positions that reflected the end points of the remodeling reaction, suggesting that those enzymes function according to the release model (Fig. 4) [6].

Many proteins in the nucleus, including several remodelers are highly mobile as revealed by fluorescence recovery after photobleaching (FRAP) experiments. For proteins that do not

inducing gene silencing and heterochromatin formation [56].

**5. Targeting remodelers: Search mechanism**

by the remodelers are discussed in this chapter.

**5.2. The continuous sampling mechanism**

**5.1. Release/termination model**

*4.2.2. Poly(ADP-ribose) polymer*

damage [88].

An ATRX (α-thalassemia X-linked mental retardation protein) – Daxx (death domain associ‐ ated protein) complex can effectively assemble H3.3-containing nucleosomes in murine embryonic stem cells. It was shown that ATRX recruits Daxx to telomeres, and both complex subunits are required for H3.3 deposition at telomeric chromatin [79]. Chd1 in *Drosophila* embryos is required for the incorporation of the H3.3 variant into the male pronucleus, enabling the paternal genome to participate in zygotic mitosis [80]. The exchange of H2A.Z for H2A by the yeast SWR1 complex is in mechanistical terms the best described model system. H2A.Z replacement studied *in vitro* occurs in a stepwise and unidirectional fashion, exchang‐ ing one H2A.Z-H2B dimer at a time. Thereby heterotypic nucleosomes, containing one H2A.Z and one H2A molecule are established as intermediates and the homotypic H2A.Z nucleo‐ somes as end products are generated in a second exchange step. The ATPase activity of SWR1 is specifically stimulated by H2A-containing nucleosomes without active displacement of histone H2A. Remarkably, the addition of free H2A.Z-H2B dimers results in a further stimu‐ lation of its ATPase activity and the combined eviction of nucleosomal H2A-H2B and depo‐ sition of H2A.Z-H2B. These results suggest that the combination of H2A-containing nucleosome and the presence of free H2A.Z-H2B dimer act as effector and substrate for SWR1 to govern the specificity and outcome of the replacement reaction [81]. Chromatin remodeling enzymes are also involved in the modification and dynamics of the histone variant H2A.X, which is phosphorylated upon DNA damage and repair. The WICH (WSTF-Snf2H) chromatin remodeling complex exhibits a novel kinase domain capable to phosphorylate Y142 on H2A.X. Both proteins, WSTF and Snf2H were also shown to bind to H2A.X in co-immunoprecipitation experiments [82]. In addition, it was recently shown that the activity of the Lsh remodeling enzyme is necessary for the efficient phosphorylation of H2A.X at DNA double-strand breaks and the successful repair of DNA damage [83].

#### **4.2. Indirect chromatin targets**

#### *4.2.1. Sequence specific DNA binding proteins*

The DNA-sequence dependent recruitment of remodelers is not necessarily mediated by the remodeling complex subunits themselves but can also occur via transient interactions with other sequence specific DNA binding proteins. For example, the NuRD complex is recruited to the various promoters of the target genes via interaction with several transcription factors and co-regulators such as NAB2, Ikaros, FOG1, BCL11B and several other factors described by Brehm and colleagues [26]. Genome wide expression, genetic and biochemical analysis established that TramTrack69, MEP1, and the *Drosophila* remodeling enzyme Mi-2 cooperate to control transcription levels of target genes [84]. It was also shown that Mi-2 binds to SUMO and to SUMO-ylated proteins giving rise to the hypothesis that this is a common signal for the Mi-2 recruitment. Similarly, Brg1 containing complexes are targeted via Sox10 to two key target genes in the Schwann cells [85]. Recruitment of SWI/SNF to the target genes of ERα requires the nuclear receptor co-activator protein Flightless-I, which then directly binds to both, the ER and the BAF53 subunit of the SWI/SNF complex [86]. The ISWI subfamily containing remod‐ eling complex NoRC is directly recruited to the rRNA gene by the transcription factor TTF-I, inducing gene silencing and heterochromatin formation [56].

#### *4.2.2. Poly(ADP-ribose) polymer*

were incorporated into the transplanted nuclei, while the incorporation of H2A and H2A.Z was minimal or not detected. The incorporation of these variant histones was independent of DNA replication suggesting an active process depending on the remodeling complexes [78].

An ATRX (α-thalassemia X-linked mental retardation protein) – Daxx (death domain associ‐ ated protein) complex can effectively assemble H3.3-containing nucleosomes in murine embryonic stem cells. It was shown that ATRX recruits Daxx to telomeres, and both complex subunits are required for H3.3 deposition at telomeric chromatin [79]. Chd1 in *Drosophila* embryos is required for the incorporation of the H3.3 variant into the male pronucleus, enabling the paternal genome to participate in zygotic mitosis [80]. The exchange of H2A.Z for H2A by the yeast SWR1 complex is in mechanistical terms the best described model system. H2A.Z replacement studied *in vitro* occurs in a stepwise and unidirectional fashion, exchang‐ ing one H2A.Z-H2B dimer at a time. Thereby heterotypic nucleosomes, containing one H2A.Z and one H2A molecule are established as intermediates and the homotypic H2A.Z nucleo‐ somes as end products are generated in a second exchange step. The ATPase activity of SWR1 is specifically stimulated by H2A-containing nucleosomes without active displacement of histone H2A. Remarkably, the addition of free H2A.Z-H2B dimers results in a further stimu‐ lation of its ATPase activity and the combined eviction of nucleosomal H2A-H2B and depo‐ sition of H2A.Z-H2B. These results suggest that the combination of H2A-containing nucleosome and the presence of free H2A.Z-H2B dimer act as effector and substrate for SWR1 to govern the specificity and outcome of the replacement reaction [81]. Chromatin remodeling enzymes are also involved in the modification and dynamics of the histone variant H2A.X, which is phosphorylated upon DNA damage and repair. The WICH (WSTF-Snf2H) chromatin remodeling complex exhibits a novel kinase domain capable to phosphorylate Y142 on H2A.X. Both proteins, WSTF and Snf2H were also shown to bind to H2A.X in co-immunoprecipitation experiments [82]. In addition, it was recently shown that the activity of the Lsh remodeling enzyme is necessary for the efficient phosphorylation of H2A.X at DNA double-strand breaks

The DNA-sequence dependent recruitment of remodelers is not necessarily mediated by the remodeling complex subunits themselves but can also occur via transient interactions with other sequence specific DNA binding proteins. For example, the NuRD complex is recruited to the various promoters of the target genes via interaction with several transcription factors and co-regulators such as NAB2, Ikaros, FOG1, BCL11B and several other factors described by Brehm and colleagues [26]. Genome wide expression, genetic and biochemical analysis established that TramTrack69, MEP1, and the *Drosophila* remodeling enzyme Mi-2 cooperate to control transcription levels of target genes [84]. It was also shown that Mi-2 binds to SUMO and to SUMO-ylated proteins giving rise to the hypothesis that this is a common signal for the Mi-2 recruitment. Similarly, Brg1 containing complexes are targeted via Sox10 to two key target genes in the Schwann cells [85]. Recruitment of SWI/SNF to the target genes of ERα requires

and the successful repair of DNA damage [83].

*4.2.1. Sequence specific DNA binding proteins*

**4.2. Indirect chromatin targets**

14 Chromatin Remodelling

Several studies demonstrated the targeting of Chd4 to sites of DNA double strand breaks in a PARP dependent manner [87]. The enzyme was shown to bind to the poly(ADP-ribose) polymer *in vitro*. Also ALC1 binds to PAR via its macrodomain and is recruited to sites of DNA damage [88].

#### **5. Targeting remodelers: Search mechanism**

The human genome is packaged into some 30 millions of nuclesosomes that have to be organized into functional chromatin domains with specific local structures. In order to identify target sites or to detect nucleosomes that have to be repositioned, the remodeling complexes have to detect such sites in chromatin very quickly. Potential genome screening mechanisms by the remodelers are discussed in this chapter.

#### **5.1. Release/termination model**

In the seventies, JJ Hopfield introduced the kinetic proofreading mechanism for reducing errors in biological systems. He used Michaelis Menten kinetics to explain how enzymes discriminate between different substrates [89]. A similar kinetic proofreading mechanism can be used to describe the action of remodelers, where "good" substrates are characterized by a high affinity of the remodeler for the nucleosome substrate (low value of Michaelis-Menten constant *K*M) and a high catalytic conversion rate *k*cat, efficiently moving the nucleosome to the end position of the translocation reaction. Thus, the *k*cat/*K*M ratio is high as expected for an efficient catalytic process. The opposite would be true for "bad" nucleosomal substrates, i. e. having a low *k*cat/*K*<sup>M</sup> ratio. According to this model, remodeler bind to "good" substrates and move them as long, as they are converted to "bad" substrates, exhibiting a lower affinity for the remodeler. The remodelers are released from the low affinity substrates, a mechanism termed "release model" (Fig. 4). In an alternative ''arrest model", all nucleosomal substrates are recognized with similar affinities, but remodeler has a slow translocation rate on a ''bad'' substrate. *In vitro* binding assays showed that the Chd1 and ACF complexes were bound with lower afiinity to the nucleosomes at positions that reflected the end points of the remodeling reaction, suggesting that those enzymes function according to the release model (Fig. 4) [6].

#### **5.2. The continuous sampling mechanism**

Many proteins in the nucleus, including several remodelers are highly mobile as revealed by fluorescence recovery after photobleaching (FRAP) experiments. For proteins that do not

events seem to be unproductive, meaning that the remodeling reaction does not occur. From the experimentally determined relatively high remodeling enzyme concentrations (in the range of μM) and short chromatin bound residence times around 100 ms, average sampling times of tens of seconds to minutes were calculated for Snf2H containing remodelers to probe 99% of all genomic nucleosomes. Thus, a combination of high remodeler concentrations, short residence times in the chromatin bound state and fast 3D diffusive translocations in the intervening periods appears to be an efficient mechanism to keep nucleosomes in place [90,92].

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**Figure 5.** Genome-wide search for nucleosomal targets by remodeling enzymes. A) Continuous sampling mechanism. It is a diffusion-driven, rapid sampling of nonspecific sites with the remodeling enzymes binding only transiently to the nucleosomes. Most binding events are non-productive, as the nucleosomes are well positioned. B) Immobilization mechanism. Remodelers are recruited to the particular sites where they change nucleosomal positions. Targeting is achieved upon recognition of specific signals like histone modifications, chromatin-associated proteins, structural fea‐

In parallel with the continuous sampling mechanism, remodeling complexes are engaged by specific recruitment or immobilization at specific target sites. The respective mechanisms are described in chapter 4. For example, when cells were treated with dexamethasone, BRG1 and BRMwere concentratedina single spotinthenucleus, as revealedby immunofluorescence.The site coincided with the multimerized MMTV DNA and RNA FISH signals, showing that the enzymes are recruited to the MMTV array in a hormone-dependent manner. In this case the recruitment of the SWI/SNF machine results in the maintenance of an active chromatin struc‐ ture that is compatible with transcription [93]. In other cases, like the nucleolar remodeling complex NoRC recruitment to the rRNA genes, continuous targeting results in gene repres‐

tures of the chromatin environment or even by small molecules such as hormones.

**5.3. Immobilization**

**Figure 4.** Model describing the affinity of remodelers to nucleosomes at different positions on the DNA. A) In the re‐ lease model, the remodeling complex has a weaker binding affinity to the end-positioned nucleosome in comparison to any other nucleosome. In the arrest model, the remodeler binds all nucleosomes with similar affinity, but the trans‐ location rate constant is much slower on a nucleosome present in the final position. B) Chd1 positions nucleosomes according to the release mechanism. Nucleosome position-dependent differences in the affinity of the remodeling complexes to the nucleosomal substrate were analyzed by bandshift assays. Remodeling reaction of Chd1 on mono‐ nucleosomal substrates reconstituted on a 350 bp DNA fragment containing hsp70 promoter region. Chd1 positions nulceosomes to the N3 and N2 positions. C) Binding reaction of Chd1 to the nucleosomes. The position of the DNA– Chd1 (D/C) and the nucleosome–Chd1 (N/C) complexes are indicated. The position of the N3 nucleosome is shown by a black box. Nucleosomes positioned at this site are bound by Chd1 with the lowest affinity. This position is at the same time the preferred endpoint of the remodeling reaction [6].

interact with any cellular structures, FRAP kinetics are a direct reflection of their translational motion properties. In contrast, proteins that bind to immobile structures such as chromatin, exhibit a slower overall mobility. The mobility of ISWI family remodelers Snf2H, Snf2L and Snf2L+13 (an ATPase inactive variant of the Snf2L) was studied in living U2OS cells. During G1/2 phase only 1-4% of the enzymes were immobilized [90], whereas the rest could be fitted by the free-diffusion model, suggesting only transient binding events. Additionally, chip-seq experiments with remodeling enzymes support the transient binding events. These experi‐ ments revealed that the localization pattern of wild-type Isw2p did not correlate with known sites of Isw2 function *in vivo*. In contrast, the catalytically inactive Isw2p–K215R was prefer‐ entially enriched at the known Isw2 target sites. This suggests, that in the absence of ATP hydrolysis the target sites remain high affinity binding sites, whereas the ATPase active enzyme does not bind to the remodeled nucleosomes [91]. These results indicate a continuous sampling mechanism (Fig. 5), by which the remodeler continuously screens the genomic nucleosomes for "good" substrates, converting them into the "bad" ones. Most of the binding events seem to be unproductive, meaning that the remodeling reaction does not occur. From the experimentally determined relatively high remodeling enzyme concentrations (in the range of μM) and short chromatin bound residence times around 100 ms, average sampling times of tens of seconds to minutes were calculated for Snf2H containing remodelers to probe 99% of all genomic nucleosomes. Thus, a combination of high remodeler concentrations, short residence times in the chromatin bound state and fast 3D diffusive translocations in the intervening periods appears to be an efficient mechanism to keep nucleosomes in place [90,92].

**Figure 5.** Genome-wide search for nucleosomal targets by remodeling enzymes. A) Continuous sampling mechanism. It is a diffusion-driven, rapid sampling of nonspecific sites with the remodeling enzymes binding only transiently to the nucleosomes. Most binding events are non-productive, as the nucleosomes are well positioned. B) Immobilization mechanism. Remodelers are recruited to the particular sites where they change nucleosomal positions. Targeting is achieved upon recognition of specific signals like histone modifications, chromatin-associated proteins, structural fea‐ tures of the chromatin environment or even by small molecules such as hormones.

#### **5.3. Immobilization**

interact with any cellular structures, FRAP kinetics are a direct reflection of their translational motion properties. In contrast, proteins that bind to immobile structures such as chromatin, exhibit a slower overall mobility. The mobility of ISWI family remodelers Snf2H, Snf2L and Snf2L+13 (an ATPase inactive variant of the Snf2L) was studied in living U2OS cells. During G1/2 phase only 1-4% of the enzymes were immobilized [90], whereas the rest could be fitted by the free-diffusion model, suggesting only transient binding events. Additionally, chip-seq experiments with remodeling enzymes support the transient binding events. These experi‐ ments revealed that the localization pattern of wild-type Isw2p did not correlate with known sites of Isw2 function *in vivo*. In contrast, the catalytically inactive Isw2p–K215R was prefer‐ entially enriched at the known Isw2 target sites. This suggests, that in the absence of ATP hydrolysis the target sites remain high affinity binding sites, whereas the ATPase active enzyme does not bind to the remodeled nucleosomes [91]. These results indicate a continuous sampling mechanism (Fig. 5), by which the remodeler continuously screens the genomic nucleosomes for "good" substrates, converting them into the "bad" ones. Most of the binding

same time the preferred endpoint of the remodeling reaction [6].

16 Chromatin Remodelling

**Figure 4.** Model describing the affinity of remodelers to nucleosomes at different positions on the DNA. A) In the re‐ lease model, the remodeling complex has a weaker binding affinity to the end-positioned nucleosome in comparison to any other nucleosome. In the arrest model, the remodeler binds all nucleosomes with similar affinity, but the trans‐ location rate constant is much slower on a nucleosome present in the final position. B) Chd1 positions nucleosomes according to the release mechanism. Nucleosome position-dependent differences in the affinity of the remodeling complexes to the nucleosomal substrate were analyzed by bandshift assays. Remodeling reaction of Chd1 on mono‐ nucleosomal substrates reconstituted on a 350 bp DNA fragment containing hsp70 promoter region. Chd1 positions nulceosomes to the N3 and N2 positions. C) Binding reaction of Chd1 to the nucleosomes. The position of the DNA– Chd1 (D/C) and the nucleosome–Chd1 (N/C) complexes are indicated. The position of the N3 nucleosome is shown by a black box. Nucleosomes positioned at this site are bound by Chd1 with the lowest affinity. This position is at the

> In parallel with the continuous sampling mechanism, remodeling complexes are engaged by specific recruitment or immobilization at specific target sites. The respective mechanisms are described in chapter 4. For example, when cells were treated with dexamethasone, BRG1 and BRMwere concentratedina single spotinthenucleus, as revealedby immunofluorescence.The site coincided with the multimerized MMTV DNA and RNA FISH signals, showing that the enzymes are recruited to the MMTV array in a hormone-dependent manner. In this case the recruitment of the SWI/SNF machine results in the maintenance of an active chromatin struc‐ ture that is compatible with transcription [93]. In other cases, like the nucleolar remodeling complex NoRC recruitment to the rRNA genes, continuous targeting results in gene repres‐

sion via changes of the promoter nucleosome positioning that are incompatible with transcrip‐ tion initiation factor binding and further leads to the heterochromatin formation [20,94].

#### **5.4. Nuclear dynamics of chromatin remodeling enzymes**

Cells express a plethora of different remodeling complexes that act simultaneously on the cellular chromatin. The remodeler complexes diffuse freely through the nucleus, searching for "good" nucleosomes. "Good" nucleosomal substrates for the one machine may represent "bad" substrates for the other machine, suggesting that an active, free diffusing pool of remodeling complexes continuously changes the local chromatin structure. Upon specific signals individual machines are recruited to the specific sites to establish local chromatin structures correlating with a persistent activation or repression of certain DNA dependent processes. We hypothesize that the mixture of remodeling complexes in the cell, with their complex-specific remodeling patterns would continuously changes local chromatin structures, depending on complex that is currently recruited to such sites. Overall the action of the diverse remodeling complexes suggests that chromatin is continuously switching local nucleosome positions according to the levels, activity and set of remodeling complexes in a given cell [95].

**Acetylation.** The acetyltransferase MOF acetylates TIP5, the largest subunit of NoRC, at position K633, adjacent to the TIP5 RNA-binding domain, and that the NAD(+)-dependent deacetylase SIRT1 removes the acetyl group. Acetylation regulates the interaction of NoRC with pRNA, which in turn affects heterochromatin formation, nucleosome positioning and rDNA silencing. Significantly, NoRC acetylation is responsive to the intracellular energy status and fluctuates during S-phase. Activation of SIRT1 on glucose deprivation leads to deacety‐ lation of K633, enhanced pRNA binding and an increase in heterochromatic histone marks [99]. The acetylation of yeast Rsc4 does not significantly affect RSC catalytic activity or its ability to recognize acetylated nucleosomes, but K25 acetylation mark plays a key role in resistance to DNA damage, in a manner that appears to be regulated by its interaction with bromodomain 1. Moreover, Rsc4 acetylation acts in parallel with the INO80-remodeling complex to promote S-phase progression in cells subject to replication stress [100]. *Drosophila* ISWI is acetylated at position K753 *in vivo* and *in vitro* by the histone acetyltransferase GCN5. The acetylated form of ISWI represents a minor species presumably associated with the nucleosome remodeling factor NURF and may contribute during metaphase chromosome condensation [101]. Human Brm was shown to be acetylated at multiple locations, but two sites, clustered in the C-terminal region, appear to play a central role in the regulation. Mutation of these sites into nonacetylatable versions creates a Brm protein with increased activity in terms of inhibition of

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19

**PARylation.** In *Drosophila*, ISWI is poly-ADP-ribosylated (PARylated) by the enzyme PARP. PARylated ISWI binds weaker to the nucleosomes and DNA and displays weak nucleosomestimulated ATPase activity. Moreover, the amount of ISWI bound to chromatin is affected by PARP activity, suggesting that PARP and ISWI might compete for common chromatin target sites and antagonize on chromosome condensation [103]. A different scenario is reported in the nucleolus of human embryonic kidney cell line, where PARP1/ARTD1-mediated paryla‐ tion of TIP5, a noncatalytic subunit of NoRC complex, promotes the silencing of rDNA chromatin during replication. It is reported that upon of pRNA binding TIP5 undergoes

colony formation and transcriptional activation [102].

**Figure 6.** Different regulation possibilities of remodeler activity.

#### **6. Regulation of remodeler activity**

As mentioned above, the individual accessory proteins of the remodeling complexes contain a diverse set of histones, DNA and nucleosome recognition motifs and these proteins change the outcome of nucleosome remodeling reactions. Accordingly, these proteins significantly determine the targeting to genomic regions and the qualitative outcome of a remodeling reaction. In this chapter, we want to focus on the regulation of the overall activity of remodeling enzymes by metabolites and modifications. Subunits of chromatin remodeling complexes often contain domains capable of recognizing specific posttranslational modifications on histone tails. However, significantly less is known about the functions of posttranslational modifications on remodeling complexes themselves and our understanding of its role is only beginning to emerge.

**Phosphorylation.** The first example of phosphoregulation of a remodeler was the mitotic phosphorylation of human SWI/SNF, which inhibits remodeling activity, with subsequent dephosphorylation by hPP2A restoring remodeling activity. It was suggested that the phos‐ phorylated form would promote global repression of chromatin remodeling during mitosis [96]. In *Drosophila*, Mi-2 undergoes constitutive phosphorylation at N-terminus and CK2 was identified as a major kinase. Dephosphorylated Mi-2 displays increased affinity for the nucleosomal substrate, which in turn leads to an increased nucleosome-stimulated ATPase and remodeling activity. It was even postulated that it might be a common regulatory mechanism for CHD family remodelers [97]. Whether and how the phosphorylation alters the biochemical activity of INO80 is not known, but upon exposure to DNA damage, it was found that yeast INO80 complex is phosphorylated on the Les4 subunit in a Mec1/Tel1-dependent manner [98].

**Figure 6.** Different regulation possibilities of remodeler activity.

sion via changes of the promoter nucleosome positioning that are incompatible with transcrip‐ tion initiation factor binding and further leads to the heterochromatin formation [20,94].

Cells express a plethora of different remodeling complexes that act simultaneously on the cellular chromatin. The remodeler complexes diffuse freely through the nucleus, searching for "good" nucleosomes. "Good" nucleosomal substrates for the one machine may represent "bad" substrates for the other machine, suggesting that an active, free diffusing pool of remodeling complexes continuously changes the local chromatin structure. Upon specific signals individual machines are recruited to the specific sites to establish local chromatin structures correlating with a persistent activation or repression of certain DNA dependent processes. We hypothesize that the mixture of remodeling complexes in the cell, with their complex-specific remodeling patterns would continuously changes local chromatin structures, depending on complex that is currently recruited to such sites. Overall the action of the diverse remodeling complexes suggests that chromatin is continuously switching local nucleosome positions according to the levels, activity and set of remodeling complexes in a given cell [95].

As mentioned above, the individual accessory proteins of the remodeling complexes contain a diverse set of histones, DNA and nucleosome recognition motifs and these proteins change the outcome of nucleosome remodeling reactions. Accordingly, these proteins significantly determine the targeting to genomic regions and the qualitative outcome of a remodeling reaction. In this chapter, we want to focus on the regulation of the overall activity of remodeling enzymes by metabolites and modifications. Subunits of chromatin remodeling complexes often contain domains capable of recognizing specific posttranslational modifications on histone tails. However, significantly less is known about the functions of posttranslational modifications on remodeling complexes themselves and our understanding of its role is only

**Phosphorylation.** The first example of phosphoregulation of a remodeler was the mitotic phosphorylation of human SWI/SNF, which inhibits remodeling activity, with subsequent dephosphorylation by hPP2A restoring remodeling activity. It was suggested that the phos‐ phorylated form would promote global repression of chromatin remodeling during mitosis [96]. In *Drosophila*, Mi-2 undergoes constitutive phosphorylation at N-terminus and CK2 was identified as a major kinase. Dephosphorylated Mi-2 displays increased affinity for the nucleosomal substrate, which in turn leads to an increased nucleosome-stimulated ATPase and remodeling activity. It was even postulated that it might be a common regulatory mechanism for CHD family remodelers [97]. Whether and how the phosphorylation alters the biochemical activity of INO80 is not known, but upon exposure to DNA damage, it was found that yeast INO80 complex is phosphorylated on the Les4 subunit in a Mec1/Tel1-dependent

**5.4. Nuclear dynamics of chromatin remodeling enzymes**

**6. Regulation of remodeler activity**

beginning to emerge.

18 Chromatin Remodelling

manner [98].

**Acetylation.** The acetyltransferase MOF acetylates TIP5, the largest subunit of NoRC, at position K633, adjacent to the TIP5 RNA-binding domain, and that the NAD(+)-dependent deacetylase SIRT1 removes the acetyl group. Acetylation regulates the interaction of NoRC with pRNA, which in turn affects heterochromatin formation, nucleosome positioning and rDNA silencing. Significantly, NoRC acetylation is responsive to the intracellular energy status and fluctuates during S-phase. Activation of SIRT1 on glucose deprivation leads to deacety‐ lation of K633, enhanced pRNA binding and an increase in heterochromatic histone marks [99]. The acetylation of yeast Rsc4 does not significantly affect RSC catalytic activity or its ability to recognize acetylated nucleosomes, but K25 acetylation mark plays a key role in resistance to DNA damage, in a manner that appears to be regulated by its interaction with bromodomain 1. Moreover, Rsc4 acetylation acts in parallel with the INO80-remodeling complex to promote S-phase progression in cells subject to replication stress [100]. *Drosophila* ISWI is acetylated at position K753 *in vivo* and *in vitro* by the histone acetyltransferase GCN5. The acetylated form of ISWI represents a minor species presumably associated with the nucleosome remodeling factor NURF and may contribute during metaphase chromosome condensation [101]. Human Brm was shown to be acetylated at multiple locations, but two sites, clustered in the C-terminal region, appear to play a central role in the regulation. Mutation of these sites into nonacetylatable versions creates a Brm protein with increased activity in terms of inhibition of colony formation and transcriptional activation [102].

**PARylation.** In *Drosophila*, ISWI is poly-ADP-ribosylated (PARylated) by the enzyme PARP. PARylated ISWI binds weaker to the nucleosomes and DNA and displays weak nucleosomestimulated ATPase activity. Moreover, the amount of ISWI bound to chromatin is affected by PARP activity, suggesting that PARP and ISWI might compete for common chromatin target sites and antagonize on chromosome condensation [103]. A different scenario is reported in the nucleolus of human embryonic kidney cell line, where PARP1/ARTD1-mediated paryla‐ tion of TIP5, a noncatalytic subunit of NoRC complex, promotes the silencing of rDNA chromatin during replication. It is reported that upon of pRNA binding TIP5 undergoes conformational change [67] which might favour the association of PARP1 and subsequently Tip5 is parylated. It was postulated that PARP1 enzymatic activity facilitates formation of silent rDNA chromatin and transcriptional silencing [104].

[3] Strahl BD, Allis CD. The language of covalent histone modifications. Nature. 2000

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21

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#### **7. Conclusion**

Global chromatin structure is a result of the combination of chromatin remodelers present in the cell. The ability to form various complexes with different activities and the concentration of the remodelers influences the nucleosomal positions genome-wide. Much data have been accumulated from *in vitro* experiments addressing the mechanistical questions of chromatin remodelers, but the recent studies have begun to reveal how these proteins find their place of action in the cell. From our current knowledge it seems that the local chromatin structures undergo a continuous change due to a continuous and random binding of different remodeling complexes. A large fraction of the remodeling complexes diffuse freely through the nucleus and act on nucleosomal substrates. In addition, the specific cellular signals are responsible for the fast recruitment of the individual machines to the specialized DNA sites correlating with a persistent activation or repression of particular DNA dependent processes, establishing persistent changes in chromatin structure.

#### **Acknowledgements**

We apologize to all colleagues whose work could not be cited due to space limitations. Work in the G.L. laboratory is funded by the DFG, EraSysBio+ and Baygene. Funding for open access charge: Regensburg University Library.

#### **Author details**

Laura Manelyte and Gernot Längst

University of Regensburg, Regensburg, Germany

#### **References**


[3] Strahl BD, Allis CD. The language of covalent histone modifications. Nature. 2000 Jan. 6;403(6765):41–45.

conformational change [67] which might favour the association of PARP1 and subsequently Tip5 is parylated. It was postulated that PARP1 enzymatic activity facilitates formation of silent

Global chromatin structure is a result of the combination of chromatin remodelers present in the cell. The ability to form various complexes with different activities and the concentration of the remodelers influences the nucleosomal positions genome-wide. Much data have been accumulated from *in vitro* experiments addressing the mechanistical questions of chromatin remodelers, but the recent studies have begun to reveal how these proteins find their place of action in the cell. From our current knowledge it seems that the local chromatin structures undergo a continuous change due to a continuous and random binding of different remodeling complexes. A large fraction of the remodeling complexes diffuse freely through the nucleus and act on nucleosomal substrates. In addition, the specific cellular signals are responsible for the fast recruitment of the individual machines to the specialized DNA sites correlating with a persistent activation or repression of particular DNA dependent processes, establishing

We apologize to all colleagues whose work could not be cited due to space limitations. Work in the G.L. laboratory is funded by the DFG, EraSysBio+ and Baygene. Funding for open access

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**Chapter 2**

**SUMO Tasks in Chromatin Remodeling**

Post-translational modifications implicate attachment of diverse molecules to proteins after translation. These modifications are essential for many biological processes as they are involved in their regulation. From relatively simple molecules to small polypetides are common covalent modifiers of proteins. Sumoylation consists in the post-translational modification of proteins by attachment of the small polypeptide SUMO (small ubiquitin-like modifier). This post-translational modification was identified two decades ago and has been very actively investigated to date. Sumoylation has consequences on protein structure and regulation. This modification controls many processes in the eukaryotic cell and is essential

From the discovery of ubiquitin in 1975, a number of ubiquitin-like proteins (UBLs) have been identified in eukaryotes and it has been shown that many of them are able to covalently attach to other proteins (reviewed in [1]). Several aspects are common to most UBLs: they are small polypeptides (less than 200 amino acids) capable of attaching to other macromolecules in a covalent way, present common structural features and use similar modification pathways. These characteristics strongly support duplication and diversification during evolution as the origin of the different pathways. Ubiquitin and UBLs are characterized by the presence of the ß-grasp fold, which also appears in ubiquitin-like domains of several other proteins of the ubiquitin system and in numerous non-related proteins (reviewed in [2]). The ß-grasp fold seems to have emerged in prokaryotes as a translation-related RNA-binding module, which diversified structurally and biochemically before to dramatically expand in eukaryotes [2]. Besides ubiquitin and SUMO, examples of UBLs are NEDD8, FUBI, FAT10, ISG15, UFM1,

The first report of a protein being modified by SUMO occurred in the nineties and concerned the mammalian nuclear pore-associated GTPase activating protein RanGAP1 [3, 4]. Subse‐

and reproduction in any medium, provided the original work is properly cited.

© 2013 Mario; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

for viability of all the organisms studied so far.

Atg8, Atg12 and Urm1 (reviewed in [1]).

Garcia-Dominguez Mario

http://dx.doi.org/10.5772/55395

**1. Introduction**


### **Chapter 2**

## **SUMO Tasks in Chromatin Remodeling**

### Garcia-Dominguez Mario

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55395

#### **1. Introduction**

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28 Chromatin Remodelling

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Post-translational modifications implicate attachment of diverse molecules to proteins after translation. These modifications are essential for many biological processes as they are involved in their regulation. From relatively simple molecules to small polypetides are common covalent modifiers of proteins. Sumoylation consists in the post-translational modification of proteins by attachment of the small polypeptide SUMO (small ubiquitin-like modifier). This post-translational modification was identified two decades ago and has been very actively investigated to date. Sumoylation has consequences on protein structure and regulation. This modification controls many processes in the eukaryotic cell and is essential for viability of all the organisms studied so far.

From the discovery of ubiquitin in 1975, a number of ubiquitin-like proteins (UBLs) have been identified in eukaryotes and it has been shown that many of them are able to covalently attach to other proteins (reviewed in [1]). Several aspects are common to most UBLs: they are small polypeptides (less than 200 amino acids) capable of attaching to other macromolecules in a covalent way, present common structural features and use similar modification pathways. These characteristics strongly support duplication and diversification during evolution as the origin of the different pathways. Ubiquitin and UBLs are characterized by the presence of the ß-grasp fold, which also appears in ubiquitin-like domains of several other proteins of the ubiquitin system and in numerous non-related proteins (reviewed in [2]). The ß-grasp fold seems to have emerged in prokaryotes as a translation-related RNA-binding module, which diversified structurally and biochemically before to dramatically expand in eukaryotes [2]. Besides ubiquitin and SUMO, examples of UBLs are NEDD8, FUBI, FAT10, ISG15, UFM1, Atg8, Atg12 and Urm1 (reviewed in [1]).

The first report of a protein being modified by SUMO occurred in the nineties and concerned the mammalian nuclear pore-associated GTPase activating protein RanGAP1 [3, 4]. Subse‐

© 2013 Mario; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

quently, more than a hundred proteins have been identified as SUMO substrates. Although similarities with ubiquitin are notable [5], SUMO plays many regulatory functions in the cell that significantly differ from the major role displayed by ubiquitin: labeling proteins to target them for proteasomal degradation [6]. A variety of consequences derived from protein sumoylation (new interaction surfaces, modulation of protein affinity and binding capacities to other molecules, modulation of protein activity, blocking of protein domains, steric hindrance, crosstalk or interference with other post-translational modifications) account for the many roles attributed to SUMO (reviewed in [7]). A major role of SUMO is associated with RanGAP1 and thereby with the nuclear pore complex. Thus, involvement of SUMO in nucleocytoplasmic transport of proteins has been well established [8]. SUMO has been also implicated in chromosome dynamics in mitosis and meiosis (condensation, cohesion, separation) and genome integrity, as many proteins involved in DNA replication, repair and recombination are modulated by SUMO modification (reviewed in [7]). Other roles attributed to SUMO are related to enzyme regulation, protein stability and cellular structure (reviewed in [9, 10]). However, the most prominent function of SUMO concerns transcriptional regulation, and specially transcription repression (reviewed in [11, 12]). The role of SUMO in transcription, in the context of chromatin structure and dynamics, is analyzed in this chapter.

is homologous to its C terminus [13]. Both monomers work together and are not found separately [14]. E1 activation of mature SUMO involves ATP hydrolysis and formation of a thiolester bond between E1 and the C terminus of SUMO before being transferred to the E2. While several E2 have been described for ubiquitination, UBC9 is the only E2 known for sumoylation [15, 16]. Thus, UBC9 is the conjugating enzyme directly involved in attachment of SUMO to the different substrates. This second step of the sumoylation reaction involves the formation of a thiolester bond between SUMO and UBC9 upon transfer from the E1. The region surrounding the active site cysteine (C93 in mammals) in UBC9 is able to directly interact with

**Enzyme Protein Activity References**

RanBP2 [22] Polycomb-2 (Pc2) [23] TOPORS [24] Class IIa HDACs [25] KAP-1 [26] RHES [27] Krox20 [28]

SUMO Tasks in Chromatin Remodeling http://dx.doi.org/10.5772/55395 31

DeSI-1 [32]

E1 (activating) AOS1/UBA2 ATP-mediated activation of SUMO [13] E2 (conjugating) UBC9 SUMO conjugation to target [15, 16] E3 (ligase) PIAS1-4 Facilitates transfer to target [20, 21]

protease SENP1-3, 5-7 Maturation/recycling [29-31]

SUMO ligases are involved in facilitating the SUMO attachment to substrates (reviewed in [33]). To date, few ligases have been described for sumoylation, in contrast to ubiquitination, where lots of them are known to play an essential role and mediate substrate specificity. In fact, SUMO ligases were undervalued at the beginning, since certain substrates are sumoylated in vitro, provided that E1 and E2 are present at the adequate concentrations. Since UBC9 is able to directly interact with sumoylation consensus sequence in substrates, it is able to render sumoylation in the absence of a ligase. However, a number of proteins, which augmented the efficiency of SUMO conjugation, were identified. The list of SUMO ligases progressively increases and essential roles for these have been described in vivo (see [34]). Although mechanisms of action of SUMO ligases have not been completely elucidated, it is obvious that many ligases facilitate transfer of SUMO by bringing together SUMO-loaded UBC9 and the target protein. Thus, similar to the RING domain-containing E3 ligases involved in ubiquiti‐ nation, SUMO ligases do not establish a covalent bond with SUMO. In this context, a SUMO ligase should normally i) interact with the substrate, ii) interact with UBC9, iii) facilitate SUMO

sumoylation consensus sequence (see below) in target proteins [17-19].

**Table 1.** Enzymes involved in SUMO conjugation.

#### **2. The modification pathway**

#### **2.1. Enzymes involved**

Modification by SUMO involves the ATP-dependent activation of mature SUMO (C terminus of SUMO needs to be excised by proteolysis) by the E1 enzyme, transfer to the E2 enzyme UBC9 and conjugation to the target protein, often mediated by a SUMO ligase or E3 (Figure 1 and Table 1) (reviewed in [9]). Maturation of the SUMO precursor, as well as removal of SUMO from targets is displayed by SUMO specific proteases.

**Figure 1.** The sumoylation pathway. Cleavage of the SUMO C terminus enables ATP-mediated activation and binding to the E1 enzyme to be transferred to the E2 conjugating enzyme UBC9, which mediates target modification with the concourse of an E3 SUMO ligase. Recycling of SUMO is performed by the same proteases involved in maturation.

SUMO E1 activity is performed by the SAE1/UBA2 heterodimer in human, in contrast to the ubiquitination pathway where the E1 activity is displayed by a monomeric enzyme. However, the SAE1 subunit is homologous to the N-terminal part of ubiquitin E1, while the UBA2 subunit is homologous to its C terminus [13]. Both monomers work together and are not found separately [14]. E1 activation of mature SUMO involves ATP hydrolysis and formation of a thiolester bond between E1 and the C terminus of SUMO before being transferred to the E2. While several E2 have been described for ubiquitination, UBC9 is the only E2 known for sumoylation [15, 16]. Thus, UBC9 is the conjugating enzyme directly involved in attachment of SUMO to the different substrates. This second step of the sumoylation reaction involves the formation of a thiolester bond between SUMO and UBC9 upon transfer from the E1. The region surrounding the active site cysteine (C93 in mammals) in UBC9 is able to directly interact with sumoylation consensus sequence (see below) in target proteins [17-19].


**Table 1.** Enzymes involved in SUMO conjugation.

quently, more than a hundred proteins have been identified as SUMO substrates. Although similarities with ubiquitin are notable [5], SUMO plays many regulatory functions in the cell that significantly differ from the major role displayed by ubiquitin: labeling proteins to target them for proteasomal degradation [6]. A variety of consequences derived from protein sumoylation (new interaction surfaces, modulation of protein affinity and binding capacities to other molecules, modulation of protein activity, blocking of protein domains, steric hindrance, crosstalk or interference with other post-translational modifications) account for the many roles attributed to SUMO (reviewed in [7]). A major role of SUMO is associated with RanGAP1 and thereby with the nuclear pore complex. Thus, involvement of SUMO in nucleocytoplasmic transport of proteins has been well established [8]. SUMO has been also implicated in chromosome dynamics in mitosis and meiosis (condensation, cohesion, separation) and genome integrity, as many proteins involved in DNA replication, repair and recombination are modulated by SUMO modification (reviewed in [7]). Other roles attributed to SUMO are related to enzyme regulation, protein stability and cellular structure (reviewed in [9, 10]). However, the most prominent function of SUMO concerns transcriptional regulation, and specially transcription repression (reviewed in [11, 12]). The role of SUMO in transcription, in

the context of chromatin structure and dynamics, is analyzed in this chapter.

SUMO from targets is displayed by SUMO specific proteases.

Modification by SUMO involves the ATP-dependent activation of mature SUMO (C terminus of SUMO needs to be excised by proteolysis) by the E1 enzyme, transfer to the E2 enzyme UBC9 and conjugation to the target protein, often mediated by a SUMO ligase or E3 (Figure 1 and Table 1) (reviewed in [9]). Maturation of the SUMO precursor, as well as removal of

**Figure 1.** The sumoylation pathway. Cleavage of the SUMO C terminus enables ATP-mediated activation and binding to the E1 enzyme to be transferred to the E2 conjugating enzyme UBC9, which mediates target modification with the concourse of an E3 SUMO ligase. Recycling of SUMO is performed by the same proteases involved in maturation.

SUMO E1 activity is performed by the SAE1/UBA2 heterodimer in human, in contrast to the ubiquitination pathway where the E1 activity is displayed by a monomeric enzyme. However, the SAE1 subunit is homologous to the N-terminal part of ubiquitin E1, while the UBA2 subunit

**2. The modification pathway**

**2.1. Enzymes involved**

30 Chromatin Remodelling

SUMO ligases are involved in facilitating the SUMO attachment to substrates (reviewed in [33]). To date, few ligases have been described for sumoylation, in contrast to ubiquitination, where lots of them are known to play an essential role and mediate substrate specificity. In fact, SUMO ligases were undervalued at the beginning, since certain substrates are sumoylated in vitro, provided that E1 and E2 are present at the adequate concentrations. Since UBC9 is able to directly interact with sumoylation consensus sequence in substrates, it is able to render sumoylation in the absence of a ligase. However, a number of proteins, which augmented the efficiency of SUMO conjugation, were identified. The list of SUMO ligases progressively increases and essential roles for these have been described in vivo (see [34]). Although mechanisms of action of SUMO ligases have not been completely elucidated, it is obvious that many ligases facilitate transfer of SUMO by bringing together SUMO-loaded UBC9 and the target protein. Thus, similar to the RING domain-containing E3 ligases involved in ubiquiti‐ nation, SUMO ligases do not establish a covalent bond with SUMO. In this context, a SUMO ligase should normally i) interact with the substrate, ii) interact with UBC9, iii) facilitate SUMO transfer to the substrate. Ligases of the PIAS family (PIAS1 to 4) have been extensively studied [35]. They present a type of RING finger domain, the SP-RING (Siz/PIAS RING), for UBC9 interaction, although Ubc9 binding to a PHD domain in plant PIAS proteins has been also described [36]. Other SUMO ligases described so far are RanBP2 [22], the Polycomb-2 (Pc2) protein [23], class IIa histone deacetylases (HDACs) [25], topoisomerase I-binding RING finger protein (TOPORS) [24], the PHD containing protein KAP-1 [26], Ras homologue enriched in striatum (RHES) [27] and the transcription factor Krox20 [28]. In contrast to most ubiquitin ligases, SUMO ligases may display significant promiscuity, as many of them enhance sumoy‐ lation of a variety of substrates.

of processes. Thus, the modification pathway is able to differentially conjugate the various

SUMO Tasks in Chromatin Remodeling http://dx.doi.org/10.5772/55395 33

Covalent attachment of SUMO occurs through the ε-amino group of a lysine residue in target proteins. In many cases the Lys (K) residue is the core of the consensus sequence ΨKxE, being Ψ a large hydrophobic residue and x any amino acid. Extended consensus (phosphorylationdependent SUMO motif (PDSM) and negatively charged residues-dependent SUMO motif (NDSM)) and variations have been described as well (Table 2) (reviewed in [34]). However, sumoylation also occurs at non-consensus sequences. As mentioned above, the consensus sequence is directly contacted by UBC9. Thus, it is possible that when sumoylation occurs at non-consensus sequences, certain amino acid residues, otherwise dispersed in the primary structure of the target protein, bring together in the three-dimensional structure to mimic a consensus-like environment. It is worth noting that conversely, sumoylation consensus sequences in a protein are not always substrate for SUMO attachment, indicating that addi‐ tional structural features regulate and enable modification by SUMO. Besides covalent attachement of SUMO, many proteins can associate with SUMO in a different way involving a non-covalent interaction (reviewed in [48]). This occurs through SUMO interacting motifs (SIMs) in proteins. SIMs are usually characterized by the presence of a short hydrophobic region surrounded by negatively charged residues (Table 2) [49]. The non-covalent interaction of proteins with SUMO has been revealed essential in the regulation of several processes. In a variety of cases function of the system relies in the combinatorial occurrence of sumoylation sites and SIMs in a given protein or in different subunits of a complex, which determines its macromolecular architecture (Figure 2 and see below). This situation is exemplified by the promyelocytic leukaemia protein (PML), in which combination of sumoylation sites and SIMs dictates the formation of PML nuclear bodies and the recruitment of additional proteins [48].

Despite that certain SUMO targets appear constitutively sumoylated, it is obvious that sumoylation, as a signaling pathway needs to be regulated. A striking feature of SUMO modification consists in the so-called "SUMO enigma" [9]. It has been observed that many SUMO targets are difficult to detect at the sumoylated state, but mutation of the acceptor lysine has severe consequences in the process involved. In other words, at the steady state, only a low proportion of the whole pool of a given target appears sumoylated, although sumoylation results essential for function of the target. Thus, sumoylation has been suggested to be a highly dynamic and transient modification that permanently marks targets for specific fates even though the SUMO moiety has been removed [9]. This can be explained by viewing sumoylation as a temporal facilitator for the establishment of protein interactions, other protein modifica‐

Sumoylation can be regulated at different levels (reviewed in [34, 50]). First level of regulation in SUMO modification relies in the nature of target proteins, as target sequence, structural features, and other protein modifications affect attachment of SUMO. The other way to

SUMO molecules depending on the substrate or the regulatory process [47].

**2.3. Sumoylation consensus motifs and SUMO interacting motifs**

**2.4. Regulation of sumoylation**

tions, or sub-cellular localization (Figure 2).

SUMO proteases are involved in maturation of the SUMO precursor by exposing two glycine residues at the C terminus for binding to E1 [37]. In addition, they are also involved in SUMO recycling by excising the SUMO moiety from substrates. Yeast Ulp1p was the first SUMO protease identified [29]. Sequence analysis revealed that it corresponded to a protease of the C48 cysteine group, not related to deubiquitylating enzymes but similar to adenovirus proteases. Mammalian SUMO proteases are represented by the SENP (sentrin-specific protease) family. It comprises six members, SENP1 to 3, and SENP5 to 7 [38]. A seventh member, initially identified as SENP4, resulted to actually correspond to SENP3. Besides SENP1 to 7, an additional family member has been reported, SENP8. However, this protease does not act on SUMO, but on another UBL, NEDD8 [39, 40]. Very recently, a new type of SUMO protease has been described, the desumoylating isopeptidase 1 (DeSI-1) [32]. The different SUMO proteases show diverse cellular localization and different specificities for the various SUMO molecules and substrates (reviewed in [38]).

#### **2.2. SUMO molecules**

Four different SUMO molecules have been described in mammals: SUMO1 to 4. SUMO1 has been implicated in regulation of many processes, while SUMO2 and SUMO3 are highly related with the response to stress. Consequently, a significant pool of free SUMO2 and SUMO3 is detected in the cell, which is rapidly mobilized after exposure to a variety of stress conditions. In contrast, most of SUMO1 appears conjugated to proteins [41]. SUMO2 and SUMO3 are usually referred as SUMO2/3, as they share 97% identity and antibodies hardly differentiate the two forms. By contrast, SUMO1 only shares about 50% identity with SUMO2/3. Despite the low similarity showed between ubiquitin and SUMO (about 18% identity with SUMO1), structurally they are quite similar, excepting the N-terminal region of SUMO not present in ubiquitin [42]. A remarkable difference between SUMO1 and SUMO2/3 is the ability of this last to form poly-SUMO chains in vitro as well as in vivo, due to the presence of a sumoylation consensus sequence in the molecule [43]. SUMO4 is the last SUMO molecule identified. It shows a restricted expression pattern [44] and several data bring into question its capacity to be conjugated to proteins [45]. However, a polymorphism found in human SUMO4 correlates with type 1 diabetes [46]. The different SUMO molecules share a common modification pathway and the existence of functional redundancy has been suggested. However, specific modification by the different SUMO paralogs has been implicated in the regulation of a variety of processes. Thus, the modification pathway is able to differentially conjugate the various SUMO molecules depending on the substrate or the regulatory process [47].

#### **2.3. Sumoylation consensus motifs and SUMO interacting motifs**

Covalent attachment of SUMO occurs through the ε-amino group of a lysine residue in target proteins. In many cases the Lys (K) residue is the core of the consensus sequence ΨKxE, being Ψ a large hydrophobic residue and x any amino acid. Extended consensus (phosphorylationdependent SUMO motif (PDSM) and negatively charged residues-dependent SUMO motif (NDSM)) and variations have been described as well (Table 2) (reviewed in [34]). However, sumoylation also occurs at non-consensus sequences. As mentioned above, the consensus sequence is directly contacted by UBC9. Thus, it is possible that when sumoylation occurs at non-consensus sequences, certain amino acid residues, otherwise dispersed in the primary structure of the target protein, bring together in the three-dimensional structure to mimic a consensus-like environment. It is worth noting that conversely, sumoylation consensus sequences in a protein are not always substrate for SUMO attachment, indicating that addi‐ tional structural features regulate and enable modification by SUMO. Besides covalent attachement of SUMO, many proteins can associate with SUMO in a different way involving a non-covalent interaction (reviewed in [48]). This occurs through SUMO interacting motifs (SIMs) in proteins. SIMs are usually characterized by the presence of a short hydrophobic region surrounded by negatively charged residues (Table 2) [49]. The non-covalent interaction of proteins with SUMO has been revealed essential in the regulation of several processes. In a variety of cases function of the system relies in the combinatorial occurrence of sumoylation sites and SIMs in a given protein or in different subunits of a complex, which determines its macromolecular architecture (Figure 2 and see below). This situation is exemplified by the promyelocytic leukaemia protein (PML), in which combination of sumoylation sites and SIMs dictates the formation of PML nuclear bodies and the recruitment of additional proteins [48].

#### **2.4. Regulation of sumoylation**

transfer to the substrate. Ligases of the PIAS family (PIAS1 to 4) have been extensively studied [35]. They present a type of RING finger domain, the SP-RING (Siz/PIAS RING), for UBC9 interaction, although Ubc9 binding to a PHD domain in plant PIAS proteins has been also described [36]. Other SUMO ligases described so far are RanBP2 [22], the Polycomb-2 (Pc2) protein [23], class IIa histone deacetylases (HDACs) [25], topoisomerase I-binding RING finger protein (TOPORS) [24], the PHD containing protein KAP-1 [26], Ras homologue enriched in striatum (RHES) [27] and the transcription factor Krox20 [28]. In contrast to most ubiquitin ligases, SUMO ligases may display significant promiscuity, as many of them enhance sumoy‐

SUMO proteases are involved in maturation of the SUMO precursor by exposing two glycine residues at the C terminus for binding to E1 [37]. In addition, they are also involved in SUMO recycling by excising the SUMO moiety from substrates. Yeast Ulp1p was the first SUMO protease identified [29]. Sequence analysis revealed that it corresponded to a protease of the C48 cysteine group, not related to deubiquitylating enzymes but similar to adenovirus proteases. Mammalian SUMO proteases are represented by the SENP (sentrin-specific protease) family. It comprises six members, SENP1 to 3, and SENP5 to 7 [38]. A seventh member, initially identified as SENP4, resulted to actually correspond to SENP3. Besides SENP1 to 7, an additional family member has been reported, SENP8. However, this protease does not act on SUMO, but on another UBL, NEDD8 [39, 40]. Very recently, a new type of SUMO protease has been described, the desumoylating isopeptidase 1 (DeSI-1) [32]. The different SUMO proteases show diverse cellular localization and different specificities for the

Four different SUMO molecules have been described in mammals: SUMO1 to 4. SUMO1 has been implicated in regulation of many processes, while SUMO2 and SUMO3 are highly related with the response to stress. Consequently, a significant pool of free SUMO2 and SUMO3 is detected in the cell, which is rapidly mobilized after exposure to a variety of stress conditions. In contrast, most of SUMO1 appears conjugated to proteins [41]. SUMO2 and SUMO3 are usually referred as SUMO2/3, as they share 97% identity and antibodies hardly differentiate the two forms. By contrast, SUMO1 only shares about 50% identity with SUMO2/3. Despite the low similarity showed between ubiquitin and SUMO (about 18% identity with SUMO1), structurally they are quite similar, excepting the N-terminal region of SUMO not present in ubiquitin [42]. A remarkable difference between SUMO1 and SUMO2/3 is the ability of this last to form poly-SUMO chains in vitro as well as in vivo, due to the presence of a sumoylation consensus sequence in the molecule [43]. SUMO4 is the last SUMO molecule identified. It shows a restricted expression pattern [44] and several data bring into question its capacity to be conjugated to proteins [45]. However, a polymorphism found in human SUMO4 correlates with type 1 diabetes [46]. The different SUMO molecules share a common modification pathway and the existence of functional redundancy has been suggested. However, specific modification by the different SUMO paralogs has been implicated in the regulation of a variety

lation of a variety of substrates.

32 Chromatin Remodelling

**2.2. SUMO molecules**

various SUMO molecules and substrates (reviewed in [38]).

Despite that certain SUMO targets appear constitutively sumoylated, it is obvious that sumoylation, as a signaling pathway needs to be regulated. A striking feature of SUMO modification consists in the so-called "SUMO enigma" [9]. It has been observed that many SUMO targets are difficult to detect at the sumoylated state, but mutation of the acceptor lysine has severe consequences in the process involved. In other words, at the steady state, only a low proportion of the whole pool of a given target appears sumoylated, although sumoylation results essential for function of the target. Thus, sumoylation has been suggested to be a highly dynamic and transient modification that permanently marks targets for specific fates even though the SUMO moiety has been removed [9]. This can be explained by viewing sumoylation as a temporal facilitator for the establishment of protein interactions, other protein modifica‐ tions, or sub-cellular localization (Figure 2).

Sumoylation can be regulated at different levels (reviewed in [34, 50]). First level of regulation in SUMO modification relies in the nature of target proteins, as target sequence, structural features, and other protein modifications affect attachment of SUMO. The other way to


**Table 2.** Sumoylation sites and SUMO interacting motifs (SIMs). Ψ represents a large hydrophobic residue. Sumoylated Lys (K) is frequently close to a hydrophobic residue and to negatively charged environment, either acidic residues Asp/Glu (D/E) or phosphorylation sites (SP). SIMs usually consist in a stretch of 4 amino acids, containing at least 3 hydrophobic residues, close to an acidic region (Asp/Glu) (D/E) or putative phosphorylation sites (Ser/Thr) (S/T). Examples of SIMs with acidic/phosphorylation region N-terminal to the hydrophobic core (ZNF198), C-terminal (with spacer (PIAS1), without spacer (SP100)), at both sides (PML), SUMO1 specific (RanBP2) and SUMO2/3 specific (CoREST1), are shown.

indicating that sumoylation mainly associates with transcription repression. Examples from different organisms have argued in favor of such a role. A characteristic of silenced genes is that they correlate with low levels of histone acetylation, while active genes usually display high histone acetylation. It has been described in yeast that temperature-sensitive mutation in Ubc9 leads to an increase in global histone acetylation [57]. In addition, in fission yeast, it has been shown that SUMO is required for the maintenance of heterochromatin stability [58]. Early evidence of the involvement of the SUMO pathway in maintenance of the heterochromatin came from Drosophila, as a PIAS mutant was identified as a suppressor of position effect variegation, that is, as a mutant releasing heterochromatin-induced gene silencing [59]. A mechanism that clearly account for the repressive role of SUMO is explained by the ability of SUMO to recruit histone deacetylases [56]. For many transcription factors sumoylation has been linked to transcription repression. Additional examples to Elk-1 are NAB proteins [28], c-Jun [60], p53 [61], IκBα [62], C/EBP [63], Sp3 [64] and MEF2 [65]. It is worth noting that in many cases sumoylation turns activators into repressors, as it is the case of p300 and CREB binding protein (CBP) [66, 67]. However, beyond SUMO modification of transcription factors, SUMO association with architecture and function of chromatin-associated repressor com‐ plexes is recently getting increased importance. This has been reviewed in [68, 69] and is

**Figure 2.** Sumoylation and SIMs are involved in complex architecture and function. Schematic representation of some examples for SUMO-SIM interactions involved in recruiting proteins to a particular subcellular localization, in the archi‐ tecture of PML aggregates and association to Daxx, and in recruitment of different repressor complexes to the chro‐

SUMO Tasks in Chromatin Remodeling http://dx.doi.org/10.5772/55395 35

Despite the clear association of SUMO with gene repression, several reports illustrate the involvement of sumoylation in transcription activation. Examples of transcription factors whose activity is stimulated by SUMO are TCF4 [70], GATA4 [71], Pax6 [72], p45 [73], Smad4

described below.

**3.2. Transcription activation**

matin through sumoylated Sp3 for transcription repression.

regulate sumoylation depends on the modification pathway. Availability of the different SUMO paralogs when sumoylation is required, or acting on the E1 and E2 enzymes, represents a global way to regulate sumoylation. For instance, stress conditions normally leads to SUMO2/3 conjugation, as SUMO2/3 is freely available in the cell [41]. On the other hand, it has been shown that expression of the Gam1 protein by the CELO adenovirus leads to E1 and E2 degradation and thereby to inhibition of sumoylation [51]. Finally, a more selective way of regulating sumoylation is given through the activity of SUMO ligases and proteases. Thus, localization or spatiotemporal regulation of the expression of these proteins has consequences in target sumoylation.

#### **3. SUMO in transcription**

#### **3.1. Transcription repression**

It is of significance that the sumoylation consensus sequence, before being established to be the site for SUMO attachment, was initially identified as a negative regulatory sequence in several transcription factors [52]. This scenario is exemplified by the transcription factor Elk-1 (Ets (E twenty-six)-like kinase 1), where a repressive domain, the R motif, was identified as an acceptor region for SUMO attachment [53]. Targeting SUMO or the SUMO conjugation enzyme UBC9 to promoters through a Gal4-based system efficiently represses transcription [54-56],

**Figure 2.** Sumoylation and SIMs are involved in complex architecture and function. Schematic representation of some examples for SUMO-SIM interactions involved in recruiting proteins to a particular subcellular localization, in the archi‐ tecture of PML aggregates and association to Daxx, and in recruitment of different repressor complexes to the chro‐ matin through sumoylated Sp3 for transcription repression.

indicating that sumoylation mainly associates with transcription repression. Examples from different organisms have argued in favor of such a role. A characteristic of silenced genes is that they correlate with low levels of histone acetylation, while active genes usually display high histone acetylation. It has been described in yeast that temperature-sensitive mutation in Ubc9 leads to an increase in global histone acetylation [57]. In addition, in fission yeast, it has been shown that SUMO is required for the maintenance of heterochromatin stability [58]. Early evidence of the involvement of the SUMO pathway in maintenance of the heterochromatin came from Drosophila, as a PIAS mutant was identified as a suppressor of position effect variegation, that is, as a mutant releasing heterochromatin-induced gene silencing [59]. A mechanism that clearly account for the repressive role of SUMO is explained by the ability of SUMO to recruit histone deacetylases [56]. For many transcription factors sumoylation has been linked to transcription repression. Additional examples to Elk-1 are NAB proteins [28], c-Jun [60], p53 [61], IκBα [62], C/EBP [63], Sp3 [64] and MEF2 [65]. It is worth noting that in many cases sumoylation turns activators into repressors, as it is the case of p300 and CREB binding protein (CBP) [66, 67]. However, beyond SUMO modification of transcription factors, SUMO association with architecture and function of chromatin-associated repressor com‐ plexes is recently getting increased importance. This has been reviewed in [68, 69] and is described below.

#### **3.2. Transcription activation**

regulate sumoylation depends on the modification pathway. Availability of the different SUMO paralogs when sumoylation is required, or acting on the E1 and E2 enzymes, represents a global way to regulate sumoylation. For instance, stress conditions normally leads to SUMO2/3 conjugation, as SUMO2/3 is freely available in the cell [41]. On the other hand, it has been shown that expression of the Gam1 protein by the CELO adenovirus leads to E1 and E2 degradation and thereby to inhibition of sumoylation [51]. Finally, a more selective way of regulating sumoylation is given through the activity of SUMO ligases and proteases. Thus, localization or spatiotemporal regulation of the expression of these proteins has consequences

**Table 2.** Sumoylation sites and SUMO interacting motifs (SIMs). Ψ represents a large hydrophobic residue. Sumoylated Lys (K) is frequently close to a hydrophobic residue and to negatively charged environment, either acidic residues Asp/Glu (D/E) or phosphorylation sites (SP). SIMs usually consist in a stretch of 4 amino acids, containing at least 3 hydrophobic residues, close to an acidic region (Asp/Glu) (D/E) or putative phosphorylation sites (Ser/Thr) (S/T). Examples of SIMs with acidic/phosphorylation region N-terminal to the hydrophobic core (ZNF198), C-terminal (with spacer (PIAS1), without spacer (SP100)), at both sides (PML), SUMO1 specific (RanBP2) and SUMO2/3 specific

**SUMO binding Type Sequence** Sumoylation site Consensus ΨKxE

SIM ZNF198 DDDDDDD VVFI

Extended consensus PDSM ΨKxExxSP

Iverted consensus ExKΨ Phosphorylated Ser ΨKxS

PIAS1 VEVI DLTI DSSSDEEEEE SP100 IIVI SSEDSEGSTDVD PML EE R VVVI SSSEDSD RanBP2 SDSPSDDD VLIV CoREST1 EESEDELEE ANGNNP IDIEV

NDSM ΨKxExx[D/E]<sup>n</sup>

It is of significance that the sumoylation consensus sequence, before being established to be the site for SUMO attachment, was initially identified as a negative regulatory sequence in several transcription factors [52]. This scenario is exemplified by the transcription factor Elk-1 (Ets (E twenty-six)-like kinase 1), where a repressive domain, the R motif, was identified as an acceptor region for SUMO attachment [53]. Targeting SUMO or the SUMO conjugation enzyme UBC9 to promoters through a Gal4-based system efficiently represses transcription [54-56],

in target sumoylation.

(CoREST1), are shown.

34 Chromatin Remodelling

**3. SUMO in transcription**

**3.1. Transcription repression**

Despite the clear association of SUMO with gene repression, several reports illustrate the involvement of sumoylation in transcription activation. Examples of transcription factors whose activity is stimulated by SUMO are TCF4 [70], GATA4 [71], Pax6 [72], p45 [73], Smad4 [74], Oct4 [75], p53 [76], myocardin [77], PEA3 [78], NFAT1 [79] and HSF1 and 2 [80, 81]. Intriguingly, p53 has been reported both to be activated and repressed by SUMO [76, 82]. Since sumoylation may compete other post-translational modifications, a mechanism proposed for SUMO-mediated activation of transcription consists in avoiding degradation, and thereby in stabilization, of the transcription factor, as it has been proposed for Oct4 [83]. Otherwise, SUMO modification may interfere with association of repressors with the transcription factor, as occurs for Ikaros, whose sumoylation avoid interaction with histone deacetylase complexes [84]. Recently, two publications have brought into consideration the general assumption that SUMO globally associates with transcription repression. It has been reported in yeast that SUMO is detected at all the constitutively transcribed genes tested and in inducible genes upon activation [85]. However, Ubc9 inactivation results in increased transcription of inducible genes, although sumoylation at promoters is reduced, suggesting a role for SUMO in the silencing of inducible genes. In sum, authors conclude that while SUMO associates with repression in some contexts, other properties of SUMO come into play under normal constit‐ utive transcription [85]. More recently, a study performed in HeLa cells has revealed that from G1 to S phase of the cell cycle SUMO1 marks chromatin at the proximal promoter region on many of the most active housekeeping genes [86]. SUMO1 depletion results in reduced expression of these genes. However, this occurs for half of the active genes and the nature of the sumoylated proteins at the promoters remains unknown [86]. Taken together, all these data indicate that although SUMO may intrinsically associate with transcription repression, many other general processes, including constitutive transcription, may also depend on sumoylation, structurally or as a signaling pathway.

**4.2. Involvement of SUMO in recognition of histone modifications**

H3R2) [88].

As explained before, sumoylation of histone tails may affect the way in which different proteins recognize other histone modifications. Conversely, sumoylation of a chromatin-associated factor may modulate its capacity to recognize a specific histone modification. For instance, it has been reported that sumoylation of the bromodomain GTE3 protein, a BET (bromodomain and extra terminal domain) family member, interferes with the capacity of this protein to associate with acetylated histone tails [36]. A surprising link between the sumoylation pathway and recognition of histone modifications is illustrated by a recent and intriguing report describing the capacity of the PHD domain of plant PIAS proteins to directly recognize histone modifications such as methylated Lys4 and Arg2 on histone H3 (methyl-H3K4 and methyl-

SUMO Tasks in Chromatin Remodeling http://dx.doi.org/10.5772/55395 37

Polycomb group (PcG) proteins are involved in regulation of gene transcription and chromatin structure especially during development. These transcriptional repressors regulate lineage choice during development and differentiation by establishing long-term heritable gene silencing of relevant genes, for instance *Hox* genes. Thus, they are tightly linked to stem cell biology and cancer [89]. Two main complexes assembling PcG proteins have been described [90]. The polycomb repressive complex 2 (PRC2) contains the histone methyl transferase Enhancer of Zeste (EZH2) and is involved in methylation of H3K27. The PRC1 complex contains the Polycomb protein, which is involved in recognition of the repressive mark trimethyl-H3K27 through a chromodomain. Recruitment of PRC1 to the chromatin results in ubiquitination of histone H2A. Hence, coordinated action of both complexes is involved in the establishment of a compact chromatin structure, which results in gene silencing. One of the mammalian orthologs of Drosophila Polycomb is Polycomb-2 (Pc2), which has been shown to display SUMO ligase activity, as previously mentioned [23]. Interestingly, two SIMs have been described in Pc2, one of them has been shown to be relevant for the several functions attributed to Pc2 [91]. Among the SUMO substrates identified for Pc2 are the kinase HIPK2 and the corepressor CtBP1 (see also below), sumoylation of which results in enhanced transcription repression [92-94]. CtBP has been shown to colocalize with Pc2 in nuclear foci called PcG bodies, which contain several PcG proteins [95]. Other Pc2 substrates for sumoylation are ZEB2, DNMT3A and centrin-2 [96, 97]. It has been recently reported that Pc2 mediates sumoylation and recruitment of BMI1 at sites of DNA lesions, linking Pc2 ligase activity with the DNA damage response [98]. Several polycomb subunits have been shown to be sumoy‐ lated, for instance SUZ12, EZH2 and YY1, although Pc2 has not been involved in the process [99, 100]. A clear role of sumoylation in PcG proteins-mediated repression came from studies in *C. elegans*. The SOP-2 protein is related to Drosophila and vertebrate PRC1-associated PcG proteins Polyhomeotic and Sex combs on midleg (Scm). It has been shown that sumoylation of SOP-2 is required for repression of *Hox* genes in *C. elegans* [101]. Indeed, impaired sumoy‐ lation leads to ectopic *Hox* gene expression and homeotic transformations, resulting in a phenotype similar to that provoked by *sop-2* mutations. Additional evidence of SUMO involvement in PcG-mediated repression in vertebrates has been more recently reported. It was previously shown that Pc2 is a target of SUMO [23]. Later, Kang et al demonstrated that sumoylated Pc2 is a target for the SUMO protease SENP2 [102]. In *Senp2* knockout mice,

#### **4. Histone modification and chromatin remodeling**

#### **4.1. Histone sumoylation**

Regarding histone modification, sumoylation has been implicated in both, direct modification of histones and deposition/recognition of other histone marks, such as acetylation and methylation. Histone sumoylation has been demonstrated in both yeast and mammal cells [55, 57]. All core histones and the H2A.Z variant have been shown to be sumoylated in yeast [57, 87], while work on mammal cells has been centered on histone H4 [55]. The N-terminal tail of canonical histones is the target for sumoylation, indicating that sumoylation may interplay with other histone modifications at this region, like acetylation, methylation and phosphory‐ lation. Interfering with the sumoylation pathway significantly reduces the level of histone sumoylation in yeast [57]. Histone sumoylation has been associated with transcription repression. Indeed, mutation of sumoylation sites in histone H2B in yeast leads to increased basal expression of several non-induced genes [57]. A more specific role in Rad51-labeling of persistent DNA double strand breaks has been attributed to sumoylation of the histone variant H2A.Z in yeast [87]. However, which is the real impact of histone sumoylation in transcription in vivo and whether it is a common feature all along the genome need to be clarified.

#### **4.2. Involvement of SUMO in recognition of histone modifications**

[74], Oct4 [75], p53 [76], myocardin [77], PEA3 [78], NFAT1 [79] and HSF1 and 2 [80, 81]. Intriguingly, p53 has been reported both to be activated and repressed by SUMO [76, 82]. Since sumoylation may compete other post-translational modifications, a mechanism proposed for SUMO-mediated activation of transcription consists in avoiding degradation, and thereby in stabilization, of the transcription factor, as it has been proposed for Oct4 [83]. Otherwise, SUMO modification may interfere with association of repressors with the transcription factor, as occurs for Ikaros, whose sumoylation avoid interaction with histone deacetylase complexes [84]. Recently, two publications have brought into consideration the general assumption that SUMO globally associates with transcription repression. It has been reported in yeast that SUMO is detected at all the constitutively transcribed genes tested and in inducible genes upon activation [85]. However, Ubc9 inactivation results in increased transcription of inducible genes, although sumoylation at promoters is reduced, suggesting a role for SUMO in the silencing of inducible genes. In sum, authors conclude that while SUMO associates with repression in some contexts, other properties of SUMO come into play under normal constit‐ utive transcription [85]. More recently, a study performed in HeLa cells has revealed that from G1 to S phase of the cell cycle SUMO1 marks chromatin at the proximal promoter region on many of the most active housekeeping genes [86]. SUMO1 depletion results in reduced expression of these genes. However, this occurs for half of the active genes and the nature of the sumoylated proteins at the promoters remains unknown [86]. Taken together, all these data indicate that although SUMO may intrinsically associate with transcription repression, many other general processes, including constitutive transcription, may also depend on

sumoylation, structurally or as a signaling pathway.

**4.1. Histone sumoylation**

36 Chromatin Remodelling

**4. Histone modification and chromatin remodeling**

Regarding histone modification, sumoylation has been implicated in both, direct modification of histones and deposition/recognition of other histone marks, such as acetylation and methylation. Histone sumoylation has been demonstrated in both yeast and mammal cells [55, 57]. All core histones and the H2A.Z variant have been shown to be sumoylated in yeast [57, 87], while work on mammal cells has been centered on histone H4 [55]. The N-terminal tail of canonical histones is the target for sumoylation, indicating that sumoylation may interplay with other histone modifications at this region, like acetylation, methylation and phosphory‐ lation. Interfering with the sumoylation pathway significantly reduces the level of histone sumoylation in yeast [57]. Histone sumoylation has been associated with transcription repression. Indeed, mutation of sumoylation sites in histone H2B in yeast leads to increased basal expression of several non-induced genes [57]. A more specific role in Rad51-labeling of persistent DNA double strand breaks has been attributed to sumoylation of the histone variant H2A.Z in yeast [87]. However, which is the real impact of histone sumoylation in transcription

in vivo and whether it is a common feature all along the genome need to be clarified.

As explained before, sumoylation of histone tails may affect the way in which different proteins recognize other histone modifications. Conversely, sumoylation of a chromatin-associated factor may modulate its capacity to recognize a specific histone modification. For instance, it has been reported that sumoylation of the bromodomain GTE3 protein, a BET (bromodomain and extra terminal domain) family member, interferes with the capacity of this protein to associate with acetylated histone tails [36]. A surprising link between the sumoylation pathway and recognition of histone modifications is illustrated by a recent and intriguing report describing the capacity of the PHD domain of plant PIAS proteins to directly recognize histone modifications such as methylated Lys4 and Arg2 on histone H3 (methyl-H3K4 and methyl-H3R2) [88].

Polycomb group (PcG) proteins are involved in regulation of gene transcription and chromatin structure especially during development. These transcriptional repressors regulate lineage choice during development and differentiation by establishing long-term heritable gene silencing of relevant genes, for instance *Hox* genes. Thus, they are tightly linked to stem cell biology and cancer [89]. Two main complexes assembling PcG proteins have been described [90]. The polycomb repressive complex 2 (PRC2) contains the histone methyl transferase Enhancer of Zeste (EZH2) and is involved in methylation of H3K27. The PRC1 complex contains the Polycomb protein, which is involved in recognition of the repressive mark trimethyl-H3K27 through a chromodomain. Recruitment of PRC1 to the chromatin results in ubiquitination of histone H2A. Hence, coordinated action of both complexes is involved in the establishment of a compact chromatin structure, which results in gene silencing. One of the mammalian orthologs of Drosophila Polycomb is Polycomb-2 (Pc2), which has been shown to display SUMO ligase activity, as previously mentioned [23]. Interestingly, two SIMs have been described in Pc2, one of them has been shown to be relevant for the several functions attributed to Pc2 [91]. Among the SUMO substrates identified for Pc2 are the kinase HIPK2 and the corepressor CtBP1 (see also below), sumoylation of which results in enhanced transcription repression [92-94]. CtBP has been shown to colocalize with Pc2 in nuclear foci called PcG bodies, which contain several PcG proteins [95]. Other Pc2 substrates for sumoylation are ZEB2, DNMT3A and centrin-2 [96, 97]. It has been recently reported that Pc2 mediates sumoylation and recruitment of BMI1 at sites of DNA lesions, linking Pc2 ligase activity with the DNA damage response [98]. Several polycomb subunits have been shown to be sumoy‐ lated, for instance SUZ12, EZH2 and YY1, although Pc2 has not been involved in the process [99, 100]. A clear role of sumoylation in PcG proteins-mediated repression came from studies in *C. elegans*. The SOP-2 protein is related to Drosophila and vertebrate PRC1-associated PcG proteins Polyhomeotic and Sex combs on midleg (Scm). It has been shown that sumoylation of SOP-2 is required for repression of *Hox* genes in *C. elegans* [101]. Indeed, impaired sumoy‐ lation leads to ectopic *Hox* gene expression and homeotic transformations, resulting in a phenotype similar to that provoked by *sop-2* mutations. Additional evidence of SUMO involvement in PcG-mediated repression in vertebrates has been more recently reported. It was previously shown that Pc2 is a target of SUMO [23]. Later, Kang et al demonstrated that sumoylated Pc2 is a target for the SUMO protease SENP2 [102]. In *Senp2* knockout mice, sumoylated Pc2 accumulates, resulting in increased occupancy at promoters of PcG target genes, such as *Gata4* and *Gata6*. As a result, expression of these genes is reduced during development, which leads to embryonic heart defects among other disorders [102]. Chromatin occupancy by PRC2 subunits and levels of trimethyl-H3K27 seem not to be affected, suggesting that Pc2 sumoylation has a role in recognition of H3K27 methylation, which is released by SENP2.

is also reported. In correlation with loss of H3K9 methylation, HP1γ disappears from the *Foxp3*

(SETDB1, MBD1, MCAF1) H3K9 tri-methylation (SETDB1)

**Complex (subunits) Activity Recruiting factor**

(L3MBTL1, HP1) Methyl-histone recognition (L3MBTL1) Sp3

(dMi-2, dMEP-1) Nucleosome remodeling (dMi-2) Sp3

(EZH2, EED, SUZ12, RbAp46/48) H3K27 methylation (EZH2) various

(Pc2, PHC, RNF1/2, SCMH) trimethyl-H3K27 recognition (Pc2) –

The histone demethylase LSD1 mediates gene repression by removing methyl groups from mono- or di-methyl-H3K4, which are marks of active transcription [108]. LSD1 works in a corepressor complex together with HDACs and CoREST1 [109, 110]. It has been shown that the LSD1/CoREST complex mediates SUMO-dependent repression of neuronal-specific genes, such as *SCN1A* and *SCN3A*, in non-neuronal cells [111]. Recruitment to the chromatin and repression depends on SUMO2/3 and is mediated by a specific SIM in CoREST. SUMO deconjugation by the SUMO protease SENP3 provokes increased levels of di-methyl-H3K4 and acetyl-H3, which leads to gene activation. Different subunits of the LSD1/CoREST complex have been shown to be sumoylated and/or to contain SIMs (reviewed in [68]). It has been recently shown that sumoylation of the LSD1/CoREST complex subunit BRAF35 controls neuronal differentiation [112]. Overexpression of BRAF35, but not of a sumoylation mutant, strongly impairs neuronal differentiation promoted by neurogenic factors in the vertebrate neural tube. Interestingly, iBRAF, a paralogue of BRAF35 ocasionally associated to the LSD1/ CoREST complex, is not sumoylated but is able to dimerize with BRAF35, inhibiting BRAF35 sumoylation and binding to the LSD1/CoREST complex. The LSD1/CoREST complex usually

**Table 3.** SUMO associated repressor complexes. Table summarizes some repressor complexes whose function depends on sumoylation. Examples of different transcription factors involved in recruitment of these complexes are

H3K4 demethylation (LSD1) Histone deacetylation (HDAC1/2)

Nucleosome remodeling (CHD3) Histone deacetylation (HDAC1)

CtBP1

39

SUMO Tasks in Chromatin Remodeling http://dx.doi.org/10.5772/55395

KAP-1

KAP-1 Sp3

promoter in the absence of PIAS1 [107].

LSD1/CoREST (LSD1, CoREST, BHC80, HDAC1/2, BRAF35, ZEB1, ZNF217/198)

NurD (CHD3/4, HDAC1/2, RbAp46/48, MTA1/2, MBD3/2)

SETDB1

L3MBTL1

dMEC

PCR2

PCR1

*4.3.2. Histone demethylation*

also shown.

#### **4.3. SUMO-mediated regulation of histone modifications**

As previously mentioned, the major impact of sumoylation on histone modification is linked to the role of SUMO in the architecture and function of several chromatin-associated complexes involved in histone modification. Sumoylation by itself may condition the way other histone marks are deposited. However, it has been unambiguously demonstrated that sumoylation is essential for function of a variety of complexes implicated in histone modification, which mostly associate with transcription repression [68, 69]. It has been previously indicated that SUMO is required for the maintenance of constitutive heterochromatin in fission yeast [58]. However, increased evidence of SUMO involvement in the establishment of heterochromatinlike structures in euchromatin loci (facultative heterochromatin) has emerged during the last years. Facultative heterochromatin, besides displaying significant DNA methylation, is characterized by low levels of histone acetylation and histone H3 methylated at Lys4 (H3K4), and high levels of histone H3 methylated at Lys27 (H3K27) and Lys9 (H3K9, di- or trimethylated), and histone H4 methylated at Lys20 (H4K20, mono-, di- or tri-methylated) [103]. Some of the complexes involved in the establishment of these marks are compiled in Table 3 and described below.

#### *4.3.1. Histone methylation*

The histone methyltransferase SETDB1 is involved in tri-methylation of H3K9, a repressive histone mark. The methyl CpG binding protein MBD1 and MCAF1 associate to SETDB1 in a complex, linking DNA methylation to histone methylation. This complex is recruited to the KAP-1 (KRAB associated protein-1) corepressor in a SUMO-dependent manner [26]. In its turn, sumoylated KAP-1 recruits the SETDB1 complex to the chromatin through the zinc finger protein KRAB. This is mediated by a SIM in SETDB1 [26]. In addition, another SIM has been reported in MCAF1, and both MCAF1 and MBD1 are sumoylated [104, 105]. Interestingly, a PHD domain in KAP-1 displays an E3 ligase activity, which promotes intramolecular sumoy‐ lation of the adjacent bromodomain [26]. The SETDB1 complex, as explained below, is also recruited to the transcription factor Sp3 in a SUMO dependent manner for transcription repression [106].

Recently, the SUMO ligase PIAS1 has been involved in maintaining an epigenetic repressive state, as studied at the *Foxp3* locus, that restricts differentiation of natural occurring thymusderived regulatory T cells [107]. Knocking down of *PIAS1* leads to reduced DNA methylation and loss of the repressive mark methyl-H3K9 on the *Foxp3* promoter. A prominent role of PIAS1 in recruitment and association to the DNA methyltransferases DNMT3A and DNMT3B is also reported. In correlation with loss of H3K9 methylation, HP1γ disappears from the *Foxp3* promoter in the absence of PIAS1 [107].


**Table 3.** SUMO associated repressor complexes. Table summarizes some repressor complexes whose function depends on sumoylation. Examples of different transcription factors involved in recruitment of these complexes are also shown.

#### *4.3.2. Histone demethylation*

sumoylated Pc2 accumulates, resulting in increased occupancy at promoters of PcG target genes, such as *Gata4* and *Gata6*. As a result, expression of these genes is reduced during development, which leads to embryonic heart defects among other disorders [102]. Chromatin occupancy by PRC2 subunits and levels of trimethyl-H3K27 seem not to be affected, suggesting that Pc2 sumoylation has a role in recognition of H3K27 methylation, which is released by

As previously mentioned, the major impact of sumoylation on histone modification is linked to the role of SUMO in the architecture and function of several chromatin-associated complexes involved in histone modification. Sumoylation by itself may condition the way other histone marks are deposited. However, it has been unambiguously demonstrated that sumoylation is essential for function of a variety of complexes implicated in histone modification, which mostly associate with transcription repression [68, 69]. It has been previously indicated that SUMO is required for the maintenance of constitutive heterochromatin in fission yeast [58]. However, increased evidence of SUMO involvement in the establishment of heterochromatinlike structures in euchromatin loci (facultative heterochromatin) has emerged during the last years. Facultative heterochromatin, besides displaying significant DNA methylation, is characterized by low levels of histone acetylation and histone H3 methylated at Lys4 (H3K4), and high levels of histone H3 methylated at Lys27 (H3K27) and Lys9 (H3K9, di- or trimethylated), and histone H4 methylated at Lys20 (H4K20, mono-, di- or tri-methylated) [103]. Some of the complexes involved in the establishment of these marks are compiled in Table 3

The histone methyltransferase SETDB1 is involved in tri-methylation of H3K9, a repressive histone mark. The methyl CpG binding protein MBD1 and MCAF1 associate to SETDB1 in a complex, linking DNA methylation to histone methylation. This complex is recruited to the KAP-1 (KRAB associated protein-1) corepressor in a SUMO-dependent manner [26]. In its turn, sumoylated KAP-1 recruits the SETDB1 complex to the chromatin through the zinc finger protein KRAB. This is mediated by a SIM in SETDB1 [26]. In addition, another SIM has been reported in MCAF1, and both MCAF1 and MBD1 are sumoylated [104, 105]. Interestingly, a PHD domain in KAP-1 displays an E3 ligase activity, which promotes intramolecular sumoy‐ lation of the adjacent bromodomain [26]. The SETDB1 complex, as explained below, is also recruited to the transcription factor Sp3 in a SUMO dependent manner for transcription

Recently, the SUMO ligase PIAS1 has been involved in maintaining an epigenetic repressive state, as studied at the *Foxp3* locus, that restricts differentiation of natural occurring thymusderived regulatory T cells [107]. Knocking down of *PIAS1* leads to reduced DNA methylation and loss of the repressive mark methyl-H3K9 on the *Foxp3* promoter. A prominent role of PIAS1 in recruitment and association to the DNA methyltransferases DNMT3A and DNMT3B

**4.3. SUMO-mediated regulation of histone modifications**

SENP2.

38 Chromatin Remodelling

and described below.

repression [106].

*4.3.1. Histone methylation*

The histone demethylase LSD1 mediates gene repression by removing methyl groups from mono- or di-methyl-H3K4, which are marks of active transcription [108]. LSD1 works in a corepressor complex together with HDACs and CoREST1 [109, 110]. It has been shown that the LSD1/CoREST complex mediates SUMO-dependent repression of neuronal-specific genes, such as *SCN1A* and *SCN3A*, in non-neuronal cells [111]. Recruitment to the chromatin and repression depends on SUMO2/3 and is mediated by a specific SIM in CoREST. SUMO deconjugation by the SUMO protease SENP3 provokes increased levels of di-methyl-H3K4 and acetyl-H3, which leads to gene activation. Different subunits of the LSD1/CoREST complex have been shown to be sumoylated and/or to contain SIMs (reviewed in [68]). It has been recently shown that sumoylation of the LSD1/CoREST complex subunit BRAF35 controls neuronal differentiation [112]. Overexpression of BRAF35, but not of a sumoylation mutant, strongly impairs neuronal differentiation promoted by neurogenic factors in the vertebrate neural tube. Interestingly, iBRAF, a paralogue of BRAF35 ocasionally associated to the LSD1/ CoREST complex, is not sumoylated but is able to dimerize with BRAF35, inhibiting BRAF35 sumoylation and binding to the LSD1/CoREST complex. The LSD1/CoREST complex usually associates with the corepressor CtBP (C-terminal binding protein), which in turn is recruited to the chromatin by a variety of transcription factors [113]. Two CtBPs have been reported in vertebrates, CtBP1 and CtBP2. CtBP1 mediates repression by recruiting a number of repression factors that in addition to LSD1 and HDACs, includes the H3K9 histone methyl-transferase G9a. CtBP1-mediated repression depends on sumoylation [92]. Besides direct interaction of CtBP1 with UBC9, CtBP1 sumoylation is also determined by the SUMO ligases PIAS1, PIAS2 and Pc2 [23, 92, 114]. One of the transcription factors recruiting CtBP1 to the chromatin is the Krüpel-like zinc finger DNA-binding repressor ZEB1, which is also a target for sumoylation [115]. Attachment of SUMO to ZEB1 is required for this factor to display full repression activity [94]. Another zinc finger protein that has been associated with the LSD1/CoREST complex is ZNF198. This factor is both able to be sumoylated and to non-covalently interact with SUMO through a SIM [116-118]. Altogether, these data indicate that SUMO is involved on several functional aspects of the LSD1/CoREST complex: it mediates recruitment of the complex to the chromatin, but also is involved in the architecture of the complex, as different subunits associate to the complex in a sumoylation/SIM-dependent manner.

class III SIRT1 deacetylases to several factors. Sumoylation of the coactivator p300 medi‐ ates recruitment of class II HDAC6 and class III SIRT1 deacetylases [66, 126]. HDAC1 recruitment to sumoylated Groucho, p68 and reptin has also been described [121, 127, 128]. Moreover, a SUMO-histone H4 fusion has been shown to precipitate HDAC1 [55]. De‐ spite these data, it is not clear at present whether SUMO-dependent recruitment of HDACs involves direct binding of HDAC to SUMO or whether HDACs associate through cofac‐ tors recruited in a SUMO-dependent manner, as indicated for the LSD1/CoREST and NuRD complexes. Another example of SUMO-dependent recruitment of HDAC is depicted by the Daxx-mediated recruitment of HDAC2 to sumoylated CBP [67]. In this context, it is worth noting that in a variety of cases, HDAC recruitment does not account for full repression activity mediated by SUMO, as inhibition of HDACs does not relieve SUMO-dependent repression as expected. For instance, it has been shown in a reporter system that repres‐ sion mediated by a Gal4-SUMO fusion is not sensitive to HDAC inhibition [56], as also occurs for SUMO-dependent Sp3-mediated repression [64, 129]. Despite HDAC2 recruit‐ ment by sumoylated Elk-1, HDAC2 knockdown only partially alleviates SUMO-depend‐ ent Elk-1-mediated repression [56]. Therefore, histone deacetylases are recruited in a SUMOdependent manner through repressor complexes, together with additional repressor components, to account for full repression activity of the complex. Conversely, HDAC displacement by target sumoylation has been less reported, but examples have been described. Thus, sumoylation of the Prospero-related homeobox 1 (Prox1) and the de novo DNA methyltransferase DNMT3A disrupts association to HDAC3 and HDAC1/2, respec‐ tively [130, 131]. On the other hand, HDACs have also been shown to be substrates for SUMO, which regulates HDAC activity. Then, mutation of the sumoylation sites in HDAC1 has been shown to dramatically reduce its repression activity in a reporter assay [122]. It has been reported that the protease SENP1 is able to remove SUMO from sumoylated HDAC1, which leads to enhanced transcription activity by the androgen receptor [132]. Interestingly, the viral protein Gam1 interferes with HDAC1 sumoylation [133]. The RanBP2 ligase has been demonstrated to promote sumoylation of HDAC4 [134], and a relevant role for SUMO chain formation on HDAC4 has been attributed to the non-covalent interaction between SUMO and UBC9 [135]. Paradoxically, while HDAC1 sumoylation seems to be essential for its repression activity [122], SUMO attachment to HDAC1 impairs associa‐ tion to the CoREST repressor [116]. As previously mentioned, class IIa HDACs have been reported as SUMO ligases. HDAC4 and other class IIa HDACs promote SUMO2/3 attach‐ ment to the myocyte enhancer factor 2 family members MEF2D and MEF2C, which leads to repression of target genes [25]. Conversely, ligase activity is inhibited by HDAC4 sumoylation. HDAC4 ligase activity has been also demonstrated on liver X receptors sumoylation by SUMO2/3 [136] and on HIC1 sumoylation by SUMO1 [137], while en‐

SUMO Tasks in Chromatin Remodeling http://dx.doi.org/10.5772/55395 41

hanced sumoylation of PML protein has been attributed to HDAC7 [138].

**4.4. Multiple complexes contribute to SUMO-dependent Sp3-mediated repression**

Sp3 belongs to the specificity protein (Sp) family of transcription factors, which regulate multiple genes involved in housekeeping, development and cell cycle. Sp3 is expressed ubiquitously and can act either as an activator or a repressor depending on the promoter

#### *4.3.3. Histone deacetylation*

It has been indicated that repression activity of the LSD1/CoREST complex is in part displayed through HDACs. Indeed, HDAC1 and HDAC2 are components of the LSD1/CoREST complex [110]. Another complex involved in HDAC recruitment to the chromatin is the NuRD (nucle‐ osome remodeling and deacetylation) complex [119]. The core component of the mammalian NuRD complex is the ATP-dependent nucleosome remodeling enzyme CHD3. In addition, this complex includes one or two type I HDACs, histone binding proteins RbAp46 and RbAp48, a methylated DNA-binding protein (MBD2 or MBD3), and members of the MTA and p66 families of proteins [119]. A screening in Drosophila cell cultures identified the CHD3 homologue dMi-2, as a factor required for SUMO dependent repression by Sp3 [64]. dMi-2/ CHD3 both sumoylates and is able to interact with SUMO-modified transcription factors through a SIM [26, 64]. Thus, CHD3 also interacts with sumoylated KAP-1 [26]. However, it has been demonstrated that phosphorylation of Ser824 in the C terminus of KAP-1, directly impairs interaction between the CHD3 SIM and the SUMO molecule attached to KAP-1 [120]. Therefore, KAP-1 sumoylation is not affected, but recognition of SUMO by the SIM in CHD3. KAP-1 phosphorylation has a role in double-strand break repair, as displaces the chromatin barrier imposed by CHD3-dependent nucleosome-remodeling activity. Additional compo‐ nents of the NuRD complex have been shown to be sumoylated and/or to contain SIMs: MTA1/2, HDAC1, RbAp48 and p66 [111, 121-123]. Interestingly, phenotype of certain vulval mutants in *C. elegans*, which associate with genes coding for NuRD components [124], is quite similar to that of *SUMO* and *UBC9* mutants [125], indicating that function of the NuRD complex is linked to sumoylation.

SUMO directly associates with HDACs in a variety of ways. As previously indicated a wellestablished link between SUMO and HDACs is illustrated by the SIM-mediated recruit‐ ment of HDACs to sumoylated proteins [56, 121]. It was first demonstrated for HDAC2 recruitment to sumoylated Elk-1 [56], and subsequently for HDACs 1, 3, 4, 5 and 6, and

class III SIRT1 deacetylases to several factors. Sumoylation of the coactivator p300 medi‐ ates recruitment of class II HDAC6 and class III SIRT1 deacetylases [66, 126]. HDAC1 recruitment to sumoylated Groucho, p68 and reptin has also been described [121, 127, 128]. Moreover, a SUMO-histone H4 fusion has been shown to precipitate HDAC1 [55]. De‐ spite these data, it is not clear at present whether SUMO-dependent recruitment of HDACs involves direct binding of HDAC to SUMO or whether HDACs associate through cofac‐ tors recruited in a SUMO-dependent manner, as indicated for the LSD1/CoREST and NuRD complexes. Another example of SUMO-dependent recruitment of HDAC is depicted by the Daxx-mediated recruitment of HDAC2 to sumoylated CBP [67]. In this context, it is worth noting that in a variety of cases, HDAC recruitment does not account for full repression activity mediated by SUMO, as inhibition of HDACs does not relieve SUMO-dependent repression as expected. For instance, it has been shown in a reporter system that repres‐ sion mediated by a Gal4-SUMO fusion is not sensitive to HDAC inhibition [56], as also occurs for SUMO-dependent Sp3-mediated repression [64, 129]. Despite HDAC2 recruit‐ ment by sumoylated Elk-1, HDAC2 knockdown only partially alleviates SUMO-depend‐ ent Elk-1-mediated repression [56]. Therefore, histone deacetylases are recruited in a SUMOdependent manner through repressor complexes, together with additional repressor components, to account for full repression activity of the complex. Conversely, HDAC displacement by target sumoylation has been less reported, but examples have been described. Thus, sumoylation of the Prospero-related homeobox 1 (Prox1) and the de novo DNA methyltransferase DNMT3A disrupts association to HDAC3 and HDAC1/2, respec‐ tively [130, 131]. On the other hand, HDACs have also been shown to be substrates for SUMO, which regulates HDAC activity. Then, mutation of the sumoylation sites in HDAC1 has been shown to dramatically reduce its repression activity in a reporter assay [122]. It has been reported that the protease SENP1 is able to remove SUMO from sumoylated HDAC1, which leads to enhanced transcription activity by the androgen receptor [132]. Interestingly, the viral protein Gam1 interferes with HDAC1 sumoylation [133]. The RanBP2 ligase has been demonstrated to promote sumoylation of HDAC4 [134], and a relevant role for SUMO chain formation on HDAC4 has been attributed to the non-covalent interaction between SUMO and UBC9 [135]. Paradoxically, while HDAC1 sumoylation seems to be essential for its repression activity [122], SUMO attachment to HDAC1 impairs associa‐ tion to the CoREST repressor [116]. As previously mentioned, class IIa HDACs have been reported as SUMO ligases. HDAC4 and other class IIa HDACs promote SUMO2/3 attach‐ ment to the myocyte enhancer factor 2 family members MEF2D and MEF2C, which leads to repression of target genes [25]. Conversely, ligase activity is inhibited by HDAC4 sumoylation. HDAC4 ligase activity has been also demonstrated on liver X receptors sumoylation by SUMO2/3 [136] and on HIC1 sumoylation by SUMO1 [137], while en‐ hanced sumoylation of PML protein has been attributed to HDAC7 [138].

associates with the corepressor CtBP (C-terminal binding protein), which in turn is recruited to the chromatin by a variety of transcription factors [113]. Two CtBPs have been reported in vertebrates, CtBP1 and CtBP2. CtBP1 mediates repression by recruiting a number of repression factors that in addition to LSD1 and HDACs, includes the H3K9 histone methyl-transferase G9a. CtBP1-mediated repression depends on sumoylation [92]. Besides direct interaction of CtBP1 with UBC9, CtBP1 sumoylation is also determined by the SUMO ligases PIAS1, PIAS2 and Pc2 [23, 92, 114]. One of the transcription factors recruiting CtBP1 to the chromatin is the Krüpel-like zinc finger DNA-binding repressor ZEB1, which is also a target for sumoylation [115]. Attachment of SUMO to ZEB1 is required for this factor to display full repression activity [94]. Another zinc finger protein that has been associated with the LSD1/CoREST complex is ZNF198. This factor is both able to be sumoylated and to non-covalently interact with SUMO through a SIM [116-118]. Altogether, these data indicate that SUMO is involved on several functional aspects of the LSD1/CoREST complex: it mediates recruitment of the complex to the chromatin, but also is involved in the architecture of the complex, as different subunits

It has been indicated that repression activity of the LSD1/CoREST complex is in part displayed through HDACs. Indeed, HDAC1 and HDAC2 are components of the LSD1/CoREST complex [110]. Another complex involved in HDAC recruitment to the chromatin is the NuRD (nucle‐ osome remodeling and deacetylation) complex [119]. The core component of the mammalian NuRD complex is the ATP-dependent nucleosome remodeling enzyme CHD3. In addition, this complex includes one or two type I HDACs, histone binding proteins RbAp46 and RbAp48, a methylated DNA-binding protein (MBD2 or MBD3), and members of the MTA and p66 families of proteins [119]. A screening in Drosophila cell cultures identified the CHD3 homologue dMi-2, as a factor required for SUMO dependent repression by Sp3 [64]. dMi-2/ CHD3 both sumoylates and is able to interact with SUMO-modified transcription factors through a SIM [26, 64]. Thus, CHD3 also interacts with sumoylated KAP-1 [26]. However, it has been demonstrated that phosphorylation of Ser824 in the C terminus of KAP-1, directly impairs interaction between the CHD3 SIM and the SUMO molecule attached to KAP-1 [120]. Therefore, KAP-1 sumoylation is not affected, but recognition of SUMO by the SIM in CHD3. KAP-1 phosphorylation has a role in double-strand break repair, as displaces the chromatin barrier imposed by CHD3-dependent nucleosome-remodeling activity. Additional compo‐ nents of the NuRD complex have been shown to be sumoylated and/or to contain SIMs: MTA1/2, HDAC1, RbAp48 and p66 [111, 121-123]. Interestingly, phenotype of certain vulval mutants in *C. elegans*, which associate with genes coding for NuRD components [124], is quite similar to that of *SUMO* and *UBC9* mutants [125], indicating that function of the NuRD

SUMO directly associates with HDACs in a variety of ways. As previously indicated a wellestablished link between SUMO and HDACs is illustrated by the SIM-mediated recruit‐ ment of HDACs to sumoylated proteins [56, 121]. It was first demonstrated for HDAC2 recruitment to sumoylated Elk-1 [56], and subsequently for HDACs 1, 3, 4, 5 and 6, and

associate to the complex in a sumoylation/SIM-dependent manner.

*4.3.3. Histone deacetylation*

40 Chromatin Remodelling

complex is linked to sumoylation.

#### **4.4. Multiple complexes contribute to SUMO-dependent Sp3-mediated repression**

Sp3 belongs to the specificity protein (Sp) family of transcription factors, which regulate multiple genes involved in housekeeping, development and cell cycle. Sp3 is expressed ubiquitously and can act either as an activator or a repressor depending on the promoter context [106, 139]. Sp3-mediated repression depends on Sp3 sumoylation, and as previously indicated, this repression activity is not affected by HDAC inhibitors [129, 140].

repeats. In contrast to Scm, Sfmbt together with Pleiohomeotic, integrates in the polycomb complex PhoRC. Thus, different polycomb complexes include MBT-containing subunits, which might be involved in recognition of mono- and dimethylated histones to facilitate

SUMO Tasks in Chromatin Remodeling http://dx.doi.org/10.5772/55395 43

In sum, Sp3 constitutes a paradigm of SUMO-dependent transcription repression through a variety of factors and chromatin-associated complexes. Clear evidence of SUMO involvement in Sp3-mediated repression came from the generation of knock-in mice with a non-sumoylat‐ able version of Sp3 [147]. As Lys residues are targets for other modifications different of sumoylation, for instance acetylation, authors, instead of mutating core Lys551 to Arg changed the acidic residue at the sumoylation site. Interestingly, they substituted Glu by Asp, which abrogated sumoylation, despite for may authors it is assumed the consensus ΨKxE/D. Mutation did not affected Sp3 protein levels. However, spermatocyte-specific genes *Dmc1* and *Dnahc8*, and neuronal genes *Paqr6*, *Rims3* and *Robo3* appeared derepressed in non-testicular and extra-neural tissues and in mouse embryonic fibroblasts [147]. This correlated with loss of the repressive heterochromatin marks trimethyl-H3K9 and trimethyl-H4K20 and affected recruitment of repressor proteins, such as HP1, SETDB1, CHD3, and L3MBTL1/2, to the corresponding promoters. Surprisingly, homozygous knock-in mice born at expected mende‐ lian frequency, were fertile and exhibited no obvious phenotype, in contrast to mice lacking Sp3 [148], suggesting that additional mechanisms may control protein expression from the

Sumoylation results essential for development and growth of all the investigated eukaryotes. In mice, embryos lacking the SUMO conjugating enzyme Ubc9 die at the early postimplanta‐ tion stage, highlighting the relevance of SUMO conjugation during development [149]. The SUMO pathway is conserved from yeast to human and, together with ubiquitination, appears to be the most utilized pathway in post-translational modifications by UBLs. Despite similar structural features and a common evolutionary origin of SUMO and ubiquitin, they have significantly diverged from a functional point of view. In fact, a complete machinery has evolved around SUMO for specific conjugation/deconjugation of this molecule. Compared with ubiquitin, about 20 N-terminal extra amino acids are present in SUMO, which should account for the different and specific SUMO roles. From the many examples of protein modification by SUMO, structural, regulatory, signaling, and scaffold roles are inferred for this molecule. All these aspects convene to reveal SUMO modification as an important posttranslational modification involved in transcription repression. Therefore, SUMO prefigures as an adaptor molecule essential for correct assembly and function of a variety of chromatinassociated repressor complexes. This does not exclude that involvement of SUMO in various systems results in transcriptional activation. A number of SUMO-dependent histone modifi‐ cations and chromatin remodeling activities have been summarized in this chapter (Table 3). They include, HDACs, HMTs and histone demethylase activities, associated to the NuRD, LSD1/CoREST, SETDB1, dMec, L3MBTL1 and Polycomb complexes, which result in chromatin

trimethylation by recruiting other subunits with histone methyltransferase activity.

aberrantly induced transcripts.

**5. Conclusions**

A genome-wide RNAi screen in Drosophila cell cultures revealed that multiple complexes were involved in SUMO-dependent repression by Sp3 [64]. Among the genes identified whose knockdown impaired SUMO-dependent transcription repression were genes encoding the ATP-dependent chromatin remodeler dMi-2, the Drosophila ortholog of the nematode protein MEP-1 and the polycomb protein Sfmbt. Biochemical analyses indicated that dMi-2, MEP-1 and Sfmbt interacted with each other, bound to SUMO and were recruited to the chromatin in a SUMO-dependent manner. In addition, chromatin immunoprecipitation experiments showed that sumoylated Sp3 recruits a number of heterochromatin associated proteins, including dMi-2, the H3K9 histone methyl transferase (HMT) SETDB1, the H4K20 histone methyl transferase SUV4-20H, heterochromatin protein 1 (HP1) α, ß and γ, and MBT-domain proteins [141].

It has been previously indicated that dMi-2 is the core component of NuRD complex, a complex with associated HDACs. However, Sp3-SUMO-mediated repression is not sensistive to HDACs inhibitors, indicating either that dMi-2 mediates repression outside the NuRD complex or that there is a redundancy in the mechanisms driving Sp3-SUMO-mediated repression. In fact, several data indicate that dMi-2 is also part of another complex lacking HDAC activity. This complex, dMec, is composed by dMi-2 and the Drosophila homolog of the *C. elegans* protein MEP-1 (dMEP-1), and works as a corepressor of proneural genes [142]. Knockdown of dMEP-1 leads to derepression of Sp3 target genes, which is in contrast to functional redundancy among the different repression mechanisms recruited to Sp3 [64]. It is worth noting that MEP-1 was previously shown to contribute to SUMO-dependent repression in *C. elegans* [143]. Thus, sumoylated LIN-1 recruits MEP-1 for repression and inhibition of vulval cell fate. As LIN-1 is homologous to the human Elk-1, it is tempting to speculate that a similar mechanism may account for the SUMO-mediated HDAC-independent repression by Elk-1, despite the absence of a clear MEP-1 homolog in vertebrates.

As formerly mentioned, two HMTs were also recruited to SUMO-Sp3: SETDB1 and SUV4-20H, while the HMT SUV39H1 was not associated [141]. These HMTs were shown to be recruited to the *Dhfr* promoter in a sumoylatable Sp3-dependent manner. Knocking down of *SETDB1* and *SUV4-20H* resulted in reduced trimethylation of H3K9 and H4K20 at the *Dhfr* promoter.

Finally, polycomb protein Sfmbt and the corresponding mammalian orthologs L3MBTL1 and L3MBTL2 also associate to sumoylated Sp3 [64, 141]. These proteins contain repeats of the malignant brain tumor (MBT) domain, which is structurally related to the chromodomain and the Tudor domain, and like these, is able to recognize methylated histones. However, MBTs associate with higher affinity to mono- and di- than to trimethylated histones [144]. It has been shown that L3MBTL1 binds HP1γ and compacts chromatin in a mono- and dimethylated H4K20 and H1bK26-dependent manner [145]. Therefore, this association provides a way to explain L3MBTL1-mediated repression. Binding of HP1α, ß and γ to Sp3 depends on sumoy‐ lation [141]. Sumoylated histone H4 recruits HP1γ [55], and HP1α has also been shown to preferentially bind sumoylated SP100 [146], suggesting that, as occurs for HDACs, SUMO mediates HP1 recruitment. As Sfmbt, the PRC2-associated PcG protein Scm also contains MBT repeats. In contrast to Scm, Sfmbt together with Pleiohomeotic, integrates in the polycomb complex PhoRC. Thus, different polycomb complexes include MBT-containing subunits, which might be involved in recognition of mono- and dimethylated histones to facilitate trimethylation by recruiting other subunits with histone methyltransferase activity.

In sum, Sp3 constitutes a paradigm of SUMO-dependent transcription repression through a variety of factors and chromatin-associated complexes. Clear evidence of SUMO involvement in Sp3-mediated repression came from the generation of knock-in mice with a non-sumoylat‐ able version of Sp3 [147]. As Lys residues are targets for other modifications different of sumoylation, for instance acetylation, authors, instead of mutating core Lys551 to Arg changed the acidic residue at the sumoylation site. Interestingly, they substituted Glu by Asp, which abrogated sumoylation, despite for may authors it is assumed the consensus ΨKxE/D. Mutation did not affected Sp3 protein levels. However, spermatocyte-specific genes *Dmc1* and *Dnahc8*, and neuronal genes *Paqr6*, *Rims3* and *Robo3* appeared derepressed in non-testicular and extra-neural tissues and in mouse embryonic fibroblasts [147]. This correlated with loss of the repressive heterochromatin marks trimethyl-H3K9 and trimethyl-H4K20 and affected recruitment of repressor proteins, such as HP1, SETDB1, CHD3, and L3MBTL1/2, to the corresponding promoters. Surprisingly, homozygous knock-in mice born at expected mende‐ lian frequency, were fertile and exhibited no obvious phenotype, in contrast to mice lacking Sp3 [148], suggesting that additional mechanisms may control protein expression from the aberrantly induced transcripts.

#### **5. Conclusions**

context [106, 139]. Sp3-mediated repression depends on Sp3 sumoylation, and as previously

A genome-wide RNAi screen in Drosophila cell cultures revealed that multiple complexes were involved in SUMO-dependent repression by Sp3 [64]. Among the genes identified whose knockdown impaired SUMO-dependent transcription repression were genes encoding the ATP-dependent chromatin remodeler dMi-2, the Drosophila ortholog of the nematode protein MEP-1 and the polycomb protein Sfmbt. Biochemical analyses indicated that dMi-2, MEP-1 and Sfmbt interacted with each other, bound to SUMO and were recruited to the chromatin in a SUMO-dependent manner. In addition, chromatin immunoprecipitation experiments showed that sumoylated Sp3 recruits a number of heterochromatin associated proteins, including dMi-2, the H3K9 histone methyl transferase (HMT) SETDB1, the H4K20 histone methyl transferase SUV4-20H, heterochromatin protein 1 (HP1) α, ß and γ, and MBT-domain

It has been previously indicated that dMi-2 is the core component of NuRD complex, a complex with associated HDACs. However, Sp3-SUMO-mediated repression is not sensistive to HDACs inhibitors, indicating either that dMi-2 mediates repression outside the NuRD complex or that there is a redundancy in the mechanisms driving Sp3-SUMO-mediated repression. In fact, several data indicate that dMi-2 is also part of another complex lacking HDAC activity. This complex, dMec, is composed by dMi-2 and the Drosophila homolog of the *C. elegans* protein MEP-1 (dMEP-1), and works as a corepressor of proneural genes [142]. Knockdown of dMEP-1 leads to derepression of Sp3 target genes, which is in contrast to functional redundancy among the different repression mechanisms recruited to Sp3 [64]. It is worth noting that MEP-1 was previously shown to contribute to SUMO-dependent repression in *C. elegans* [143]. Thus, sumoylated LIN-1 recruits MEP-1 for repression and inhibition of vulval cell fate. As LIN-1 is homologous to the human Elk-1, it is tempting to speculate that a similar mechanism may account for the SUMO-mediated HDAC-independent repression by

As formerly mentioned, two HMTs were also recruited to SUMO-Sp3: SETDB1 and SUV4-20H, while the HMT SUV39H1 was not associated [141]. These HMTs were shown to be recruited to the *Dhfr* promoter in a sumoylatable Sp3-dependent manner. Knocking down of *SETDB1* and *SUV4-20H* resulted in reduced trimethylation of H3K9 and H4K20 at the *Dhfr* promoter. Finally, polycomb protein Sfmbt and the corresponding mammalian orthologs L3MBTL1 and L3MBTL2 also associate to sumoylated Sp3 [64, 141]. These proteins contain repeats of the malignant brain tumor (MBT) domain, which is structurally related to the chromodomain and the Tudor domain, and like these, is able to recognize methylated histones. However, MBTs associate with higher affinity to mono- and di- than to trimethylated histones [144]. It has been shown that L3MBTL1 binds HP1γ and compacts chromatin in a mono- and dimethylated H4K20 and H1bK26-dependent manner [145]. Therefore, this association provides a way to explain L3MBTL1-mediated repression. Binding of HP1α, ß and γ to Sp3 depends on sumoy‐ lation [141]. Sumoylated histone H4 recruits HP1γ [55], and HP1α has also been shown to preferentially bind sumoylated SP100 [146], suggesting that, as occurs for HDACs, SUMO mediates HP1 recruitment. As Sfmbt, the PRC2-associated PcG protein Scm also contains MBT

Elk-1, despite the absence of a clear MEP-1 homolog in vertebrates.

indicated, this repression activity is not affected by HDAC inhibitors [129, 140].

proteins [141].

42 Chromatin Remodelling

Sumoylation results essential for development and growth of all the investigated eukaryotes. In mice, embryos lacking the SUMO conjugating enzyme Ubc9 die at the early postimplanta‐ tion stage, highlighting the relevance of SUMO conjugation during development [149]. The SUMO pathway is conserved from yeast to human and, together with ubiquitination, appears to be the most utilized pathway in post-translational modifications by UBLs. Despite similar structural features and a common evolutionary origin of SUMO and ubiquitin, they have significantly diverged from a functional point of view. In fact, a complete machinery has evolved around SUMO for specific conjugation/deconjugation of this molecule. Compared with ubiquitin, about 20 N-terminal extra amino acids are present in SUMO, which should account for the different and specific SUMO roles. From the many examples of protein modification by SUMO, structural, regulatory, signaling, and scaffold roles are inferred for this molecule. All these aspects convene to reveal SUMO modification as an important posttranslational modification involved in transcription repression. Therefore, SUMO prefigures as an adaptor molecule essential for correct assembly and function of a variety of chromatinassociated repressor complexes. This does not exclude that involvement of SUMO in various systems results in transcriptional activation. A number of SUMO-dependent histone modifi‐ cations and chromatin remodeling activities have been summarized in this chapter (Table 3). They include, HDACs, HMTs and histone demethylase activities, associated to the NuRD, LSD1/CoREST, SETDB1, dMec, L3MBTL1 and Polycomb complexes, which result in chromatin compaction and gene silencing. However, many questions remain open. For instance, whether proteins with intrinsic repression activity like HDACs are directly recruited by SUMO or instead, relevant repression activity in vivo results from association of HDACs to repressor complexes recruited in a SUMO-dependent manner, needs to be clarified. In addition, although HDACs have intrinsic repression activity, it has been shown that sumoylation of HDAC1 accounts for its full repression activity [122], raising the question whether SUMO modulates its activity or is recruiting additional repressors. Another intriguing aspect concerns functional redundancy among the different repressors recruited to a locus via SUMO. A number of repressors are recruited to the chromatin through a Gal4-SUMO2 fusion [123], but it has been shown that individually knocking down of these factors has little consequences in SUMO2 displayed repression, which may be explained by functional redundancy of the multiple repressors associated. In a similar way, downregulation of CHD3 (mammalian dMi-2) or L3MBTL1/2 does not impair Sp3-SUMO-mediated repression in vertebrate cells [64, 141]. However, mutation of *dMi-2* or *Sfmbt* in Drosophila has a significant impact in Sp3-SUMOdependent repression [64], suggesting that promoter context and local features account for the level of functional redundancy of SUMO-associated repressors. In addition, an important aspect of the SUMO modification concerns the fleeting nature of the modification in many cases, which means that SUMO-SIM interactions may have permanent consequences despite they are not further detected, a notion that implies a kind of memory and that thereby links SUMO to epigenetics. Interestingly, mutation of the SUMO2 SIM in CoREST is sufficient to abrogate repression of some neuronal specific genes in non-neuronal cells [111], highlighting the relevance of the non-covalent interaction of proteins with SUMO in regulating SUMOdependent repression. In this context, SIMs and sumoylation sites have been described in many subunits within a repressor complex (reviewed in [68]), which rises the question about how the appropriate connections are established.

PDSM, phosphorylation-dependent SUMO motif

PIAS, protein inhibitor of activated STAT PML, promyelocytic leukaemia protein

SENP, sentrin-specific protease SIM, SUMO interacting motif

SUMO, small ubiquitin-like modifier

and F Juarez-Vicente for critical reading of this chapter.

Address all correspondence to: mario.garcia@cabimer.es

Work in the Garcia-Dominguez laboratory is supported by the Spanish National Ministry of Economy and Competitiveness grant BFU2012-37304/BFI. I thank JC Reyes, P Garcia-Gutierrez

SUMO Tasks in Chromatin Remodeling http://dx.doi.org/10.5772/55395 45

Stem Cells Department. Andalusian Center for Molecular Biology and Regenerative Medi‐

[1] Hochstrasser M (2009) Origin and function of ubiquitin-like proteins. *Nature* 458:

[2] Burroughs AM, Iyer LM, Aravind L (2012) The natural history of ubiquitin and ubiq‐

[3] Mahajan R, Delphin C, Guan T, Gerace L, Melchior F (1997) A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein

cine (CABIMER) & High Council for Scientific Research (CSIC), Seville, Spain

uitin-related domains. *Frontiers in Bioscience* 17: 1433-1460.

UBLs, ubiquitin-like proteins

**Acknowledgements**

**Author details**

**References**

422-429.

RanBP2. *Cell* 88: 97-107.

Garcia-Dominguez Mario

Sp3, specificity protein 3 SP-RING, Siz/PIAS-RING

#### **Abbreviations**

CBP, CREB binding protein DeSI-1, desumoylating isopepyidase-1 EZH2, Enhancer of Zeste HDAC, histone deacetylase HMT, histone methyl transferase KAP-1, KRAB associated protein-1 NDSM, negatively charged residues-dependent SUMO motif Pc2, polycomb-2 PcG, polycomb group PCR1/2, polycomb repressive complex 1/2

PDSM, phosphorylation-dependent SUMO motif PIAS, protein inhibitor of activated STAT PML, promyelocytic leukaemia protein SENP, sentrin-specific protease SIM, SUMO interacting motif Sp3, specificity protein 3 SP-RING, Siz/PIAS-RING SUMO, small ubiquitin-like modifier UBLs, ubiquitin-like proteins

#### **Acknowledgements**

compaction and gene silencing. However, many questions remain open. For instance, whether proteins with intrinsic repression activity like HDACs are directly recruited by SUMO or instead, relevant repression activity in vivo results from association of HDACs to repressor complexes recruited in a SUMO-dependent manner, needs to be clarified. In addition, although HDACs have intrinsic repression activity, it has been shown that sumoylation of HDAC1 accounts for its full repression activity [122], raising the question whether SUMO modulates its activity or is recruiting additional repressors. Another intriguing aspect concerns functional redundancy among the different repressors recruited to a locus via SUMO. A number of repressors are recruited to the chromatin through a Gal4-SUMO2 fusion [123], but it has been shown that individually knocking down of these factors has little consequences in SUMO2 displayed repression, which may be explained by functional redundancy of the multiple repressors associated. In a similar way, downregulation of CHD3 (mammalian dMi-2) or L3MBTL1/2 does not impair Sp3-SUMO-mediated repression in vertebrate cells [64, 141]. However, mutation of *dMi-2* or *Sfmbt* in Drosophila has a significant impact in Sp3-SUMOdependent repression [64], suggesting that promoter context and local features account for the level of functional redundancy of SUMO-associated repressors. In addition, an important aspect of the SUMO modification concerns the fleeting nature of the modification in many cases, which means that SUMO-SIM interactions may have permanent consequences despite they are not further detected, a notion that implies a kind of memory and that thereby links SUMO to epigenetics. Interestingly, mutation of the SUMO2 SIM in CoREST is sufficient to abrogate repression of some neuronal specific genes in non-neuronal cells [111], highlighting the relevance of the non-covalent interaction of proteins with SUMO in regulating SUMOdependent repression. In this context, SIMs and sumoylation sites have been described in many subunits within a repressor complex (reviewed in [68]), which rises the question about how

the appropriate connections are established.

**Abbreviations**

44 Chromatin Remodelling

Pc2, polycomb-2

PcG, polycomb group

CBP, CREB binding protein

EZH2, Enhancer of Zeste HDAC, histone deacetylase

DeSI-1, desumoylating isopepyidase-1

HMT, histone methyl transferase KAP-1, KRAB associated protein-1

PCR1/2, polycomb repressive complex 1/2

NDSM, negatively charged residues-dependent SUMO motif

Work in the Garcia-Dominguez laboratory is supported by the Spanish National Ministry of Economy and Competitiveness grant BFU2012-37304/BFI. I thank JC Reyes, P Garcia-Gutierrez and F Juarez-Vicente for critical reading of this chapter.

#### **Author details**

Garcia-Dominguez Mario

Address all correspondence to: mario.garcia@cabimer.es

Stem Cells Department. Andalusian Center for Molecular Biology and Regenerative Medi‐ cine (CABIMER) & High Council for Scientific Research (CSIC), Seville, Spain

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**Section 2**

**Chromatin Remodeling in Regulating Gene**

**Expression**


**Chromatin Remodeling in Regulating Gene Expression**

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56 Chromatin Remodelling

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**Chapter 3**

**SWI/SNF Chromatin Remodeling**

Nadezhda E. Vorobyeva, Marina U. Mazina and

Additional information is available at the end of the chapter

Semen A. Doronin

**1. Introduction**

transcription regulation [3].

unit [6].

http://dx.doi.org/10.5772/55877

**Complex Involved in RNA Polymerase II**

**Elongation Process in** *Drosophila melanogaster*

After more than a decade of studying the chromatin remodeling, better view of the function mechanisms of the chromatin remodeling complexes has been developed. It was found that chromatin remodeling complexes facilitate transcription of genes by reducing the nucleosome density on specific genomic regions, such as enhancers and promoters, and increasing their affinity to activators and activator-binding complexes. Moreover, the importance of the chromatin remodeling complexes for transcriptional repression has been shown recently [1, 2]. Therefore chromatin remodeling complexes appear to be involved in nearly all aspects of

At present, the SWI/SNF chromatin remodeling complex is considered to be a significant player in the process of RNA Polymerase II transcription initiation. Recruitment of the complex precedes other transcriptional events and is important for the binding of the general tran‐ scriptional machinery [4]. The interplay between chromatin remodeling and general tran‐ scriptional factors is so close, that these complexes may unite into physically stable formations termed supercomplexes [5]. An example of such cooperation has been demonstrated for the *Drosophila* SWI/SNF (dSWI/SNF) and TFIID complexes with the SAYP coactivator as a linchpin

Recently, abundant evidence concerning SWI/SNF participation in the process of RNA Polymerase II elongation has been reported. It has been demonstrated that the SWI/SNF complex does not leave the promoter after general transcriptional factors recruitment but is involved in transcription elongation and co-transcriptional events. In addition SWI/SNF direct

and reproduction in any medium, provided the original work is properly cited.

© 2013 Vorobyeva et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,
