**7. Epigenetic and FSHD: Role of methylation and chromatine structure**

Several clinical features, such as penetrance variability, gender bias in severity, asymmetric muscle wasting, and discordance in monozygotic twins, suggest that FSHD development involves epigenetic factors that can influence gene expression through local modification of chromatin structure.

#### **7.1 DNA methylation**

C5 methylation of cytosine, the most common epigenetic modification of mammalian DNA is known to be involved in development, X-chromosome inactivation, imprinting, and gene silencing (Robertson and Wolffe, 2000). CpG methylation can affect occupancy of specific genomic regions since several transcription factors and chromatin-binding proteins, such as CTCF and Yin Yang 1 (YY1), are sensitive to it (Hark et al., 2000; Kim et al., 2003). Each D4Z4 repeat unit harbors two classes of GC-rich sequences, namely the low copy-repeats hhspm3 and LSau. This type of repetitive DNA is predominantly found in heterochromatic regions of the genome (Hewitt et al., 1994). On this basis, it has been hypothesized that D4Z4 repeat reduction might induce changes in chromatin conformation leading to inappropriate expression of 4q35 genes. The first study of DNA methylation at the D4Z4 repeat array did not show a change in this epigenetic marker in FSHD tissues. D4Z4 was found highly methylated in both normal and FSHD lymphoblasts, as well as in somatic tissues, including skeletal muscle. Nevertheless the study did not discriminate between the methylation status of the repeat array at chromosome 4 or chromosome 10 (Tsien et al., 2001). A subsequent study, revealed a significant hypomethylation of three different CpG dinucleotides of the D4Z4-reduced allele in lymphoblasts and muscle biopsies from FSHD patients (van Overveld et al., 2003). Importantly, low methylation levels at D4Z4 were observed at both chromosome 4 and 10 in the so-called phenotypic FSHD patients. These patients are clinically indistinguishable from 4q-linked FSHD patients but do not carry any D4Z4-reduced (de Greef et al, 2009). However, methylation levels can vary substantially between individuals. Generally, patients with residual repeat sizes between 10 and 19 kb (1- 3 D4Z4 units) are severely affected and show very low DNA methylation levels, whereas FSHD patients with repeat sizes between 20 and 31 kb (4-6 D4Z4 units) show interindividual variation in both clinical severity and D4Z4 hypomethylation (van Overveld et al., 2005). In addition, non-penetrant carriers show the same D4Z4 hypomethylation as their affected relatives and strong D4Z4 hypomethylation is also reported in patients with immunodeficiency, centromeric instability and facial anomalies syndrome (ICF) without any myopatic symptoms (Xu et al., 1999; Kondo et al., 2000; van Overveld et al., 2003). Currently, the role of D4Z4 hypomethylation in FSHD pathogenesis remains elusive.

#### **7.2 Histone modification**

Chromatin conformation results from the interaction between DNA and histone proteins and the involvement of other chromosomal proteins. The basic structural unit of chromatin

comparison to healthy controls (Laoudj-Chenivesse et al., 2005). Even though both increase of oxidative stress and *ANT1* overexpression are proposed to be early events in the development of FSHD, it remains unclear if these are sequential or parallel processes

Several clinical features, such as penetrance variability, gender bias in severity, asymmetric muscle wasting, and discordance in monozygotic twins, suggest that FSHD development involves epigenetic factors that can influence gene expression through local modification of

C5 methylation of cytosine, the most common epigenetic modification of mammalian DNA is known to be involved in development, X-chromosome inactivation, imprinting, and gene silencing (Robertson and Wolffe, 2000). CpG methylation can affect occupancy of specific genomic regions since several transcription factors and chromatin-binding proteins, such as CTCF and Yin Yang 1 (YY1), are sensitive to it (Hark et al., 2000; Kim et al., 2003). Each D4Z4 repeat unit harbors two classes of GC-rich sequences, namely the low copy-repeats hhspm3 and LSau. This type of repetitive DNA is predominantly found in heterochromatic regions of the genome (Hewitt et al., 1994). On this basis, it has been hypothesized that D4Z4 repeat reduction might induce changes in chromatin conformation leading to inappropriate expression of 4q35 genes. The first study of DNA methylation at the D4Z4 repeat array did not show a change in this epigenetic marker in FSHD tissues. D4Z4 was found highly methylated in both normal and FSHD lymphoblasts, as well as in somatic tissues, including skeletal muscle. Nevertheless the study did not discriminate between the methylation status of the repeat array at chromosome 4 or chromosome 10 (Tsien et al., 2001). A subsequent study, revealed a significant hypomethylation of three different CpG dinucleotides of the D4Z4-reduced allele in lymphoblasts and muscle biopsies from FSHD patients (van Overveld et al., 2003). Importantly, low methylation levels at D4Z4 were observed at both chromosome 4 and 10 in the so-called phenotypic FSHD patients. These patients are clinically indistinguishable from 4q-linked FSHD patients but do not carry any D4Z4-reduced (de Greef et al, 2009). However, methylation levels can vary substantially between individuals. Generally, patients with residual repeat sizes between 10 and 19 kb (1- 3 D4Z4 units) are severely affected and show very low DNA methylation levels, whereas FSHD patients with repeat sizes between 20 and 31 kb (4-6 D4Z4 units) show interindividual variation in both clinical severity and D4Z4 hypomethylation (van Overveld et al., 2005). In addition, non-penetrant carriers show the same D4Z4 hypomethylation as their affected relatives and strong D4Z4 hypomethylation is also reported in patients with immunodeficiency, centromeric instability and facial anomalies syndrome (ICF) without any myopatic symptoms (Xu et al., 1999; Kondo et al., 2000; van Overveld et al., 2003). Currently,

the role of D4Z4 hypomethylation in FSHD pathogenesis remains elusive.

Chromatin conformation results from the interaction between DNA and histone proteins and the involvement of other chromosomal proteins. The basic structural unit of chromatin

**7. Epigenetic and FSHD: Role of methylation and chromatine structure** 

(Winokur et al., 2003a).

chromatin structure.

**7.1 DNA methylation** 

**7.2 Histone modification** 

is the nucleosome that consists of 146 bp of DNA wrapped around a protein octamer of core histone proteins (Kornberg et al., 1974; Finch et al., 1977). Histone proteins may be posttranslational modified, by acetylation, methylation, phosphorylation, ubiquitination, SUMOylation and ADP-ribosylation (Bernstein et al., 2007). Modified histones are likely to control the structure and/or function of the chromatin fiber, with different modifications yielding distinct functional consequences. Furthermore, recruitment of chromatinassociating proteins may depend upon the recognition of a specific histone modification pattern (Strahl and Allis, 2000; Peterson and Laniel, 2004). Extracellular and intracellular stimuli may change these patterns of modification, making the chromatin itself an integrator of various signaling pathways, ultimately affecting basic cellular processes such as transcription or replication (Cheung et al., 2000; Nightingale et al., 2006). *In vivo*, chromatin exists as fibers with differing degrees of compaction. The morphologically distinct classes of chromatin within the nucleus of higher eukaryotes are heterochromatin, which is more compacted and generally transcriptionally inactive, and euchromatin, wich is less compacted and generally transcriptionally active (Frenster et al., 1963). Although D4Z4 unit harbors two classes of repetitive DNA, hhspm3 and LSau, both of which are found predominantly in heterochromatic domains of the genome, FSHD locus at 4qter does not share some of the common properties of heterochromatin. For instance it does not colocalize with DAPI-intense loci or it does not replicate in late S-phase. A recent study on D4Z4 histone modification seems to indicate that the repeat array may be organized in distinct domains, some characterized by transcriptionally repressive heterochromatin and others by transcriptionally permissive euchromatin (Zeng et al., 2009). These results indicate that the D4Z4 locus might display a chromatin structure more similar to euchromatin and favor the hypothesis that this region might be more dynamic than expected. Interestingly loss of marks of unexpressed heterochromatin such as histone H3K9me3 was observed in both FSHD with or without D4Z4 contraction. This phenomenon seems to be strictly associated with FSHD phenotype; in fact it was not found in ICF syndrome, despite its apparent similarity to FSHD with regard to D4Z4 DNA hypomethylation, or in other types of muscular dystrophies tested (Zeng et al., 2009). H3K9 methylation at D4Z4 is specifically mediated by the histone methyltransferase SUV39H1 (Zeng et al., 2009), which interacts with MyoD to suppress MyoD-dependent muscle gene expression (Mal, 2006). Interestingly, the heterochromatin binding protein HP1, which mediates transcriptional silencing (Bannister et al., 2001; Bernard et al., 2001), and the sister chromatid cohesion complex, cohesin, bind to D4Z4 in an H3K9me3-dependent manner and their recruitment is seriously compromised in FSHD (Zeng et al., 2009). These data support the indirect mechanism (Figure 5 C) where loss of repeats generates structural and functional modification, possibly through epigenetic changes in the histone pattern, which in turn might have an effect on transcriptional regulation in *cis* and/or in *trans*. It is reasonable to anticipate that future studies on the possible chromatin organization involving D4Z4 and its changes in FSHD may provide critical insight into the mechanism of FSHD pathogenesis.

#### **7.3 Long distance effect: A repressor complex binding D4Z4**

The alteration of 4q35 gene expression observed in FSHD affected muscle (Gabellini et al., 2002) raised the question whether D4Z4 was directly involved in transcriptional control of 4q35 genes. The analysis of the interaction between D4Z4 and nuclear proteins revealed the presence of a 27 bp binding site (DBE, D4Z4 Binding Element) able to recruit a multi-protein

Facioscapulohumeral Muscular Dystrophy: From Clinical Data to Molecular Genetics and Return 41

transcriptional activity, replication timing, and chromosome size (Sun HB et al., 2000; Tanabe et al., 2002). The nucleoplasm is separated from the cytoplasm by the nuclear envelope (NE), consisting of an inner (INM) and outer nuclear membrane (ONM), (Gerace et al., 1988 ). Chromosome 4qter is preferentially localized in the outer nuclear periphery (Masny et al., 2004; Tam et al., 2004) although mammalian telomeres, including 10qter, are usually dispersed in the inner part of the nucleus (Ludérus et al., 1996; Nagele et al., 2001; Amrichová et al., 2003; Weierich et al., 2003). Sequences proximal to D4Z4, and not the repeat array itself, seem to be required to localize the 4q telomere at the periphery (Masny et al., 2004). These sequences are not found at 10qter and this may explain the different nuclear localization of 10qter. Recently, Ottaviani et al. (2009) identified an 80-bp sequence inside the D4Z4 unit that can trigger perinuclear positioning of artificial telomeres in a CTCF- and lamin A–dependent manner. Furthermore in cells lacking the *lamin A* gene, chromosome 4 telomeres are dispersed (Masny et al., 2004). In addition, lamin A is shown to be associated with D4Z4 in vivo by chromatin immunoprecipitation and the perinuclear localization of 4qter is largely lost in fibroblasts lacking lamin A/C (Ottaviani et al., 2009). Although Fluoresece In Situ Hybridization (FISH) analyses showed no change in the chromosome 4 localization, between FSHD and healthy subjects, the peripheral environment of the FSHD 4q35 allele may be altered because of modification in chromatin structure at D4Z4. This nuclear lamina alteration might produce a change in the binding of specific proteins, thereby contribute to the aberrant 4q35 gene expression reported in FSHD (Masny et al.,

D4Z4 repeat contraction in patients with FSHD was discovered almost 20 years ago, nevertheless the exact molecular mechanism causing the FSHD phenotype has still not been elucidated and the search for a unifying model that can explain all the clinical features that have been observed in time has been frustrated. No histological or biochemical markers are available to independently confirm a specific FSHD diagnosis that remains mainly clinical. The molecular test primarily used for FSHD diagnosis was based on the initial observation that 95% of FSHD patients carry a reduction of integral numbers of D4Z4 repeats at 4q35 with full penetrance (Van Deutekom et al., 2003). However the wide use of this test revealed several exceptions to the original assumption. Through the years the threshold size of D4Z4 alleles has been increased from the original 28 kb (6 D4Z4 repeats) (Wimenga et al., 1999b) to 35 kb (8 D4Z4 repeats) (Van Deutekom et al., 2003), with FSHD cases carrying D4Z4 alleles of 38-41 kb (9-11 D4Z4 repeats) considered borderline alleles (Butz et al., 2003; Vitelli et al., 1999). A further analysis of genotype-phenotype correlation led in time to the identification of subjects carrying D4Z4 reduced alleles with no sign of muscle weakness in FSHD families (Ricci et al., 1999; Tonini et al., 2004) as well as in normal controls (Van Overveld et al., 2000; Weiffenbach et al., 1992). The genotype-phenotype correlation conducted more recently on a large scale using a standardized method of evaluation allowed to estimate that 1) 20% of FSHD patients carry full-length D4Z4 alleles, 2) over 25% of relatives carrying D4Z4 reduced alleles do not have FSHD, 3) 3% of healthy subjects from the general population carry D4Z4 reduced alleles 4) no specific 4q haplotype is uniquely associated with FSHD. Remarkably, these studies established as a general rule rather than an exception that detection of a D4Z4 reduced allele is not sufficient to predict FSHD (Scionti et al., 2012a; Scionti et al., 2012b). Over the years, the molecular etiology of FSHD

2004; Tam et al., 2004; Ottaviani et al., 2009).

**8. Conclusions** 

complex in vitro and in vivo comprising of YY1, HMGB2 and nucleolin, termed D4Z4 Recognition Complex (DRC) (Gabellini et al., 2002). The ubiquitous transcription factor Yin Yang 1 (YY1) is a recruiter of polycomb group proteins (PcG), which are responsible for chromatin remodelling and epigenetic silencing in many fundamental biological processes. YY1, exerts its effects on genes involved in normal biologic processes such as embryogenesis, differentiation, replication, and cellular proliferation. Its ability to initiate, activate, or repress transcription depends upon context (Gordon et al., 2006). Furthermore, the activity of YY1 is modulated by histone deacetylases and histone acetyltransferases (Yao et al., 2001). HMGB2 is a member of one of the three families of high mobility group (HMG) proteins (Bustin, 1999; Bianchi and Beltrame, 2000;Agresti and Bianchi, 2003). It has been proposed that HMGB2 might be involved in the organization and/or maintenance of heterochromatic regions through the SP100-mediated interaction with HP1 (Lehming et al., 1998). The third component of the DRC, nucleolin, is an abundant nucleolar protein, which has been implicated in chromatin structure, ribosomal RNA (rRNA) transcription, rRNA maturation, ribosome assembly and nucleo-cytoplasmic transport. To address whether the level of the DRC components influenced transcription of 4q35 genes, antisense experiments to decrease intracellular levels of DRC components were performed. These experiments showed that depletion of YY1, HMGB2 or nucleolin results in overexpression of the 4q35 gene *FRG2*, which is silent in normal cells and tissues (Gabellini et al., 2002). Accumulating evidences indicate that gene regulation can be affected by physical interaction between two distant chromosomal regions in *cis* and in *trans* in mammalian cells (Tolhuis et al., 2002; Horike et al., 2005; Spilianakis et al., 2005; Lomvardas et al., 2006). Thus the DRC might exerts is inhibitory activity either modifying the chromatin structure or acting directly on 4q35 genes promoters through a physical interaction mediated by the formation of a cromatin loop (Gabellini et al., 2002; Pirozhkova et al., 2008). The physical interaction between D4Z4 and FRG1 has been demonstrated (Pirozhkova et al., 2008; Bodega et al., 2009) in normal myoblast by Chromosome conformation capture (3C), which is a technique that identifies long distance intra- and inter-chromosomal interactions (Dekker et al., 2002). Interestingly chromatin seems to undergo remodeling during myogenic differentiation. It has been shown that in normal myoblasts, the *FRG1* gene is repressed and its promoter physically interacts with the D4Z4 array; upon differentiation, *FRG1* gene is expressed and the chromatin loop between FRG1 promoter and D4Z4 is relaxed (Bodega et al.2009). Consistent with the observed mis-regulation of FRG1, a small reduction in the D4Z4–FRG1 promoter interaction was observed in FSHD myoblasts compared with controls (Bodega et al., 2009). Different findings obtained with 3C analysis described the formation of loops between other elements in the FSHD locus (DUX4c and the 4qA/B marker) and the FRG1 promoter (Pirozhkova et al., 2008). These data indicate that the tridimensional structure of the FSHD locus is complex and composite, probably more than one sequence elements (for example, D4Z4, DUX4c,4qA/B) or more than one chromatin modification factor might be required to obtain a fine regulation of *FRG1* gene expression during muscle differentiation (Petrov et al., 2006; Pirozhkova et al., 2008).

#### **7.4 Subnuclear localization of 4q35**

The nucleoplasm is a high defined and structured compartment and chromosomes occupy specific and distinct territories. These chromosome territories are related to gene density, transcriptional activity, replication timing, and chromosome size (Sun HB et al., 2000; Tanabe et al., 2002). The nucleoplasm is separated from the cytoplasm by the nuclear envelope (NE), consisting of an inner (INM) and outer nuclear membrane (ONM), (Gerace et al., 1988 ). Chromosome 4qter is preferentially localized in the outer nuclear periphery (Masny et al., 2004; Tam et al., 2004) although mammalian telomeres, including 10qter, are usually dispersed in the inner part of the nucleus (Ludérus et al., 1996; Nagele et al., 2001; Amrichová et al., 2003; Weierich et al., 2003). Sequences proximal to D4Z4, and not the repeat array itself, seem to be required to localize the 4q telomere at the periphery (Masny et al., 2004). These sequences are not found at 10qter and this may explain the different nuclear localization of 10qter. Recently, Ottaviani et al. (2009) identified an 80-bp sequence inside the D4Z4 unit that can trigger perinuclear positioning of artificial telomeres in a CTCF- and lamin A–dependent manner. Furthermore in cells lacking the *lamin A* gene, chromosome 4 telomeres are dispersed (Masny et al., 2004). In addition, lamin A is shown to be associated with D4Z4 in vivo by chromatin immunoprecipitation and the perinuclear localization of 4qter is largely lost in fibroblasts lacking lamin A/C (Ottaviani et al., 2009). Although Fluoresece In Situ Hybridization (FISH) analyses showed no change in the chromosome 4 localization, between FSHD and healthy subjects, the peripheral environment of the FSHD 4q35 allele may be altered because of modification in chromatin structure at D4Z4. This nuclear lamina alteration might produce a change in the binding of specific proteins, thereby contribute to the aberrant 4q35 gene expression reported in FSHD (Masny et al., 2004; Tam et al., 2004; Ottaviani et al., 2009).
