**3. Nuclear localization and function of MAPKs**

In resting cells, MAPK components are usually located in the cytoplasm through their interaction with different anchor proteins, scaffolds, or phosphatases. Upon stimulation, MAPK signaling cascades rapidly transmit information into the nucleus to ensure the appropriate transcriptional response (**Figure 2A**). Across eukaryotes, this process is often initiated by transient accumulation of the MAPKs within the nucleus. The duration and the type of stimuli affect the nuclear localization of MAPK signaling proteins and play an important role in determination of the transcriptional output. Translocation of MAPK molecules requires specialized transport elements to travel through the nuclear pore complex (NPC).

#### **Figure 2.**

*MAPK regulatory roles on gene expression: From transcription initiation to translation. (***A***) Activated MAPK is released from its cytoplasmic anchor and translocated to the nucleus. (***B***) From top and clockwise, MAPK regulation on different targets is represented by a black arrow; MAPKs are known to activate transcription factors (TFs) through phosphorylation and to recruit PolII to initiate transcription. Moreover, MAPKs also target several chromatin remodelers (Ch Rem) and histone modifiers (Hist mod) to regulate chromatin structure and histone eviction. MAPK interacts with transcription elongating (TEF) and termination factors to enhance transcription rate. mRNA is shown as a green line with a 5*′ *cap (green dot) and the polyA at the 3*′ *end. MAPKs also regulate several stabilizing RNA-binding proteins (RBPs), target miRNA processing through the microprocessor complex (MC), nuclear exporters, and splicing factors. Finally, MAPKs are also known to regulate translation elongation initiation factors (eIFs) to stimulate rapid mRNA translation. Overall, all these mechanisms aim to promote a rapid and efficient response for maximal cell adaptation.*

Nucleoplasmic shuttling of active MAPK can be mediated mainly by three strategies: (1) active regulation of import-export through the NPC; (2) escape from cytoplasmic anchors and/or sequestration by nuclear components; and (3) passive diffusion.

Canonical nuclear localization is an active process during which nuclear α/β importin complexes deliver cargo containing mono- or bi-partite stretches of basic residues (nuclear localization signals—NLSs) to the nucleus. Once in the nucleus, targeted proteins dissociate from importins by interacting with RanGTP. For example, in its inactive state, a nuclear export signal (NES) is exposed in ERK2, which confines it to the cytoplasm. Upon activation of ERK2, a conformational change disrupts its N- and C-terminal interactions, thereby exposing a NLS that sends the kinase into the nucleus. Similarly, activation of ERK1/2 allows their interaction with importin-7 and their nuclear accumulation [30]. Not only ERK1/2 nuclear accumulation is mediated through Ran as direct interaction, but also phosphorylation of nucleoporins (NUP50) facilitates translocation through importin-β [31]. The mechanisms by which p38 and JNK translocate into the nucleus are far less well understood. Recently, the motifs for interaction of both p38 and JNK with importins 3, 7, and 9 have been mapped at their N-terminal region. Ablated interaction of p38/ JNK with their importins selectively impairs their nuclear accumulation and phosphorylation of their nuclear but not their cytosolic targets [32]. Nuclear translocation of the budding yeast Hog1 requires both Ran (GSP1) and importin-β (NMD5). Phosphorylation of Hog1 by its MAP2K Pbs2 is essential for its translocation, while MAPK activity is dispensable for its import. Similar to mammalian MAPKs, transcription factors such as Msn2/4, Hot1, Sko1, and the nuclear phosphatase Ptp2 contribute to the nuclear retention of Hog1. Dephosphorylated Hog1 is exported out of the nucleus through an importin-β homolog, XPO1. Blocking its nuclear export traps Hog1 in the nucleus but does not prevent its dephosphorylation [33].

An increasing body of knowledge supports the presence of other upstream kinases: MEK1/2, MEK5, MEKK2/3, and MKK6 in the nucleus [3, 6, 34, 35]. The role of upstream signaling components in transcriptional regulation has not received much attention and requires deeper understanding.

#### **4. MAPK-regulated gene expression**

Nuclear localized MAPKs have the capacity to rewire the transcriptional architecture by controlling several layers of mRNA biogenesis (**Figure 2B**). Nuclear localized MAPKs are competent to govern the transcription cycle by acting on several layers of the process. Temporal integration of MAPK signaling into transcription is generally mediated by the phosphorylation of hundreds of transcription-related targets. How this transcriptional control is achieved will be discussed in this section.

#### **4.1 Genes regulated by MAPK activation: global induction/repression patterns**

MAPK activation overrides the homeostatic transcriptional program by transiently governing the simultaneous upregulation and downregulation of gene expression. Activation of different MAPK cascades leads to a pathway-specific transcriptional landscape. This stimuli-specific response is required to redefine the demands of each condition and involves the regulation of all RNA species. Unbiased approaches such as tiling arrays and RNA-seq have further extended the type of MAPK-regulated transcripts to noncoding RNA (ncRNA), long noncoding RNA (lncRNA), and, specifically in higher eukaryotes, the expression of miRNA.

**27**

*Shaping the Transcriptional Landscape through MAPK Signaling*

cycle- and growth-related genes (cyclins, tRNAs, and rRNAs).

**4.2 MAPK as components of the transcriptional machinery**

ing their nuclear localization, protein stability, or affinity to DNA [38].

*4.2.1 Transcription initiation: transcription factor modification*

through its noncatalytic region [3].

The MAPK-induced transcriptional response encompasses not only stimulispecific genes but also a set of well-defined genes that respond to multiple signals, providing coping mechanisms for adaptation. The transcriptional program induced by MAPK activation is classically described in two stages: A primary response is independent of protein synthesis and triggers the expression of immediate and delayed early genes (IEG and DEG, respectively). Then, the secondary response follows in a protein synthesis-dependent manner to induce the expression of secondary response genes [36]. Here, we will focus our attention on the mechanisms that

During this early or primary response, cells have to be able to repress cell cycle and growth genes while upregulating several transcription factor genes, which, once translated, will amplify the signal to generate a secondary or late response [37]. Thus, while a selected group of genes are upregulated, the rest of the transcriptome is transiently downregulated. Understanding of the mechanisms of MAPK-mediated gene repression has lagged behind when compared to the activating mechanisms, but some well understood prominent targets of repression are cell

MAPKs localize and interact with all of the regulatory regions of their target genes to control gene expression through similar principles but through distinct molecular mechanisms. These mechanisms include the coordinated control of transcription initiation, elongation, and termination together with modulation of chromatin architecture to ensure proper transcription through its target genes. MAPK phosphorylation of chromatin-related factors alters their activity by regulat-

Transcription initiation is the first step in governing gene expression and can be either directly or indirectly regulated by MAPKs. The most common regulatory mechanism involves the control of promoters by the regulation of an intricate network of transcription factors usually through direct phosphorylation and/ or by induction of their expression [39]. Transcription factors serve as anchoring platforms for the recruitment of MAPKs to chromatin. Chromatin-tethered MAPK nucleates the key signaling components to promoters and other regulatory elements to form a competent pre-initiation complex (PIC). Examples of "hubs" in the transcription factor network that facilitate the recruitment of active MAPK to chromatin are Elk-1, c-Jun and c-Fos for p38, ERKs, and JNKs. ERK5 is a rare MAPK that contains a transcriptional coactivator domain and has the capability of stimulating transcription through transcription factors or by direct binding to DNA

One of the best characterized transcription factors is c-Jun upon which stimulation is phosphorylated by JNK in its transactivator domain, which is required for induction of its maximal transcriptional activity and increased protein stability [39]. A single-transcription factor can serve to integrate signals from different MAPKs, or several MAPKs can cooperate in regulation of the same target. In response to UV light, both p38 and ERK contribute to the activation of c-Fos. On the other hand, efficient Elk1 phosphorylation is achieved by its differential interaction with ERK1/2, p38, and JNK. Activated Elk1 induces the expression of c-Fos and c-Jun transcription factors that will subsequently regulate a second transcriptional wave that includes other transcription factors and phosphatases [38]. Alternatively,

*DOI: http://dx.doi.org/10.5772/intechopen.80634*

promote gene induction.

*Gene Expression and Control*

diffusion.

Nucleoplasmic shuttling of active MAPK can be mediated mainly by three strategies: (1) active regulation of import-export through the NPC; (2) escape from cytoplasmic anchors and/or sequestration by nuclear components; and (3) passive

Canonical nuclear localization is an active process during which nuclear α/β importin complexes deliver cargo containing mono- or bi-partite stretches of basic residues (nuclear localization signals—NLSs) to the nucleus. Once in the nucleus, targeted proteins dissociate from importins by interacting with RanGTP. For example, in its inactive state, a nuclear export signal (NES) is exposed in ERK2, which confines it to the cytoplasm. Upon activation of ERK2, a conformational change disrupts its N- and C-terminal interactions, thereby exposing a NLS that sends the kinase into the nucleus. Similarly, activation of ERK1/2 allows their interaction with importin-7 and their nuclear accumulation [30]. Not only ERK1/2 nuclear accumulation is mediated through Ran as direct interaction, but also phosphorylation of nucleoporins (NUP50) facilitates translocation through importin-β [31]. The mechanisms by which p38 and JNK translocate into the nucleus are far less well understood. Recently, the motifs for interaction of both p38 and JNK with importins 3, 7, and 9 have been mapped at their N-terminal region. Ablated interaction of p38/ JNK with their importins selectively impairs their nuclear accumulation and phosphorylation of their nuclear but not their cytosolic targets [32]. Nuclear translocation of the budding yeast Hog1 requires both Ran (GSP1) and importin-β (NMD5). Phosphorylation of Hog1 by its MAP2K Pbs2 is essential for its translocation, while MAPK activity is dispensable for its import. Similar to mammalian MAPKs, transcription factors such as Msn2/4, Hot1, Sko1, and the nuclear phosphatase Ptp2 contribute to the nuclear retention of Hog1. Dephosphorylated Hog1 is exported out of the nucleus through an importin-β homolog, XPO1. Blocking its nuclear export

traps Hog1 in the nucleus but does not prevent its dephosphorylation [33].

much attention and requires deeper understanding.

**4. MAPK-regulated gene expression**

An increasing body of knowledge supports the presence of other upstream kinases: MEK1/2, MEK5, MEKK2/3, and MKK6 in the nucleus [3, 6, 34, 35]. The role of upstream signaling components in transcriptional regulation has not received

Nuclear localized MAPKs have the capacity to rewire the transcriptional architecture by controlling several layers of mRNA biogenesis (**Figure 2B**). Nuclear localized MAPKs are competent to govern the transcription cycle by acting on several layers of the process. Temporal integration of MAPK signaling into transcription is generally mediated by the phosphorylation of hundreds of transcription-related targets. How this transcriptional control is achieved will be discussed

**4.1 Genes regulated by MAPK activation: global induction/repression patterns**

MAPK activation overrides the homeostatic transcriptional program by transiently governing the simultaneous upregulation and downregulation of gene expression. Activation of different MAPK cascades leads to a pathway-specific transcriptional landscape. This stimuli-specific response is required to redefine the demands of each condition and involves the regulation of all RNA species. Unbiased approaches such as tiling arrays and RNA-seq have further extended the type of MAPK-regulated transcripts to noncoding RNA (ncRNA), long noncoding RNA (lncRNA), and, specifically in higher eukaryotes, the expression of miRNA.

**26**

in this section.

The MAPK-induced transcriptional response encompasses not only stimulispecific genes but also a set of well-defined genes that respond to multiple signals, providing coping mechanisms for adaptation. The transcriptional program induced by MAPK activation is classically described in two stages: A primary response is independent of protein synthesis and triggers the expression of immediate and delayed early genes (IEG and DEG, respectively). Then, the secondary response follows in a protein synthesis-dependent manner to induce the expression of secondary response genes [36]. Here, we will focus our attention on the mechanisms that promote gene induction.

During this early or primary response, cells have to be able to repress cell cycle and growth genes while upregulating several transcription factor genes, which, once translated, will amplify the signal to generate a secondary or late response [37]. Thus, while a selected group of genes are upregulated, the rest of the transcriptome is transiently downregulated. Understanding of the mechanisms of MAPK-mediated gene repression has lagged behind when compared to the activating mechanisms, but some well understood prominent targets of repression are cell cycle- and growth-related genes (cyclins, tRNAs, and rRNAs).

#### **4.2 MAPK as components of the transcriptional machinery**

MAPKs localize and interact with all of the regulatory regions of their target genes to control gene expression through similar principles but through distinct molecular mechanisms. These mechanisms include the coordinated control of transcription initiation, elongation, and termination together with modulation of chromatin architecture to ensure proper transcription through its target genes. MAPK phosphorylation of chromatin-related factors alters their activity by regulating their nuclear localization, protein stability, or affinity to DNA [38].

#### *4.2.1 Transcription initiation: transcription factor modification*

Transcription initiation is the first step in governing gene expression and can be either directly or indirectly regulated by MAPKs. The most common regulatory mechanism involves the control of promoters by the regulation of an intricate network of transcription factors usually through direct phosphorylation and/ or by induction of their expression [39]. Transcription factors serve as anchoring platforms for the recruitment of MAPKs to chromatin. Chromatin-tethered MAPK nucleates the key signaling components to promoters and other regulatory elements to form a competent pre-initiation complex (PIC). Examples of "hubs" in the transcription factor network that facilitate the recruitment of active MAPK to chromatin are Elk-1, c-Jun and c-Fos for p38, ERKs, and JNKs. ERK5 is a rare MAPK that contains a transcriptional coactivator domain and has the capability of stimulating transcription through transcription factors or by direct binding to DNA through its noncatalytic region [3].

One of the best characterized transcription factors is c-Jun upon which stimulation is phosphorylated by JNK in its transactivator domain, which is required for induction of its maximal transcriptional activity and increased protein stability [39]. A single-transcription factor can serve to integrate signals from different MAPKs, or several MAPKs can cooperate in regulation of the same target. In response to UV light, both p38 and ERK contribute to the activation of c-Fos. On the other hand, efficient Elk1 phosphorylation is achieved by its differential interaction with ERK1/2, p38, and JNK. Activated Elk1 induces the expression of c-Fos and c-Jun transcription factors that will subsequently regulate a second transcriptional wave that includes other transcription factors and phosphatases [38]. Alternatively,

a more indirect method to promote transcription is to activate downstream kinases that will themselves activate other transcription factors. For example, p38 activates two downstream kinases, mitogen- and stress-activated kinase 1/2 (MSK1–2), that activate another set of transcription factors STAT1/3, CREB, ATF1, and NF-ĸB [40].

In yeast, Fus3 and Kss1 MAPKs activate the transcription factor Ste12 that induces the expression of over 200 genes, including its own gene [41]. For example, in yeast, the combination of deletions of transcription factors and genome-wide analyses has been especially useful in providing a detailed view of the circuitry activated by the Hog1 or Fus3 MAPKs [42, 43].

The interrelationship between transcription factors and MAPKs is conserved throughout evolution, although the number of players and their functions has increased over time. MEF2 family transcription factors are substrates for several ERKs and in particular for p38 [44]. In yeast, it has been widely reported that the different transcription factors relevant for osmoresponsive gene expression are phosphorylated and recruited to target genes in a Hog1-dependent manner [45, 46]. Targeted recruitment of the MAPK activation machinery can also include recruitment of upstream MAPK-regulatory kinases. Examples of such in mammals are the recruitment of MEK1/2 to ERK-dependent genes [47] and the recruitment of MKK6 to p38 targeted regions in a MAPK-dependent manner [35]. Yeast upstream MAPK components have received far less attention than those of mammals, although Ste5 also associates with chromatin upon pheromone stimulation [48].

Besides controlling transcription factors, MAPKs control several other enzymatic activities, protein complexes, and targets that contribute to the formation of a transcriptionally competent Pre-Initiation Complex (PIC) (SAGA, Mediator, Ubp3) [49, 50]. A critical downstream node for MEK1/2 and ERK1/2 signaling upon the induction of EGF responsive genes is the integrator complex, a transcriptional coactivator. The binding of integrator to chromatin depends on catalytically active ERK1/2. Indeed, inhibition of the MAPK resulted in diminished association of integrator and RNA Pol II to chromatin [51].

#### *4.2.2 Transcription elongation*

Our knowledge of MAPK-regulated transcriptional control extends far beyond its control of transcription initiation and mainly originates from analysis of yeast MAPKs. The detection of MAPKs at the coding regions of their target genes suggested a far more extensive role for MAPKs as crucial components of the transcription regulatory complex. Seminal work regarding this phenomenon has been done in *S. cerevisiae* in which the association of Hog1, Fus3, and Mpk1 MAPKs with the coding regions of their target genes has been reported. Mpk1 elicits elongation of stress-responsive genes in a catalytic-independent manner by its interaction with the Paf1 elongation complex. Mpk1 is tethered to its target genes through binding to Paf1 that serves as a scaffold to escort Mpk1 into the elongating RNA Pol II. This binding requires the presence of the cell cycle transcriptional regulator SBF. The loss of this interaction restricts Mpk1 to the promoter region, which impairs both transcription and cell viability upon stress [52]. In response to osmotic stress, Hog1 and Paf1 interact through an unknown region, but the function and outcome of the Paf1 complex are kinase-specific.

The majority of genes targeted by Hog1 display an enrichment of the MAPK throughout the coding region [53, 54] that is mediated by the 3'UTR and is independent of promoter association. ORF-bound Hog1 behaves as a selective elongation factor by traveling and interacting with phosphorylated RNA Pol II (Rpb1). As RNA Pol II moves across the gene, it regulates chromatin structure through the recruitment of chromatin remodelers and chromatin-modifying enzymes (Section 4.3).

**29**

reviewed in [38].

*Shaping the Transcriptional Landscape through MAPK Signaling*

Moreover, Hog1 phosphorylates the Spt4 elongation factor to regulate RNA Pol II processivity to stimulate elongation efficiency at stress-responsive genes [55]. As happens during initiation, Hog1 recruits other protein complexes with specific enzymatic activities such as deubiquitinase (Ubp3) to ensure the proper production of stress-responsive genes [50]. Further studies in mammalian cells also corroborated p38 binding to coding regions of genes not only in response to osmotic stress but also during skeletal muscle differentiation, suggesting that the mechanism and purposes of Hog1/p38 transcriptional regulation are conserved throughout

Transcription elongation rates for many genes depend on the entangled interplay

of factors and complexes that regulate RNA Pol II. During elongation, a number of positive and negative elongation factors (P-TEFs and N-TEFs, respectively) have been shown to accelerate or attenuate Pol II, and, not surprisingly, these factors are targeted by MAPKs at stress-responsive genes. In response to hormone stimulation, MEK1 and ERK1/2 promote elongation and abolish pausing of RNA Pol II [56].

Unlike initiation and elongation, transcription termination can be carried out through different pathways depending on the coding or noncoding nature of the transcript. The two best defined termination pathways that are also highly conserved are the polyA-dependent pathway for protein coding and the Sen1-

One of the best studied examples of the involvement of MAPKs in the control of transcription termination is that of the role of Mpk1 in transcription termination during heat stress in yeast. As mentioned before, Paf1 and Mpk1 interact at heat responsive genes; this association prevents the recruitment of the Sen1-Nrd1-Nab3 termination machinery (NNS). Interestingly, the same study showed that human ERK5 and human Paf1 complex expressed in yeast also regulated termination in response to cell wall stress [52]. Mpk1 has recently been shown to directly phosphorylate Tyr1 in the RNA Pol II CTD as it traverses the coding region with the elongating machinery. This phosphorylation occurs in a stress-dependent manner and prevents early termination through the NNS pathway [57]. Deep sequencing of osmotically stressed neuronal cell lines identified a new set of transcripts termed downstream of gene-containing transcripts (DoGs). These noncoding transcripts span large region downstream of annotated gene features (>45 Kb) and are actively

MAPKs facilitate the abovementioned transcription activity by also regulating several chromatin remodelers to generate the proper chromatin environment for the transcription machinery. For induction of gene expression, chromatin must be accessible to allow the assembly of transcription factors, RNA Pol II and other factors, during initiation, elongation, and termination. These chromatin remodelers have been studied in both yeast and mammalian models as has been extensively

There are numerous examples of MAPKs interacting with chromatin remodelers. For instance, both Hog1 and p38 govern the recruitment of the remodeling complex SWI/SNF to target genes [38]. On the other hand, MAPK regulation goes beyond the substrate phosphorylation. As described in previous sections, MAPKs can also regulate chromatin remodeling through direct protein-protein interactions. This is the case with ERK2, which contacts PolyADP-ribose polymerase (PARP1), thereby

*DOI: http://dx.doi.org/10.5772/intechopen.80634*

dependent pathway for noncoding transcripts.

regulated through IP3 signaling [58].

**4.3 MAP kinases and their effects on chromatin**

evolution.

*4.2.3 Termination*

#### *Shaping the Transcriptional Landscape through MAPK Signaling DOI: http://dx.doi.org/10.5772/intechopen.80634*

Moreover, Hog1 phosphorylates the Spt4 elongation factor to regulate RNA Pol II processivity to stimulate elongation efficiency at stress-responsive genes [55]. As happens during initiation, Hog1 recruits other protein complexes with specific enzymatic activities such as deubiquitinase (Ubp3) to ensure the proper production of stress-responsive genes [50]. Further studies in mammalian cells also corroborated p38 binding to coding regions of genes not only in response to osmotic stress but also during skeletal muscle differentiation, suggesting that the mechanism and purposes of Hog1/p38 transcriptional regulation are conserved throughout evolution.

Transcription elongation rates for many genes depend on the entangled interplay of factors and complexes that regulate RNA Pol II. During elongation, a number of positive and negative elongation factors (P-TEFs and N-TEFs, respectively) have been shown to accelerate or attenuate Pol II, and, not surprisingly, these factors are targeted by MAPKs at stress-responsive genes. In response to hormone stimulation, MEK1 and ERK1/2 promote elongation and abolish pausing of RNA Pol II [56].

#### *4.2.3 Termination*

*Gene Expression and Control*

activated by the Hog1 or Fus3 MAPKs [42, 43].

integrator and RNA Pol II to chromatin [51].

*4.2.2 Transcription elongation*

Paf1 complex are kinase-specific.

a more indirect method to promote transcription is to activate downstream kinases that will themselves activate other transcription factors. For example, p38 activates two downstream kinases, mitogen- and stress-activated kinase 1/2 (MSK1–2), that activate another set of transcription factors STAT1/3, CREB, ATF1, and NF-ĸB [40]. In yeast, Fus3 and Kss1 MAPKs activate the transcription factor Ste12 that induces the expression of over 200 genes, including its own gene [41]. For example, in yeast, the combination of deletions of transcription factors and genome-wide analyses has been especially useful in providing a detailed view of the circuitry

The interrelationship between transcription factors and MAPKs is conserved throughout evolution, although the number of players and their functions has increased over time. MEF2 family transcription factors are substrates for several ERKs and in particular for p38 [44]. In yeast, it has been widely reported that the different transcription factors relevant for osmoresponsive gene expression are phosphorylated and recruited to target genes in a Hog1-dependent manner [45, 46]. Targeted recruitment of the MAPK activation machinery can also include recruitment of upstream MAPK-regulatory kinases. Examples of such in mammals are the recruitment of MEK1/2 to ERK-dependent genes [47] and the recruitment of MKK6 to p38 targeted regions in a MAPK-dependent manner [35]. Yeast upstream MAPK components have received far less attention than those of mammals, although Ste5

Besides controlling transcription factors, MAPKs control several other enzymatic activities, protein complexes, and targets that contribute to the formation of a transcriptionally competent Pre-Initiation Complex (PIC) (SAGA, Mediator, Ubp3) [49, 50]. A critical downstream node for MEK1/2 and ERK1/2 signaling upon the induction of EGF responsive genes is the integrator complex, a transcriptional coactivator. The binding of integrator to chromatin depends on catalytically active ERK1/2. Indeed, inhibition of the MAPK resulted in diminished association of

Our knowledge of MAPK-regulated transcriptional control extends far beyond its control of transcription initiation and mainly originates from analysis of yeast MAPKs. The detection of MAPKs at the coding regions of their target genes suggested a far more extensive role for MAPKs as crucial components of the transcription regulatory complex. Seminal work regarding this phenomenon has been done in *S. cerevisiae* in which the association of Hog1, Fus3, and Mpk1 MAPKs with the coding regions of their target genes has been reported. Mpk1 elicits elongation of stress-responsive genes in a catalytic-independent manner by its interaction with the Paf1 elongation complex. Mpk1 is tethered to its target genes through binding to Paf1 that serves as a scaffold to escort Mpk1 into the elongating RNA Pol II. This binding requires the presence of the cell cycle transcriptional regulator SBF. The loss of this interaction restricts Mpk1 to the promoter region, which impairs both transcription and cell viability upon stress [52]. In response to osmotic stress, Hog1 and Paf1 interact through an unknown region, but the function and outcome of the

The majority of genes targeted by Hog1 display an enrichment of the MAPK throughout the coding region [53, 54] that is mediated by the 3'UTR and is independent of promoter association. ORF-bound Hog1 behaves as a selective elongation factor by traveling and interacting with phosphorylated RNA Pol II (Rpb1). As RNA Pol II moves across the gene, it regulates chromatin structure through the recruitment of chromatin remodelers and chromatin-modifying enzymes (Section 4.3).

also associates with chromatin upon pheromone stimulation [48].

**28**

Unlike initiation and elongation, transcription termination can be carried out through different pathways depending on the coding or noncoding nature of the transcript. The two best defined termination pathways that are also highly conserved are the polyA-dependent pathway for protein coding and the Sen1 dependent pathway for noncoding transcripts.

One of the best studied examples of the involvement of MAPKs in the control of transcription termination is that of the role of Mpk1 in transcription termination during heat stress in yeast. As mentioned before, Paf1 and Mpk1 interact at heat responsive genes; this association prevents the recruitment of the Sen1-Nrd1-Nab3 termination machinery (NNS). Interestingly, the same study showed that human ERK5 and human Paf1 complex expressed in yeast also regulated termination in response to cell wall stress [52]. Mpk1 has recently been shown to directly phosphorylate Tyr1 in the RNA Pol II CTD as it traverses the coding region with the elongating machinery. This phosphorylation occurs in a stress-dependent manner and prevents early termination through the NNS pathway [57]. Deep sequencing of osmotically stressed neuronal cell lines identified a new set of transcripts termed downstream of gene-containing transcripts (DoGs). These noncoding transcripts span large region downstream of annotated gene features (>45 Kb) and are actively regulated through IP3 signaling [58].

#### **4.3 MAP kinases and their effects on chromatin**

MAPKs facilitate the abovementioned transcription activity by also regulating several chromatin remodelers to generate the proper chromatin environment for the transcription machinery. For induction of gene expression, chromatin must be accessible to allow the assembly of transcription factors, RNA Pol II and other factors, during initiation, elongation, and termination. These chromatin remodelers have been studied in both yeast and mammalian models as has been extensively reviewed in [38].

There are numerous examples of MAPKs interacting with chromatin remodelers. For instance, both Hog1 and p38 govern the recruitment of the remodeling complex SWI/SNF to target genes [38]. On the other hand, MAPK regulation goes beyond the substrate phosphorylation. As described in previous sections, MAPKs can also regulate chromatin remodeling through direct protein-protein interactions. This is the case with ERK2, which contacts PolyADP-ribose polymerase (PARP1), thereby

increasing its activating activity on chromatin remodelers [59]. Apart from chromatin remodelers, MAPKs govern a cohort of histone modifiers that not only destabilize nucleosomes but also, in a more complex manner, generate selective marks that dictate nucleosome dynamics. An example of this type of regulation is the Hog1 dependent gene recruitment of Rpd3, a histone deacetylase, that induces gene expression by promoting the eviction of histones at osmoresponsive genes [60] and the regulation of H3K4 monomethylation to dictate specificity of chromatin remodelers [61]. During elongation, as Hog1 travels with the elongating RNA polymerase, it recruits the RSC remodeling complex, thereby facilitating transcription along the gene body [62]. In mammals, ERKs, p38, and JNK promote the phosphorylation of H3S10 either directly or through their downstream kinases [38, 63]. p38 also phosphorylates the transcription factor MEF2D, which, in turn, leads to recruitment of the Ash2L-containing methyltransferase complex that generates an increase in the activating mark H3K4me3 [64]. These examples highlight the relevance of MAPK-mediated histone modification to generate an efficient chromatin remodeling robustly achieved through different mechanisms.

MAPKs also regulate gene silencing through chromatin remodelers. ERK1/2 directly interacts with the histone deacetylase 4 (HDAC4) that removes acetyl groups leading to chromatin condensation [6]. Similarly, Hog1 promotes the transcription of *PNC1*, which encodes an activator of Sir2, a histone acetyltransferase that protects sensible rRNA-coding regions from DNA damage [65]. In these two cases, MAPKs act as repressing elements of chromatin remodeling.
