European Journal of Cell Biology 92 (2013) 237–246

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Review

Sin3: Insight into its transcription regulatory functions Rama Kadamb 1 , Shilpi Mittal 1 , Nidhi Bansal, Harish Batra, Daman Saluja ∗ Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007, India

a r t i c l e

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Article history: Received 20 May 2013 Received in revised form 27 August 2013 Accepted 11 September 2013 Keywords: Sin3A Sin3B Histone deacetylase Co-repressor complex Transcriptional regulation Protein stability

a b s t r a c t Sin3, a large acidic protein, shares structural similarity with the helix-loop-helix dimerization domain of proteins of the Myc family of transcription factors. Sin3/HDAC corepressor complex functions in transcriptional regulation of several genes and is therefore implicated in the regulation of key biological processes. Knockdown studies have confirmed the role of Sin3 in cellular proliferation, differentiation, apoptosis and cell cycle regulation, emphasizing Sin3 as an essential regulator of critical cellular events in normal and pathological processes. The present review covers the diverse functions of this master transcriptional regulator as well as illustrates the redundant and distinct functions of its two mammalian isoforms. © 2013 Elsevier GmbH. All rights reserved.

Introduction Eukaryotic gene expression is a highly orchestrated process that involves coordinated activation and silencing of genes mediated by complex interactions between the chromatin and transcription regulatory proteins. The transcription regulatory proteins include DNA-binding transcription factors along with various coactivator and co-repressor complexes that serve as both ‘readers’ and ‘modifiers’ of the chromatin architecture (Kuo and Allis, 1998). Co-repressors, for instance, mediate transcriptional silencing via on-site recruitment of chromatin remodeling activities that can either inhibit or stall the basal transcription machinery (Burke and Baniahmad, 2000). The Sin3/HDAC co-repressor complex is one such multiprotein complex that mediates gene repression through chromatin compaction. Sin3, through its interaction with DNA-binding factors, acts as a scaffold protein that recruits histone deacetylases (Class I HDACs; HDAC1 and HDAC2) and other chromatin-modifying enzymes onto the target promoters (Silverstein and Ekwall, 2005). Presence of several paralogs and alternatively spliced isoforms of Sin3 in different organisms potentially contribute to the complexity and diversity of Sin3 functions (Silverstein and Ekwall, 2005; Sharma et al., 2008). In mammals two paralogs of Sin3 exist, Sin3A and Sin3B, that share high sequence similarity and overlapping expression pattern; in fact, recent reports suggest distinct and non-overlapping roles for

∗ Corresponding author. Tel.: +91 93 1001 8699; fax: +91 11 2766 6248. E-mail addresses: [email protected] (R. Kadamb), [email protected] (D. Saluja). 1 These authors contributed equally. 0171-9335/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ejcb.2013.09.001

each isoform (David et al., 2008; Grandinetti et al., 2009; van Oevelen et al., 2008). In the last decade intensive research efforts have unraveled diverse roles of the Sin3 complex. These include normal cellular events of development, growth, differentiation and senescence as well as oncogenic transformation in pathological conditions. This review strings together the role of Sin3 in diverse cellular processes and attempts to present a concise overview of our current understanding of Sin3-mediated regulation of its target genes.

Structural overview Sin3 shows similarity and interaction with helix-loop-helix proteins of the Myc (Mad-Max1) family of transcriptional factors (Ayer et al., 1995; Schreiber-Agus et al., 1995). Sin3 interacts with a large number of transcriptional factors through its six conserved domains that include four Paired Amphipathic Helices (PAH 1–4), one Histone Deacetylase Interaction Domain (HID) and one Highly Conserved Region (HCR). The emerging view describes Sin3 as a modular protein where PAH1 and PAH2 are reserved for interactions with various transcription factors while the regions spanning PAH3, HID and PAH4 serve a scaffolding function by interacting with other subunits of the co-repressor complex (Grzenda et al., 2009). PAH1-3 domains form pre-folded binding modules on fulllength Sin3, like a beads-on-string model (Le Guezennec et al., 2006). Unlike the other PAH domains, PAH4 most likely does not fold as a four-helix bundle, but instead adapts a distinct fold (van Ingen et al., 2006). Structural studies have revealed that the PAH2 domain of mammalian Sin3A exhibits conformational heterogeneity that enables Sin3A to regulate its interaction with diverse

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protein targets (Brubaker et al., 2000; Swanson et al., 2004; Zhang et al., 2006). Although, there is a high degree of similarity between the PAH1 and PAH2 domains, PAH1 recognizes different sequence motifs as compared to PAH2 thereby enabling a high degree of target specificity (Le Guezennec et al., 2006; Sahu et al., 2008). The PAH2 domain of mSin3B interacts with its targets in a manner similar to that of mSin3A but these two paralogs differ in their non-interactive forms (He and Radhakrishnan, 2008; Spronk et al., 2000; van Ingen et al., 2004, 2006). The apo-mSin3A PAH2 domain homodimerizes and exists in unfolded state but the apomSin3B PAH2 domain is monomeric and is fully folded (He and Radhakrishnan, 2008; Sahu et al., 2008); in fact, results of an in-silico modeling experiment in our laboratory suggest a flexible nature of the N-terminal region of hSin3B (PAH1-3). Based on this study, we predict that this flexibility of the PAH 1–3 domains of hSin3B can provide additional new surfaces for protein-protein interactions (unpublished results). Furthermore, non-conserved amino acids between Sin3A and Sin3B may contribute toward differential functions and specificity of the two paralogs in choosing their interacting partners. Isoforms of Sin3 Sin3 protein is highly conserved from yeast to mammals and has varied numbers of isoforms in these metazoans. Saccharomyces cerevisiae has only one isoform of Sin3 whereas Schizosaccharomyces pombe has three isoforms viz. Pst1, Pst2 and Pst3, each encoded by a separate gene (Dang et al., 1999; Silverstein and Ekwall, 2005). In Drosophila, three isoforms (Sin3 187, Sin3 190 and Sin3 220) are produced by alternate splicing which differ in their carboxy-terminal region. The high-molecular-weight isoform (Sin3 220) is principally expressed in proliferating cells whereas the low-molecular-weight Sin3 187 is primarily found in differentiated tissues. The Sin3 190 isoform is expressed only in embryos and adult females implicating that this isoform may be maternally deposited during oogenesis, providing a possible explanation for its presence in the embryo (Fig. 1). Differences in spatial temporal expression of Sin3 isoforms in Drosophila support the notion that each isoform may regulate a distinct set of genes at the time of embryonic development and therefore can perform non-overlapping functions (Sharma et al., 2008). Multiple Sin3 isoforms are also documented in three species of mosquitoes which are conserved at splice sites and have a similar protein sequence to that of Drosophila Sin3 187 and Sin3 220 (Sharma et al., 2008). In mammals two isoforms, Sin3A and Sin3B, are encoded by two separate genes and are considered to be the result of gene duplication (Ayer et al., 1995). Both isoforms have spliced variants in both mice and humans. The mouse Sin3A is more closely related to human Sin3A than to mouse Sin3B. Mammalian Sin3A and Sin3B proteins are approximately 57% identical throughout the length of their polypeptide chains with the highest degree of homology localized in the PAH and HID regions (Alland et al., 1997; Yang et al., 2000; Silverstein and Ekwall, 2005). Both proteins are widely expressed (frequently within the same cells and tissues) and bind common as well as distinct transcriptional repressors and complexes (Brubaker et al., 2000; Jelinic et al., 2011; Spronk et al., 2000). Identification of unique regulatory functions for each Sin3 isoform justifies the concept of gene duplication as a part of the evolutionary process to achieve complete functional flexibility. Sin3/HDAC core complex The Sin3/HDAC co-repressor complex is a multiprotein complex comprised of several proteins like HDAC1, HDAC2, RBBP4/7, (Sin3 Associated Proteins) SAP30, SAP18, CpG methylated binding

protein (MeCP2) and Rb-binding protein (RBP1) (Hassig et al., 1997; Laherty et al., 1997; Lai et al., 2001; Nan et al., 1998; Zhang et al., 1997). Further characterization of this complex revealed other associated proteins like SAP180, BRMS1, SAP130, SAP25 and ING1/2 that support and stabilize interactions with the histones and other components of the complex (Fleischer et al., 2003; Lai et al., 2001; Meehan et al., 2004). Homologs of SAP have also been found in yeast, worms, flies, amphibians and Arabidopsis indicating a likely structural and functional conservation of the Sin3/HDAC complex in eukaryotes (Choy et al., 2007; Hill et al., 2008; Tsai et al., 1999; Vermaak et al., 1999). With no recognized DNA-binding activity, Sin3 is known to act like a scaffold, bringing together chromatin remodelers like HDAC1/2, histone lysine methylases (e.g., ESET) and demethylases (e.g., RBP2/JARID1A), methylated DNA-binding proteins and various other transcription regulators (p53, Rb, E2F, SMAR1 and REST/NRSF), which restructure the chromatin for gene transcription (Brehm et al., 1998; Grzenda et al., 2009; Laherty et al., 1997; Murphy et al., 1999; Rampalli et al., 2005; Rayman et al., 2002; Roopra et al., 2000; Silverstein and Ekwall, 2005). Initially, a single Sin3/HDAC complex was documented but recent studies have revealed the existence of subsets of this complex (Carrozza et al., 2005; Keogh et al., 2005; Li et al., 2007; Jelinic et al., 2011). In yeast (S. cerevisiae), two distinct complexes, large Rpd3L and small Rpd3S are present (Carrozza et al., 2005). These two complexes share some common subunits such as Sin3, Rpd3 and Ume1 whereas other subunits are specific for each complex (Fig. 1). Rxt1, Rxt2, Dep1, Sds3, Pho23 and Sap30 are specific for the Rpd3L complex whereas Eaf3 and Rco1 are unique components of the Rpd3S complex (Keogh et al., 2005) (Fig. 1). The mechanism of repression for each complex is also distinct. The Rpd3L complex carries out its trans-repression function by binding to the promoter through DNA-binding factors or other co-repressors while the mechanism of repression by Rpd3S involves recognition of methylated H3K36 by the chromodomain of Eaf3 and subsequent recruitment of the core complex which brings about deacetylation of nucleosomes at the actively transcribed loci. The Rpd3S complex reverses the process of histone acetylation that occurs at the time of RNA polymerase-mediated elongation and thereby prevents transcription initiation from intragenic cryptic sites (Li et al., 2007). Existence of different complexes is also reported in another strain of yeast, i.e., S. pombe, which is known to have three homologs Pst1, Pst2 and Pst3. However, out of the three isoforms, Pst1 and Pst2 form two distinct complexes, i.e., Complex I and Complex II involving similar as well as distinct protein members and follow repression mechanisms similar to Rpd3L and Rpd3S, respectively. Complex I contains Pst1 and Sds3 while Complex II comprises Pst2, Alp13, Cph1 and Cph2 as distinct subunits. Clr6 and Prw1 are common to both the complexes (Nicolas et al., 2007) (Fig. 1). Likewise, in Drosophila, Sin3 187 and Sin3 220 isoforms form two distinct HDAC complexes localized on discrete regions of polytene chromosomes (Spain et al., 2010). Recently, a homolog of the Rpd3S complex was reported in mammals that mediates restoration of repressed chromatin structure at actively transcribed regions. Designated as the SHMP complex, the tetrameric complex is localized at discrete loci approximately 1 kb downstream of transcription start sites and is comprised of Sin3B, HDAC1, Mrg15 and the PHD finger-containing Pf1 protein (Jelinic et al., 2011).

Regulation of Sin3 protein It is ironic that although numerous Sin3-regulated gene promoters have been identified, the transcriptional regulation of this master scaffold remains a mystery with no clear definition of even its promoter elements. A glimpse of post-translational regulation of Sin3 protein, however, was provided by a yeast two-hybrid screen

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Fig. 1. Schematic representation of Sin3 isoforms and distinct Sin3/HDAC complexes present in yeast, Drosophila and mammals. Each Sin3 isoform can form a unique complex to carry out its function.

in which Sin3B was identified as a target of a novel ubiquitin ligase, RNF220 (RING finger protein 220) (Kong et al., 2010). Sin3 not only colocalizes with RNF220, but also physically associates with it, both in vivo and in vitro. Using deletion constructs it was shown that both the N- and C-terminus of Sin3 are ubiquitinated. Interestingly, the Sin3 PAH2 domain interacts with RNF 220 but is itself not ubiquitinated. Although the mechanism is not clear, it is postulated that RNF220 acts as an E3 ubiquitin ligase for Sin3B and can promote its ubiquitination and subsequent proteasomal degradation (Kong et al., 2010). Recently, it was shown that Sin3A is under

post-transcriptional regulation by microRNA, (miR-138), through binding to its 3 UTR (Ramachandran et al., 2012). Sin3: Transcription repressor or transcription activator? The mainstay of transcription regulation by Sin3 is its ability to provide a multifaceted platform for interaction with and recruitment of diverse transcription factors and the chromatin remodeling machinery at the target promoters (Table 1) (Silverstein and Ekwall, 2005). Classically the Sin3/HDAC complex targets the

Table 1 Mode of sin3 mediated gene regulation through interaction with various transcription factors. The Sin3/HDAC complex associates with a number of DNA-binding proteins and mediates either positive or negative regulation of genes involved in diverse biological functions. Representative examples illustrating the dual mode of transcription regulation by Sin3 are shown. Associated protein

Genes regulated

Mode of gene regulation

Functions

References

Cti6 ER␣

Aft1, Tup1 ESR1

Repression Repression

Puig et al. (2004) Ellison-Zelski et al. (2009)

Zbtb4 EKLF

P21CIP1 ␤-globin

Repression Repression

Sox2 SMAR1 HAP1 FAM60A Foxk1 KLF11

Nanog CyclinD1 HAP1 CyclinD1 p21 A2␣

Activation Repression Activation Repression Repression Repression

E2F4 Ebp1

ttn, ttnc1, acta1, dmpk E2F1, PSF1

Activation Repression

Mnt Hog1

CyclinD2 Hog1

Repression Repression

p53

HSPA8, MAD1, CRYZ, MAP4 STATHMIN

Repression

N/A N/A

GAM3 STA1

Activation Activation

Fe-Cu metabolism Regulation of E2 signaling Cell cycle regulator Regulates cell proliferation Cellular proliferation Cell cycle regulator Heme regulation Cell cycle regulator Cell cycle regulator Inhibits prostaglandin synthesis Muscle differentiation Mediates differentiation Quiescence Survival upon osmostress Heat shock protein, Cytoskeletal regulator, Cell cycle regulator Normal growth Encode for extracellular lucoamylase

Weber et al. (2008) Chen and Bieker (2004) Baltus et al. (2009) Rampalli et al. (2005) Xin et al. (2007) ˜ et al. (2012) Munoz Shi et al. (2012) Buttar et al. (2010) van Oevelen et al. (2010) Zhang et al. (2005a,b) Popov et al. (2005) De Nadal et al. (2004) Bansal et al. (2011) and Murphy et al. (1999)

Yoshimoto et al. (1992) Yoshimoto et al. (1992)

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Fig. 2. Chromatin remodeling and transcription regulation by the Sin3/HDAC complex. The modular structure of Sin3 provides a flexible platform for interaction with and on-site recruitment of transcription factors with diverse enzymatic activities needed for precise gene regulation. (a) Sin3 is capable of chromatin remodeling through recruitment of numerous transcription factors, histone methyltransferase and demethylases which result in chromatin compaction (van Oevelen et al., 2008; Silverstein and Ekwall, 2005). (b) DNA damage, leading to induction of double strand breaks (DSB), results in Sin3/Rpd3-dependent reduction in the levels of histone 4 acetylation at lysine 16 (H4K16Ac) residues (Jazayeri et al., 2004). (c) Sin3 through recruitment of O-GlcNAc transferase (OGT) via PAH4 leads to glycosylation of RNA polymerase II and consequent stalling of transcription elongation (Kelly et al., 1993; Yang et al., 2002).

deacetylation of histones H3 and H4 which in combination with dynamically coordinated methylation and demethylation of key histone lysines lead to Sin3-mediated gene repression (Bansal et al., 2011; Das et al., 2013; van Oevelen et al., 2010) (Fig. 2). Deacetylation of non-histone proteins is now also recognized to contribute toward Sin3/HDAC-mediated target gene repression. Recently Icardi and coworkers demonstrated that deacetylation of STAT3 by the Sin3A/HDAC complex is essential for STAT3 targeted repression of genes like FGG, AGT and CD14 (Icardi et al., 2012). It is rather interesting that once viewed as a transcriptional repressor, Sin3 is now being also put forward as a transcriptional activator in both yeast and mammals (Table 1). The first report of Sin3 as an activator was published in the 1990s wherein it was found to activate GAM3/STA1, a gene encoding an extracellular glucoamylase in yeast. Sin3 (also called GAM2) acts as a positive transcriptional regulator of GAM3/ADR6 which is required for transcriptional activation of genes like STA1 and ADH2. In contrast, other genes like HO, SPO11, SPO13, SPO16 and TRK2 are repressed by Sin3 in yeast (Yoshimoto et al., 1992; Vidal et al., 1991). Another interesting example is the heat stress-induced Sin3-mediated gene activation (CTT1, ALD3, PNS1, and TPS1) and repression (GAR1, RPL16A, KRL1, MAK5) through transcription factor MSN2/4 (Ruiz-Roig et al., 2010). It is important to note that the Rpd3L complex has an explicit role in heat stress-mediated gene induction as shown by mutations in unique components of the Rpd3S complex which fails to show changes in gene expression

upon heat stress (Ruiz-Roig et al., 2010). Similarly, Rpd3 interacts with Hog1 upon osmotic stress and recruits the Rpd3-Sin3 complex onto the promoters of osmo-responsive genes to induce their expression (De Nadal et al., 2004). Though several mechanisms explain Sin3-mediated gene repression, gene activation by Sin3 is still enigmatic (Fig. 2). One of the possible mechanism of gene activation by Sin3 could be explained by recruitment of Rpd3 at the promoters of heat-responsive genes that facilitates RNA polymerase II (RNA Pol II) entry and/or Sin3-mediated deacetylation of nucleosomes downstream of the promoter which in turn may support efficient elongation by RNA Pol II (De Nadal et al., 2004; Kim and Buratowski, 2009). The aforementioned reports of transcription activation by the yeast Sin3-Rpd3 complex have now been extended to higher eukaryotes, although the molecular mechanism is not corroborated. In Drosophila, Pile and co-workers demonstrated the requirement of Sin3 for enhanced expression of several genes that are required for mitochondrial activity like glutathione transferases (CG11784 and CG1742), Rough deal (Rod), thymidylate synthase (TS), cyclin B (CycB) and String (STG) (Pile et al., 2003). In another study, conditional knockdown of Sin3 in mouse embryonic fibroblasts resulted in downregulation of various genes (like ttn, ttnc, acta1 and dmpk) involved in muscle development; these results suggest that gene activation processes by Sin3 may regulate sarcomere functions (van Oevelen et al., 2010). The role of Sin3 in gene activation is further strengthened by another report in Drosophila where Sin3A positively regulates almost 70% of target

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genes involved in cell invasion/migration (Das et al., 2013). Interestingly, the Sin3-activated loci are accompanied by chromatin activation marks like H3K4me3, H3K9ac and H4K12ac, suggestive of HDAC independent mechanisms for activation by Sin3 and existence of multiple transcription complexes (Das et al., 2013). At this point we want to specifically highlight the regulation of the Nanog promoter by the Sin3/HDAC complex in proliferating embryonic stem cells. The expression of Nanog is upregulated by Sox2-dependent recruitment of the Sin3A/HDAC complex as opposed to Sin3A-mediated p53-dependent silencing of Nanog during differentiation (Baltus et al., 2009; Lin et al., 2005). These studies also suggest the existence of a common set of Sin3 target genes that can be differentially regulated by the Sin3 complex in a context-dependent manner. In brief, Sin3 can very well be appreciated as a dual regulator of transcription, albeit the molecular and cellular conditions that determine the direction of gene regulation currently remain elusive. Sin3: A dual regulator of protein stability Sin3 has been shown to regulate protein stability through PAH domain-mediated protein–protein interactions. For instance, under normal conditions, levels of p53 are tightly regulated by MDM2-mediated degradation. However, in the event of cellular insult, there is an increase in the p53 protein level due to Sin3Amediated posttranslational stabilization of p53 (Zilfou et al., 2001). The Sin3 interaction domain (SID) of p53 lies within its prolinerich domain that also contains motifs for protein degradation. Interaction of p53 with Sin3A effectively masks this motif and protects p53 from proteasome-mediated degradation in a manner independent of MDM2. It was also observed that only the Sin3Abound fraction of p53 gets stabilized by Sin3 whereas unbound p53 degrades rapidly (Zilfou et al., 2001). Thus, under conditions of cellular and/or genotoxic stress, Sin3A mediates stabilization of p53. Sin3B also directly interacts with the proline-rich domain of p53; however its contribution in increasing p53 protein halflife has not been elucidated (Bansal et al., 2011). Another example where Sin3 regulates protein stability is the Drosophila SMRTER protein. Decreased expression of SMRTER upon deletion of Sin3 was attributed to proteasome-mediated degradation since loss of Sin3 did not affect the mRNA level of SMRTER. Furthermore, the protein-stabilizing function of Sin3 is carried out as a component of the Sin3/Rpd3 complex and deacetylase activity of this complex is crucial for this function (Pile et al., 2002). Sin3 isoforms (Sin3A and Sin3B) along with c-Myc regulatory proteins have been found to stabilize both exogenous as well as endogenous Mad4 protein levels in glioblastoma multiforme (GBM) cell lines. Mad4 is targeted by c-IAP1 for degradation, and Sin3 through its interaction with the destabilizing protein c-IAP1 abrogates its interaction with Mad4, thereby protecting the latter from degradation (Yang et al., 2012). Sin3 also regulates the stability of ␤-catenin by controlling the levels of PP1␤, an important component of the ␤-catenin degradation complex (Das et al., 2013). In contrast to these reports, Nascimento and coworkers reported Sin3 as a negative regulator of protein stability by describing its involvement in deacetylation and consequent destabilization of c-Myc protein (Nascimento et al., 2011). Post-translational regulation of proteins is significant in cellular signaling and as illustrated by the above examples, it is justified to assume Sin3 as an important regulatory node in diverse signaling networks. Sin3: A key developmental regulator The complex process of development involves precise transcriptional regulation through the orchestrated coordination and fine tuning between transcriptional regulators and components of the

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general transcriptional machinery (Young, 2011). Sin3 is emerging as a key regulator in development ranging from organ development in Drosophila to muscle maintenance, T cell regulation, germ cell lineage and bone development in higher mammals (Cowley et al., 2005; David et al., 2008; Pellegrino et al., 2012; Silverstein and Ekwall, 2005; Singh et al., 2005; Swaminathan and Pile, 2010). Clues to the possible role of Sin3 in development came from studies in Drosophila showing requirement of Sin3 in eye development (Neufeld et al., 1998). Sin3 binds to SMRTER and colocalizes with it onto the promoters of ecdysone-regulated target genes resulting in development-specific modulation of gene expression in response to hormone signaling (Pile and Wassarman, 2000; Tsai et al., 1999). Although, Sin3 is not essential for viability in S. cerevisiae, Sin3 null mutants die during larval development stages in Drosophila (Pile et al., 2003; Sharma et al., 2008). It was further shown that different isoforms of dSin3 are expressed in a stage- and tissue-specific manner. Conditional lethal knockdown of Sin3 in Drosophila further supported the contention that Sin3 is required at multiple stages of development and each isoform might regulate distinct sets of genes required for specific developmental pathways (Pennetta and Pauli, 1998; Sharma et al., 2008). Development of the Drosophila head requires anterior determinant Bicoid protein that interacts with SAP18 (earlier identified as Bicoid-interacting protein). At the time of head development, there is retraction of the anterior most segment and this retraction is accomplished by reduced expression of Bicoid-dependent genes like hunchback. The mechanism of retraction involves interaction of Bicoid with SAP18 leading to the recruitment of the Sin3/Rpd3 complex and thereby switching Bicoid from an activator to a repressor (Zhu et al., 2001). In another study, reduced expression of Sin3 in wing imaginal disc resulted in progenies with overall reduced size and curly wings. Such Sin3depleted progenies also showed defects in the venation pattern including thinner veins and an incomplete L5 vein (Swaminathan and Pile, 2010). The mechanism for this abnormal wing phenotype was attributed to deregulation of String (STG) expression, a cell cycle regulator which is under positive regulation by Sin3 (Pile et al., 2002). In mammals, knockdown studies in mice have demonstrated the possible contribution of Sin3 in reproductive health. Deletion of Sin3A in mouse germ cell lineage resulted in reduced testicular size and weight along with impaired spermatogenesis and male sterility (Pellegrino et al., 2012). In the development of immune cells, Sin3A deletion was shown to affect thymic cellularity and count of CD8SP T cells (Cowley et al., 2005). Similarly, a role of Sin3 in kidney development was indicated from studies using heterozygous pups with reduced expression of mSin3A that showed splenomegaly and membranous glomerulopathy (Cowley et al., 2005). In contrast, pups nullizygous for Sin3B showed reduction in overall size due to defects in bone to cartilage ratio. Sin3B null mutants also possessed poorly differentiated erythrocytes which were detected by the presence of Howell-jolly bodies (David et al., 2008). Recently it was shown that Sin3 plays a pivotal role in muscle biology. Primary myotubes and myoblast depleted for only Sin3A or both Sin3A and Sin3B showed gross defects in muscle structure and sarcomere functions resulting in loss of muscle integrity (van Oevelen et al., 2010). Sin3A and Sin3B directly contribute to activation of a subset of genes encoding components of sarcomeric and extracellular matrix (ECM) compartments as well as genes involved in the maintenance of muscle structure and sarcomere function. Interestingly, although the two isoforms interact with each other, Sin3A alone or the Sin3A/B heterodimer but not Sin3B could modulate the expression of genes involved in sarcomeric functions. However, at the phenotypic level, deletion of both Sin3A and Sin3B led to an enhanced mutant phenotype which was not observed upon deletion of only Sin3B (van Oevelen et al., 2010). The authors suggested a coordinated recruitment of the two isoforms on the

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promoters of genes which might be involved in maintaining muscle integrity. These observations once again accentuate that there can be functional differences between the two isoforms to regulate distinct or similar subsets of genes. In spite of the fact that all these studies strongly advocate the regulatory role of Sin3 in eukaryotic developmental pathways, the mechanistic details are far from clear. Future studies should be directed to unravel the various components which associate with Sin3 and help in contributing toward growth and developmental pathways. Sin3: A protein controlling senescence and heterochromatinization events Senescence is a stable cell cycle arrest activated by oncogenic signaling and is characterized by the formation of senescenceassociated heterochromatic foci (SAHFs) (Garcia and Pereira-Smith, 2008; Narita, 2007). The onset of senescence has been associated with several histone modifications including an increase in the levels of H3K9 trimethylation (H3K9me3) and macro-H2A. On the contrary, levels of linker H1 are downregulated (Narita et al., 2003, 2006; Rogakou and Sekeri-Pataryas, 1999; Zhang et al., 2005a,b). The key proteins which are identified as regulators of senescence include several tumor suppressor proteins like TP53, E2F and retinoblastoma (Rb). Interaction of Sin3 with the aforementioned proteins is now well-documented (Bansal et al., 2011; Brehm et al., 1998; Murphy et al., 1999; Rayman et al., 2002). Sin3B is a potential regulator of chromatin modifications in both transient and stable gene silencing of the promoters undergoing heterochromatinization and induction of senescence. The first evidence toward this point was provided by David and co-workers by demonstrating the association of Sin3-containing complexes with the formation of pericentric heterochromatin. Association of Sin3B with RBP2 through SAP30 and recruitment of the histone methyltransferase SUV39H1, results in trimethylation of hypoacetylated H3K9 that provides a binding platform for the chromodomain of heterochromatic protein 1 (HP1). This tethering of Sin3B/RBP2/HP1 leads to chromatin compaction and stable gene silencing while loss of mSds3 (a member of the Sin3 complex) causes impairment of pericentric histone deacetylation thereby preventing the cascade of histone modification events required for the establishment of a functional pericentric heterochromatin structure (David et al., 2003, 2008). Furthermore, genetic inactivation of Sin3B in mouse embryonic fibroblasts (MEFs) impairs the transcriptional repression of E2F-responsive genes that make the cells refractory to replicative and oncogenic-induced senescence (Grandinetti et al., 2009). Several studies also highlight different roles of Sin3A and Sin3B in senescence. Oncogenic stress selectively increases the expression of Sin3B consistent with the senescence program wherein the enrichment of heterochromatin marks at E2F promoters requires Sin3B (Grandinetti et al., 2009). In contrast, levels of Sin3A decreased in WI-38 fibroblasts undergoing senescence, though the levels of Sin3B protein remained unchanged (Grandinetti et al., 2009; Kyrylenko et al., 2000). Recently, a new member of the Sin3 complex, Morf4-Related Gene 15 (MRG15) has been identified which belongs to the MRG family of proteins (Jelinic et al., 2011). MRG15 is known to interact with a number of proteins including tumor suppressor Rb, HDAC1/2, RBP2 and NuA4-KAT5 (Carrozza et al., 2005; Eisen et al., 2001; Hayakawa et al., 2007; Pardo et al., 2002). In yeast, MRG15 interacts with the Sin3-Rpd3 (HDAC) complex in a manner similar to the Inhibitor of Growth (ING) family of proteins implicated in senescence (Han et al., 2006). MRG15 is also associated with age-specific high-molecular-weight complexes which contain senescence-associated proteins like Rb and HDAC1/2. The role of MRG15 in cellular proliferation and development is well established. Based on these studies, MRG15 has emerged as an important player in the regulation of oncogenic

induced senescence and/or tissue aging. Being a stable component of both HAT and HDAC complex, MRG15 can be important for repression of specific target genes affecting induction of senescence (Garcia and Pereira-Smith, 2008). Together, it is clear that Sin3 along with the other complex members, like MRG15, contribute to several histone modifications that can lead to heterochromatinization, gene silencing and senescence. Sin3: Required for cell proliferation and maintenance of cell survival Sin3A null MEFs undergo cell cycle arrest and show defects in DNA damage response due to deregulation of genes involved in cell cycle regulation, DNA damage response pathways and mitochondrial metabolism (Cowley et al., 2005; Dannenberg et al., 2005). Interaction of Sin3 with regulators of the cell cycle and proliferation like the Myc-Mad network, the pRb-E2F complex and p53 is well established (Bansal et al., 2011; Dannenberg et al., 2005; Murphy et al., 1999; Rayman et al., 2002). Ablation of Sin3A in myotubes caused perinatal lethality signifying the role of Sin3A in viability of differentiated cells (van Oevelen et al., 2010). Furthermore, a role of Sin3A in proliferation of embryonic stem (ES) cells and germ cells has been recently established (McDonel et al., 2012). Knockdown of Sin3A led to impaired proliferation and spermatogenesis which emphasizes the importance of Sin3 for survival of undifferentiated cells (McDonel et al., 2012). However, the mechanisms of gene regulation for differentiated and undifferentiated cells seem to be distinct since the genes de-repressed following Sin3 deletion in differentiated cells are actually highly expressed in Sin3-positive undifferentiated embryonic stem (ES) cells. In ES cells, Sin3A is required for the elevated expression of cell cycle regulators like E2f1, Ccn1e and Ccn1b (McDonel et al., 2012). Sin3 also increases the levels of Mcm2 that protects pluripotent stem cells against double strand breaks (DSBs) acquired during replication. Elevation of these genes by Sin3 is indirect and is mediated by activating transcriptional activators of these genes, i.e., Myc and E2F through repression of the Myc/E2F repressor (McDonel et al., 2012). Loss of Sin3A in male germ cells led to reduced survival and germ cell apoptosis during postnatal mitosis and before the initiation of meiosis (Pellegrino et al., 2012). On the contrary, it has also been shown that Sin3A expression can inhibit c-Myc-dependent proliferation in skin, testis and salivary glands. Loss of Sin3A but not Sin3B in mouse led to unusual increases in the thickness of the interfollicular epidermis (IFE) and sebaceous glands (Nascimento et al., 2011). In contrast to the effects of Sin3A, Sin3B null cells undergo normal proliferation (David et al., 2008). Selective involvement of Sin3A, but not Sin3B, in cell proliferation strengthens the notion that these two isoforms perform distinct functions, and that loss of one isoform cannot be compensated by the presence of the other (David et al., 2008; Grzenda et al., 2009; van Oevelen et al., 2010). Sin3: Controller of cell cycle progression and exit Various studies have illustrated the function of Sin3B in controlling cell cycle progression and cell cycle entry/exit through recruitment of p107/p130 proteins onto E2F-responsive promoters (David et al., 2008; Rayman et al., 2002; van Oevelen et al., 2008). Sin3B provides a platform for the assembly of E2F4, pocket proteins p130/p107 and HDAC1/2 leading to repression of cell cycle-responsive genes through reduced acetylation of histones H3 and H4. The recruitment of this co-repressor complex is cell phase specific as it binds to E2F-responsive genes during G0 /G1 phase and mediates histone deacetylation and chromatin compaction that prevents the expression of genes required for constitutive cell division. However, progression of the cell cycle to S phase disassembles this co-repressor complex from the target promoters and

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further mediates transcriptional activation through recruitment of activator proteins like E2Fs and histone acetyl transferases (HAT) (Rayman et al., 2002). Sin3 binds specifically to E2F4 and E2F5, both of which are involved in transcriptional repression as compared to other members of the E2F family namely, E2F1, E2F2 and E2F3 which are involved in gene activation (Humbert et al., 2000; Leone et al., 1998; Wu et al., 2001). Sin3 is involved not only in the normal cell cycle but has a significant role in mediating quiescence as well as differentiation of cells through binding to E2F-responsive genes. However, the mechanism for regulation of genes under quiescence is different from repression of genes during the cell cycle (Balciunaite et al., 2005). Sin3 recruits p130 and p107 onto the promoters of genes involved in cell cycling whereas only p130 and Sin3 are recruited onto the promoters of cells undergoing quiescence. It is however striking that Sin3 is recruited onto a distinct set of promoters under quiescence and proliferation implicating highly regulated modes of Sin3 functions. The E2F/Rb pathways, involved in cellular differentiation, get deregulated following genetic inactivation of Sin3B in mouse, signifying Sin3B as an essential factor for cell cycle exit (David et al., 2008). The phenotype observed upon deletion of Sin3B is similar to Rb/E2F mutant strains, suggesting a plausible interaction between these two proteins and their role in mediating cellular differentiation. Interaction of Sin3B with transcription factor E2F4 coordinates chromatin modifications for stable silencing of cell cycle genes to mediate cellular differentiation. Loss of Sin3B in mouse embryos was shown to be lethal due to multiple differentiation defects established by the presence of immature blood cells, endochondral ossification and decreased bone-to-cartilage ratio of hind limbs (David et al., 2008). The suggested mechanism for differentiation involves recruitment of Sin3/E2F4 along with the lysine demethylase RBP2, downstream of the transcription start site (TSS) at an early stage of differentiation that leads to decreased levels of H3 acetylation and H3K4 tri-methylation along with increased H3K27 di/tri-methylation, a marker of differentiation (van Oevelen et al., 2008). Sin3B null cells fail to tether E2F4 and HDAC1 on the promoters of E2F-responsive genes like Cdc2a, suggesting Sin3 as an essential factor for the recruitment of this complex (van Oevelen et al., 2008). This further supports the notion that Sin3 plays a critical role in recruitment of chromatin modifiers onto E2F target genes and in regulating the process of cell cycle progression and cell cycle exit (David et al., 2008; van Oevelen et al., 2008). Sin3: Role in oncogenic transformation The role of Sin3 in cancer is ambiguous as independent studies have reported Sin3A functions to be both oncogenic and tumor suppressive (Dannenberg et al., 2005; Das et al., 2013; Ellison-Zelski and Alarid, 2010; Farias et al., 2010). Sin3A has been shown to be essential for survival of transformed cells (Dannenberg et al., 2005). Furthermore, decreased expression of Sin3A was associated with increased apoptosis of tumor cells and enhanced expression of several genes involved in both intrinsic and extrinsic death signaling pathways (Ellison-Zelski and Alarid, 2010). Interference with Sin3A functions in triple negative breast cancer cells has been shown to induce epigenetic reprogramming and differentiation (Farias et al., 2010). In contrast, a recent study in Drosophila revealed Sin3A as a negative regulator for tumor progression where reduction in levels of Sin3A in tumor cells lead to activation of Src, Jnk, Rho1 and Arm/␤ catenin pathways that promote cell migration and cell invasion (Das et al., 2013). In breast cancer the functional involvement of Sin3A in BRMS1-mediated metastasis suppression has also been implicated (Hurst, 2012; Hurst et al., 2013). The divergent role of Sin3A in cellular proliferation across different cell types and the functions of Sin3A in tumorigenesis remain equivocal. For Sin3B, its levels are known to be upregulated in response to oncogenic stress

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that is thought to drive the cells toward senescence (Grandinetti et al., 2009). Beyond this, the role of Sin3 specifically in tumor progression/regression has not been investigated. Sin3: An essential protein for regulation of metabolic pathways Energy metabolism and generation of biomass for nextgeneration cells are important components of cellular homeostasis that involve complex regulation of gene expression. Detailed gene expression analysis revealed that almost 3% of Drosophila genes are altered upon deletion of Sin3B. Interestingly, a significant fraction of the genes which showed altered expression participate in the regulation of genes involved in mitochondrial and cellular protein synthesis, glucose metabolism, oxidative metabolism, fatty acid oxidation, oxidative phosphorylation and mitochondrial biogenesis (Pile et al., 2003). Loss of Sin3 resulted in enhanced expression of mitochondrial genes, i.e., mt:ATPase. mt:CoI, mt:cytb and mt:MD1, an effect attributed to an increased expression of mitochondrial RNA polymerase upon Sin3 deletion. Additionally, increased mitochondrial biogenesis due to increased expression of ribosomal translation and elongation proteins is observed in Sin3-deleted cells (Pile et al., 2003). Sin3 null mutants in yeast also showed defects in carbon metabolism and exhibited low levels of ATP and respiration rate compared to wild type (Barnes et al., 2010). Loss of RPD3 also resulted in altered mitochondrial and gene activity similar to that observed upon Sin3 deletion (Chen et al., 2008; Foglietti et al., 2006). Thus, a substantial fraction of genes induced by loss of Sin3 is involved in cyotosolic and mitochondrial energy-dependent pathways (Barnes et al., 2010). Cumulatively, these studies give an insight into the functional and evolutionary conserved role of Sin3 in control of cellular energy production. Sin3A and Sin3B: Functionally redundant or functionally distinct? The similarity of mouse Sin3A (mSin3A) to human Sin3A (hSin3A) is higher than to mSin3B. This indicates that divergence of Sin3A and Sin3B from Sin3 during evolution occurred before speciation (Silverstein and Ekwall, 2005; Halleck et al., 1995). Although the six domains of Sin3 are conserved both in Sin3A and Sin3B, it is reasonable to assume that nature must have reserved some unique and specialized functions for each isoform. The functional redundancy and specificity of the two paralogs of Sin3 is currently an area of active research. In the past, characterization of Sin3 complex formation and protein–protein interactions had been limited to mSin3A and it is only recently that research on Sin3B is gaining momentum. The PAH and HID regions are conserved in mSin3B, therefore it is expected that mSin3B may possess similar scaffolding capabilities as mSin3A (Alland et al., 1997; Koipally et al., 1999). However, it is also anticipated that at least some functions of each isoform are unique and specialized, which would allow greater flexibility during embryonic development and normal cell functioning (Silverstein and Ekwall, 2005). DNA-binding transcription factors like Mad1, KLF, REST, and ESET interact with Sin3A as well as Sin3B. On the one hand, transcription regulators like SMRT and MeCP2 have been shown to associate with mSin3A while on the other hand the master regulator of MHC II, CIITA, associates with HDAC2/Sin3B that mediate IFN-␥-induced repression of collagen type I gene transcription (Nagy et al., 1997; Nan et al., 1998; Xu et al., 2008). Rayman and coworkers, identified that Sin3B was associated with repression of a subset of E2F-target genes while both Sin3A and Sin3B have been shown to be associated with p53-mediated gene repression (Hoffman et al., 2002; Murphy et al., 1999; Rayman et al., 2002; Zilfou et al., 2001). Sin3B is an essential factor in promoting the cell cycle via the E2F–Rb pathway (David et al., 2008; Grandinetti et al., 2009). Sin3B also acts as an important player for regulation of cell cycle exit and in

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This review in a nutshell attempts to summarize the functional significance of Sin3 in several biological pathways (Fig. 3). Outlook

Fig. 3. Diagrammatic representation of involvement of the Sin3/HDAC complex in regulation of biological functions to maintain cellular homeostasis. Various subunits of the core complex participate in helping Sin3 to carry out its functions.

repression of E2F target genes, along with a role in differentiation of erythrocytes and chondrocytes (David et al., 2008). A subsequent paper by the same group demonstrated that Sin3B is upregulated during oncogenic stress, signaled by Ras overexpression and is required for cellular senescence (Grandinetti et al., 2009). In contrast, Sin3A removal has no effect on cell cycle progression instead it promotes apoptosis of MCF7 breast cancer cells (Hurst et al., 2013). Using decoy molecules, Farias and co-workers (Farias et al., 2010), selectively hampered Sin3 functions and induced a substantial degree of differentiation in triple negative breast cancer cell lines. Coupled with previous reports from Dannenberg and group (Dannenberg et al., 2005), it is now recognized that the mSin3A co-repressor regulates diverse transcriptional networks governing normal and neoplastic growth and survival. Based on these observations, it will not be inappropriate to expect several unique targets of each isoform in the cell. Conclusion Sin3, first discovered in yeast as a scaffolding protein, is now well established as an important protein involved in diverse cellular functions such as chromosome segregation, rDNA silencing, DNA damage repair, cell cycle, senescence, organ development, oncogenesis and many more. Although shown to interact with several proteins, the molecular mechanism of Sin3 action in such diverse cellular activities is far from clear. The distinction and extent of overlap in the functions of the two mammalian isoforms (Sin3A and Sin3B) also remain unclear. Recent studies have provided an insight into the role of Sin3 in cellular senescence and heterochromatinization through the recruitment of histone demethylases onto gene promoters. Sin3, along with DNA-binding proteins, forms a complex, providing a paradigm for the coordinated modification of chromatin marks, ultimately leading to the formation of heterochromatin and permanent gene silencing in differentiated cells. Sin3 provides a support for multiple peripheral enzymatic activities by flexibly interacting with different DNA-binding proteins, and therefore controlling gene expression. Though the mechanism is not clear, Sin3 unequivocally regulates complex cellular pathways such as energy metabolism, cellular proliferation and cell survival.

In spite of active research into mechanisms of gene regulation in general, the role of Sin3 remains enigmatic. Sin3 has been widely studied as a transcriptional corepressor and is known to repress the expression of genes involved in varied cellular functions. However, recent studies have also put forward Sin3 as a transcriptional activator. Existence of two Sin3 paralogs in higher eukaryotes poses the important question of whether these isoforms perform redundant or dissimilar functions. The available evidence suggests that both Sin3 isoforms bind to similar or overlapping regions of their interacting partners; however, the conditions and mechanisms by which the specific recruitment of one Sin3 isoform occurs over the other need to be investigated in more detail. Structure–function analysis of Sin3A and Sin3B with different interacting proteins may provide further insight into the ir mechanisms of action. Currently, a plethora of proteins are known to interact with Sin3; however, additional binding partners remain to be identified that may provide further insight into the diverse functions of Sin3. Investigating the role of Sin3 in oncogenesis is another area of interest that can translate into the design of novel cancer therapeutics. Future efforts must be devoted to better understand the complexity of the interactions of the Sin3 core complex and identify the distinct and exclusive functions of the two mammalian Sin3 isoforms. Acknowledgement We are grateful to Prof. Samuel Waxman and Dr. Eduardo F. Farias at Mount Sinai School of Medicine, New York, USA for their critical comments, suggestions and in depth reading of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ejcb.2013.09.001. References Alland, L., Muhle, R., Hou, H., Potes, J., Chin, L., Schreiber-Agus, N., DePinho, R.A., 1997. Role for N-CoR and histone deacetylase in Sin3-mediated transcriptional repression. Nature 387, 49–55. Ayer, D.E., Lawrence, Q.A., Eisenman, R.N., 1995. Mad-Max transcriptional repression is mediated by ternary complex formation with mammalian homologs of yeast repressor Sin3. Cell 80, 767–776. Balciunaite, E., Spektor, A., Lents, N.H., Cam, H., Te Riele, H., Scime, A., Rudnicki, M.A., Young, R., Dynlacht, B.D., 2005. Pocket protein complexes are recruited to distinct targets in quiescent and proliferating cells. Mol. Cell. Biol. 25, 8166–8178. Baltus, G.A., Kowalski, M.P., Tutter, A.V., Kadam, S., 2009. A positive regulatory role for the mSin3A-HDAC complex in pluripotency through Nanog and Sox2. J. Biol. Chem. 284, 6998–7006. Bansal, N., Kadamb, R., Mittal, S., Vig, L., Sharma, R., Dwarakanath, B.S., Saluja, D., 2011. Tumor suppressor protein p53 recruits human Sin3B/HDAC1 complex for down-regulation of its target promoters in response to genotoxic stress. PLoS ONE 6, e26156. Barnes, V.L., Strunk, B.S., Lee, I., Hüttemann, M., Pile, L.A., 2010. Loss of the SIN3 transcriptional corepressor results in aberrant mitochondrial function. BMC Biochem. 11, 26. Brehm, A., Miska, E.A., McCance, D.J., Reid, J.L., Bannister, A.J., Kouzarides, T., 1998. Retinoblastoma protein recruits histone deacetylase to repress transcription. Nature 391, 597–601. Brubaker, K., Cowley, S.M., Huang, K., Loo, L., Yochum, G.S., Ayer, D.E., Eisenman, R.N., Radhakrishnan, I., 2000. Solution structure of the interacting domains of the Mad-Sin3 complex: implications for recruitment of a chromatin-modifying complex. Cell 103, 655–665. Burke, L.J., Baniahmad, A., 2000. Co-repressors 2000. FASEB J. 13, 1876–1888. Buttar, N.S., DeMars, C.J., Lomberk, G., Rizvi, S., Bonilla-Velez, J., Achra, S., Rashtak, S., Wang, K.K., Fernandez-Zapico, M.E., Urrutia, R., 2010. Distinct role of

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