Biosci. Rep. (2015) / 35 / art:e00234 / doi 10.1042/BSR20150122

Stress-mediated Sin3B activation leads to negative regulation of subset of p53 target

Rama Kadamb*1, Shilpi Mittal*, Nidhi Bansal* and Daman Saluja*1

*Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi-110007, India

Synopsis The multiprotein SWI-independent 3 (Sin3)–HDAC (histone deacetylase) complex mediates re- pression through its interaction with DNA-binding factors and recruitment of chromatin-modifying on to the promoters of target gene. Previously, an increased expression of Sin3B and tumour suppressor , p53 has been established upon adriamycin treatment. We, now provide evidence that Sin3B expression is significantly up-regulated under variety of stress conditions and this response is not stress-type specific. We observed that Sin3B expression is significantly up-regulated both at transcript and at protein level upon DNA damage induced by bleomycin drug, a radiomimetic agent. This increase in Sin3B expression upon stress is found to be p53-dependent and is associated with enhanced interaction of Sin3B with Ser15 phosphorylated p53. Binding of Sin3–HDAC repressor complex on to the promoters of p53 target genes influences gene regulation by altering histone modifications (H3K9me3 and H3K27me3) at target genes. Furthermore, knockdown of Sin3B by shRNA severely compromises p53-mediated gene repression under stress conditions. Taken together, these results suggest that stress-induced Sin3B activation is p53-dependent and is essential for p53-mediated repression of its selective target genes. The present study has an implication in understanding the transrepression mechanism of p53 under DNA damaging conditions. Key words: gene regulation and bleomycin-induced stress, genotoxic stress, p53, Sin3B, Sin3–histone deacetylase (HDAC) complex, transrepression.

Cite this article as: Bioscience Reports (2015) 35, e00234, doi:10.1042/BSR20150122

INTRODUCTION Sin3 has no DNA-binding domain and interacts with various transcription factors through Sin3 interaction domain (SID) and thereby gets recruited on to the promoters of target genes [1,3,4]. Gene regulation in eukaryotes is achieved through fine tuning of Once recruited to the promoters, Sin3 executes gene repression activator and repressor complexes that mediate relaxed and con- by altering chromatin state through associated HDACs, such as densed chromatin state respectively. In general, two counteracting Rpd3 (reduced potasium deficiency 3) in yeast and HDAC1 and enzymes control the primarily: histone acetyl- HDAC2 in mammals [5,6]. transferases (HATs) that add acetyl groups to the histone moieties In mammals, Sin3 has two paralogues; Sin3A and Sin3B which and thus mediate gene activation; and the histone deacetylases are known to perform varied functions under different cellular (HDACs) which deacetylate histone proteins and thus contribute conditions [4,7]. Knockout studies in mice demonstrate distinct to gene repression [1]. These enzymes do not work individually, functions and differential requirement of the two isoforms dur- instead, they are recruited to the promoters of genes as multi- ing development. Loss of Sin3A is lethal during the early stages subunit protein complexes where other proteins function as chro- of development in mice and is required for T-cell development and matin remodellers and recruit additional cofactors or perform maintenance of sarcomere structure [8,9]. In contrast, Sin3B is unknown functions [2]. Sin3, a negative regulator of gene tran- needed at later stages of development and is required for skeletal scription, is a component of HDAC co-repressor complex that is development and cell-cycle progression [10]. However, overlap- known to be involved in scaffolding of genome [3]. Like HDACs, ping functions between these two paralogues cannot be ruled

...... Abbreviations: ANLN, anillin; BC, bleomycin control; BT, bleomycin treated; CDC25c, cell division cycle 25C; CRYZ, crystallin, zeta; HDAC, histone deacetylase; HBSS, Hank’s balanced salt solution; HP1, heterochromatin-binding protein 1; HSPA8, heat-shock 70 kDa protein 8; IP, immunoprecipitation; KDB, knockdown Sin3B; MAD1, mitotic arrest deficient-like 1; PBST, PBS with Tween 20; PTM, post-translational modification; qPCR, quantitative real-time PCR; RBP2, retinol-binding protein 2; RE, response element; RIPA, radioimmunoprecipitation assay; RNF220, ring finger protein 220; Rpd3, reduced potassium dependency; RT, real time; SAP, Sin3-associated protein.; Sin3, SWI-independent 1 Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).

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out as Sin3A is found to perform compensatory functions in plasmid (Santa Cruz Biotechnology) using plasmid transfection the absence of Sin3B suggesting that there can be various com- reagent according to manufacturer’s protocol (Santa Cruz Bi- plexes present in cell with different combinations of Sin3A/Sin3B otechnology). Forty-eight hours after transfection, the positive [11–13]. Sin3 has recently been discovered to be involved in clones were selected by growing cells in puromycin containing various cellular functions ranging from T-cell development, cell- medium (ant-pr-1, Invivogen). KB, A549 and Saos2 cell lines cycle progression, differentiation, senescence, protein stabiliza- were procured from National Center for Cell Sciences (NCCS) tion, energy metabolism and cell survival [4,8,9,12,14–20]. Apart and HCT116 (wild-type and null for p53) and H1299 cell lines from the above mentioned functions, role of Sin3 under DNA were provided as a kind gift from Dr Bert Vogelstein from John damage conditions has previously been studied in Saccharomy- Hopkins University and Dr Sanjeev Das from National Institute ces cerevisiae where it mediates non-homologous end joining of Immunology respectively. DNA repair [21]. However, the involvement of Sin3 under stress conditions is not well studied in higher organisms which promp- ted us to study the regulation of Sin3 in humans upon bleomy- Cell treatment cin treatment, a radiomimetic drug that causes double strand Sub-confluent cultures were treated with different stress-inducing breakage [22,23]. Sin3 interacts with expanding array of DNA- agents: cells were incubated with 400 μg/ml and 20 ng/ml of binding transcription factors and our previous study affirmed bleomycin and colchicine respectively, for indicated time points. interaction of Sin3B with p53, a stress-related protein [14,24– For γ radiation treatment, cells at a confluency of 70%–80% 27]. p53 is exemplified as the ‘guardian of genome’ which gets were irradiated with γ radiation source (60Co; Eldorad78) in rapidly activated upon exposure of cells to various stress signals Hank’s balanced salt solution (HBSS) and, after treatment, the [28,29]. p53 is a sequence-specific transcription factor that can cells were incubated at 37 ◦C for 1 and 4 h respectively. either transactivate or transrepress various genes in a context- dependent manner [30]. The mechanism of p53-mediated gene activation and its role in regulation of damage/stress response Western blotting pathways is well established [29,30]. However, the mechanism CellsculturedinT25cm2 flasks were subjected to different drug underlying p53-mediated transcriptional repression remains elu- treatments for desired time intervals and harvested for analysing sive. Previously, Sin3 has been demonstrated to play an imperat- protein expression. Cell lysates were prepared by re-suspending ive role in p53-mediated gene regulation [14,26]. Nevertheless, the cells in RIPA (radioimmunoprecipitation assay) buffer for it is believed that different stress signals can lead to different, 45 min at 4 ◦C. The soluble protein fractions were collected by as well as independent pathways for p53 activation, which in centrifugation at 15 777 g. Total protein was estimated using turn can mediate different responses [31]. In the present study, BCA protein estimation kit (Bangalore Genei) and equal amount we looked into the insight of mechanism of Sin3B activation and of proteins (30 μg) were resolved on SDS/PAGE (either 8% or p53-mediated negative gene regulation under bleomycin-induced 10% gel) and transferred on to immunoblot- PVDF membranes DNA damaging conditions. The present study shows that under (Santa Cruz Biotechnology). Following blocking with 5% BSA bleomycin-induced stress, expression of Sin3B gets up-regulated for 1 h at room temperature and washing with PBS with Tween 20 and it gets recruited by p53 at its target promoters. Knockdown of (PBST), the PVDF membranes were incubated overnight at 4 ◦C Sin3B leads to impaired negative regulation of p53 target genes in primary antibody (1:1000) diluted in PBST containing 1% and thus exemplifies Sin3B as a critical player in down-regulation BSA. Membranes were subsequently incubated with secondary of p53 subset target genes. antibodies conjugated with horseradish peroxidase (1:5000) for 1 h at room temperature. The blots were probed with the en- hanced chemiluminescence (ECL) western blot detection system (Biogene) according to manufacturer’s instructions. MATERIALS AND METHODS

Co-immunoprecipitation Cell culture and establishment of stably Sub-confluent cultures of untreated and bleomycin-treated cells transfected cell lines were harvested and lysed in RIPA buffer (buffer composition as HCT116 (human colorectal carcinoma), KB (head and neck given in the Supplementary Table S1) supplemented with pro- squamous carcinoma), A549 (human lung carcinoma), Saos2 tease inhibitor cocktail (Sigma–Aldrich). Equal amount of pro- (osteosarcoma) and H1299 (non small cell lung carcinoma) tein (1.5 mg) from each sample was immunoprecipitated with ◦ cell lines were cultured at 37 Cin5%CO2 humidi- 1 μg of desired antibody. For collection of the immunoprecip- fied atmosphere. Cells were grown in high glucose Dul- itates, 30 μl of protein A/G agarose beads were added to each becco modified Eagle’s medium (DMEM; Sigma; Pan Bi- tube and the samples were incubated for 2–3 h at 4 ◦Conrota- otech) supplemented with 10% FBS (Gibco) and penicillin tion. Each immunoprecipitate was washed thrice in RIPA buffer (60 units/ml) and streptomycin (50 μg/ml). For transfection, and eluted with 2× loading dye, fractionated on 8% SDS/PAGE HCT116 cells were seeded into six-well tissue culture and transferred overnight on to immuno-blot PVDF membrane plates and transfected with 1 μg of control/Sin3B shRNA (Santa Cruz Biotechnology). Immunodetection was done with

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anti-Sin3B antibody, p53 and phospho Ser15-p53 at a dilution of 45 ◦C. DNA from eluted samples was recovered with PureLink 1:1000 for Figures 3(A) and 3(B) respectively. Ten percent of the PCR purification Kit (Invitrogen). PCR and quantitative real input was saved as a positive control from the total lysate. time PCR were performed using gene-specific primers for pro- moter regions. Composition of the buffers and primer pairs used are given in Supplementary Tables S1 and S3. RNA purification, cDNA synthesis and qPCR Untreated/control and bleomycin-treated cells were processed for RNA purification using RNeasy kit (Qiagen) according to man- Statistical analysis ufacturer’s instructions. One μg of RNA was used for cDNA Student’s t-test was used to evaluate the significance of the differ- synthesis using Verso cDNA kit (Thermo Scientific) according ences between control and drug-treated samples in all pertinent to the manufacturer’s protocol. Semi-quantitative and quantitat- experiments; a P-values < 0.05 and 0.01 was considered signi- ive real-time PCR (qPCR) reactions were carried out using gene ficant and highly significant respectively. specific primers. Real time gene expression analysis was carried out using MESA Green qPCR Mastermix plus for SYBR assay (Eurogenetec) on Applied Biosystems 7300 RT-PCR system and RESULTS data were collected and exported with SDS 2.2.2 version. Rel- ative expression was calculated using 2 − Ct method and 18S rRNA was used as an endogenous control. Sequence of primer Sin3B expression is induced upon different cellular pairs used for semi-quantitative and real-time PCR are listed in stress conditions and is not cell-type specific Supplementary Table S2. Cell lines of different origins were subjected to genotoxic (bleo- mycin and γ -radiation) and non-genotoxic stress (colchicine) to study their effect on the expression of human Sin3B and Cell-cycle analysis stress-related protein, p53. Initially, the dose of bleomycin which To prepare cells for FACS analysis, 1 × 106 (control and treated) can induce cellular stress and thus elicit p53 activation was cells were washed with 1× PBS, resuspended in 0.5 ml of 1× standardized. It was found that 400 μg/ml of bleomycin drug PBS and incubated with 10 μl of RNase A (10 mg/ml) at 37 ◦C is sufficient to activate p53 levels and lead to arrest of cells

in a water bath for 30 min, followed by addition of 4 μg/ml of in the G2-phase of the cell cycle (Supplementary Figures S1A Propidium Iodide in dark on ice. Stained cells were analysed on and S1B). Western blot analysis of lysates from untreated and Becton Dickinson FACScan machine and the data were analysed treated cells depicted time-dependent increase in the expression with either CELLQuest Pro or FlowJo software. of Sin3B and p53 upon bleomycin drug treatment as compared with control cells (Figure 1A, left panel). The expression of both Sin3B and p53 showed significant increase at protein level ChIP (chromatin-immunoprecipitation) within 20 min after bleomycin treatment and these high levels of HCT116 cells were treated with bleomycin drug prior to form- protein expression were maintained up to 24 h post drug treat- aldehyde fixation. Formaldehyde fixed cells lysed in 0.1% SDS ment. The expression levels of p53 and Sin3B at 24 h after lysis buffer were sonicated (20 cycles of 30-s pulse with 1-min bleomycin treatment was approximately 5- and 2.5-fold higher interval) at 90% power using Bioruptor sonicator (Diagenode). respectively as compared with controls (Supplementary Figure Samples were centrifuged for 10 min at 18516 g at 4 ◦C and were S1C). This increase in expression of Sin3B was not isoform spe- diluted ten times with dilution buffer except for the input sample. cific as we observed similar increase in the levels of another Sin3 The diluted supernatants were collected and pre-cleared with pro- isoform, i.e. Sin3A in bleomycin-treated cells (Supplementary tein A/G Agarose beads containing 1 mg/ml salmon sperm DNA Figure S1D). Similarly, exposure of cells to 5 Gy of γ -radiation for 3–4 h at 4 ◦C with agitation. Samples were spun down at 7012 dose lead to enhanced Sin3B expression at 1 and 4 h with a g for 5 min at 4 ◦C and incubated overnight with specific anti- concomitant increase in the p53 levels (Figure 1B). bodies. Samples were incubated with 30 μl of pre-blocked beads Under conditions of non-genotoxic stress, induced by col- (protein A/G agarose/salmon sperm DNA) for 3 h, spun at 986 chicine (mitotic poison), we observed that expression of Sin3B g at 4 ◦C for 3 min and washed sequentially with the following began to increase within 30 min after treatment with colchicine buffers at 4 ◦C: once with 0.1% SDS lysis buffer, twice with low drug at a concentration of 20 ng/ml and time-dependent increase salt-wash buffer, twice with high salt-wash buffer, twice with LiCl was observed till 16 h of drug incubation (Figure 1A, right panel). wash buffer and twice with TE buffer. For de-cross-linking and In concordance to published reports, we also observed that expos- PCR, samples were eluted twice with 100 μl of elution buffer, ure of cells to different kinds of stress stimuli was accompanied vortexed briefly and incubated at room temperature for 15 min by an increase in p53 protein level. We also demonstrate that with rotation. Eluates were pooled (200 μl) and 8 μlof5M increased expression of Sin3B, upon bleomycin treatment, is not NaCl added. De-cross-linking was performed at 65 ◦Cfor6hto cell-type specific as cell lines of different origins (head and neck overnight. Following elution, samples were treated with 4 μlof squamous carcinoma and non-small cell lung carcinoma) treated 0.5 M EDTA, 4 μl of 1 M Tris, pH 6.8, 2 μl of 10 mg/ml pro- with bleomycin showed similar increase in Sin3B expression teinase K, 2 μl of 10 mg/ml RNAase A and incubated for 3 h at (Figure 1C).

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Figure 1 Expression of Sin3B increases under conditions of cellular stress in a p53-dependent manner HCT116 wild-type (HCT116 p53 + / + ) cells were treated with different stress inducers that are known to elicit p53 activation and response. (A) Cells were subjected to bleomycin (radiomimetic agent) and colchicine (mitotic poison) drug treatment at 400 μg/ml and 20 ng/ml dose respectively, at indicated time points. (B) KB cells were subjected to γ -radiation at a dose of 5 Gy and incubated for 1 and 4 h in HBSS. (C)Western blots showing expression of Sin3B and p53 protein in KB and A549 cell lines. (D) Western blot analysis showing no effect on the expression of Sin3B in p53 null cells (HCT116 p53 − / − ). Following different drug treatments, the expression of p53 and Sin3B were checked by performing western blot analysis using specific antibodies. For all the experiments, β-actin was used as endogenous control.

Further, to gain insight into the role of p53 in Sin3B regulation, (Figures 2Aand2B). To rule out the possibility of ineffective drug p53 null cells were subjected to similar conditions of stress and treatment for insignificant changes in p53 expression, transcript analysed for protein expression. Importantly, p53 null cells did levels of p21 gene were also analysed simultaneously. p21 is a not show any significant increase in Sin3B expression upon bleo- well-known p53 target gene which gets up-regulated under vari- mycin drug treatment neither at protein nor at transcript levels ety of stress conditions [29]. We observed significant increase in (Figure 1D; Supplementary Figure S2). These results evinced p21 transcript levels following bleomycin treatment, confirming that increase in Sin3B expression level upon stress conditions is that the drug treatment is indeed effective (Supplementary Figure a general phenomenon that can occur in a variety of cell type re- S3). In the current study, no significant changes in the p53 mRNA gardless of the stimulus types that initiate this process and p53 is levels (Figure 2A) are consistent with previous studies that sug- required for Sin3B activation. gest p53 expression is typically enhanced at post-translational level under stress conditions [32]. In contrast, a significant in- crease was observed for Sin3B mRNA levels within 20 min of Increased Sin3B expression is due to increased bleomycin drug treatment that further increased up to 4-fold in a transcript level and post-translational stabilization time-dependent manner (Figure 2B). To ascertain whether increase in Sin3B protein upon cellular We next checked whether augmentation of Sin3B protein upon stress is a result of increased transcription, total RNA from con- stress was a result of increased translational product alone (due to trol and bleomycin-treated samples were isolated and reverse increase in mRNA for Sin3B) or post-translational stabilization transcribed. Semi-quantitative and qPCR indicated a significant has any role to play in increasing Sin3B protein levels. Previously, increase in the levels of Sin3B transcript; however, no changes RNF220 (ring finger protein 220) protein has been identified as a were observed for p53 transcript levels upon bleomycin treatment novel ubiquitin ligase of Sin3B [33]. We, therefore, compared the

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Figure 2 Human Sin3B transcript levels are increased upon bleomycin treatment Total RNA was isolated and reverse transcribed at various time point as indicated in the figure. The cDNA (control and treated samples) was used as a template for semi-quantitative PCR as well as for qPCR using gene-specific primers. (A and B) represents the expression of p53 and Sin3B transcripts respectively as detected by qPCR (upper panels) and semi-quantitative PCR (lower panels) in HCT116 cell line after bleomycin treatment at different time points. Relative expression levels were calculated by taking transcript level in untreated cells as one. The Ct value for each gene was normalized with 18S rRNA which was used as an endogenous control. Error bar shows S.D. and the plots have been + plotted with − S.E.M. *P < 0.05 and **P < 0.01. For semi-quantitative PCR, p53 and Sin3B were amplified for 28 and 30 cycles respectively. The results are representative of three independent experiments. (C) Western blot analysis of cell lysates from bleomycin-untreated and -treated samples at various time points as indicated in the figure for checking the expression levels of Sin3B and RNF220, a ubiquitin ligase. β-actin was used a protein loading control.

expression levels of RNF220 protein along with Sin3B protein ing p53 and phospho Ser15-p53 antibody followed by immun- under similar stress conditions. Western blot analysis showed that oblotting with Sin3B-specific antibody. Co-immunoprecipitation expression of RNF220 decreased as a function of time of bleo- assay with total pool of p53 showed no increased association of mycin treatment with a concomitant increase in the expression Sin3B under bleomycin-induced stress; however, immunoprecip- of Sin3B protein (Figure 2C). Thus, our results suggest that in- itation of the complex with phospho Ser15-p53 antibody followed creased expression of Sin3B at protein level is a cumulative effect by detection with anti-Sin3B antibody showed enhanced interac- of increased transcript synthesis and increased stabilization due tion between Ser15 phosphorylated p53 and Sin3B (Figure 3A). to decreased RNF220 expression upon bleomycin treatment. In contrast, no significant band was observed in the isotype (mock) lane that confirmed the specificity of interaction. Re- ciprocal immunoprecipitation-western experiments using anti- Increased association of human Sin3B with p53 Sin3B antibody for immunoprecipitation of the complex and phosphorylated at Ser15 anti-p53 antibody for immunodetection demonstrated that Sin3B Human p53 is known to undergo several post-translational modi- could also co-immunoprecipitate p53 under similar conditions. fications (PTMs) through activation of several kinases. Using This, further, affirmed the specific interaction between these two co-immunoprecipitation and yeast two-hybrid studies, we earlier proteins (Figure 3B). reported that p53 directly interacts with Sin3B under normal conditions without any increase in association between the two proteins under adriamycin-induced stress conditions [14]. Out of Sin3–HDAC complex docks on to the promoter of the many types of phosphorylation, p53 phosphorylation on Ser15 subset of p53 target genes residues occurs following DNA damage induced by γ -radiation To decipher the functional significance of human p53–Sin3B and several chemotherapeutics agents [34–36]. To study the ef- interaction and to understand the role played by Sin3B in p53- fect of PTM of p53 on its interaction with Sin3B, under stress mediated gene regulation upon bleomycin treatment, the recruit- conditions, co-immunoprecipitation assays were performed. Ini- ment of human Sin3B on to the five selected p53 responsive tially, expression of Ser15 phosphorylated p53 was investigated promoters was investigated. Whereas Sin3 has been shown in control and 24 h drug-treated HCT116 cells. Western blot ana- to stabilize p53 for mediating its transrepression functions lysis showed that levels of Ser15 phosphorylated at p53 residues [20,26], there has been no comprehensive study regarding the increased after treatment of cells with bleomycin (Supplement- role of Sin3B in p53-mediated negative gene regulation under ary Figure S4). Following confirmation of p53 modification at bleomycin-induced DNA damaging situations. Ser15 residues after bleomycin treatment, protein lysates from To study the recruitment of Sin3B on to p53 target promoters, untreated and drug-treated cells were immunoprecipitated us- ChIP assays were performed with cellular extracts from untreated

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Figure 3 Increased physical association occur between Sin3B and phosphorylated pool of p53 (Ser15) in bleomycin- treated cells (A) Cell lysates from untreated and 24 h bleomycin drug-treated cells were immunoprecipitated (IP) with antibodies specific for p53 and phospho Ser15-p53 followed by immunoblotting (IB) with antibody specific to Sin3B. (B) Reciprocal co-immunoprecipitation in HCT116 wild-type cells using anti-Sin3B antibody for immunoprecipitation of the immune complex and anti-p53 antibody for immunodetection. Input corresponding to 10 % of the total cell lysate was used as a positive control whereas mouse and rabbit IgG were used as negative controls.

and 24-h bleomycin-treated HCT116 cells, using promoter- reduction in the expression of Sin3B as compared with untrans- specific primers designed across the p53-response element (RE). fected cells both at protein and at RNA level (Figure 5A). Dens- In the initial screening phase, we observed significant enrich- itometric analysis revealed that Sin3B shRNA treatment resulted ment of human Sin3B on five out of eight p53-target promoters, in approximately 80% reduction in the expression of Sin3B in viz. MAD1 (mitotic arrest deficient-like 1), HSPA8 (heat-shock transfected cells as compared with untransfected cells. 70 kDa protein 8), CRYZ (crystallin, zeta),ANLN(anillin) and Knockdown of Sin3B in Drosophila S2 cells is known to cause

CDC25c (cell division cycle 25C; result not shown) and hence, arrest of cells in the G2-phase of the cell cycle [17]. Therefore, we focused at these five promoters only for a detailed dissection also investigated whether knockdown of Sin3B in HCT116 cell of the involvement of Sin3B in p53-mediated gene repression. line causes any cell-cycle perturbation. Using Propidium Iodide Using specific antibodies against p53, Sin3B and HDAC1, sub- staining, cell cycle profile of untransfected and Sin3B shRNA stantial recruitment of these three proteins at the region spanning transfected cells were analysed under normal and bleomycin- the p53 REs were observed for the five p53 target genes under induced DNA damage conditions. Cell cycle analysis revealed

both normal and stressed conditions. The strength of the specific that significant fraction of cells entered G2-phase of cell cycle binding of antibodies was evident when compared with isotype in cells ablated for Sin3B as compared with untransfected cells control (Figure 4A). upon bleomycin treatment. However, no significant changes in qPCR assay was performed to further investigate that whether the cell cycle patterns were observed between untransfected and any enrichment occurs for p53, Sin3B and HDAC1 at the p53 Sin3B transfected cells under normal cycling conditions/without target promoters in the presence and absence of bleomycin drug drug treatment (Figure 5B). We next checked for the effect of treatment. For this, ChIP DNA obtained after immunoprecip- Sin3B knockdown on the expression of p53 target genes fol- itation with specific antibody (Figure 4B) from both untreated lowing bleomycin drug treatment. We observed significant dere- and drug-treated cells was used as a template and amplified us- pression of the p53 target genes in the cells transfected with ing gene-specific primers. Our qPCR results were in agreement Sin3B shRNA upon bleomycin treatment. In contrast, untrans- with ChIP PCR results that showed no significant changes in the fected cells showed significant repression of the same target genes recruitment of p53, Sin3B and HDAC1 at selective p53 target under similar conditions (Figure 5C). promoters in bleomycin-treated cells as compared with untreated Thus, our results highlight the importance of Sin3B in neg- cells (Figure 4B). atively regulating p53 gene repression under bleomycin-induced stress conditions.

Negative regulation of subset of p53 target genes is Sin3B dependent Induction of epigenetic modifications on promoters To further validate the role of Sin3B in stress-induced p53- of p53 target genes mediated gene repression of its target genes, we transfected the Sin3B–HDAC complex is associated with enzymes involved in HCT116 cells with Sin3B shRNA to knockdown Sin3B (KDB) histone methylation and such PTMs at histones play critical expression. Cells transfected with shRNA showed significant role in gene regulation [10,37]. We, therefore, tested whether

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Figure 4 Recruitment of p53–Sin3B–HDAC1 occurs on subset of p53 target genes ChIP assays were performed in HCT116 cells in untreated and cells treated with bleomycin for 24 h. Equal amounts of cross-linked chromatin were pre-cleared and incubated with 1μg of antibody for anti-p53, anti-Sin3B, anti-HDAC1 as indicated above each lane. Following DNA precipitation, samples were analysed by PCR using specific primers for HSPA8, MAD1, CRYZ, CDC25c and ANLN. Input corresponding to 10 % of the total chromatin used as positive control for each immunoprecipitation reaction and IgG was used as isotype control. * Indicates primer dimers. (B) For RT-PCR assays, ChIP DNA recovered from each immunoprecipitation using specific antibodies for p53, Sin3B and HDAC1 was used as a template and amplified using gene-specific primers with SYBR green dye on ABI-7300 system. Amplification in the mock (isotype) was used as control. Percent input was calculated for enrichment of DNA on to the respective target promoters in bleomycin control and bleomycin-treated conditions.

bleomycin-mediated stress alters the methylation marks on his- and H3K27me3 (Figure 6). These observations suggested that tones at promoters of p53 target genes. To better understand this differential hypermethylation of histones occur following bleo- phenomenon, HCT116 wild-type cells were treated with bleomy- mycin treatment and such changes are responsible for bleomycin- cin and the endogenous promoter occupancy was examined on mediated p53 gene repression of its target genes. formaldehyde cross-linked chromatin by using specific antibod- ies for H3K4me3 as activation mark and H3K9me3/H3K27me3 as repression marks. To monitor the specificity of reaction, IgG was used as an isotype control. ChIP assay revealed DISCUSSION that exposure of cells to bleomycin drug did not cause any change in the enrichment of H3K4me3 except for MAD1 gene (Figure 6). However, significant increase in hypermethylation Several lines of evidence suggest that p53 modulates gene expres- of H3K9me3/H3K27me3, repression marks, was observed after sion under diverse environmental, genotoxic and developmental bleomycin drug treatment. At the promoter of HSPA8 gene, we stimuli by different mechanisms of transrepression [14,38,39]. observed increased hypermethylation of H3K9me3 residues with Furthermore, the mechanism of transrepression being context- no significant change in trimethylation of His3 at Lys27 residues. dependent, interaction and cross-talk between these pathways In contrast, CDC25c promoter showed increased hypermethyla- cannot be ruled out. It is also well established that several cellular tion for H3K27me3 residue without any significant changes for insults/stimuli induce p53 expression [32]. However, the regula- the other repression mark, i.e. H3K9me3. However, at MAD1, tion/response of Sin3B under such scenario is still not clear. We, CRYZ and ANLN promoter sites, we observed an increased now provide evidence that expression of Sin3B increases in p53- hypermethylation of both the repression marks, i.e. H3K9me3 dependent manner under variety of stress conditions. Though,

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Figure 5 Knockdown of Sin3B inhibits p53-mediated repression of its target genes (A) HCT116 cells were transfected with control and Sin3B shRNA and the expression of Sin3B was analysed in control and bleomycin-treated transfected cells through western blotting (left panel) and semi-quantitative PCR (right panel). (B) Cell cycle profile of HCT116 cell line in control shRNA (upper panel) and upon transfection with Sin3B shRNA (lower panel) in untreated and drug-treated cells. (C) Expression levels of p53 target genes were analysed in control and Sin3B shRNA transfected cells under both untreated and bleomycin-treated conditions. Relative expression levels for each gene were calculated by considering transcript levels of genes in untreated conditions as one. 18s rRNA was used as endogenous + control. Error bar shows S.D. and the plots have been plotted with − S.E.M. *P < 0.05 and **P < 0.01. Abbreviations: BC, bleomycin control; BT, bleomycin treated.

increase in Sin3B expression was also observed upon Rat Sar- translational stabilization by RNF220. In contrast, the expression coma (Ras) oncogene activation and adriamycin treatment to cells of p53 increased at protein level only with no significant change [14,40]; the mechanism of Sin3 up-regulation is still far from in transcript level upon bleomycin treatment. Thus, our obser- clear. Previously, RNF220, a ubiquitin ligase, has been shown to vation further supports the fact that p53 is post-translationally associate with Sin3B and promotes its proteasomal degradation stabilized under stress [32]. Interestingly, we did not find stress- [33]. In the present study, we found that expression of RNF220 induced expression of Sin3B in p53 null cells neither at protein decreased with a concomitant increase in Sin3B expression upon nor at transcript levels (Figure 1D; Supplementary Figure S4). bleomycin treatment. At the RNA level, our qPCR results indicate These findings, though, imply that increase in Sin3B expression significant increase in the Sin3B transcripts in a time-dependent might be regulated through p53 (directly or indirectly); the mech- manner following bleomycin treatment. These observations sug- anism for gene regulation cannot be explained as of this writing gest that increased Sin3B expression at protein level is a cu- as promoter of Sin3B is yet not identified. We further looked mulative effect of both increased transcript synthesis and post- into the Sin3B gene in search for any consensus p53-binding

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Figure 6 Epigenetic modifications occur at the promoters of p53 target genes upon bleomycin treatment HCT116 wild-type untreated and 24 h bleomycin drug-treated cells were harvested for ChIP assay. Equal amounts of cross-linked chromatin were pre-cleared and incubated with antibodies methylated at different residues of His3 as indicated in the figure. Following DNA precipitation, samples were analysed by qPCR using specific primers for HSPA8, MAD1, CRYZ, + CDC25c and ANLN promoters. Error bar shows standard deviation and the plots have been plotted with − S.E.M. *P < 0.05 and **P < 0.01. Percent input was calculated for enrichment of DNA on to the respective target promoters in bleomycin control and bleomycin treated conditions.

site using TRANSFAC program. However, we did not find any Our previous studies suggested that Sin3B interacts with N- p53-binding site upstream of the putative transcription start site terminal region of p53 under normal as well as stress conditions of Sin3B gene. Thus, in future, due attention is required to study [14]. However, the interaction status between p53 and Sin3B does the transcriptional and post-translational regulation of Sin3B by not change upon adriamycin drug treatment [14]. Like adriamy- p53, especially under the conditions of stress. cin, we found that bleomycin also does not cause any change

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in the interaction status of Sin3B with total pool of p53. Inter- target genes as compared with untransfected cells. The derepres- estingly, in the present study, we observed that, association of sion of p53 target genes was seen only under bleomycin-induced Sin3B is increased with Ser15 phosphorylated p53 under stress DNA damaging conditions. Untransfected and Sin3B shRNA conditions. In response to DNA damage, Ser15 and Ser20 residues transfected cells showed no significant changes under the nor- of p53 primarily get phosphorylated and blocks its interaction mal cell cycling conditions. However, following Sin3B shRNA with mouse double minute 2 (MDM2), a negative regulator of treatment we could not observe complete derepression of the p53 p53 [41–43]. Ser15 phosphorylation has been shown to enhance target genes. This lack of complete derepression after transfec- the interaction of p53 with various transcription factors. Phos- tion of Sin3B shRNA could be attributed to the fact that Sin3 phorylation of p53 at Ser15 residues is reported to result in in- gene has two paralogues, i.e. Sin3A and Sin3B. Both paralogues creased association of p53 with CREB (cAMP-RE-binding pro- show structural similarity with each other and regulate unique, as tein) under DNA damage conditions. In fact, phosphorylation well as overlapping subset of target genes [3,7,20,26,48,49]. It is of the p53 at N-terminus is shown to be involved in controlling believed that in the absence of Sin3B, the other isoform, namely protein interaction, regulating transcription of downstream genes Sin3A, can substitute for it and take over the function. Sin3A and it may also contribute in determining the response of cells associates with transcription regulators like methyl CpG binding to DNA damage [44]. Thus, our results are in concordance with protein 2 (MeCP2) and silencing of retinoic acid and published reports that propose phosphorylation of p53 in medi- thyroid hormone receptor (SMRT) whereas on the other hand ating increased interaction with other proteins [44]. Although, it Sin3B preferentially associates with MHC Class II Transactiv- remains unclear that whether this increase in p53 Ser15–Sin3B ator (CIITA) and major histocompatibility complex (MHC II) interaction represents an increase in binding affinity or binding [50–52]. Previously, Sin3A isoform has been shown to be in- stoichiometry and thus in future needs to be explored in detail. volved in negative regulation of microtubule-associated protein In order to check whether the association of p53 with Sin3B 4(MAP4) and stathmin (STMN1) genes [20,26]. Studies by van results in modulating the expression of downstream target genes, Oevelen et al. [12] have shown that both the isoforms are required recruitment of Sin3B–HDAC1 was analysed on to selective pro- for the maintenance of sarcomere functions and deletion of any moters of p53 target genes. A consistent recruitment of p53– one of the paralogues cannot rescue the normal phenotype [3]. Sin3B–HDAC1 on to the promoters of p53 repressed genes Interplay of DNA and histone modifications via histone methyl was observed both before and after DNA damaging conditions. transferases are prerequisite for regulation of gene expression. Ceribelli and co-workers showed that for genes, like ataxia tel- Sin3 associates with RBP2 (retinol-binding protein 2) through angiectasia and Rad3-related protein (ATR), casitas B-lineage SAP30 (Sin3-associated protein 30) and recruits histone methyl- lymphoma (CBL), clusterin (CLU), HMG-coA reductase degrad- transferase Suv39H1 (suppressor of variegation 3–9 homologue ation protien 1 (HRD1) and replication factor C (RFC4), the levels 1) that results in trimethylation of hypoacetylated H3K9 and of p53 bound to the target promoters do not seem to be affected provides a binding platform for the chromodomain of HP1 following adriamycin treatment as compared with untreated cells (heterochromatin-binding protein 1) protein [10,37,53,54]. This [45]. Further, p53 and nuclear transcription factor-Y (NF-Y) tran- tethering of Sin3B–RBP2–HP1 leads to chromatin compaction. scriptional factors are known to be recruited/associated on to the The Sin3 core complex contains Sin3A/Sin3B, HDAC1/HDAC2, promoters of cyclin B2, CDC25c and cyclin dependent protein Sin3 associated protein 18 (SAP18), ING1/2 (inhibitor of growth kinase 2 (CDC2) genes before and after DNA damage [14,45– protein 1/2) and retinoblastoma-associated proteins (RbAps) that 47]. Thus, our results are in agreement with literature that sug- serves as a scaffold for additional moieties with enzymatic func- gests binding of p53 under normal and cellular stress conditions. tions, such as nucleosomal remodelling, N-acetylglucosamine p53 is a sequence-specific transcription factor that can bind to transferase, DNA methylation and histone methylation [10,37]. several target promoters but in few cases it has been suggested In the present study, we observed that the mechanism of p53- that mere binding of a transcription factor to a specific genomic mediated gene repression is via the recruitment of Sin3–HDAC sequence does not necessarily imply direct transcriptional regu- complex-associated epigenetic modifications on to p53 target lation. Therefore, we also checked the expression of p53 target gene promoters. We suggest that differential hypermethylation in genes in HCT116 (p53 + / + ) cell line to observe the downstream response to bleomycin-induced DNA damage for different p53 effect of p53–Sin3B–HDAC1 recruitment on to the target pro- target genes are responsible for gene regulation. Interestingly, we moters upon bleomycin treatment. We found that although we observed increased hypermethylation of H3K4, a well-known did not observe any difference in the enrichment of p53–Sin3B– activation marker on to MAD1 promoter along with H3K9 and HDAC1 on to the promoters but significant down regulation of H3K27 repression marks. Trimethylation of His3 at Lys4 residues the same target genes was observed in bleomycin-treated cells as predominantly acts in transcription up-regulation activities. How- compared with control cells. ever, Shi et al. [55] showed that trimethylated H3K4 recruits Since, we observed the up-regulation of Sin3B following ex- Sin3–HDAC complex through plant homeodomain (PHD) do- posure of cells to any kind of stress stimuli and recruitment of main of ING 2, a subunit of Sin3–HDAC complex in response Sin3B on to p53 target promoters, therefore we performed knock- to DNA damage and brings about gene repression. Apart from down assays to find out the role of Sin3B in modulation of p53 methylation, role of other PTMs of histones and DNA in regu- target genes. Our data in cells ablated for Sin3B using Sin3B lating the transcriptional activities of these genes cannot be ruled shRNA showed highly significant derepression of subset of p53 out.

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In the present report, we investigated the mechanism of p53- 3 Silverstein, R.A. and Ekwall, K. (2005) Sin3: a flexible regulator of mediated repression of target promoters, active in the regulation global gene expression and genome stability. Curr. Genet. 47, of heat-shock maintenance, cell cycle and cytoskeleton regulation 1–17 CrossRef PubMed 4 Kadamb, R., Mittal, S., Bansal, N., Batra, H. and Saluja, D. (2013) under bleomycin-induced DNA damaging conditions. We came Sin3: insight into its transcription regulatory functions. Eur. J. Cell to the following conclusions: (i) Sin3B acts as a stress-related Biol. 92, 237–246 CrossRef PubMed protein whose expression increases upon exposure to a variety of 5 Kadosh, D. and Struhl, K. (1997) Repression by Ume6 involves cellular stimuli; (ii) Interaction of Sin3B with p53 phosphorylated recruitment of a complex containing Sin3 corepressor and Rpd3 histone deacetylase to target promoters. Cell 89, 365–371 at Ser15 residues is enhanced under stress conditions; (iii) Pres- CrossRef PubMed ence of Sin3B is crucial for repression of selective p53 target 6 Laherty, C.D., Yang, W.M., Sun, J.M., Davie, J.R., Seto, E. and genes under stress conditions. Based on all these observations, Eisenman, R.N. (1997) Histone deacetylases associated with the we propose that direct binding of p53 to its REs is associated mSin3 corepressor mediate mad transcriptional repression. Cell with recruitment of Sin3–HDAC complex. However, in the ab- 89, 349–356 CrossRef PubMed 7 Ayer, D.E., Lawrence, Q.A. and Eisenman, R.N. (1995) Mad-Max sence of any external stimuli, this complex is unable to induce transcriptional repression is mediated by ternary complex any chromatin modifications. On the other hand, once a cell is formation with mammalian homologs of yeast repressor Sin3. Cell subjected to DNA damaging stimuli (such as bleomycin), the 80, 767 – -776 CrossRef Sin3–HDAC complex can bring about the histone modifications 8 Cowley, S.M., Iritani, B.M., Mendrysa, S.M., Xu, T., Cheng, P.F., on to the promoters of selective p53 target genes that mediates Yada, J., Liggitt, H.D. and Eisenman, R.N. (2005) The mSin3A chromatin-modifying complex is essential for embryogenesis and chromatin compaction resulting in gene repression. T-cell development. Mol. Cell. Biol. 25, 6990–7004 CrossRef PubMed AUTHORS CONTRIBUTION 9 Dannenberg, J.H., David, G., Zhong, S., van der Torre, J., Wong, W.H. and Depinho, R.A. (2005) mSin3A corepressor regulates Rama Kadamb designed and performed the experiments, analysed diverse transcriptional networks governing normal and neoplastic the data and wrote the manuscript. Shilpi Mittal was involved in growth and survival. Genes Dev 19, 1581–1595 performing qPCR and ChIP experiments. Nidhi Bansal was involved CrossRef PubMed in experiments during early phase of this work. Daman Saluja con- 10 David, G., Grandinetti, K.B., Finnerty, P.M., Simpson, N., Chu, G.C. and Depinho, R.A. (2008) Specific requirement of the chromatin ceived the project, designed the experiments, supervised and wrote modifier mSin3B in cell cycle exit and cellular differentiation. Proc. the manuscript. Natl. Acad. Sci. U.S.A. 105, 4168–4172 CrossRef PubMed 11 Grzenda, A., Lomberk, G., Zhang, J.S. and Urrutia, R. (2009) Sin3: ACKNOWLEDGEMENTS master scaffold and transcriptional corepressor. Biochim. Biophys. We thank Dr B.S. Dwarakanth for his critical suggestions and for Acta 1789, 443–450 CrossRef PubMed 12 van Oevelen, C., Bowman, C., Pellegrino, J., Asp, P., Cheng, J., permitting use of γ -radiation source at Institute of Nuclear Medi- Parisi, F., Micsinai, M., Kluger, Y., Chu, A., Blais, A. et al. (2010) cine and Allied Sciences. We also thank Dr Bert Vogelstein and Dr The mammalian Sin3 proteins are required for muscle Sanjeev Das for providing us HCT116 and H1299 cells from John development and sarcomere specification. Mol. Cell. Biol. 30, Hopkins University and National Institute of Immunology respect- 5686–5697 CrossRef PubMed ively. We appreciate the help of Mr Prateek Arora for his assistance 13 van Oevelen, C., Wang, J., Asp, P., Yan, Q., Kaelin, Jr, W.G., Kluger, Y. and Dynlacht, B.D. (2008) A role for mammalian Sin3 in in cell cycle analysis. permanent gene silencing. Mol. Cell. 32, 359–370 CrossRef PubMed 14 Bansal, N., Kadamb, R., Mittal, S., Vig, L., Sharma, R., FUNDING Dwarakanath, B.S. and Saluja, D. (2011) Tumor suppressor protein This work was supported by the Council of Scientific and Indus- p53 recruits human Sin3B/HDAC1 complex for down-regulation of trial Research [grant number 37/1431/10/EMR-II]; and the De- its target promoters in response to genotoxic stress. PLoS One 6, e26156 CrossRef PubMed partment of Science, Government of India [grant number R/SO/ 15 Das, T.K., Sangodkar, J., Negre, N., Narla, G. and Cagan, R.L. BB-55/2004 (to D.S.)]; the Council of Scientific and Industrial Re- (2013) Sin3a acts through a multi-gene module to regulate search (to R.K. and N.B.); and the University Grants Commission invasion in Drosophila and human tumors.. Oncogene 32, (to S.M.). 3184–3197 CrossRef PubMed 16 Nascimento, E.M., Cox, C.L., MacArthur, S., Hussain, S., Trotter, M., Blanco, S., Suraj, M., Nichols, J., Kubler, B., Benitah, S.A. et al. (2011) The opposing transcriptional functions of Sin3a and c-Myc are required to maintain tissue homeostasis. Nat. Cell Biol. REFERENCES 13, 1395–1405 CrossRef PubMed 17 Pile, L.A., Schlag, E.M. and Wassarman, D.A. (2002) The SIN3/RPD3 deacetylase complex is essential for G(2) phase cell cycle progression and regulation of SMRTER corepressor levels. 1 Kuo, M.H. and Allis, C.D. (1998) Roles of histone Mol. 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Received 12 May 2015/3 June 2015; accepted 19 June 2015 Published as Immediate Publication 25 June 2015, doi 10.1042/BSR20150122

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