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

Stress-mediated Sin3B activation leads tonegative regulation of subset of p53 target genesRama Kadamb*1, Shilpi Mittal*, Nidhi Bansal* and Daman Saluja*1

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

SynopsisThe multiprotein SWI-independent 3 (Sin3)–HDAC (histone deacetylase) corepressor complex mediates gene re-pression through its interaction with DNA-binding factors and recruitment of chromatin-modifying proteins on to thepromoters of target gene. Previously, an increased expression of Sin3B and tumour suppressor protein, p53 has beenestablished upon adriamycin treatment. We, now provide evidence that Sin3B expression is significantly up-regulatedunder variety of stress conditions and this response is not stress-type specific. We observed that Sin3B expressionis significantly up-regulated both at transcript and at protein level upon DNA damage induced by bleomycin drug, aradiomimetic agent. This increase in Sin3B expression upon stress is found to be p53-dependent and is associatedwith enhanced interaction of Sin3B with Ser15 phosphorylated p53. Binding of Sin3–HDAC repressor complex onto the promoters of p53 target genes influences gene regulation by altering histone modifications (H3K9me3 andH3K27me3) at target genes. Furthermore, knockdown of Sin3B by shRNA severely compromises p53-mediated generepression under stress conditions. Taken together, these results suggest that stress-induced Sin3B activation isp53-dependent and is essential for p53-mediated repression of its selective target genes. The present study has animplication 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

Gene regulation in eukaryotes is achieved through fine tuning ofactivator and repressor complexes that mediate relaxed and con-densed chromatin state respectively. In general, two counteractingenzymes control the gene expression primarily: histone acetyl-transferases (HATs) that add acetyl groups to the histone moietiesand thus mediate gene activation; and the histone deacetylases(HDACs) which deacetylate histone proteins and thus contributeto gene repression [1]. These enzymes do not work individually,instead, they are recruited to the promoters of genes as multi-subunit protein complexes where other proteins function as chro-matin remodellers and recruit additional cofactors or performunknown functions [2]. Sin3, a negative regulator of gene tran-scription, is a component of HDAC co-repressor complex that isknown to be involved in scaffolding of genome [3]. Like HDACs,

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: ANLN, anillin; BC, bleomycin control; BT, bleomycin treated; CDC25c, cell division cycle 25C; CRYZ, crystallin, zeta; HDAC, histone deacetylase; HBSS, Hank’sbalanced salt solution; HP1, heterochromatin-binding protein 1; HSPA8, heat-shock 70 kDa protein 8; IP, immunoprecipitation; KDB, knockdown Sin3B; MAD1, mitotic arrestdeficient-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-independent1 Correspondence may be addressed to either of these authors (email dsalujach59@gmail.com or ramakadamb@gmail.com).

Sin3 has no DNA-binding domain and interacts with varioustranscription factors through Sin3 interaction domain (SID) andthereby gets recruited on to the promoters of target genes [1,3,4].Once recruited to the promoters, Sin3 executes gene repressionby altering chromatin state through associated HDACs, such asRpd3 (reduced potasium deficiency 3) in yeast and HDAC1 andHDAC2 in mammals [5,6].

In mammals, Sin3 has two paralogues; Sin3A and Sin3B whichare known to perform varied functions under different cellularconditions [4,7]. Knockout studies in mice demonstrate distinctfunctions and differential requirement of the two isoforms dur-ing development. Loss of Sin3A is lethal during the early stagesof development in mice and is required for T-cell development andmaintenance of sarcomere structure [8,9]. In contrast, Sin3B isneeded at later stages of development and is required for skeletaldevelopment and cell-cycle progression [10]. However, overlap-ping functions between these two paralogues cannot be ruled

c© 2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. 1

R. Kadamb and others

out as Sin3A is found to perform compensatory functions inthe absence of Sin3B suggesting that there can be various com-plexes present in cell with different combinations of Sin3A/Sin3B[11–13]. Sin3 has recently been discovered to be involved invarious cellular functions ranging from T-cell development, cell-cycle progression, differentiation, senescence, protein stabiliza-tion, energy metabolism and cell survival [4,8,9,12,14–20]. Apartfrom the above mentioned functions, role of Sin3 under DNAdamage conditions has previously been studied in Saccharomy-ces cerevisiae where it mediates non-homologous end joiningDNA repair [21]. However, the involvement of Sin3 under stressconditions is not well studied in higher organisms which promp-ted us to study the regulation of Sin3 in humans upon bleomy-cin treatment, a radiomimetic drug that causes double strandbreakage [22,23]. Sin3 interacts with expanding array of DNA-binding transcription factors and our previous study affirmedinteraction of Sin3B with p53, a stress-related protein [14,24–27]. p53 is exemplified as the ‘guardian of genome’ which getsrapidly activated upon exposure of cells to various stress signals[28,29]. p53 is a sequence-specific transcription factor that caneither transactivate or transrepress various genes in a context-dependent manner [30]. The mechanism of p53-mediated geneactivation and its role in regulation of damage/stress responsepathways is well established [29,30]. However, the mechanismunderlying p53-mediated transcriptional repression remains elu-sive. Previously, Sin3 has been demonstrated to play an imperat-ive role in p53-mediated gene regulation [14,26]. Nevertheless,it is believed that different stress signals can lead to different,as well as independent pathways for p53 activation, which inturn can mediate different responses [31]. In the present study,we looked into the insight of mechanism of Sin3B activation andp53-mediated negative gene regulation under bleomycin-inducedDNA damaging conditions. The present study shows that underbleomycin-induced stress, expression of Sin3B gets up-regulatedand it gets recruited by p53 at its target promoters. Knockdown ofSin3B leads to impaired negative regulation of p53 target genesand thus exemplifies Sin3B as a critical player in down-regulationof p53 subset target genes.

MATERIALS AND METHODS

Cell culture and establishment of stablytransfected cell linesHCT116 (human colorectal carcinoma), KB (head and necksquamous carcinoma), A549 (human lung carcinoma), Saos2(osteosarcoma) and H1299 (non small cell lung carcinoma)cell lines were cultured at 37 ◦C in 5 % CO2 humidi-fied atmosphere. Cells were grown in high glucose Dul-becco modified Eagle’s medium (DMEM; Sigma; Pan Bi-otech) supplemented with 10 % FBS (Gibco) and penicillin(60 units/ml) and streptomycin (50 μg/ml). For transfection,HCT116 cells were seeded into six-well tissue cultureplates and transfected with 1 μg of control/Sin3B shRNA

plasmid (Santa Cruz Biotechnology) using plasmid transfectionreagent according to manufacturer’s protocol (Santa Cruz Bi-otechnology). Forty-eight hours after transfection, the positiveclones were selected by growing cells in puromycin containingmedium (ant-pr-1, Invivogen). KB, A549 and Saos2 cell lineswere procured from National Center for Cell Sciences (NCCS)and HCT116 (wild-type and null for p53) and H1299 cell lineswere provided as a kind gift from Dr Bert Vogelstein from JohnHopkins University and Dr Sanjeev Das from National Instituteof Immunology respectively.

Cell treatmentSub-confluent cultures were treated with different stress-inducingagents: cells were incubated with 400 μg/ml and 20 ng/ml ofbleomycin and colchicine respectively, for indicated time points.For γ radiation treatment, cells at a confluency of 70 %–80 %were irradiated with γ radiation source (60Co; Eldorad78) inHank’s balanced salt solution (HBSS) and, after treatment, thecells were incubated at 37 ◦C for 1 and 4 h respectively.

Western blottingCells cultured in T25 cm2 flasks were subjected to different drugtreatments for desired time intervals and harvested for analysingprotein expression. Cell lysates were prepared by re-suspendingthe cells in RIPA (radioimmunoprecipitation assay) buffer for45 min at 4 ◦C. The soluble protein fractions were collected bycentrifugation at 15 777 g. Total protein was estimated usingBCA protein estimation kit (Bangalore Genei) and equal amountof proteins (30 μg) were resolved on SDS/PAGE (either 8 % or10 % gel) and transferred on to immunoblot- PVDF membranes(Santa Cruz Biotechnology). Following blocking with 5 % BSAfor 1 h at room temperature and washing with PBS with Tween 20(PBST), the PVDF membranes were incubated overnight at 4 ◦Cin primary antibody (1:1000) diluted in PBST containing 1 %BSA. Membranes were subsequently incubated with secondaryantibodies conjugated with horseradish peroxidase (1:5000) for1 h at room temperature. The blots were probed with the en-hanced chemiluminescence (ECL) western blot detection system(Biogene) according to manufacturer’s instructions.

Co-immunoprecipitationSub-confluent cultures of untreated and bleomycin-treated cellswere harvested and lysed in RIPA buffer (buffer composition asgiven in the Supplementary Table S1) supplemented with pro-tease inhibitor cocktail (Sigma–Aldrich). Equal amount of pro-tein (1.5 mg) from each sample was immunoprecipitated with1 μg of desired antibody. For collection of the immunoprecip-itates, 30 μl of protein A/G agarose beads were added to eachtube and the samples were incubated for 2–3 h at 4 ◦C on rota-tion. Each immunoprecipitate was washed thrice in RIPA bufferand eluted with 2× loading dye, fractionated on 8 % SDS/PAGEand transferred overnight on to immuno-blot PVDF membrane(Santa Cruz Biotechnology). Immunodetection was done with

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 c© 2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0.

Sin3B regulates p53 target genes

anti-Sin3B antibody, p53 and phospho Ser15-p53 at a dilution of1:1000 for Figures 3(A) and 3(B) respectively. Ten percent of theinput was saved as a positive control from the total lysate.

RNA purification, cDNA synthesis and qPCRUntreated/control and bleomycin-treated cells were processed forRNA purification using RNeasy kit (Qiagen) according to man-ufacturer’s instructions. One μg of RNA was used for cDNAsynthesis using Verso cDNA kit (Thermo Scientific) accordingto the manufacturer’s protocol. Semi-quantitative and quantitat-ive real-time PCR (qPCR) reactions were carried out using genespecific primers. Real time gene expression analysis was carriedout using MESA Green qPCR Mastermix plus for SYBR assay(Eurogenetec) on Applied Biosystems 7300 RT-PCR system anddata were collected and exported with SDS 2.2.2 version. Rel-ative expression was calculated using 2− ��Ct method and 18SrRNA was used as an endogenous control. Sequence of primerpairs used for semi-quantitative and real-time PCR are listed inSupplementary Table S2.

Cell-cycle analysisTo prepare cells for FACS analysis, 1 × 106 (control and treated)cells were washed with 1× PBS, resuspended in 0.5 ml of 1×PBS and incubated with 10 μl of RNase A (10 mg/ml) at 37 ◦Cin a water bath for 30 min, followed by addition of 4 μg/ml ofPropidium Iodide in dark on ice. Stained cells were analysed onBecton Dickinson FACScan machine and the data were analysedwith either CELLQuest Pro or FlowJo software.

ChIP (chromatin-immunoprecipitation)HCT116 cells were treated with bleomycin drug prior to form-aldehyde fixation. Formaldehyde fixed cells lysed in 0.1 % SDSlysis buffer were sonicated (20 cycles of 30-s pulse with 1-mininterval) at 90 % power using Bioruptor sonicator (Diagenode).Samples were centrifuged for 10 min at 18 516 g at 4 ◦C and werediluted ten times with dilution buffer except for the input sample.The diluted supernatants were collected and pre-cleared with pro-tein A/G Agarose beads containing 1 mg/ml salmon sperm DNAfor 3–4 h at 4 ◦C with agitation. Samples were spun down at 7012g for 5 min at 4 ◦C and incubated overnight with specific anti-bodies. Samples were incubated with 30 μl of pre-blocked beads(protein A/G agarose/salmon sperm DNA) for 3 h, spun at 986g at 4 ◦C for 3 min and washed sequentially with the followingbuffers at 4 ◦C: once with 0.1 % SDS lysis buffer, twice with lowsalt-wash buffer, twice with high salt-wash buffer, twice with LiClwash buffer and twice with TE buffer. For de-cross-linking andPCR, samples were eluted twice with 100 μl of elution buffer,vortexed briefly and incubated at room temperature for 15 minwith rotation. Eluates were pooled (200 μl) and 8 μl of 5 MNaCl added. De-cross-linking was performed at 65 ◦C for 6 h toovernight. Following elution, samples were treated with 4 μl of0.5 M EDTA, 4 μl of 1 M Tris, pH 6.8, 2 μl of 10 mg/ml pro-teinase K, 2 μl of 10 mg/ml RNAase A and incubated for 3 h at

45 ◦C. DNA from eluted samples was recovered with PureLinkPCR purification Kit (Invitrogen). PCR and quantitative realtime PCR were performed using gene-specific primers for pro-moter regions. Composition of the buffers and primer pairs usedare given in Supplementary Tables S1 and S3.

Statistical analysisStudent’s t-test was used to evaluate the significance of the differ-ences between control and drug-treated samples in all pertinentexperiments; a P-values < 0.05 and 0.01 was considered signi-ficant and highly significant respectively.

RESULTS

Sin3B expression is induced upon different cellularstress conditions and is not cell-type specificCell 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 andstress-related protein, p53. Initially, the dose of bleomycin whichcan induce cellular stress and thus elicit p53 activation wasstandardized. It was found that 400 μg/ml of bleomycin drugis sufficient to activate p53 levels and lead to arrest of cellsin the G2-phase of the cell cycle (Supplementary Figures S1Aand S1B). Western blot analysis of lysates from untreated andtreated cells depicted time-dependent increase in the expressionof Sin3B and p53 upon bleomycin drug treatment as comparedwith control cells (Figure 1A, left panel). The expression ofboth Sin3B and p53 showed significant increase at protein levelwithin 20 min after bleomycin treatment and these high levels ofprotein expression were maintained up to 24 h post drug treat-ment. The expression levels of p53 and Sin3B at 24 h afterbleomycin treatment was approximately 5- and 2.5-fold higherrespectively as compared with controls (Supplementary FigureS1C). This increase in expression of Sin3B was not isoform spe-cific as we observed similar increase in the levels of another Sin3isoform, i.e. Sin3A in bleomycin-treated cells (SupplementaryFigure S1D). Similarly, exposure of cells to 5 Gy of γ -radiationdose lead to enhanced Sin3B expression at 1 and 4 h with aconcomitant increase in the p53 levels (Figure 1B).

Under conditions of non-genotoxic stress, induced by col-chicine (mitotic poison), we observed that expression of Sin3Bbegan to increase within 30 min after treatment with colchicinedrug at a concentration of 20 ng/ml and time-dependent increasewas observed till 16 h of drug incubation (Figure 1A, right panel).In concordance to published reports, we also observed that expos-ure of cells to different kinds of stress stimuli was accompaniedby an increase in p53 protein level. We also demonstrate thatincreased expression of Sin3B, upon bleomycin treatment, is notcell-type specific as cell lines of different origins (head and necksquamous carcinoma and non-small cell lung carcinoma) treatedwith bleomycin showed similar increase in Sin3B expression(Figure 1C).

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c© 2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. 3

R. Kadamb and others

Figure 1 Expression of Sin3B increases under conditions of cellular stress in a p53-dependent mannerHCT116 wild-type (HCT116 p53+ / + ) cells were treated with different stress inducers that are known to elicit p53 activationand response. (A) Cells were subjected to bleomycin (radiomimetic agent) and colchicine (mitotic poison) drug treatmentat 400 μg/ml and 20 ng/ml dose respectively, at indicated time points. (B) KB cells were subjected to γ -radiation at adose of 5 Gy and incubated for 1 and 4 h in HBSS. (C)Western blots showing expression of Sin3B and p53 protein in KBand A549 cell lines. (D) Western blot analysis showing no effect on the expression of Sin3B in p53 null cells (HCT116p53− / − ). Following different drug treatments, the expression of p53 and Sin3B were checked by performing western blotanalysis 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,p53 null cells were subjected to similar conditions of stress andanalysed for protein expression. Importantly, p53 null cells didnot show any significant increase in Sin3B expression upon bleo-mycin drug treatment neither at protein nor at transcript levels(Figure 1D; Supplementary Figure S2). These results evincedthat increase in Sin3B expression level upon stress conditions isa general phenomenon that can occur in a variety of cell type re-gardless of the stimulus types that initiate this process and p53 isrequired for Sin3B activation.

Increased Sin3B expression is due to increasedtranscript level and post-translational stabilizationTo ascertain whether increase in Sin3B protein upon cellularstress is a result of increased transcription, total RNA from con-trol and bleomycin-treated samples were isolated and reversetranscribed. Semi-quantitative and qPCR indicated a significantincrease in the levels of Sin3B transcript; however, no changeswere observed for p53 transcript levels upon bleomycin treatment

(Figures 2A and 2B). To rule out the possibility of ineffective drugtreatment for insignificant changes in p53 expression, transcriptlevels of p21 gene were also analysed simultaneously. p21 is awell-known p53 target gene which gets up-regulated under vari-ety of stress conditions [29]. We observed significant increase inp21 transcript levels following bleomycin treatment, confirmingthat the drug treatment is indeed effective (Supplementary FigureS3). In the current study, no significant changes in the p53 mRNAlevels (Figure 2A) are consistent with previous studies that sug-gest p53 expression is typically enhanced at post-translationallevel under stress conditions [32]. In contrast, a significant in-crease was observed for Sin3B mRNA levels within 20 min ofbleomycin drug treatment that further increased up to 4-fold in atime-dependent manner (Figure 2B).

We next checked whether augmentation of Sin3B protein uponstress was a result of increased translational product alone (due toincrease in mRNA for Sin3B) or post-translational stabilizationhas any role to play in increasing Sin3B protein levels. Previously,RNF220 (ring finger protein 220) protein has been identified as anovel ubiquitin ligase of Sin3B [33]. We, therefore, compared the

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4 c© 2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0.

Sin3B regulates p53 target genes

Figure 2 Human Sin3B transcript levels are increased upon bleomycin treatmentTotal RNA was isolated and reverse transcribed at various time point as indicated in the figure. The cDNA (control andtreated 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. Relativeexpression levels were calculated by taking transcript level in untreated cells as one. The Ct value for each gene wasnormalized with 18S rRNA which was used as an endogenous control. Error bar shows S.D. and the plots have beenplotted with +− S.E.M. *P < 0.05 and **P < 0.01. For semi-quantitative PCR, p53 and Sin3B were amplified for 28 and30 cycles respectively. The results are representative of three independent experiments. (C) Western blot analysis of celllysates from bleomycin-untreated and -treated samples at various time points as indicated in the figure for checking theexpression levels of Sin3B and RNF220, a ubiquitin ligase. β -actin was used a protein loading control.

expression levels of RNF220 protein along with Sin3B proteinunder similar stress conditions. Western blot analysis showed thatexpression of RNF220 decreased as a function of time of bleo-mycin treatment with a concomitant increase in the expressionof Sin3B protein (Figure 2C). Thus, our results suggest that in-creased expression of Sin3B at protein level is a cumulative effectof increased transcript synthesis and increased stabilization dueto decreased RNF220 expression upon bleomycin treatment.

Increased association of human Sin3B with p53phosphorylated at Ser15

Human p53 is known to undergo several post-translational modi-fications (PTMs) through activation of several kinases. Usingco-immunoprecipitation and yeast two-hybrid studies, we earlierreported that p53 directly interacts with Sin3B under normalconditions without any increase in association between the twoproteins under adriamycin-induced stress conditions [14]. Out ofthe many types of phosphorylation, p53 phosphorylation on Ser15

residues occurs following DNA damage induced by γ -radiationand several chemotherapeutics agents [34–36]. To study the ef-fect of PTM of p53 on its interaction with Sin3B, under stressconditions, co-immunoprecipitation assays were performed. Ini-tially, expression of Ser15 phosphorylated p53 was investigatedin control and 24 h drug-treated HCT116 cells. Western blot ana-lysis showed that levels of Ser15 phosphorylated at p53 residuesincreased after treatment of cells with bleomycin (Supplement-ary Figure S4). Following confirmation of p53 modification atSer15 residues after bleomycin treatment, protein lysates fromuntreated and drug-treated cells were immunoprecipitated us-

ing p53 and phospho Ser15-p53 antibody followed by immun-oblotting with Sin3B-specific antibody. Co-immunoprecipitationassay with total pool of p53 showed no increased association ofSin3B under bleomycin-induced stress; however, immunoprecip-itation of the complex with phospho Ser15-p53 antibody followedby detection with anti-Sin3B antibody showed enhanced interac-tion between Ser15 phosphorylated p53 and Sin3B (Figure 3A).

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-Sin3B antibody for immunoprecipitation of the complex andanti-p53 antibody for immunodetection demonstrated that Sin3Bcould also co-immunoprecipitate p53 under similar conditions.This, further, affirmed the specific interaction between these twoproteins (Figure 3B).

Sin3–HDAC complex docks on to the promoter ofsubset of p53 target genesTo decipher the functional significance of human p53–Sin3Binteraction and to understand the role played by Sin3B in p53-mediated gene regulation upon bleomycin treatment, the recruit-ment of human Sin3B on to the five selected p53 responsivepromoters was investigated. Whereas Sin3 has been shownto stabilize p53 for mediating its transrepression functions[20,26], there has been no comprehensive study regarding therole of Sin3B in p53-mediated negative gene regulation underbleomycin-induced DNA damaging situations.

To study the recruitment of Sin3B on to p53 target promoters,ChIP assays were performed with cellular extracts from untreated

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c© 2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. 5

R. Kadamb and others

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 antibodiesspecific for p53 and phospho Ser15-p53 followed by immunoblotting (IB) with antibody specific to Sin3B. (B) Reciprocalco-immunoprecipitation in HCT116 wild-type cells using anti-Sin3B antibody for immunoprecipitation of the immune complexand anti-p53 antibody for immunodetection. Input corresponding to 10 % of the total cell lysate was used as a positivecontrol whereas mouse and rabbit IgG were used as negative controls.

and 24-h bleomycin-treated HCT116 cells, using promoter-specific primers designed across the p53-response element (RE).In the initial screening phase, we observed significant enrich-ment of human Sin3B on five out of eight p53-target promoters,viz. MAD1 (mitotic arrest deficient-like 1), HSPA8 (heat-shock70 kDa protein 8), CRYZ (crystallin, zeta), ANLN (anillin) andCDC25c (cell division cycle 25C; result not shown) and hence,focused at these five promoters only for a detailed dissectionof the involvement of Sin3B in p53-mediated gene repression.Using specific antibodies against p53, Sin3B and HDAC1, sub-stantial recruitment of these three proteins at the region spanningthe p53 REs were observed for the five p53 target genes underboth normal and stressed conditions. The strength of the specificbinding of antibodies was evident when compared with isotypecontrol (Figure 4A).

qPCR assay was performed to further investigate that whetherany enrichment occurs for p53, Sin3B and HDAC1 at the p53target promoters in the presence and absence of bleomycin drugtreatment. For this, ChIP DNA obtained after immunoprecip-itation with specific antibody (Figure 4B) from both untreatedand drug-treated cells was used as a template and amplified us-ing gene-specific primers. Our qPCR results were in agreementwith ChIP PCR results that showed no significant changes in therecruitment of p53, Sin3B and HDAC1 at selective p53 targetpromoters in bleomycin-treated cells as compared with untreatedcells (Figure 4B).

Negative regulation of subset of p53 target genesis Sin3B dependentTo further validate the role of Sin3B in stress-induced p53-mediated gene repression of its target genes, we transfected theHCT116 cells with Sin3B shRNA to knockdown Sin3B (KDB)expression. Cells transfected with shRNA showed significant

reduction in the expression of Sin3B as compared with untrans-fected cells both at protein and at RNA level (Figure 5A). Dens-itometric analysis revealed that Sin3B shRNA treatment resultedin approximately 80 % reduction in the expression of Sin3B intransfected cells as compared with untransfected cells.

Knockdown of Sin3B in Drosophila S2 cells is known to causearrest of cells in the G2-phase of the cell cycle [17]. Therefore, wealso investigated whether knockdown of Sin3B in HCT116 cellline causes any cell-cycle perturbation. Using Propidium Iodidestaining, cell cycle profile of untransfected and Sin3B shRNAtransfected cells were analysed under normal and bleomycin-induced DNA damage conditions. Cell cycle analysis revealedthat significant fraction of cells entered G2-phase of cell cyclein cells ablated for Sin3B as compared with untransfected cellsupon bleomycin treatment. However, no significant changes inthe cell cycle patterns were observed between untransfected andSin3B transfected cells under normal cycling conditions/withoutdrug treatment (Figure 5B). We next checked for the effect ofSin3B knockdown on the expression of p53 target genes fol-lowing bleomycin drug treatment. We observed significant dere-pression of the p53 target genes in the cells transfected withSin3B shRNA upon bleomycin treatment. In contrast, untrans-fected cells showed significant repression of the same target genesunder similar conditions (Figure 5C).

Thus, our results highlight the importance of Sin3B in neg-atively regulating p53 gene repression under bleomycin-inducedstress conditions.

Induction of epigenetic modifications on promotersof p53 target genesSin3B–HDAC complex is associated with enzymes involved inhistone methylation and such PTMs at histones play criticalrole in gene regulation [10,37]. We, therefore, tested whether

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6 c© 2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0.

Sin3B regulates p53 target genes

Figure 4 Recruitment of p53–Sin3B–HDAC1 occurs on subset of p53 target genesChIP assays were performed in HCT116 cells in untreated and cells treated with bleomycin for 24 h. Equal amountsof cross-linked chromatin were pre-cleared and incubated with 1μg of antibody for anti-p53, anti-Sin3B, anti-HDAC1 asindicated 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 eachimmunoprecipitation 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 atemplate 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 promotersin bleomycin control and bleomycin-treated conditions.

bleomycin-mediated stress alters the methylation marks on his-tones at promoters of p53 target genes. To better understand thisphenomenon, HCT116 wild-type cells were treated with bleomy-cin and the endogenous promoter occupancy was examined onformaldehyde cross-linked chromatin by using specific antibod-ies for H3K4me3 as activation mark and H3K9me3/H3K27me3as repression marks. To monitor the specificity of reaction,IgG was used as an isotype control. ChIP assay revealedthat exposure of cells to bleomycin drug did not cause anychange in the enrichment of H3K4me3 except for MAD1 gene(Figure 6). However, significant increase in hypermethylationof H3K9me3/H3K27me3, repression marks, was observed afterbleomycin drug treatment. At the promoter of HSPA8 gene, weobserved increased hypermethylation of H3K9me3 residues withno significant change in trimethylation of His3 at Lys27 residues.In contrast, CDC25c promoter showed increased hypermethyla-tion for H3K27me3 residue without any significant changes forthe other repression mark, i.e. H3K9me3. However, at MAD1,CRYZ and ANLN promoter sites, we observed an increasedhypermethylation of both the repression marks, i.e. H3K9me3

and H3K27me3 (Figure 6). These observations suggested thatdifferential hypermethylation of histones occur following bleo-mycin treatment and such changes are responsible for bleomycin-mediated p53 gene repression of its target genes.

DISCUSSION

Several lines of evidence suggest that p53 modulates gene expres-sion under diverse environmental, genotoxic and developmentalstimuli by different mechanisms of transrepression [14,38,39].Furthermore, the mechanism of transrepression being context-dependent, interaction and cross-talk between these pathwayscannot be ruled out. It is also well established that several cellularinsults/stimuli induce p53 expression [32]. However, the regula-tion/response of Sin3B under such scenario is still not clear. We,now provide evidence that expression of Sin3B increases in p53-dependent manner under variety of stress conditions. Though,

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c© 2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. 7

R. Kadamb and others

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 controland 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 shRNAtransfected cells under both untreated and bleomycin-treated conditions. Relative expression levels for each gene werecalculated by considering transcript levels of genes in untreated conditions as one. 18s rRNA was used as endogenouscontrol. 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-coma (Ras) oncogene activation and adriamycin treatment to cells[14,40]; the mechanism of Sin3 up-regulation is still far fromclear. Previously, RNF220, a ubiquitin ligase, has been shown toassociate with Sin3B and promotes its proteasomal degradation[33]. In the present study, we found that expression of RNF220decreased with a concomitant increase in Sin3B expression uponbleomycin treatment. At the RNA level, our qPCR results indicatesignificant increase in the Sin3B transcripts in a time-dependentmanner following bleomycin treatment. These observations sug-gest that increased Sin3B expression at protein level is a cu-mulative effect of both increased transcript synthesis and post-

translational stabilization by RNF220. In contrast, the expressionof p53 increased at protein level only with no significant changein transcript level upon bleomycin treatment. Thus, our obser-vation further supports the fact that p53 is post-translationallystabilized under stress [32]. Interestingly, we did not find stress-induced expression of Sin3B in p53 null cells neither at proteinnor at transcript levels (Figure 1D; Supplementary Figure S4).These findings, though, imply that increase in Sin3B expressionmight be regulated through p53 (directly or indirectly); the mech-anism for gene regulation cannot be explained as of this writingas promoter of Sin3B is yet not identified. We further lookedinto the Sin3B gene in search for any consensus p53-binding

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8 c© 2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0.

Sin3B regulates p53 target genes

Figure 6 Epigenetic modifications occur at the promoters of p53 target genes upon bleomycin treatmentHCT116 wild-type untreated and 24 h bleomycin drug-treated cells were harvested for ChIP assay. Equal amounts ofcross-linked chromatin were pre-cleared and incubated with antibodies methylated at different residues of His3 as indicatedin 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 < 0.05and **P < 0.01. Percent input was calculated for enrichment of DNA on to the respective target promoters in bleomycincontrol and bleomycin treated conditions.

site using TRANSFAC program. However, we did not find anyp53-binding site upstream of the putative transcription start siteof Sin3B gene. Thus, in future, due attention is required to studythe transcriptional and post-translational regulation of Sin3B byp53, especially under the conditions of stress.

Our previous studies suggested that Sin3B interacts with N-terminal region of p53 under normal as well as stress conditions[14]. However, the interaction status between p53 and Sin3B doesnot change upon adriamycin drug treatment [14]. Like adriamy-cin, we found that bleomycin also does not cause any change

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c© 2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. 9

R. Kadamb and others

in the interaction status of Sin3B with total pool of p53. Inter-estingly, in the present study, we observed that, association ofSin3B is increased with Ser15 phosphorylated p53 under stressconditions. In response to DNA damage, Ser15 and Ser20 residuesof p53 primarily get phosphorylated and blocks its interactionwith mouse double minute 2 (MDM2), a negative regulator ofp53 [41–43]. Ser15 phosphorylation has been shown to enhancethe interaction of p53 with various transcription factors. Phos-phorylation of p53 at Ser15 residues is reported to result in in-creased association of p53 with CREB (cAMP-RE-binding pro-tein) under DNA damage conditions. In fact, phosphorylationof the p53 at N-terminus is shown to be involved in controllingprotein interaction, regulating transcription of downstream genesand it may also contribute in determining the response of cellsto DNA damage [44]. Thus, our results are in concordance withpublished reports that propose phosphorylation of p53 in medi-ating increased interaction with other proteins [44]. Although, itremains unclear that whether this increase in p53 Ser15–Sin3Binteraction represents an increase in binding affinity or bindingstoichiometry and thus in future needs to be explored in detail.

In order to check whether the association of p53 with Sin3Bresults in modulating the expression of downstream target genes,recruitment of Sin3B–HDAC1 was analysed on to selective pro-moters of p53 target genes. A consistent recruitment of p53–Sin3B–HDAC1 on to the promoters of p53 repressed geneswas observed both before and after DNA damaging conditions.Ceribelli and co-workers showed that for genes, like ataxia tel-angiectasia and Rad3-related protein (ATR), casitas B-lineagelymphoma (CBL), clusterin (CLU), HMG-coA reductase degrad-ation protien 1 (HRD1) and replication factor C (RFC4), the levelsof p53 bound to the target promoters do not seem to be affectedfollowing adriamycin treatment as compared with untreated cells[45]. Further, p53 and nuclear transcription factor-Y (NF-Y) tran-scriptional factors are known to be recruited/associated on to thepromoters of cyclin B2, CDC25c and cyclin dependent proteinkinase 2 (CDC2) genes before and after DNA damage [14,45–47]. Thus, our results are in agreement with literature that sug-gests binding of p53 under normal and cellular stress conditions.p53 is a sequence-specific transcription factor that can bind toseveral target promoters but in few cases it has been suggestedthat mere binding of a transcription factor to a specific genomicsequence does not necessarily imply direct transcriptional regu-lation. Therefore, we also checked the expression of p53 targetgenes in HCT116 (p53+ / + ) cell line to observe the downstreameffect of p53–Sin3B–HDAC1 recruitment on to the target pro-moters upon bleomycin treatment. We found that although wedid not observe any difference in the enrichment of p53–Sin3B–HDAC1 on to the promoters but significant down regulation ofthe same target genes was observed in bleomycin-treated cells ascompared with control cells.

Since, we observed the up-regulation of Sin3B following ex-posure of cells to any kind of stress stimuli and recruitment ofSin3B on to p53 target promoters, therefore we performed knock-down assays to find out the role of Sin3B in modulation of p53target genes. Our data in cells ablated for Sin3B using Sin3BshRNA showed highly significant derepression of subset of p53

target genes as compared with untransfected cells. The derepres-sion of p53 target genes was seen only under bleomycin-inducedDNA damaging conditions. Untransfected and Sin3B shRNAtransfected cells showed no significant changes under the nor-mal cell cycling conditions. However, following Sin3B shRNAtreatment we could not observe complete derepression of the p53target genes. This lack of complete derepression after transfec-tion of Sin3B shRNA could be attributed to the fact that Sin3gene has two paralogues, i.e. Sin3A and Sin3B. Both paraloguesshow structural similarity with each other and regulate unique, aswell as overlapping subset of target genes [3,7,20,26,48,49]. It isbelieved that in the absence of Sin3B, the other isoform, namelySin3A, can substitute for it and take over the function. Sin3Aassociates with transcription regulators like methyl CpG bindingprotein 2 (MeCP2) and silencing mediator of retinoic acid andthyroid hormone receptor (SMRT) whereas on the other handSin3B preferentially associates with MHC Class II Transactiv-ator (CIITA) and major histocompatibility complex (MHC II)[50–52]. Previously, Sin3A isoform has been shown to be in-volved in negative regulation of microtubule-associated protein4 (MAP4) and stathmin (STMN1) genes [20,26]. Studies by vanOevelen et al. [12] have shown that both the isoforms are requiredfor the maintenance of sarcomere functions and deletion of anyone of the paralogues cannot rescue the normal phenotype [3].

Interplay of DNA and histone modifications via histone methyltransferases are prerequisite for regulation of gene expression.Sin3 associates with RBP2 (retinol-binding protein 2) throughSAP30 (Sin3-associated protein 30) and recruits histone methyl-transferase Suv39H1 (suppressor of variegation 3–9 homologue1) that results in trimethylation of hypoacetylated H3K9 andprovides a binding platform for the chromodomain of HP1(heterochromatin-binding protein 1) protein [10,37,53,54]. Thistethering of Sin3B–RBP2–HP1 leads to chromatin compaction.The Sin3 core complex contains Sin3A/Sin3B, HDAC1/HDAC2,Sin3 associated protein 18 (SAP18), ING1/2 (inhibitor of growthprotein 1/2) and retinoblastoma-associated proteins (RbAps) thatserves as a scaffold for additional moieties with enzymatic func-tions, such as nucleosomal remodelling, N-acetylglucosaminetransferase, DNA methylation and histone methylation [10,37].In the present study, we observed that the mechanism of p53-mediated gene repression is via the recruitment of Sin3–HDACcomplex-associated epigenetic modifications on to p53 targetgene promoters. We suggest that differential hypermethylation inresponse to bleomycin-induced DNA damage for different p53target genes are responsible for gene regulation. Interestingly, weobserved increased hypermethylation of H3K4, a well-knownactivation marker on to MAD1 promoter along with H3K9 andH3K27 repression marks. Trimethylation of His3 at Lys4 residuespredominantly acts in transcription up-regulation activities. How-ever, Shi et al. [55] showed that trimethylated H3K4 recruitsSin3–HDAC complex through plant homeodomain (PHD) do-main of ING 2, a subunit of Sin3–HDAC complex in responseto DNA damage and brings about gene repression. Apart frommethylation, role of other PTMs of histones and DNA in regu-lating the transcriptional activities of these genes cannot be ruledout.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

10 c© 2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0.

Sin3B regulates p53 target genes

In the present report, we investigated the mechanism of p53-mediated repression of target promoters, active in the regulationof heat-shock maintenance, cell cycle and cytoskeleton regulationunder bleomycin-induced DNA damaging conditions. We cameto the following conclusions: (i) Sin3B acts as a stress-relatedprotein whose expression increases upon exposure to a variety ofcellular stimuli; (ii) Interaction of Sin3B with p53 phosphorylatedat Ser15 residues is enhanced under stress conditions; (iii) Pres-ence of Sin3B is crucial for repression of selective p53 targetgenes under stress conditions. Based on all these observations,we propose that direct binding of p53 to its REs is associatedwith recruitment of Sin3–HDAC complex. However, in the ab-sence of any external stimuli, this complex is unable to induceany chromatin modifications. On the other hand, once a cell issubjected to DNA damaging stimuli (such as bleomycin), theSin3–HDAC complex can bring about the histone modificationson to the promoters of selective p53 target genes that mediateschromatin compaction resulting in gene repression.

AUTHORS CONTRIBUTION

Rama Kadamb designed and performed the experiments, analysedthe data and wrote the manuscript. Shilpi Mittal was involved inperforming qPCR and ChIP experiments. Nidhi Bansal was involvedin experiments during early phase of this work. Daman Saluja con-ceived the project, designed the experiments, supervised and wrotethe manuscript.

ACKNOWLEDGEMENTS

We thank Dr B.S. Dwarakanth for his critical suggestions and forpermitting use of γ -radiation source at Institute of Nuclear Medi-cine and Allied Sciences. We also thank Dr Bert Vogelstein and DrSanjeev Das for providing us HCT116 and H1299 cells from JohnHopkins University and National Institute of Immunology respect-ively. We appreciate the help of Mr Prateek Arora for his assistancein cell cycle analysis.

FUNDING

This work was supported by the Council of Scientific and Indus-trial Research [grant number 37/1431/10/EMR-II]; and the De-partment of Science, Government of India [grant number R/SO/BB-55/2004 (to D.S.)]; the Council of Scientific and Industrial Re-search (to R.K. and N.B.); and the University Grants Commission(to S.M.).

REFERENCES

1 Kuo, M.H. and Allis, C.D. (1998) Roles of histoneacetyltransferases and deacetylases in gene regulation. Bioessays20, 615–626 CrossRef PubMed

2 Hayakawa, T. and Nakayama, J. (2011) Physiological roles of classI HDAC complex and histone demethylase. J. Biomed. Biotechnol.2011, 129383 CrossRef PubMed

3 Silverstein, R.A. and Ekwall, K. (2005) Sin3: a flexible regulator ofglobal gene expression and genome stability. Curr. Genet. 47,1–17 CrossRef PubMed

4 Kadamb, R., Mittal, S., Bansal, N., Batra, H. and Saluja, D. (2013)Sin3: insight into its transcription regulatory functions. Eur. J. CellBiol. 92, 237–246 CrossRef PubMed

5 Kadosh, D. and Struhl, K. (1997) Repression by Ume6 involvesrecruitment of a complex containing Sin3 corepressor and Rpd3histone deacetylase to target promoters. Cell 89, 365–371CrossRef PubMed

6 Laherty, C.D., Yang, W.M., Sun, J.M., Davie, J.R., Seto, E. andEisenman, R.N. (1997) Histone deacetylases associated with themSin3 corepressor mediate mad transcriptional repression. Cell89, 349–356 CrossRef PubMed

7 Ayer, D.E., Lawrence, Q.A. and Eisenman, R.N. (1995) Mad-Maxtranscriptional repression is mediated by ternary complexformation with mammalian homologs of yeast repressor Sin3. Cell80, 767 – -776 CrossRef

8 Cowley, S.M., Iritani, B.M., Mendrysa, S.M., Xu, T., Cheng, P.F.,Yada, J., Liggitt, H.D. and Eisenman, R.N. (2005) The mSin3Achromatin-modifying complex is essential for embryogenesis andT-cell development. Mol. Cell. Biol. 25, 6990–7004CrossRef PubMed

9 Dannenberg, J.H., David, G., Zhong, S., van der Torre, J., Wong,W.H. and Depinho, R.A. (2005) mSin3A corepressor regulatesdiverse transcriptional networks governing normal and neoplasticgrowth and survival. Genes Dev 19, 1581–1595CrossRef PubMed

10 David, G., Grandinetti, K.B., Finnerty, P.M., Simpson, N., Chu, G.C.and Depinho, R.A. (2008) Specific requirement of the chromatinmodifier mSin3B in cell cycle exit and cellular differentiation. Proc.Natl. Acad. Sci. U.S.A. 105, 4168–4172CrossRef PubMed

11 Grzenda, A., Lomberk, G., Zhang, J.S. and Urrutia, R. (2009) Sin3:master scaffold and transcriptional corepressor. Biochim. Biophys.Acta 1789, 443–450 CrossRef PubMed

12 van Oevelen, C., Bowman, C., Pellegrino, J., Asp, P., Cheng, J.,Parisi, F., Micsinai, M., Kluger, Y., Chu, A., Blais, A. et al. (2010)The mammalian Sin3 proteins are required for muscledevelopment and sarcomere specification. Mol. Cell. Biol. 30,5686–5697 CrossRef PubMed

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 inpermanent gene silencing. Mol. Cell. 32, 359–370CrossRef PubMed

14 Bansal, N., Kadamb, R., Mittal, S., Vig, L., Sharma, R.,Dwarakanath, B.S. and Saluja, D. (2011) Tumor suppressor proteinp53 recruits human Sin3B/HDAC1 complex for down-regulation ofits target promoters in response to genotoxic stress. PLoS One 6,e26156 CrossRef PubMed

15 Das, T.K., Sangodkar, J., Negre, N., Narla, G. and Cagan, R.L.(2013) Sin3a acts through a multi-gene module to regulateinvasion in Drosophila and human tumors.. Oncogene 32,3184–3197 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 andc-Myc are required to maintain tissue homeostasis. Nat. Cell Biol.13, 1395–1405 CrossRef PubMed

17 Pile, L.A., Schlag, E.M. and Wassarman, D.A. (2002) TheSIN3/RPD3 deacetylase complex is essential for G(2) phase cellcycle progression and regulation of SMRTER corepressor levels.Mol. Cell. Biol. 22, 4965–4976 CrossRef PubMed

18 Pile, L.A., Spellman, P.T., Katzenberger, R.J. and Wassarman, D.A.(2003) The SIN3 deacetylase complex represses genes encodingmitochondrial proteins: implications for the regulation of energymetabolism. J. Biol. Chem. 278, 37840–37848CrossRef PubMed

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c© 2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. 11

R. Kadamb and others

19 Yang, W., Yang, X., David, G. and Dorsey, J.F. (2012) Dissecting thecomplex regulation of Mad4 in glioblastoma multiforme cells.Cancer Biol. Ther. 13, 1339–1348 CrossRef PubMed

20 Zilfou, J.T., Hoffman, W.H., Sank, M., George, D.L. and Murphy, M.(2001) The corepressor mSin3a interacts with the proline-richdomain of p53 and protects p53 from proteasome-mediateddegradation. Mol. Cell. Biol. 21, 3974–3985CrossRef PubMed

21 Jazayeri, A., McAinsh, A.D. and Jackson, S.P. (2004)Saccharomyces cerevisiae Sin3p facilitates DNA double-strandbreak repair. Proc. Natl. Acad. Sci. U.S.A. 101, 1644–1649CrossRef PubMed

22 Keller, T.J. and Oppenheimer, N.J. (1987) Enhancedbleomycin-mediated damage of DNA opposite charged nicks. Amodel for bleomycin-directed double strand scission of DNA. J.Biol. Chem. 262, 15144–15150

23 Mirabelli, C.K., Ting, A., Huang, C.-H., Mong, S. and Crooke, S.T.(1982) Bleomycin and talisomycin sequence-specific strandscission of DNA: a mechanism of double-strand cleavage. CancerRes 42, 2779–2785 PubMed

24 De Nadal, E., Zapater, M., Alepuz, P.M., Sumoy, L., Mas, G. andPosas, F. (2004) The MAPK Hog1 recruits Rpd3 histonedeacetylase to activate osmoresponsive genes. Nature 427,370–374 CrossRef PubMed

25 Munoz, I.M., MacArtney, T., Sanchez-Pulido, L., Ponting, C.P.,Rocha, S. and Rouse, J. (2012) Family with sequence similarity60A (FAM60A) protein is a cell cycle-fluctuating regulator of theSIN3-HDAC1 histone deacetylase complex. J. Biol. Chem. 287,32346–32353 CrossRef PubMed

26 Murphy, M., Ahn, J., Walker, K.K., Hoffman, W.H., Evans, R.M.,Levine, A.J. and George, D.L. (1999) Transcriptional repression bywild-type p53 utilizes histone deacetylases, mediated byinteraction with mSin3a. Genes Dev 13, 2490–2501CrossRef PubMed

27 Weber, A., Marquardt, J., Elzi, D., Forster, N., Starke, S., Glaum, A.,Yamada, D., Defossez, P.A., Delrow, J., Eisenman, R.N. et al.(2008) Zbtb4 represses transcription of P21CIP1 and controls thecellular response to p53 activation. EMBO J 27, 1563–1574CrossRef PubMed

28 Funk, W.D., Pak, D.T., Karas, R.H., Wright, W.E. and Shay, J.W.(1992) A transcriptionally active DNA-binding site for human p53protein complexes. Mol. Cell. Biol. 12, 2866–2871 PubMed

29 Laptenko, O. and Prives, C. (2006) Transcriptional regulation byp53: one protein, many possibilities. Cell Death Differ 13,951–961 CrossRef PubMed

30 Riley, T., Sontag, E., Chen, P. and Levine, A. (2008) Transcriptionalcontrol of human p53-regulated genes. Nat. Rev. Mol. Cell. Biol. 9,402–412 CrossRef PubMed

31 Horn, H.F. and Vousden, K.H. (2007) Coping with stress: multipleways to activate p53. Oncogene 26, 1306–1316CrossRef PubMed

32 Kastan, M.B., Onyekwere, O., Sidransky, D., Vogelstein, B. andCraig, R.W. (1991) Participation of p53 protein in the cellularresponse to DNA damage. Cancer Res 51, 6304–6311PubMed

33 Kong, Q., Zeng, W., Wu, J., Hu, W., Li, C. and Mao, B. (2010)RNF220, an E3 ubiquitin ligase that targets Sin3B forubiquitination. Biochem. Biophys. Res. Commun. 393, 708–713CrossRef PubMed

34 Appella, E. and Anderson, C.W. (2001) Post-translationalmodifications and activation of p53 by genotoxic stresses. Eur. J.Biochem. 268, 2764–2772 CrossRef PubMed

35 Fiscella, M., Ullrich, S.J., Zambrano, N., Shields, M.T., Lin, D.,Lees-Miller, S.P., Anderson, C.W., Mercer, W.E. and Appella, E.(1993) Mutation of the serine 15 phosphorylation site of humanp53 reduces the ability of p53 to inhibit cell cycle progression.Oncogene 8, 1519–1528 PubMed

36 Siliciano, J.D., Canman, C.E., Taya, Y., Sakaguchi, K., Appella, E.and Kastan, M.B. (1997) DNA damage induces phosphorylation ofthe amino terminus of p53. Genes Dev 11, 3471–3481CrossRef PubMed

37 David, G., Turner, G.M., Yao, Y., Protopopov, A. and DePinho, R.A.(2003) mSin3-associated protein, mSds3, is essential forpericentric heterochromatin formation and chromosomesegregation in mammalian cells. Genes Dev 17, 2396–2405CrossRef PubMed

38 Koumenis, C., Alarcon, R., Hammond, E., Sutphin, P., Hoffman, W.,Murphy, M., Derr, J., Taya, Y., Lowe, S.W., Kastan, M. and Giaccia,A. (2001) Regulation of p53 by hypoxia: dissociation oftranscriptional repression and apoptosis from p53-dependenttransactivation. Mol. Cell. Biol. 21, 1297–1310CrossRef PubMed

39 Lee, K.C., Crowe, A.J. and Barton, M.C. (1999) p53-mediatedrepression of alpha-fetoprotein gene expression by specific DNAbinding. Mol. Cell. Biol. 19, 1279–1288 PubMed

40 Grandinetti, K.B., Jelinic, P., DiMauro, T., Pellegrino, J., Rodriguez,R.F., Finnerty, P.M., Ruoff, R., Bardeesy, N., Logan, S.K. and David,G. (2009) Sin3B expression is required for cellular senescenceand is up-regulated upon oncogenic stress. Cancer Res 69,6430–6437 CrossRef PubMed

41 Castedo, M., Ferri, K.F., Blanco, J., Roumier, T., Larochette, N.,Barretina, J., Amendola, A., Nardacci, R., Metivier, D., Este, J.A.,Piacentini, M. and Kroemer, G. (2001) Human immunodeficiencyvirus 1 envelope glycoprotein complex-induced apoptosis involvesmammalian target of rapamycin/FKBP12-rapamycin-associatedprotein-mediated p53 phosphorylation. J. Exp. Med. 194,1097–1110 CrossRef PubMed

42 Castedo, M., Perfettini, J.L., Roumier, T., Yakushijin, K., Horne, D.,Medema, R. and Kroemer, G. (2004) The cell cycle checkpointkinase Chk2 is a negative regulator of mitotic catastrophe.Oncogene 23, 4353–4361 CrossRef PubMed

43 O’Driscoll, M., Ruiz-Perez, V.L., Woods, C.G., Jeggo, P.A. andGoodship, J.A. (2003) A splicing mutation affecting expression ofataxia-telangiectasia and Rad3-related protein (ATR) results inSeckel syndrome. Nat. Genet. 33, 497–501 CrossRef PubMed

44 Lambert, P.F., Kashanchi, F., Radonovich, M.F., Shiekhattar, R. andBrady, J.N. (1998) Phosphorylation of p53 serine 15 increasesinteraction with CBP. J Biol Chem 273, 33048–33053CrossRef PubMed

45 Ceribelli, M., Alcalay, M., Vigano, M.A. and Mantovani, R. (2006)Repression of new p53 targets revealed by ChIP on chipexperiments. Cell Cycle 5, 1102–1110 CrossRef PubMed

46 Crosby, M.E., Oancea, M. and Almasan, A. (2004) p53 binding totarget sites is dynamically regulated before and after ionizingradiation-mediated DNA damage. J. Environ. Pathol. Toxicol. Oncol.23, 67–79 CrossRef PubMed

47 Imbriano, C., Gurtner, A., Cocchiarella, F., Di Agostino, S., Basile,V., Gostissa, M., Dobbelstein, M., Del Sal, G., Piaggio, G. andMantovani, R. (2005) Direct p53 transcriptional repression: in vivoanalysis of CCAAT-containing G2/M promoters. Mol. Cell Biol. 25,3737–3751 CrossRef PubMed

48 Rayman, J.B., Takahashi, Y., Indjeian, V.B., Dannenberg, J.-H.,Catchpole, S., Watson, R.J., te Riele, H. and Dynlacht, B.D. (2002)E2F mediates cell cycle-dependent transcriptional repressionin vivo by recruitment of an HDAC1/mSin3B corepressor complex.Genes Dev. 16, 933–947 CrossRef PubMed

49 Yang, Q., Kong, Y., Rothermel, B., Garry, D., Bassel-Duby, R. andWilliams, R. (2000) The winged-helix/forkhead protein myocytenuclear factor I2 (MNF-I2) forms a co-repressor complex withmammalian Sin3B. Biochem. J. 345, 335–343CrossRef PubMed

50 Nagy, L., Kao, H.-Y., Chakravarti, D., Lin, R.J., Hassig, C.A., Ayer,D.E., Schreiber, S.L. and Evans, R.M. (1997) Nuclear receptorrepression mediated by a complex containing SMRT, mSin3A, andhistone deacetylase. Cell 89, 373–380 CrossRef PubMed

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

12 c© 2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0.

Sin3B regulates p53 target genes

51 Nan, X., Ng, H.-H., Johnson, C.A., Laherty, C.D., Turner, B.M.,Eisenman, R.N. and Bird, A. (1998) Transcriptional repression bythe methyl-CpG-binding protein MeCP2 involves a histonedeacetylase complex. Nature 393, 386–389 CrossRef PubMed

52 Xu, Y., Harton, J.A. and Smith, B.D. (2008) CIITA mediatesinterferon-I3 repression of collagen transcription throughphosphorylation-dependent interactions with co-repressormolecules. J. Biol. Chem. 283, 1243–1256 CrossRef PubMed

53 Fleischer, T.C., Yun, U.J. and Ayer, D.E. (2003) Identification andcharacterization of three new components of the mSin3Acorepressor complex. Mol. Cell. Biol. 23, 3456–3467CrossRef PubMed

54 Lai, A., Kennedy, B.K., Barbie, D.A., Bertos, N.R., Yang, X.J.,Theberge, M.-C., Tsai, S.-C., Seto, E., Zhang, Y. and Kuzmichev, A.(2001) RBP1 recruits the mSIN3-histone deacetylase complex tothe pocket of retinoblastoma tumor suppressor family proteinsfound in limited discrete regions of the nucleus at growth arrest.Mol. Cell Biol. 21, 2918–2932CrossRef PubMed

55 Shi, X., Hong, T., Walter, K.L., Ewalt, M., Michishita, E., Hung, T.,Carney, D., Pena, P., Lan, F. and Kaadige, M.R. (2006) ING2 PHDdomain links histone H3 lysine 4 methylation to active generepression. Nature 442, 96–99CrossRef PubMed

Received 12 May 2015/3 June 2015; accepted 19 June 2015

Published as Immediate Publication 25 June 2015, doi 10.1042/BSR20150122

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c© 2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. 13