doi:10.1016/j.jmb.2009.12.058 J. Mol. Biol. (2010) 397, 1–12
Available online at www.sciencedirect.com
Human Positive Coactivator 4 ControlsHeterochromatinization and Silencing of NeuralGene Expression by Interacting with REST/NRSFand CoREST
Chandrima Das†, Shrikanth S. Gadad† and Tapas K. Kundu⁎
Transcription and DiseaseLaboratory, Molecular Biologyand Genetics Unit, JawaharlalNehru Centre for AdvancedScientific Research, Jakkur P.O.,Bangalore 560064, India
Received 20 May 2009;received in revised form29 December 2009;accepted 30 December 2009Available online18 January 2010
The highly abundant, multifunctional transcriptional positive coactivator 4(PC4) plays important roles in transcription, replication and DNA repair.Our recent work showed that PC4 is a bona fide non-histone component ofchromatin. Here, we report that knockdown of PC4 dramatically altersheterochromatin organization of the genome, accompanied by increasedH3K9 (histone H3 at lysine residue 9)/14 acetylation, H3K4 trimethylationand reduction in the level of H3K9 dimethylation. These posttranslationalmodifications of histone H3 result in overexpression of normally silencedgenes (e.g., neural genes) located in heterochromatin. The results of ChIP(chromatin immunoprecipitation) and re-ChIP assays showed that over-expression of a neuronal-specific gene is accompanied by histonehyperacetylation. We further show that PC4 interacts with heterochromatinprotein 1α, REST/NRSF (RE1-silencing transcription factor/neuron-restric-tive silencer factor) and CoREST to establish the repressed state of neuralgenes in nonneuronal cells. Thus, PC4 plays a crucial role in maintaining adynamic chromatin state and heterochromatin gene silencing.
The highly dynamic nature of the eukaryoticgenome is achieved by diverse nuclear factors,including non-histone chromatin-associated pro-teins (CAPs), histone chaperones, ATP-dependentremodeling machinery and enzymes that posttran-slationally modify chromatin proteins. The abun-dant and multifunctional non-histone chromatin
ess:
ally to this work.coactivator 4; H3K9,, chromatinencing transcriptionncer factor; CAP,heterochromatincopy; MNase,ic acid decarboxylasescarinic acid receptor;3, acetylated histone, small interfering
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proteins, such as HMGs, heterochromatin protein 1(HP1), MeCP2 and PARP1,1–4 regulate the extentof chromatin condensation and thereby exertspatiotemporal control of gene expression. Post-translational modification of chromatin proteins3–6
and/or their oligomeric association7 also regulatesthe higher-order folding of chromatin functionaldomains. The different HP1 subtypes (α, β and γ)are central players in these regulatory processes;however, the cellular interplay of HP1s is muchmore complex than was initially predicted.3 A keyevent is methylation of histone H3 at lysineresidue 9 (H3K9), which constitutes the dockingsite for HP1s. Binding of HP1 to dimethylatedH3K9 is followed by recruitment of other hetero-chromatin protein components,8 establishment of aheterochromatic domain and silencing of geneexpression. Thus, formation of the RNA polymer-ase II preinitiation complex is inhibited by HP1,9
for example.In the case of neuronal cells, MeCP2, a repressor of
the neuronal genes,6 docks onto methylated DNA atits target promoters and helps in the recruitment ofthe Sin3a–HDAC corepressor complex.10 Also,transcriptional repression of neural-specific genes
in nonneuronal cells is dependent on the REST (RE1-silencing transcription factor)–CoREST complex.8,11
Although REST is expressed ubiquitously in non-neuronal cells, it is down-regulated in neuronalprecursors and turned off in postmitotic neurons,indicating that it not only prevents extraneuronalexpression of a certain class of genes but also causesa delay in differentiation of neuronal progenitors.12
A recent report shows that REST is regulated byubiquitin-mediated proteolysis during neuronaldifferentiation.13 The binding of REST and specificCpG methylation events facilitate the recruitment ofCoREST and the subsequent assembly and spread-ing of the silencing machinery.2
Another important histone modification is phos-phorylation of H3S10 by Aurora B kinase, whichinfluences the binding of HP1 to histone H3 as cellsenter mitosis.14,15 H3S10 phosphorylation acts as apotential signal to initiate disassembly of hetero-chromatin domains by the removal of HP1.16Subsequently, the chromatin becomes hyperacety-lated (e.g., at H3K9, H3K14), resulting in activationof transcription.17 Along with hyperacetylation,histones also become methylated at specific sites(e.g., H3K4Me).18 Recently, it was shown thatextraneuronal gene silencing is mediated byMED12, linking REST with G9a-dependent H3K9dimethylation.19,20 Thus, epigenetic modificationsgovern the transition from repressed to activechromatin states.Human transcriptional positive coactivator 4
(PC4), a newly discovered member of the CAPsuperfamily, has been shown to be involved inchromatin organization.21 PC4 is an abundant,multifunctional protein in mammalian cells in-volved in regulating DNA-templated phenomenasuch as transcription, replication and repair.22–26
As a positive coactivator, PC4 interacts with TBPand TFIIA, components of the basal transcriptionmachinery, as well as with the activator, to induceactivator-dependent transcription by several fold.22
PC4 is also an activator of p53 function,27 where itwas found to induce p53-mediated gene expressionand apoptosis. Interestingly, expression of PC4 isalso controlled by p53, which established the firstknown positive loop for p53 activation.28 PC4interacts with human single-stranded DNA bind-ing protein to markedly affect the latter's replica-tion function.25 Interestingly, PC4 is also known tointeract with XPG and plays an important role inrepairing oxidative DNA damage.26 Recently, weshowed that PC4 activates nonhomologous DNAend joining and double-strand break-repair activityin vivo.29 Although the single-stranded DNAbinding ability of PC4 has been implicated inactivator-independent transcriptional repression,the in vivo significance of these observations isnot known.30
In this study, we investigated the role of PC4 inthe maintenance of the epigenetic state of chromatinand neuronal gene expression. Taken together, ourresults establish a critical role for PC4 in regulatingheterochromatin structure and gene silencing.
Results
Silencing of PC4 alters the epigenetic state ofchromatin
In view of the role of PC4 in influencing thestructure of chromatin,21 we first investigated theeffect of PC4 gene knockdown on the nuclearmorphology of HeLa cells by using atomic forcemicroscopy (AFM). The results of this analysisshow that silencing of PC4 leads to decompactionof nuclei (Fig. 1a, compare I and II of top andbottom panels; ‘PG7’ refers to cells transfected withthe PC4 gene-silencing cassette). Presumably, uponPC4 silencing, the chromatin decondenses, causingthe volume of the nucleus to increase. The surfacetopology plots clearly reflect a difference inchromatin compaction between PC4-silenced andcontrol nuclei (Fig. 1a, compare bottom panels).Decondensation of the chromatin was verified bymeasuring the susceptibility of chromatin tomicrococcal nuclease (MNase) digestion. Asexpected, upon partial MNase digestion, the nucleitransfected with the control vector remainedrelatively intact, whereas the PC4-silenced nucleiwere digested into smaller fragments (Fig. 1b,compare I and II of top and bottom panels).Taken together, these observations led us tohypothesize that PC4 knockdown may lead to anepigenetically altered, relatively open chromatinstate.We therefore investigated the alteration of
histone modifications upon PC4 silencing. Byemploying immunofluorescence (Fig. 2a, panel I)and Western blotting analysis (Fig. 2b, panel I), weobserved that histone H3 is dramatically hyper-acetylated upon PC4 silencing as compared withhistone H3 in control cells (compare panels I andIV in Fig. 2a and b). Furthermore, knocking downof PC4 gene expression led to a significant increasein H3K4 trimethylation, which is indicative of atranscriptionally active state of chromatin (Fig. 2aand b, panel II). The role of PC4 in establishing arepressed chromatin state was further investigatedby monitoring the levels of H3K9 dimethylation.Interestingly, along with histone H3 hyperacetyla-tion and H3K4 methylation, a substantial reductionof H3K9 dimethylation could be observed in PC4knockdown cells (Fig. 2a and b, panel III). Thesedata indicate that PC4 could be involved in themaintenance of the architectural integrity andepigenetic state of chromatin and thus may playa significant role in chromatin dynamics and generegulation.
Silencing of PC4 results in overexpression ofneuronal genes
The relatively open chromatin structure and thechanges in histone modification observed uponsilencing of PC4 suggest there could be global orspecific up-regulation of transcription (at least for
Fig. 1. AFM reveals altered chromatin organization in PC4-silenced cells. HeLa cells were transfected with either PG7plasmid encoding an siRNA (for PC4 silencing) or a control plasmid (empty vector, PgShinII). Transfection was confirmedby expression of GFP encoded by the vector. Upon transfection, intact (a) or partially MNase-digested (b) HeLa cell nucleiwere subjected to AFM (for details, see Materials and Methods). The top panels in (a) and (b) show the AFM image,whereas the bottom panels show the three-dimensional surface topology plot of the AFM image. Upon silencing of PC4,the intact nuclei (a) became less compact [compare (I) and (II)] and the MNase-digested nuclei (b) became more accessibleto the nuclease, as evidenced by the high level of fragmentation [compare (I) and (II)].
3Coactivator Controls Heterochromatinization
the genes located within heterochromatin). Thus, toaddress the possible involvement of PC4 in hetero-chromatic gene silencing, we investigated theexpression of three neural genes in two nonneuronalcell lines (HeLa and 293T). In HeLa cells, silencing ofPC4 dramatically induced the expression of aspecific subset of neuronal genes:20 glutamic aciddecarboxylase 1 (GAD1), sodium channel 2 (SCN2)and muscarinic acid receptor 4 (M4), as revealed byreal-time PCR analysis. The expression of GAD1,SCN2 and M4 was up-regulated by 4- to 6-fold overthe control level upon silencing of PC4 expression inHeLa cells (Fig. 3a). Knockdown of PC4 in 293T cellsresulted in fold inductions of SCN2 and M4expression similar to those in HeLa cells but notquite a 2-fold up-regulation of GAD1 (Fig. 3b).Presumably, knocking down of PC4 in these celllines disturbed the organization of heterochromatin,which permitted the expression of genes situated inheterochromatic domains. Taken together, theseresults raise the interesting possibility of involve-ment of PC4 in neural gene repression along withknown components of the neural gene repressivecomplex, REST/NRSF (neuron-restrictive silencerfactor), CoREST and HP1.
PC4 interactswithHP1α, REST/NRSFandCoREST
A FLAG-tagged PC4 construct was transfected intoHeLa cells and the native complex associated withPC4 was immuno-pulled-down using anti-FLAG-M2agarose to investigate possible functional interactionsof PC4with different proteins involved in heterochro-matin gene silencing. Interestingly, probing of thePC4-interacting complex byWestern blotting analysiswith HP1-subtype-specific antibodies revealed thatPC4 interacts with HP1α but not with HP1β or HP1γ(Fig. 4a, lane 2, panel I versus panels II and III).A coimmunofluorescence experiment was per-
formed in 293T cells to visualize the extent ofcolocalization of PC4 and HP1α in vivo. The resultsof this analysis showed a convincing associationbetween HP1α and PC4 [Fig. 4b, merged image ofHP1α (red) and PC4 (green) without Hoechst(blue)]. The third quadrant of the scatter plotshows the colocalized pixels of HP1α and PC4(Fig. 4b). The calculated weighted colocalizationcoefficients are 0.486 for PC4 (Fig. 4b, table-Ch3-T1)and 0.633 for HP1α (Fig. 4b, table-Ch2-T3); both areN0.3 and thus considered significant (according toPearson's correlation coefficients).
Fig. 2. Silencing of PC4 alters the pattern of histone modification. The levels of histone H3 modification (K9/14acetylation, K4 trimethylation and K9 dimethylation) upon silencing of PC4 (PG7) versus the control (vector) weremonitored by immunofluorescence (a) and Western blotting analysis (b).
4 Coactivator Controls Heterochromatinization
Western blotting analysis of the FLAG-PC4 pull-down complex also confirmed that both REST andCoREST are associated with PC4 (Fig. 4c, lane 3,panels I and II). In order to rule out the artifact thatthe interaction could be due to overexpression ofPC4, we pulled down the in vivo PC4 complex byanti-PC4 polyclonal antibody from HEK293Twhole-cell lysate, and the complex was analyzedby immunoblotting with highly specific polyclonalanti-REST and anti-CoREST antibodies. It was
found that PC4 could efficiently pull down RESTand CoREST (Fig. 4d, panels I and II, lane 3 versuslane 2). Because MeCP2 is often associated withneural gene silencing, we also tested for the presenceof MeCP2 with PC4. In contrast to HP1α, REST andCoREST, MeCP2 was absent from the PC4 pull-down complex (Fig. 4c, panel III, lane 3). To furtherexplore the nature of the association of PC4 withthese heterochromatin markers involved in neuralgene silencing, we carried out in vitro interaction
Fig. 3. Knockdown of PC4 induces expression of neural genes. PC4 was silenced using PG7 plasmid in two cell lines,HeLa (a) and 293T (b). Expression of neural (GAD1, SCN2 and M4) genes was monitored by real-time PCR analysis. Thedata are presented as fold increase in PG7-transfected versus empty-vector-transfected cells.
5Coactivator Controls Heterochromatinization
studies of PC4 with glutathione S-transferase (GST)-tagged, bacterially expressed constructs of HP1(HP1α, HP1β and HP1γ), REST and CoREST. TheGST pull-down assays showed that PC4 interactsstrongly with HP1α and REST but very weakly withCoREST (Fig. 4e, lanes 3, 6, 7 and 8). As expectedfrom our earlier findings, PC4 did not interact withHP1β and HP1γ (Fig. 4e, lanes 4 and 5). These datasuggest that PC4 could be an integral component ofthe protein machinery involved in heterochromatin-mediated neural gene silencing.
REST–CoREST occupancy at the SCN2 genepromoter is dependent on PC4
Although these observations suggest direct in-volvement of PC4 in REST–CoREST-mediated re-pression of neuronal gene expression in nonneuronalcells, it was not clear whether PC4 is a functionalcomponent of the heterochromatin complex recruitedto the repressed neuronal gene promoter. Wetherefore analyzed the occupancy of PC4 at theSCN2 gene promoter along with the REST–CoRESTcorepressor complex. ChIP (chromatin immunopre-cipitation) assays using anti-PC4 antibody showedthat PC4 is indeed present at the promoter of theSCN2 gene in both HeLa and 293T cells (Fig. 5a,panels I and II, lane 1). Furthermore,we also analyzedthe alteration of promoter occupancy of the REST–CoREST complex in this REST-responsive gene(SCN2) upon silencing of PC4 (Fig. 5b–d). Signifi-cantly, silencing of PC4 led to a reduction in the levelsof REST and CoREST recruited to the SCN2 gene inboth HeLa (Fig. 5b, panels II and III, lane 3) and 293T(Fig. 5c, panels II and III, lane 3) cells. REST–CoRESToccupancy at a different region of the SCN2 genepromoter31 was also investigated in order to confirmthis observation further. In agreement with the above
result, we found that there was a substantialreduction of REST–CoREST complex on the SCN2promoter (Fig. 5f) upon PC4 silencing in 293T cells.
PC4 influences the acetylation status of histoneH3 at the SCN2 promoter and gene silencing
In our initial analysis, we observed that silencing ofPC4 induces hyperacetylation of histones (Fig. 2).Therefore, we analyzed the status of histone modifi-cation at the SCN2 promoter. We found that, in bothHeLa and 293T cells, histone H3 is substantiallyhyperacetylated upon PC4 knockdown (Fig. 5b and c,panel IV, lane 2 versus lane 3), indicating the state oftranscriptionally active gene. These results werefurther quantified by real-time PCR analysis, whichshowed a significant depletion of REST and CoRESTfrom the SCN2 promoter (Fig. 5d) and concomitantenrichment of acetylated histone H3 (AcH3) (Fig. 5d)following PC4 knockdown. The levels of REST,CoREST and acetylated histones at the SCN2 pro-moterwere also checked in the background of normalexpression of PC4 by re-ChIP assays. Immuno-pull-down of REST and CoREST (Fig. 5e, lanes 1 and 2)followed by PC4 showed a high enrichment of PC4 atthe SCN2 promoter (Fig. 5e, lanes 1 and 2; see bluebar), whereas the level of AcH3 was found to be verylow (Fig. 5e, lanes 1 and 2; see maroon bars). Thereciprocal pull-downs (reciprocal re-ChIP) (i.e.,immuno-pull-down of PC4 followed by REST,CoREST or AcH3; Fig. 5e, lane 3) showed highenrichment of REST and CoREST and low levels ofAcH3. Silencing of PC4 substantially decreased thelevels of REST and CoREST at the SCN2 promoter(Fig. 5f). One possible explanation of this phenome-non could be the absence of these proteins due to PC4silencing. Significantly, knockdown of PC4 did notalter the expression of HP1 (all subtypes), REST,
6 Coactivator Controls Heterochromatinization
CoREST and MeCP2 (Fig. 5g). These results establishthat the silencing of PC4 inhibits the recruitment ofthese repressor components at the neuronal genepromoters, which leads to expression of the genes.Upon PC4 silencing, the repressive complex at the
SCN2 promoter could not be found, as confirmed byChIP assay. Furthermore,we also observed that at thehypoacetylated promoter, REST, CoREST and PC4colocalized when SCN2 expression was repressed.These observations led us to investigate the effect ofREST silencing. In 293T cells, expression of neuralgeneswas inducedby 2- to 6-folduponknockdownofREST. These genes include GAD1 and M4 (2.5-fold)and SCN2 (6-fold) (Fig. 5h). Taken together, theseresults suggest that a REST–PC4 complex is likely tobe involved in the silencing of neural genes.
Discussion
Wepreviously showed that PC4 is a bona fide non-histone CAP that directly interacts with core histones
Fig. 4 (legend
(predominantly histones H3 and H2B) and induceschromatin compaction.21 In a defined reconstitutedsystem, PC4-mediated chromatin condensation wasfound to produce chromatin structures distinctlydifferent from the 30-nm nucleosomal fibers gener-ated by histone H1. In a slow kinetic process, PC4stimulates the formation of chromatin ‘globules,’which could be an important step toward theorganization of chromatin territories.21 In the pres-ent study, down-regulation of PC4 led to massivedecondensation of chromatin (Fig. 1) and alteredglobal gene expression.21 These findings tempted usto investigate whether PC4 plays any specific role inthe organization of heterochromatin. It was foundthat knocking down of PC4 expression induces theexpression of neural genes in nonneuronal cells(HeLa and 293T) (Fig. 3a and b).The HP1 family of proteins is essential to maintain
heterochromatin integrity and thereby genesilencing.32 Of the three HP1 isoforms, α and β aremostly concentrated at pericentric heterochromatin.HP1β is found to be associated with different
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Fig. 4. PC4 interacts with HP1α, REST and CoREST. (a and c) Whole-cell extracts (WCEs) prepared from FLAG-PC4-transfected HeLa cells were subjected to pull-down by M2-agarose and probed with (a) anti-HP1-subtype-specific (I–III)and (c) REST (I), CoREST (II) and MeCP2 (III) antibodies to investigate the possible interaction of PC4 with the HP1 andneural gene repressors REST and CoREST. (b) The cellular interaction of PC4 with HP1 was further confirmed bycoimmunofluorescence of PC4 and HP1α in 293T cells (top panel). The table shows the extent of colocalization calculatedaccording to Pearson's coefficient (Carl Zeiss software) [represented by the scatter plot (quadrant 3)]; the weightedcolocalization coefficients are 0.486 for PC4 (Ch3-T1) and 0.633 for HP1α (Ch2-T3). (d) In vivo PC4 complex was pulleddown from HEK293T whole-cell extract by anti-PC4 antibody and subjected to Western blotting analysis by anti-REST (I)and CoREST (II) (lane 3) antibodies. (e) His6-tagged PC4 was mixed with recombinant GST alone or GST-tagged HP1-subtype (lanes 3–5), REST (lanes 6–7) and CoREST (lane 8) proteins to investigate the interaction of PC4; the Sepharosebeads were pulled down and analyzed by Western blotting analysis (II) using anti-His6 antibody. Because of the highermolecular weight of bacterially expressed REST and CoREST, selected domains were used in the pull-down studies: N-terminus (68–546 amino acids; lane 6), C-terminus (801–1097 amino acids; lane 7) and CoREST (109–293 amino acids; lane8). Panel (I) shows the Ponceau-stained membrane that was subjected to Western blotting. The GST-tagged proteins aremarked with asterisks (I).
7Coactivator Controls Heterochromatinization
nucleoplasmic sites, whereas HP1γ is dispersedthroughout the nucleus and often associated withtranscriptionally active chromatin.33,34 PC4 wasfound to interact with the HP1α isoform and notwith HP1β or HP1γ (Fig. 4). HP1 associates withchromatin by binding to specific histone modifica-tions, di- or trimethylated H3K9, through itschromodomain.35 Silencing of neural genes in non-neuronal cells is achieved by recruitment ofHP1 ontoH3K9-dimethylated marks (mediated by G9 methyl-transferase) at the promoter.36 Deacetylation of H3precedes H3K9 methylation, as the deacetylated tailis a better substrate for methylation.37 However,these modifications ensure that the heterochromatindomains are hypoacetylated and hyper-H3K9 (di/tri)methylated. Significantly, we have found thatmaintenance of this epigenetic mark requires PC4because silencing of PC4 expression dramaticallyreducedH3K9methylation and induced bothH3K9/
14 acetylation and H3K4 trimethylation (Fig. 2).These observations argue for a central role for PC4 inorganizing the structure of heterochromatin. Silenc-ing of PC4 turns the repressed heterochromatin to amore open, transcriptionally active chromatin. Themolecular mechanisms responsible for this phenom-enon remain to be elucidated.The effect of PC4 silencing on the transcriptional
activation of different genes was found to be moregene specific than expected. Knockdown of PC4 didnot alter the expression of HP1α, HP1β, HP1γ,REST, CoREST, MeCP2 or several housekeepinggenes (e.g., GAPDH and actin) (Fig. 5g). Significant-ly, expression of the neural-specific genes GAD1,SCN2 and M4 was up-regulated upon PC4 silenc-ing. The levels of overexpression of SCN2 and M4were comparable in both of the nonneuronal celllines (HeLa and 293T), but GAD1 expression wasenhanced by 2-fold in 293T cells and by 6-fold in
Fig. 5. Recruitment of the REST–CoREST repressor complex along with PC4 to the promoter of the neural gene SCN2and expression of neural genes upon REST silencing. (a) The presence of PC4 at the SCN2 gene promoter in HeLa (I) and293T (II) cells was investigated by ChIP assay. Recruitment of REST and CoREST proteins to and the level of AcH3 at theSCN2 promoter in the presence and upon PC4 silencing were analyzed in HeLa (b) and 293T (c) cells by ChIP. Folddepletion of REST and CoREST and enrichment of AcH3 proteins upon PC4 silencing as probed by ChIP and followed byquantitative (Q)-PCR (d). (e) The bars show the Q-PCR results of the re-ChIP pull-down: re-ChIP assays (in 293T cells)with consecutive pull-downs of REST followed by PC4 (blue bar) or AcH3 (maroon bar) (histogram group 1, left) andCoREST followed by PC4 (blue bar) or AcH3 (maroon bar) (histogram group 2, middle) were performed. Reciprocal pull-downs (PC4 followed by REST, CoREST or AcH3) of PC4 with REST (blue bar), CoREST (maroon bar) or AcH3 (greenbar) (histogram group 3, right) were also done (e). (f) Fold depletion of REST and CoREST proteins to SCN2 promoterupon PC4 silencing by siRNA in 293T cells was probed by ChIP and followed by quantitative PCR using another set ofprimers as described elsewhere.31 (g) The levels of PC4 (panel I), HP1 (α, β and γ) (panels II–IV), REST (panel V), CoREST(panel VI) and MeCP2 (panel VII) in comparison with actin (panel VIII) and GAPDH (panel IX) upon silencing PC4 wereestimated by Western blotting analysis. (h) Upon silencing of REST, expression of different neural genes (GAD1, SCN2and M4) in 293T cells was quantified by real-time PCR analysis.
8 Coactivator Controls Heterochromatinization
9Coactivator Controls Heterochromatinization
HeLa cells compared with control levels. Thisdifference in the level of gene expression betweencell types is unique to GAD1, but the mechanism isnot understood.Neural genes are silenced in nonneuronal cells due
to their association with a repressor complex in theheterochromatin domain. The classical repressorcomplex, containing REST/NRSF and CoREST, isinvolved in maintaining tissue-specific gene expres-sion. We have found that PC4 directly interacts withthe REST–CoREST complex, which is functionallyassociated with the HDACs and HMTase (G9a).Furthermore, we demonstrated that silencing ofREST has a similar effect as silencing of PC4 onneural gene expression. Knockdown of REST in 293Tcells induced the expression of GAD1 and SCN2genes, but the predominant effect was on SCN2expression. These data suggest that PC4 and othercomponents of heterochromatin components main-tain the repressive chromatin state in a cooperativemanner. Recently, it was shown that Mediator(MED12), G9a and REST complex are involved inextraneuronal gene silencing.19 PC4 could also beone of the components of this repressor complex. Inagreement with this hypothesis, we found that thelevels of H3K9 dimethylation are dramaticallyreduced upon silencing of PC4 expression.Our results suggest three salient features of the
PC4-containing repressor complex in the context ofSCN2 gene regulation: (i) presence of PC4 at theSCN2 promoter when the gene is repressed innonneuronal cells (HeLa and 293T); (ii) interactionbetween PC4 and REST–CoREST at the promoter;and (iii) reduced acetylation of histone H3 when theSCN2 gene is repressed. Originally described as atranscriptional coactivator and more recently as aDNA replication and repair factor, PC4 can now alsobe said to be a regulator of higher-order chromatinstructure. It remains to be determined how the manyfunctions of PC4 are regulated especially in neuralcell differentiation and whether mutation of PC4could be a factor in disease etiology.
Materials and Methods
AFM of PC4-silenced HeLa cells
HeLa cells were cultured in a 35-mm dish. Afterwashing with phosphate-buffered saline (PBS; 172 mMNaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.76 mM KH2PO4),the cells were resuspended in buffer A [100 mM NaCl,0.5% Triton X-100, 3 mM MgCl2, 1 mM ethylenediamine-tetraacetic acid (EDTA), 10 mM Tris–HCl, pH 7.5, 1 mMPMSF] and incubated for 10 min at 4 °C. Aftercentrifugation at 1000 rpm for 5 min at 4 °C, the cellswere fixed with 4% paraformaldehyde for 20 min at roomtemperature. For visualizing partially MNase-digestednuclei, the cell pellet was washed with buffer A and thenresuspended in buffer A without Triton X-100. A total of100 mM CaCl2 was added to a final concentration of3 mM, followed by addition of 0.6 U/μl of MNase andincubation for 20 min at 37 °C. The reaction was stoppedby adding 200 mM EDTA and chilled on ice. The MNase-
treated cells were then centrifuged at 1000 rpm for 5min at4 °C and resuspended in 4% paraformaldehyde. The fixednuclei or MNase pellet was layered onto a poly-L-lysine-coated glass cover slip, incubated for 15 min at 25 °C andwashed with PBS buffer supplemented with 0.1 μg/ml ofHoechst 33258 (Sigma). The solution was completelydrained and dried by N2 gas.AFM observation was performed with a Bioscope/
Bioscope SZ Nanoscope IIIa controller (Veeco Instru-ments, Santa Barbara, CA) using the contact mode. Thecantilever (Non-Conductive Silicon Nitride, Model DNP-20, Veeco Instruments) was 0.4–0.7 mm in length with aspring constant of 0.06 N/m. The scanning frequency was1.001 Hz, and the images were captured with the heightmode in 256×256- and 512×512-pixel formats.
PC4 knockdown
PC4 silencingwasdoneusing a vector-based system (PG7)as described elsewhere.21 HeLa or 293T cells were trans-fectedwith PG7 and control vector using Lipofectamine 2000Plus (Invitrogen) according to the manufacturer's protocol.For RT (reverse transcriptase)-PCR, total RNA was isolatedusing Trizol reagent (Invitrogen). The RNAwas subjected toRT-PCR using the enzyme Superscript II (Invitrogen) togenerate a cDNA library. Subsequently, real-time PCR wascarried out using gene-specific primers.
Small interfering RNA knockdown of PC4
The small interfering RNA (siRNA) (Sigma) used forPC4 knockdown (Fig. 5f) isPC4 siRNA #1 (sense, 5-[GCAUUGAGUUUAUAAA-
CUU]dTdT;antisense, 5-[AAGUUUAUAAACUCAAUGC]dTdT).Alexa488-conjugated all-star negative control siRNA
(Human Starter Kit, Qiagen) was used as control. siRNAtransfections in 293T cells were done for 48 h withHiPerFect Transfection Reagent (Qiagen) according to themanufacturer's instructions.
REST knockdown
REST silencing was done using an HP GenomeWidesiRNA kit (Qiagen). 293T cells were transfected with siRNAand scrambled RNA using oligofectamine (Invitrogen)according to the manufacturer's protocol. For RT-PCR, totalRNA was isolated using an RNeasy kit (Qiagen). The RNAwas subjected to RT-PCR using the enzyme Superscript II(Invitrogen) to generate a cDNA library. Subsequently, real-time PCR was carried out using gene-specific primers.
Immunofluorescence
Cells grown on poly-L-lysine-coated cover slips werepermeabilized by 1% Triton X-100 in PBS, followed byblocking in 1% fetal bovine serum. Probing was done withanti-AcH3 (K9/14), anti-H3K4Me3, anti-H3K9Me2 (Up-state), anti-H3 and anti-PC4 polyclonal antibodies fol-lowed by secondary antibodies conjugated to TRIT-C(Bangalore Genei) or Alexa488 (Invitrogen). The cells werestained with 0.1 μg/ml of Hoechst 33258 (Sigma) in PBS tovisualize the DNA. Fluorescence for TRIT-C/Alexa488and Hoechst was visualized by using different filters of anAxioskop 2 Plus confocal microscope (Carl Zeiss). Theimage was captured by an AxioCam MRc camera.
10 Coactivator Controls Heterochromatinization
Real-time PCR analysis
Real-time RT-PCR analysis was performed to quantifythe fold increase in neuronal-specific gene expressionupon silencing of PC4. The M4, SCN2 and GAD1 geneswere chosen for neuronal-specific gene expression studies.The primer sequences for each gene used in this study areas follows:
The in vivo interactions of PC4 with heterochromatincomponents were investigated by performing an anti-FLAG-M2-agarose pull-down assay on FLAG-PC4-trans-fected HeLa whole-cell extracts, followed by immunoblot-ting with anti-HP1 (α, β and γ), anti-REST, anti-CoRESTand anti-MeCP2 antibodies. The in vitro interaction studieswere carried out by GST-pull-down assays using GST-tagged HP1 (α, β and γ), C-terminal (801–1097 aminoacids) REST, REST (68–546 amino acids), CoREST (109–293amino acids) and C-terminal histidine-tagged PC4.
ChIP and re-ChIP assays
PC4 expression was silenced in HeLa and HEK293Tcells, and the cells were used for ChIP assay. The ChIPassay was performed as described elsewhere.28 Briefly,after transfection of PG7 or vector only, cross-linking wasdone with 1% formaldehyde followed by cell lysis in SDSlysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl,pH 8.0). After sonication of the chromatin (six times for10 s at 91% power setting), cold dilution buffer (0.01%SDS, 1.1% Triton X-100, 1.2 mMEDTA, 16.7 mMTris–HCl,pH 8.0, 167 mM NaCl) was added along with preblockedprotein G-Sepharose (Amersham Pharmacia) and anti-REST, anti-CoREST, anti-PC4 or anti-AcH3 antibodiesand kept for overnight binding. The sonicated sampleswere precleared prior to immunoprecipitation. Beadswere washed with low-salt buffer (0.1% SDS, 1% TritonX-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8.0, 150 mMNaCl), high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mMEDTA, 20 mM Tris–HCl, pH 8.0, 500 mM NaCl), LiClbuffer (250 mM LiCl, 1% NP40, 1% NaDOC, 1 mM EDTA,10 mM Tris–HCl, pH 8.0) and TE (10 mM Tris–HCl,pH 8.0, 1 mM EDTA) consecutively. Elution buffer (0.2%SDS, 100 mM NaHCO3) along with 200 mM NaCl wasadded to the washed beads, and the bead solution waskept overnight at 65 °C. The next day, 0.1 mg/ml ofproteinase K (Sigma) and 0.04 mg/ml of RNase A (Sigma)were added to the bead solution and the mixture wasincubated for 2 h at 55 °C. The immunoprecipitatedsamples were deproteinized, ethanol precipitated andused for real-time PCR analysis. Specific primer sets wereused for the real-time PCR analysis;for one of the REST binding sites (Fig. 5a–e):
The re-ChIP experiments were performed as describedelsewhere.38 Briefly, the immunocomplex pulled downwith the first antibody was treated with 0.05 M DTT,followed by a 20-fold dilution before performing the pulldown with the second antibody.
Coimmunofluorescence
The cells, grown on poly-L-lysine-coated cover slips,were permeabilized by 1% Triton X-100 in PBS,followed by blocking in 1% fetal bovine serum. Probingwas done with anti-HP1α monoclonal antibody (Up-state) followed by secondary antibody conjugated withAlexa568. After extensive washing, for visualizing PC4colocalization with HP1α, purified polyclonal PC4antibody followed by Alexa488-conjugated secondaryantibody was used. The cells were stained with 0.1 μg/ml of Hoechst 33258 in PBS to visualize the DNA.Fluorescence for Alexa and Hoechst was visualized byusing different filters of an Axioskop 2 Plus microscope(Carl Zeiss), and images were captured by an AxioCamMRc camera. AxioVision 3.1 software was used toprocess the images and to calculate Pearson's weightedcolocalization coefficients.
Acknowledgements
This work was supported by the Department ofBiotechnology, Government of India, and theJawaharlal Nehru Centre for Advanced ScientificResearch. We acknowledge Prof. Gail Mandel(Oregon Health & Science University, Portland,OR) for providing the REST and CoREST clones.GST-tagged HP1-subtype bacterial expression con-structs were provided by Prof. Michael Carey(Department of Biological Chemistry, University ofCalifornia, Los Angeles, CA). We also acknowledgeMrs. Suma B.S. for her assistance with the confocalmicroscopy experiments and Kunal Bose (VeecoInstruments) for suggestions during the AFMexperiments. We thank Senthil Kumar for raisingantibody against PC4. We also acknowledge Jaya-sha Shandilya and Roshan Elizabeth Rajan forproviding technical help. S.S.G. is a Senior ResearchFellow of the Council of Scientific and IndustrialResearch, Government of India.
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