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RESEARCH PAPER RESEARCH PAPER Epigenetics 5:6, 509-515; August 16, 2010; © 2010 Landes Bioscience Interplay of bromodomain and histone acetylation in the regulation of p300-dependent genes Jihong Chen,1 Feras M. Ghazawi1,2 and Qiao Li1,2,* Departments of 1Pathology and Laboratory Medicine; and of 2Cellular and Molecular Medicine; Faculty of Medicine; University of Ottawa; Ottawa, ON CA Key words: gene regulation, histone acetylation, transcription, acetyltransferase, coactivator, bromodomain
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  www.landesbioscience.com Epigenetics 509Epigenetics 5:6, 509-515; August 16, 2010; © 2010 Landes Bioscience RESEARCH PAPER RESEARCH PAPER *Correspondence to: Qiao Li; Email: qiaoli@uottawa.caSubmitted: 02/07/10; Accepted: 05/04/10Previously published online: www.landesbioscience.com/journals/epigenetics/article/12224DOI: 10.4161/epi.5.6.12224 Introduction Transcriptional coactivator p300 was identied initially asan E1A-associated protein and later as a tumor suppressor. 1,2  Mutations in p300 genes have been detected in various epithelialcancers and reintroduction o wild-type p300 leads to growthsuppression o the cancer cells. 3-5 Patients with Rubinstein-Taybisyndrome due to p300 heterozygosity have an increased predis-position to cancer. 6-8 In addition, E1A, HPV E6 oncoprotein andSV40 large T antigen target p300, urther underlining a criticalrole or p300 in events related to cellular transormation. 9-11 The p300 contains an intrinsic histone acetyltranserase(HAT) activity and multiple interaction suraces or a variety o transcription actors and components o the basal transcriptionmachinery. 12 It thus acts as either a HAT enzyme to acetylatehistones or a bridging actor to integrate dierent regulatory signalsin transcriptional activation. 5,12 One o the p300 domains is anevolutionarily conserved bromodomain which was characterizedrst or the Drosophila melanogaster  Brahma protein and later Interplay of bromodomainand histone acetylation in the regulationof p300-dependent genes Jihong Chen, 1 Feras M. Ghazawi 1,2 and Qiao Li 1,2, * Departments o  1 Pathology and Laboratory Medicine; and o  2 Cellular and Molecular Medicine; Faculty o Medicine; University o Ottawa; Ottawa, ON CA Key words: gene regulation, histone acetylation, transcription, acetyltranserase, coactivator, bromodomain  Abbreviations: HAT, histone acetyltranserase; HDAC, histone deacetylaseound in many transcription regulators and nearly all nuclearHAT proteins. 13,14 The unction o bromodomain is important orp300 to recognize specic chromatin substrates and to coordinatechromatin remodeling and transcriptional activation. 15 Onemode o bromodomain action is to serve as acetyl-lysine bindingmodule tethering the HAT activity to a dened chromosomalsite to achieve highly specic histone acetylation. 16-19 However,it is not clear whether the bromodomain is required or geneexpression o all or only a limited set o p300-dependent genes.The overall topology o the bromodomain modules is similaramong P/CAF, GCN5 and TAFII250 and the residues impor-tant or acetyl-lysine recognition are largely conserved. 17,20,21  However, there is a clear dierence in the ligand specicity o bromodomain. It is the bromodomain o P/CAF, not CBP, thatbinds specically to the acetyl-lysine 50 o HIV-1 Tat. 19 Likewise,the binding o CBP to acetyl-lysine 382 o p53 is required or p53acetylation-dependent recruitment o coactivator in responseto UV-induced DNA damage, but the association o p300 with p53 does not depend on the bromodomains-acetyl-lysine  The bromodomain is an evolutionarily conserved moti ound in many transcriptional activators including p300 whichcontains an intrinsic histone acetyltranserase activity and is a general coactivator or many transcription actors. Onemode o bromodomain action is to serve as a binding module to recognize specic acetyl-lysine residue o histonesduring chromatin remodeling and transcriptional activation. The unction o p300 is required or diverse sets o geneexpression. However, it is not known whether the p300 bromodomain is involved in the expression o all or only subseto p300-dependent genes. In this study, we examined the impact o either a wild-type or a bromo-decient p300 on theexpression o several p300-dependant genes. The eects o histone acetylation on the expression o these genes werealso assessed by targeting histone deacetylase activities with an inhibitor approach. We show that the impact o theseinhibitors on the transcriptional activation o p300-dependent genes is impaired in cells containing the bromo-decientp300, indicating that the interplay o p300 and histone acetylation in p300-dependent gene transcription requires thebromodomain. We also observed an increase in the expression o bromo-decient p300 at the level o transcriptionpossibly to compensate or the loss o p300 unction. However, the high level o bromo-decient p300 is not able tomaintain the basal level o histone acetylation. Thus, the bromodomain is important or p300 to maintain the basallevel o histone acetylation and to induce the transcriptional activation o p300-dependent genes. Nevertheless, therequirement o bromodomain and histone acetylation in p300-dependent gene transcription is determined by a genespecic manner.  510 Epigenetics Volume 5 Issue 6 genes such as p21, Egr1 and E2F1, to determine the interplay o bromodomain and histone acetylation in the regulation o p300-dependent genes. Results Bromo-decient p300 o SiHa cells. The SiHa cervical carci-noma cells contain a homozygous internal deletion o exons 15–18in the p300 gene (p300 ∆ ex15-18 ) which gives rise to a p300 proteinlacking the bromodomain (bromo-decient). 5 Most importantly,the HAT activity o the bromodomain decient p300 is consider-ably impaired, only about 30% remaining compared to the wild-type p300. 5 To probe or cellular mechanisms involved in theregulation o p300 unction, we rst characterized in details thebromo-decient p300 in SiHa cells in parallel with the wild-typep300 in HeLa cervical carcinoma cells. Western analysis demonstrated that the level o the truncatedorm o p300 in the SiHa cells was signicantly higher than the wild-type p300 in the HeLa cells, about 5-old ( Fig. 1A and B ).Quantitative real-time RT-PCR analysis revealed that indeed themRNA level o bromo-decient p300 in the SiHa cells was signi-icantly higher than the wild-type p300 in the HeLa cells, about10-old ( Fig. 1C ). In addition, a pulse-labeling protocol demon-strated that the rate o protein synthesis o the bromo-decientinteraction. 18 The bromodomain o p300 and CBP are highly homologous, yet they dier in ligand selectivity, indicating thatthe ligand selectivity is controlled by ew but very importantvariations in bromodomain sequences or the action mode o thebromodomain is not solely linked to the acetyl-lysine binding.Targeting histone deacetylase (HDAC) with inhibitorsaects gene expression through inhibiting histone deacetylation, which results in the accumulation o acetylated histones and theassociation o activator complex to activate gene transcription. 22-24  Thereore, HDAC inhibitors have emerged in recent years as a new class o cancer therapeutics since they selectively induce apoptosisin tumor cells, with limited toxicity to normal cells. 25 Thereare several classes o natural and synthetic HDAC inhibitors.One is the short-chain atty acids such as butyrate and valproicacid. 26,27 Butyrate is naturally produced by anaerobic bacteriaermenting undigested dietary carbohydrates and induces cellcycle arrest and selective apoptosis o tumor cells through alteringhistone acetylation. 23,28 Valproic acid is commonly used in thetreatment o epilepsy, bipolar disorder and migraines and alsoselectively induces transormed cells to undergo growth arrestand apoptosis. 29 Trichostatin A (TSA), a hydroxamate compoundrepresents another class o HDAC inhibitors. 30 In this study, we examined the roles o bromodomain andhistone acetylation in the expression o several p300-dependant Figure 1. Characterization o the wild-type and bromo-decient p300 in HeLa and SiHa cells. (A) Equal amounts o whole cell extracts (50 µ g) romthe HeLa or SiHa cells were subjected to western analysis or the wild-type and bromo-decient p300. The blots were then stripped and reprobedor β -actin as a loading control. (B) Quantication o the western blots was plotted as old dierence in reerence to the wild-type p300 in the HeLacells. Error bars represent the standard deviation o three independent experiments. Statistical signicance is denoted by * to indicate p < 0.05compared with the wild-type p300. (C) Relative mRNA level o bromo-decient p300 in the SiHa cells was analyzed by quantitative real-time RT-PCRand presented as old variation to the wild-type p300 transcripts in the HeLa cells. Error bars represent standard deviations o three independentexperiments in duplicate samples. (D) Relative synthesis o the bromo-decient p300 protein in the SiHa cells was determined ollowing 2 h o pulse-labeling with 35 S-methionine and expressed as old variation o the wild-type p300 in the HeLa cells. Error bars represent standard deviations o three independent experiments. (E) The HeLa cells were pulse labeled with 35 S-methionine or 2 h and chased or 6–24 h. The apparent hal-lie o thewild-type p300 protein was derived rom three independent experiments in duplicate samples. (F) The experimental procedure was as in (E) exceptthat the SiHa cells were used to determine the apparent hal-lie o the bromo-decient p300. (G) Intracellular localization o the wild-type or bromo-decient p300 protein in the HeLa or SiHa cells was determined by immunofuorescence microscopy.  www.landesbioscience.com Epigenetics 511 seen in the HeLa cells (comparing Fig. 2D with B ). Interestingly,the protein levels o E2F1 were not aected by the treatments, which is the same as seen in the HeLa cells (comparing Fig. 2D   with B ). Taken together, these data suggest that the interplay o p300 unction and histone acetylation in the expression o p300-dependent genes may require the bromodomain. Eects o HDAC inhibitors on the transcription o p300-dependent genes. To determine i the eects o these HDACinhibitors on the protein levels o p21 and Egr1 are transcriptiondependent, we proled the mRNA levels o p21 and Egr1ollowing HDAC inhibitor treatments by using quantitative realtime RT-PCR analysis. As shown in Figure 3A  , treatments o theHeLa cells with butyrate, valproic acid or TSA or 4 h resultedin a signicant increase in the levels o p21 mRNA, up to 3-old, while 16 h o treatment urther increased the augmentation toabout 6-old. Likewise, the levels o Egr1 mRNA were augmentedsignicantly to about 4-old ollowing 4 h o treatments, andincreased urther to about 21-old ollowing 16 h o treatments( Fig. 3A  ). Together, these results demonstrate that HDACinhibitors increase the expression o p300-dependent genes suchas p21 and Egr1 at the level o transcription.Next we examined the transcript levels o p21 and Egr1 inthe SiHa cells in response to these treatments. As shown in Figure 3B , ollowing 16 h o treatments, the mRNA levels o p21 were only increased up to 3-old, signicantly lower thanthe 6-old o increase observed in the HeLa cells (compare Fig.3B with  A  ). With respect to Egr1, 16 h o treatments resulted inabout 6-old o augmentation in the levels o Egr1 mRNA, againsignicantly lower than the 21-old o increase observed in theHeLa cells ( Fig. 3A and B ). Taken together, these data indicatethat the impact o these HDAC inhibitors on transcription o p300-dependent genes is impaired in the SiHa cells which harbora bromo-decient p300. Global histone acetylation and p300-dependent geneexpression. To determine the status o histone acetylation, weexamined the eects o HDAC inhibitors on the levels o globalhistone acetylation in the HeLa and SiHa cells. The cells weretreated with butyrate, valproic acid or TSA or 4 h and thensubjected to western analysis with a specic antibody againstacetylated K9 and K14 on histone H3. As shown in Figure4A and B , treatments with these HDAC inhibitors increasedthe levels o acetylated H3 similarly, about 2-old, in both theHeLa and SiHa cells ( Fig. 4A and B ). However, the basal levelo H3 acetylation was signicantly lower in the SiHa than theHeLa cells ( Fig. 4 ). These data suggest that the bromodomainis important or p300 to exert its HAT activity in histone acety-lation since the high level o bromo-decient p300 was not ableto maintain the basal level o histone acetylation in the SiHacells ( Figs. 1 and 4 ). We next examined the interplay bromodomain and histoneacetylation in p300-depedent gene expression o SiHa cells.The cells were rst transected with expression plasmid or the wild-type p300 and then treated with butyrate, valproic acidor TSA or 8 h. As shown with the western analysis, p21 levels were upregulated by butyrate and TSA, whereas Egr1 levels wereaugmented by valproic acid and TSA ( Fig. 4C ). Interestingly,p300 in the SiHa cells was signicantly higher than the wild-typep300 in the HeLa cells, about 10 old ( Fig. 1D ).To examine the impact o bromodomain on p300 turnover, we employed a pulse chase protocol to compare the metabolicstability o wild-type and bromo-decient p300. Previousstudies show that the hal lie o wild-type p300 is about 11–14 hin several cell lines. 31-33 We determined that in the HeLa cells, theapparent hal lie o wild-type p300 is about 11 h as assessed by a pulse-chase protocol ( Fig. 1E ). In the SiHa cells, the apparenthal-lie o the bromo-decient p300 was about 10 h, which is inthe similar range o the wild-type p300 in the HeLa cells (com-paring Fig. 1F with E ). In addition, immunofuorescence micros-copy showed that the bromo-decient p300, as the wild-typep300, localized predominantly to the nucleus ( Fig. 1G ). Takentogether, these data suggest that the increase in the level o thetruncated orm o p300 protein in the SiHa cells may stem roma eed-back regulation to compensate or the loss o p300 HATactivity, through the augmentation o p300 ∆ ex15-18 gene expres-sion, but not a decrease o the bromo-decient p300 turnover. HDAC inhibitors and p300-dependent gene expression.  Genome-wide survey o promoter structure occupied by p300 hasidentied a group o p300-dependent genes, such as p21, Egr1and E2F1, which contain the characteristic o GC rich region andsome common motis or transcription actor binding. 34 However,gene transcription o p21 and E2F1 in HeLa cells harboring a wild-type p300 exhibits dierential sensitivity to treatment withHDAC inhibitor butyrate. 35 To dene the roles o p300 and histone acetylation in the reg-ulation o p300-dependent genes, we rst examined the expres-sion prole o p21, Egr1 and E2F1 ollowing targeting HDACactivities with HDAC inhibitors such as butyrate, valproic acidand TSA in time course studies. As shown in Figure 2A and B ,ollowing 8 h o treatments with these HDAC inhibitors, the pro-tein levels o p21 in the HeLa cells were signicantly augmented,up to 7-old, whereas 16 h o treatments increased the levels o p21 protein urther to about 17-old. Similarly, the protein levelso Egr1 were also signicantly augmented, up to 3-old ollowing8 h o treatment, and up to 9-old ollowing 16 h o treatments( Fig. 2A and B ). On the other hand, the protein levels o E2F1 were not much aected by these treatments ( Fig. 2A and B ), which is consistent with previous report. 35 Taken together, ourdata suggest that the role o histone acetylation in the expressiono p300-dependent genes may be determined by a gene specicmanner. Gene expression in cells harboring the bromo-decient p300.  To examine the interplay o bromodomain and histone acetylationin the regulation o p300-depedent genes, we next examined theeects o butyrate, valproic acid and TSA, on the protein levelso p21, Egr1 and E2F1 in the SiHa cells containing the bromo-decient p300. Following 16 h o treatments, an increase in thelevels o p21 protein was detected by western blotting analysis( Fig. 2C and D ). However, this augmentation was only about4-old, signicantly lower than the 17-old increase seen in theHeLa cells (comparing Fig. 2D with B ). Similarly, the proteinlevels o Egr1 were only increased about 2-old in response to thetreatments ( Fig. 2C and D ), in contrast to the 9-old increase  512 Epigenetics Volume 5 Issue 6 Histone acetylation is generally associated with active geneloci and the acetylation o histone by p300 is an important ave-nue to achieve transcriptional activation. The acetylation o spe-cic lysine residues on the histone tails plays distinct roles in generegulation by marking the histone as a fag or transactivation. 36  In this event, p300 is oten recruited to the regulatory elementsby chromatin-remodeling actors and gene specic transcriptionactors. On the other hand, histone acetylation also serves as abinding signal or transcriptional coactivators such as p300. 17  In such case, the acetylated lysine residues are recognized andbound by the bromodomain to initiate the assembly o transcrip-tion actors and RNA polymerase II complex.Here, we demonstrate that although histone acetylationis generally associated with transcriptional activation, it only regulates a subset o p300-dependent genes. HDAC inhibitorsthe sensitivity o p21 or Egr1 to these HDAC inhibitors becamesimilar to the HeLa cells in which butyrate is a strong induceror p21 and valproic acid or Egr1 (comparing Fig. 4C to Fig. 2  A  ). Thus, these data suggest that the bromodomain isimportant or p300 to induce the transcriptional activation o p300-dependent genes. Discussion The major conclusion o this work is that the bromodomain isimportant or p300 to exert its HAT activity to maintain thebasal level o histone acetylation and to induce the transcrip-tional activation o p300-dependent genes. However, the roles o bromodomain and histone acetylation in p300-dependent geneexpression are determined in a gene specic manner. Figure 2. Eects o HDAC inhibitors on the expression o p300-dependent genes. (A) HeLa cells were treated with butyrate (NaB, 5 mM), valproic acid(VPA, 5 mM) or TSA (200 nM) or periods o 4, 8 or 16 h. Equal amounts o whole cell extracts (50 µ g) were subjected to western analysis o p21, Egr1or E2F1 protein. The blots were stripped and reprobed or β -actin as loading controls or quantication. (B) Quantication o the western blots wasplotted as old dierence in reerence to the untreated controls. (C and D) SiHa cells were treated and subjected to western analysis as or the HeLacells in (A and B).
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