Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in nuclear receptor transactivation
2010; Springer Nature; Volume: 30; Issue: 2 Linguagem: Inglês
10.1038/emboj.2010.318
ISSN1460-2075
AutoresQihuang Jin, Li‐Rong Yu, Lifeng Wang, Zhijing Zhang, Lawryn H. Kasper, Ji‐Eun Lee, Chaochen Wang, Paul K. Brindle, Sharon Dent, Kai Ge,
Tópico(s)Nuclear Receptors and Signaling
ResumoArticle3 December 2010free access Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in nuclear receptor transactivation Qihuang Jin Qihuang Jin Nuclear Receptor Biology Section, CEB, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Li-Rong Yu Li-Rong Yu Center of Excellence for Proteomics, Division of Systems Biology, National Center for Toxicological Research, FDA, Jefferson, AR, USA Search for more papers by this author Lifeng Wang Lifeng Wang Nuclear Receptor Biology Section, CEB, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Zhijing Zhang Zhijing Zhang Department of Biochemistry and Molecular Biology, University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Lawryn H Kasper Lawryn H Kasper Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Ji-Eun Lee Ji-Eun Lee Nuclear Receptor Biology Section, CEB, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Chaochen Wang Chaochen Wang Nuclear Receptor Biology Section, CEB, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Paul K Brindle Paul K Brindle Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Sharon Y R Dent Sharon Y R Dent Department of Biochemistry and Molecular Biology, University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Kai Ge Corresponding Author Kai Ge Nuclear Receptor Biology Section, CEB, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Qihuang Jin Qihuang Jin Nuclear Receptor Biology Section, CEB, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Li-Rong Yu Li-Rong Yu Center of Excellence for Proteomics, Division of Systems Biology, National Center for Toxicological Research, FDA, Jefferson, AR, USA Search for more papers by this author Lifeng Wang Lifeng Wang Nuclear Receptor Biology Section, CEB, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Zhijing Zhang Zhijing Zhang Department of Biochemistry and Molecular Biology, University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Lawryn H Kasper Lawryn H Kasper Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Ji-Eun Lee Ji-Eun Lee Nuclear Receptor Biology Section, CEB, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Chaochen Wang Chaochen Wang Nuclear Receptor Biology Section, CEB, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Paul K Brindle Paul K Brindle Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Sharon Y R Dent Sharon Y R Dent Department of Biochemistry and Molecular Biology, University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Kai Ge Corresponding Author Kai Ge Nuclear Receptor Biology Section, CEB, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Author Information Qihuang Jin1, Li-Rong Yu2,‡, Lifeng Wang1,‡, Zhijing Zhang3, Lawryn H Kasper4, Ji-Eun Lee1, Chaochen Wang1, Paul K Brindle4, Sharon Y R Dent3 and Kai Ge 1 1Nuclear Receptor Biology Section, CEB, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA 2Center of Excellence for Proteomics, Division of Systems Biology, National Center for Toxicological Research, FDA, Jefferson, AR, USA 3Department of Biochemistry and Molecular Biology, University of Texas MD Anderson Cancer Center, Houston, TX, USA 4Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USA ‡These authors contributed equally to this work *Corresponding author. Nuclear Receptor Biology Section, CEB, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 10, Room 8N307C, 9000 Rockville Pike, Bethesda, MD 20892, USA. Tel.: +1 301 451 1998; Fax: +1 301 480 1021; E-mail: [email protected] The EMBO Journal (2011)30:249-262https://doi.org/10.1038/emboj.2010.318 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Histone acetyltransferases (HATs) GCN5 and PCAF (GCN5/PCAF) and CBP and p300 (CBP/p300) are transcription co-activators. However, how these two distinct families of HATs regulate gene activation remains unclear. Here, we show deletion of GCN5/PCAF in cells specifically and dramatically reduces acetylation on histone H3K9 (H3K9ac) while deletion of CBP/p300 specifically and dramatically reduces acetylations on H3K18 and H3K27 (H3K18/27ac). A ligand for nuclear receptor (NR) PPARδ induces sequential enrichment of H3K18/27ac, RNA polymerase II (Pol II) and H3K9ac on PPARδ target gene Angptl4 promoter, which correlates with a robust Angptl4 expression. Inhibiting transcription elongation blocks ligand-induced H3K9ac, but not H3K18/27ac, on the Angptl4 promoter. Finally, we show GCN5/PCAF and GCN5/PCAF-mediated H3K9ac correlate with, but are surprisingly dispensable for, NR target gene activation. In contrast, CBP/p300 and their HAT activities are essential for ligand-induced Pol II recruitment on, and activation of, NR target genes. These results highlight the substrate and site specificities of HATs in cells, demonstrate the distinct roles of GCN5/PCAF- and CBP/p300-mediated histone acetylations in gene activation, and suggest an important role of CBP/p300-mediated H3K18/27ac in NR-dependent transcription. Introduction Histone modifications, in particular methylation (me) and acetylation (ac) on the lysine residues of core histones, have been implicated in regulating both global and inducible gene expression (Kouzarides, 2007; Li et al, 2007). Histone methylation associates with both gene activation and repression, depending on the specific lysine (K) residue that gets methylated and the state of methylation (me1, me2 or me3) (Barski et al, 2007; Kouzarides, 2007). For example, tri-methylations on K4 and K36 of histone H3 (H3K4me3 and H3K36me3) associate with gene activation. H3K4me3 is enriched around the transcription start sites (TSSs) and associates with serine 5-phosphorylated initiating RNA polymerase II (S5P Pol II). H3K36me3 is enriched at the 3′ end of transcribed regions and associates with serine 2-phosphorylated elongating RNA Pol II (S2P Pol II). In contrast, both di-methylation on K9 of histone H3 (H3K9me2) and tri-methylation on K27 of histone H3 (H3K27me3) are associated with gene silencing (Kouzarides, 2007). Histone acetylation generally correlates with gene activation, although the molecular mechanisms by which histone acetylation regulates transcription remain largely undetermined. Histone acetyltransferases (HATs) often are capable of acetylating multiple lysine (K) residues in vitro. Thus, the biological functions of histone acetylation are believed to largely rely on the cumulative effects (Li et al, 2007). Genome-wide analyses of histone acetylation patterns in mammalian cells have confirmed the correlation between histone acetylation and gene activation. For example, H3K9ac, H3K18ac and H3K27ac are enriched around the TSSs while acetylations on histone H4 are enriched in both the promoters and the transcribed regions of active genes (Wang et al, 2008b). However, it remains to be established whether the increased histone acetylations are a cause or a consequence of the increased transcription in mammalian cells (Roth et al, 2001). The identification of yeast GCN5 protein as the first transcription-related HAT provides strong molecular evidence to directly link histone acetylation and gene activation (Brownell et al, 1996). Yeast GCN5 is the enzymatic subunit of the SAGA complex that is capable of acetylating multiple K residues on histone H3 in vitro, including H3K9, H3K14, H3K18 and H3K23 (Grant et al, 1999). In contrast, the yeast NuA3 complex preferentially acetylates H3K14 (Lee and Workman, 2007). Mammals express two highly homologous GCN5-like paralogues: GCN5 and PCAF. Deletion of mouse GCN5 leads to early embryonic lethality, while PCAF knockout mice show no obvious phenotype. Combined loss of GCN5 and PCAF in mice leads to more severe developmental defects, suggesting a partial functional redundancy between GCN5 and PCAF in vivo (Roth et al, 2001). GCN5 and PCAF exist, in a mutually exclusive manner, in the multi-subunit mammalian SAGA (also known as STAGA and TFTC) and ATAC complexes. These two HAT complexes use GCN5 or PCAF as the acetyltransferase to specifically acetylate nucleosomal histone H3 in vitro (Wang et al, 2008a). The mammalian CBP and p300 are another pair of ubiquitously expressed, paralogous proteins that belong to a distinct family of HATs (Bedford et al, 2010). CBP and p300 are both essential for animal development as deletion of either one in mice leads to early embryonic lethality. These two HATs have been shown to function as transcription co-activators for hundreds of transcription factors including nuclear receptors (NRs) (Kraus and Wong, 2002; Bedford et al, 2010). CBP and p300 are largely functionally interchangeable in vitro and in cultured cells, but they also display unique properties in vivo (Kasper et al, 2006). In vitro, CBP/p300 are capable of acetylating multiple K residues on core histones (Kouzarides, 2007). Genome-wide mapping of HATs in human cells shows that consistent with their roles as transcription co-activators, both the GCN5/PCAF and the CBP/p300 pairs of HATs correlate with gene activation and are recruited to regions surrounding the TSSs (Wang et al, 2009). However, the substrate and site specificities of mammalian GCN5/PCAF and CBP/p300 in vivo, as well as the roles of GCN5/PCAF- and CBP/p300-mediated histone acetylations in gene activation, remain largely unclear. Ligand-induced activation of NR target genes provides a robust model system to study the molecular mechanisms underlying transcription regulation (Rosenfeld and Glass, 2001; Kraus and Wong, 2002). GCN5 has been shown to function as a transcription co-activator for several NRs such as androgen receptor, estrogen receptor α (ERα) and PPARγ (Yanagisawa et al, 2002; Zhao et al, 2008). Similarly, PCAF has been shown to function as a co-activator for ERα, retinoic acid receptor (RAR) and thyroid hormone receptor (TR) on reporter genes (Blanco et al, 1998; Korzus et al, 1998). GCN5 and PCAF are enriched on ERα target gene promoters upon ligand treatment (Metivier et al, 2003). However, the data that implicate GCN5/PCAF as NR co-activators were mostly obtained from ectopic expression of GCN5/PCAF in reporter assays. It remains to be determined whether GCN5/PCAF are required for activation of endogenous NR target genes. More importantly, the role of GCN5/PCAF-mediated histone acetylation in NR-dependent transcription is largely unclear. The roles of CBP/p300 as NR co-activators are better characterized. CBP/p300 were initially shown to function as transcription co-activators for glucocorticoid receptor, RAR and TR on reporter genes in cells (Chakravarti et al, 1996; Kamei et al, 1996). p300 acts synergistically with ligand-activated ERα and RAR to enhance transcription initiation on chromatin templates in vitro (Kraus and Kadonaga, 1998; Dilworth et al, 2000). Further, p300 requires its HAT activity to function as a co-activator for ERα and TR (Kraus et al, 1999; Li et al, 2000). CBP/p300 are also enriched on ERα target gene promoters upon ligand treatment (Metivier et al, 2003). In both primary CBP+/− cells and primary p300−/− cells, NRs show reduced transcriptional activities on reporter genes (Yao et al, 1998; Yamauchi et al, 2002). These results indicate that CBP/p300 are important for ligand-induced NR target gene activation. However, likely due to the early embryonic lethality of both CBP−/− and p300−/− mice as well as the potential functional redundancy between CBP and p300 in cells, the roles of CBP/p300 in expression of endogenous NR target genes were incompletely understood. More importantly, because the substrate and site specificities of CBP/p300 in vivo were not determined, the molecular mechanisms by which CBP/p300-mediated histone acetylations regulate NR-dependent transcription have remained largely unclear. PPARδ is a ubiquitously expressed NR. Activation of PPARδ promotes fat burning. Highly specific synthetic PPARδ ligands (agonists) such as GW501516 (GW), are promising drug candidates for obesity and diabetes (Evans et al, 2004). Endogenous PPARδ is abundantly expressed in mouse embryonic fibroblasts (MEFs), but associates with histone deacetylases and behaves as a transcriptional repressor in the absence of ligand. Upon ligand treatment, endogenous PPARδ switches from a repressor to an activator, which leads to a robust activation of target genes (Shi et al, 2002). Angiopoietin-like 4 (Angptl4, also known as PGAR and FIAF) is a direct target gene of PPARδ, with the PPAR response element (PPRE) being located at 2.3 kb downstream of the TSS in intron 3 (Mandard et al, 2004). Treating MEFs with 100 nM GW selectively activates PPARδ target genes in MEFs, with Angptl4 being the most significantly induced one (Oliver et al, 2001; Hummasti and Tontonoz, 2006). In this paper, we use the GW-induced Angptl4 expression in MEFs as a model system to initiate the investigation on the roles of GCN5/PCAF- and CBP/p300-mediated histone acetylations in NR target gene activation. We found that GW induces sequential enrichment of H3K18/27ac, Pol II, H3K9ac, and several histone methylations on the Angptl4 promoter. Using GCN5/PCAF double knockout (DKO) cells and CBP/p300 DKO cells, we determined the substrate and site specificities of these two distinct families of HATs in cells and show that GCN5/PCAF and CBP/p300 are specifically required for H3K9ac and H3K18/27ac, respectively. Surprisingly, GCN5/PCAF-mediated H3K9ac correlates with, but is dispensable for, GW-induced Angptl4 expression. In contrast, CBP/p300 and their HAT activities are essential for both GW-induced enrichment of histone modifications and Pol II on the Angptl4 promoter and GW-induced Angptl4 expression. Examination of several other endogenous NR target genes obtained similar results. Results PPARδ ligand-induced histone modifications on Angptl4 gene By quantitative reverse transcriptase–PCR (qRT–PCR) analysis of gene expression, we confirmed the PPARδ ligand GW-dependent activation of known direct PPARδ target genes Angptl4 and PDK4 in MEFs, with Angptl4 being more significantly induced (Figure 1A; Supplementary Figure S1A). Consistent with the previous report that PPARδ functions as a transcriptional repressor in the absence of ligand (Shi et al, 2002), deletion of PPARδ by retroviral Cre expression in PPARδflox/flox MEFs led to a moderate increase of the basal level of Angptl4. However, deletion of PPARδ in MEFs completely prevented the GW-induced Angptl4 and PDK4 expression, indicating that ligand-induced expression of endogenous Angptl4 and PDK4 is strictly dependent on PPARδ (Supplementary Figure S1A). Consistent with Angptl4 and PDK4 being direct target genes of PPARδ, protein synthesis inhibitor cycloheximide failed to inhibit GW-induced Angptl4 and PDK4 expression in MEFs (Supplementary Figure S1B). Figure 1.PPARδ ligand-induced histone modifications on Angptl4 gene. (A) Ligand-dependent activation of PPARδ target gene Angptl4 in MEFs. Wild-type MEFs were treated with 100 nM PPARδ-specific ligand GW501516 (GW). Samples were collected at indicated time points for analysis of Angptl4 expression by qRT–PCR. (B–E) MEFs were treated with GW or DMSO for 24 h, followed by chromatin immunoprecipitation (ChIP) analyses on Angptl4 gene. The intron/exon organization of the 6.6-kb Angptl4 gene is shown at the bottom with an arrow indicating the transcription start site. (B) ChIP of histone methylations using antibodies against H3K4me3, H3K9me2, H3K27me3, H3K36me3 and H3K79me2, respectively. (C) ChIP of histone acetylations using antibodies against H3K9ac, H3K14ac, H3K18ac, H3K27ac and H4ac, respectively. (D) ChIP of histone H3. (E) ChIP of total RNA polymerase II (Pol II), serine 5-phosphorylated initiating Pol II (S5P Pol II) and serine 2-phosphorylated elongating Pol II (S2P Pol II). All results are representative of two to four independent experiments. Quantitative PCR data in all figures are presented as mean values±s.d. Download figure Download PowerPoint As the first step towards understanding the roles of histone modifications in regulating ligand-induced NR target gene expression, MEFs were treated with GW for 24 h, followed by chromatin immunoprecipitation (ChIP) analyses of histone modifications on the Angptl4 gene. As shown in Figure 1B, GW treatment had little effect on the levels of H3K9me2 and H3K27me3 but increased H3K4me3, H3K36me3 and H3K79me2 signals on Angptl4 gene, which correlated with the GW-induced Angptl4 expression. GW-induced H3K4me3 and H3K79me2 were enriched around the TSS while GW-induced H3K36me3 was enriched at the 3′ end of the transcribed region. GW treatment increased the levels of all histone acetylations that we have examined on Angptl4 gene, including H3K9ac, H3K14ac, H3K18ac, H3K27ac and histone H4 acetylation (H4ac), with the signals peaked around the TSS (Figure 1C). The GW-induced histone methylations and acetylations were not due to change in nucleosome occupancy, as the histone H3 signal on Angptl4 gene was not affected by GW treatment (Figure 1D). We next examined the Pol II recruitment on Angptl4 gene in GW-treated MEFs (Figure 1E). In the absence of GW, the signals of total Pol II, serine 5-phosphorylated initiating Pol II (S5P Pol II) and serine 2-phosphorylated elongating Pol II (S2P Pol II) were all very low on Angptl4 gene. GW treatment led to markedly increased enrichment of all three types of Pol II on Angptl4 gene. The signal of S5P Pol II peaked around the TSS while the signal of S2P Pol II peaked at the 3′ end of the transcribed region. These results indicate that Pol II recruitment is a major regulatory step for PPARδ ligand-induced Angptl4 expression. PPARδ ligand induces sequential histone modifications on Angptl4 gene Next, we examined the time course of PPARδ ligand-induced histone modifications and Pol II recruitment on Angptl4 gene. MEFs were treated with GW for 0, 10, 30 min, 1, 4 and 24 h, followed by ChIP assays. Based on Figure 1 results, we chose the +3.7- and +6.5-kb regions on Angptl4 gene to examine GW-induced H3K36me3 and S2P Pol II, respectively. Other histone modifications and Pol II recruitment were examined at the +0.6-kb region on Angptl4 gene. As shown in Figure 2A–C, significantly increased H3K14ac, H3K18ac, H3K27ac and H4ac signals were observed on Angptl4 gene 10 min after the start of GW treatment. The increased H3K9ac and Pol II signals were observed after 30 min while the increased H3K4me3, H3K36me3 and H3K79me2 signals observed after MEFs were treated with GW for 4 h. Figure 2.PPARδ ligand induces sequential histone modifications on Angptl4 gene. (A–C) The time courses of GW-induced Pol II recruitment and histone methylations and acetylations on Angptl4 gene. Wild-type MEFs were treated with GW. Cells were collected at indicated time points for ChIP analyses of total Pol II (A), histone methylations (B) and acetylations (C) on Angptl4 gene. (D–G) Wild-type MEFs were pre-treated with DRB for 30 min, followed by treatment with GW for 4 h in the presence of DRB. Cells were collected for analysis of Angptl4 expression (D), as well as ChIP analyses of S2P Pol II (E), histone methylations (F) and acetylations (G) on Angptl4 gene. Based on Figure 1, we chose the +3.7-kb and the +6.5-kb regions on Angptl4 gene to examine GW-induced H3K36me3 and S2P Pol II, respectively. All other histone modifications and Pol II recruitment were examined at the +0.6-kb region on Angptl4 gene. All results are representative of two to four independent experiments. Download figure Download PowerPoint To verify the sequential manner of GW-induced histone modifications on Angptl4 gene, we used DRB, an inhibitor of transcription elongation (Edmunds et al, 2008). As prolonged exposure to DRB is toxic to cells, we pre-treated MEFs with DRB for 30 min, followed by GW treatment in the presence of DRB for 4 h. DRB completely blocked GW-induced Angptl4 expression (Figure 2D). Consistent with its role as an inhibitor of transcription elongation, DRB blocked enrichment of S2P Pol II to Angptl4 gene (Figure 2E). DRB completely blocked GW-induced H3K9ac, H3K4me3, H3K36me3 and H3K79me2, and decreased GW-induced H3K14ac and H4ac, but had no effect on GW-induced H3K18ac and H3K27ac on Angptl4 gene (Figure 2F and G). Thus, GW-induced H3K18ac and H3K27ac precede, while GW-induced H3K9ac, H3K4me3, H3K36me3 and H3K79me2 occur following the start of transcription elongation on Angptl4 gene. GCN5 and PCAF are specifically required for H3K9ac in cells The dynamic histone H3 methylations and H4ac during gene induction have been investigated (Edmunds et al, 2008; Hargreaves et al, 2009). We decided to focus on the role of histone H3 acetylation in regulating ligand-induced NR target gene expression. Our approach was to delete the two pairs of HATs, GCN5/PCAF and CBP/p300, in MEFs, to study the regulation of endogenous NR target gene activation by histone acetylation. Before trying to understand the role of GCN5/PCAF-mediated histone acetylation in gene activation, we sought to determine the substrate and site specificities of GCN5/PCAF in cells. Because of the lack of phenotype in PCAF null mice (Roth et al, 2001), we started with deletion of GCN5 in MEFs. Retroviruses expressing Cre were used to infect the immortalized GCN5flox/Δ MEFs carrying one floxed and one null alleles of GCN5 (Atanassov et al, 2009). Deletion of GCN5 by Cre in MEFs had no significant effect on the global levels of histone acetylations or the GW-induced Angptl4 expression (Supplementary Figure S2; Figure 3E), suggesting that GCN5 and PCAF could be functionally redundant in MEFs. Figure 3.GCN5 and PCAF are redundant and are specifically required for H3K9ac in cells. Immortalized PCAF−/−;GCN5flox/Δ MEFs were infected with retroviruses MSCVpuro expressing Cre or Vec. (A) Confirmation of deletion of GCN5 and PCAF genes by qRT–PCR. Wild-type MEFs were included as control. (B) Cell morphology under the microscope. (C) Cell growth curves. (D) Deletion of GCN5 and PCAF in MEFs vastly reduces global level of H3K9ac. Nuclear extracts were prepared for western blot analysis of histone acetylations and methylations using indicated antibodies. (E) GCN5 and PCAF are redundant and are required for the global level of H3K9ac. GCN5flox/Δ MEFs and PCAF−/−;GCN5flox/Δ MEFs were infected with MSCVpuro expressing Cre. Nuclear extracts were prepared for western blot analysis. (F) In vitro HAT assays were performed by incubating 1.5 μg GST-GCN5 protein purified from bacteria or 1 μg GCN5-associated HAT complexes (GCN5.com) purified from MEFs (Supplementary Figure S4) with 1 μg recombinant histone H3 in the presence of acetyl CoA, followed by western blot analyses. Control, mock-purified sample from MEFs. The signals in the GST and the control lanes reflect non-specific detection of recombinant histone H3 by histone acetylation antibodies. All results are representative of two to four independent experiments. Download figure Download PowerPoint Next, we sought to delete both GCN5 and PCAF in MEFs. Primary MEFs were isolated from PCAF−/−;GCN5flox/Δ mouse embryos that carried two null alleles of PCAF and one floxed and one null alleles of GCN5. After immortalization, PCAF−/−;GCN5flox/Δ MEFs were infected with retroviral Cre to generate GCN5/PCAF DKO cells. Deletion of GCN5/PCAF was confirmed at both mRNA and protein levels (Figure 3A and E). Interestingly, DKO of GCN5 and PCAF in MEFs had no significant effect on the cell morphology and only slightly decreased the cell growth rate, indicating that GCN5/PCAF are largely dispensable for the viability and growth of immortalized MEFs (Figure 3B and C). By western blot analysis of histone acetylations and methylations, we found that deletion of GCN5/PCAF in MEFs specifically and dramatically reduced the global level of H3K9ac (Figure 3D). In contrast, single knockout of either GCN5 or PCAF had no effect on the global level of H3K9ac in MEFs (Figure 3E). Western blot in a different type of cells, brown pre-adipocytes, also showed that deletion of GCN5/PCAF dramatically reduced the global level of H3K9ac but not H3K14ac (Supplementary Figure S3A). Using anti-H3K14ac antibodies from two different sources, we confirmed that deletion of GCN5/PCAF had no effect on the global level of H3K14ac in MEFs (Supplementary Figure S3B). The specific loss of H3K9ac but not H3K14ac or other histone acetylations in GCN5/PCAF DKO cells was surprising, given that the yeast GCN5 and associated SAGA complex are capable of acetylating multiple lysine residues on histone H3 and preferentially acetylate H3K14 over H3K9 in vitro (Grant et al, 1999). To investigate whether the mammalian GCN5 and associated HAT complexes are capable of acetylating H3K14, we purified recombinant mouse GCN5 from bacteria and affinity-purified GCN5-associated HAT complexes (GCN5.com) from MEFs (Supplementary Figure S4). In the in vitro HAT assays using recombinant histone H3 as substrate, both recombinant mouse GCN5 and GCN5.com strongly acetylated multiple lysine residues on histone H3, including H3K9, H3K14, H3K18, H3K23 and H3K56, with weak acetylations on H3K27 and H3K36 (Figure 3F). These results suggest that mammalian GCN5 and associated HAT complexes are capable of acetylating H3K14, but this acetylation may be compensated by other HATs in GCN5/PCAF DKO cells. To confirm the western blot results and more importantly, to provide direct evidence that mouse GCN5/PCAF are required for H3K9ac but not H3K14ac in cells, we performed mass spectrometric analysis of the total levels of H3K9ac and H3K14ac on histone H3 protein purified from MEFs (Table I). The data revealed that the total level of H3K14ac (sum of 14Kac in Table I) was over 20-fold more abundant than that of H3K9ac in MEFs and that H3K9ac always co-existed with H3K14ac on the same histone H3 molecule to form H3K9/14ac (di-acetylation on K9 and K14 of histone H3). Thus, the total H3K9ac level is represented by the H3K9/14ac level in the mass spectrometry data. Infecting PCAF−/−;GCN5flox/Δ MEFs with retroviral Cre led to over 19-fold decrease of H3K9/14ac level, but had no significant effect on the total level of H3K14ac (Table I). These results provide direct evidence to indicate that GCN5/PCAF are required for H3K9ac, but not H3K14ac in cells. Taken together, western blot and mass spectrometry data demonstrate that mouse GCN5 and PCAF are redundant and are specifically required for the global level of H3K9ac in cells. Table 1. Mass spectrometric analysis of the total acetylation levels on H3K9 and H3K14 in retroviral Vec- or Cre-infected PCAF−/−;GCN5flox/Δ MEFs Peptide MH+ ΔM (p.p.m.) Peak area ( × 106) Ratio (Vec/Cre) Vec Cre 9KSTGG14KacAPR 943.5320 −1.40 534.4 638.6 0.84 9Kme1STGG14KacAPR 957.5476 −2.06 541.0 548.0 0.99 9Kme2STGG14KacAPR 971.5633 −0.91 1658.7 1956.8 0.83 9KacSTGG14KacAPR 985.5425 −2.22 131.5 6.8 19.26 Sum of 14Kac 2865.5 3150.2 0.91 The histone H3 proteins isolated from MEFs were digested with endoproteinase Arg-C to release the 9KSTGG14KAPR peptide encompassing residues K9–R17 of histone H3. The peak area value represents the abundance of each type of modified peptides determined by mass spectrometry, with the relative standard deviation of ∼15%. Note that the peptide with acetylation on K9 alone, 9KacSTGG14KAPR, was undetectable. In other words, K9ac was always detected with co-existing K14ac on the same peptide to form 9KacSTGG14KacAPR (H3K9/14ac). Therefore, the total level of H3K9 acetylation (H3K9ac) is represented by the H3K9/14ac level. At the shown high mass measurement accuracy (ΔM (p.p.m.)), tri-methylated and acetylated lysine residues can be distinguished confidently by the mass spectrometer. GCN5/PCAF and H3K9ac are dispensable for ligand-induced NR target gene expression We next examined the effects of deletion of GCN5/PCAF and thus the dramatic reduction of H3K9ac on gene expression. Surprisingly, deletion of GCN5/PCAF had little effect
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