Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in nuclear receptor transactivation

Abstract

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.

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.

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.

Figure 3

GCN5 and PCAF are redundant and are specifically required for H3K9ac in cells. Immortalized PCAF^−/−;GCN5^flox/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. GCN5^flox/Δ MEFs and PCAF^−/−;GCN5^flox/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.

Figure 4

GCN5/PCAF and H3K9ac are dispensable for PPARδ ligand-induced Angptl4 expression. (A–D) GCN5/PCAF and GCN5/PCAF-mediated H3K9ac are dispensable for ligand-induced PPARδ target gene expression in MEFs. PCAF^−/−;GCN5^flox/Δ MEFs infected with retroviral Vec or Cre were treated with GW for 24 h, followed by ChIP of histone acetylations (A), methylations (B) and Pol II recruitment (C) on Angptl4 gene as described in Figure 2. Gene expression was analysed by qRT–PCR (D). (E) Time course of ligand-induced GCN5 and PCAF recruitment on Angptl4 promoter. Wild-type MEFs were treated with GW. Cells were collected at indicated time points for ChIP of GCN5 and PCAF at the +0.1-kb region on Angptl4 gene. (F) 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 ChIP of GCN5 and PCAF at the +0.1-kb region on Angptl4 gene. All results are representative of two to four independent experiments.

Figure 5

CBP and p300 are redundant and are specifically required for H3K18ac and H3K27ac in cells. Immortalized CBP^flox/flox;p300^flox/flox MEFs were infected with adenoviruses expressing Cre or GFP control. Two days later, cells were re-plated to remove dead cells. After 24 h, cells were subjected to the following analyses. (A) Confirmation of deletion of CBP and p300 genes by qRT–PCR using primers located in deleted regions. (B) Cell morphology under the microscope. (C) Cell growth curves. (D) Deletion of CBP and p300 in MEFs markedly reduces the global levels of H3K18ac and H3K27ac. Nuclear extracts were prepared for western blot analyses of histone acetylations and methylations. (E) CBP and p300 are redundant and are required for the global levels of H3K18ac and H3K27ac. Immortalized CBP^flox/flox MEFs and p300^flox/flox MEFs were infected with retroviruses MSCVpuro expressing Cre or Vec. Nuclear extracts were prepared for western blot analysis. (F) CBP and p300 are dispensable for heat shock-induced Hsp70 expression. Cells were incubated at 43°C for 30 min, followed by recovery at 37°C for 2 h. (G) CBP and p300 are dispensable for the cytokine interleukin 1β (IL-1β)-induced Cxcl1 and Cxcl2 expression. Cells were treated with 10 ng/ml IL-1β for 1 h. All results are representative of two to four independent experiments.

Figure 6

CBP/p300 and their HAT activities are essential for ligand-induced PPARδ target gene expression. (A, B, C, E) Immortalized CBP^flox/flox;p300^flox/flox MEFs were infected with adenoviruses expressing Cre or GFP control. Two days later, cells were re-plated. After 24 h, cells were treated with GW for 24 h, followed by ChIP assays of histone acetylations (A), methylations (B) and Pol II recruitment (C) on Angptl4 gene as described in Figure 2, as well as qRT–PCR analysis of gene expression (E). (D) Time course of total Pol II recruitment analysed by ChIP. Experiment was done as in C except that cells were treated with GW for 0, 1 and 2 h. (F, G) Wild-type MEFs were treated with GW in the presence of 50 μM curcumin for 6 h, followed by qRT–PCR analysis of gene expression (F) and ChIP analyses of histone acetylations and Pol II recruitment on Angptl4 promoter (G). (H) Time course of ligand-induced CBP and p300 recruitment on Angptl4 gene. Wild-type MEFs were treated with GW. Cells were collected at indicated time points for ChIP analyses of CBP and p300 at the +2.3-kb PPRE on Angptl4 gene. (I) 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 ChIP analysis of CBP and p300 at the +2.3-kb PPRE on Angptl4 gene. (J) Model depicting the distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in NR transactivation, using PPARδ ligand-induced Angptl4 expression as an example (see Discussion). All results are representative of two to four independent experiments.