Alteration of gene expression in differentiating C2C12 cells upon inhibition of PCAF by embelin
Ontology highlight
ABSTRACT: KAT2B (PCAF) belongs to GNAT family of lysine acetyltransferases (KAT), which specifically acetylates histone H3K9 residue and several nonhistone proteins. PCAF is also a transcription coactivator. Due to the lack of PCAF, KAT specific small molecule inhibitor, the exclusive role of the acetyltransferase activity of PCAF is not well understood. Here we report that a hydroxy benzoquinone class of natural compound, embelin, specifically inhibits H3Lys9 acetylation in mice and recombinant PCAF mediated acetylation with near complete specificity in vitro. Further, using embelin, we have identified the PCAF regulated gene networks during muscle differentiation, further highlighting the broader regulatory functions of PCAF in muscle differentiation, apart from regulation through MyoD acetylation. C2C12 cells were treated with either DMSO or 10uM embelin in GM/DM for either 24 or 48h.
Project description:KAT2B (PCAF) belongs to GNAT family of lysine acetyltransferases (KAT), which specifically acetylates histone H3K9 residue and several nonhistone proteins. PCAF is also a transcription coactivator. Due to the lack of PCAF, KAT specific small molecule inhibitor, the exclusive role of the acetyltransferase activity of PCAF is not well understood. Here we report that a hydroxy benzoquinone class of natural compound, embelin, specifically inhibits H3Lys9 acetylation in mice and recombinant PCAF mediated acetylation with near complete specificity in vitro. Further, using embelin, we have identified the PCAF regulated gene networks during muscle differentiation, further highlighting the broader regulatory functions of PCAF in muscle differentiation, apart from regulation through MyoD acetylation. HEK 293T cells were treated with DMSO (solvent control) and 20uM embelin for 24h. Cells were harvested and total RNA was extracted by Trizol method. RNA was cleaned up using RNeasy MinElute Cleanup Kit (Qiagen) and reverse transcribed, labelled and subsequently hybridized to Agilent human 4X 44k array slide.
Project description:KAT2B (PCAF) belongs to GNAT family of lysine acetyltransferases (KAT), which specifically acetylates histone H3K9 residue and several nonhistone proteins. PCAF is also a transcription coactivator. Due to the lack of PCAF, KAT specific small molecule inhibitor, the exclusive role of the acetyltransferase activity of PCAF is not well understood. Here we report that a hydroxy benzoquinone class of natural compound, embelin, specifically inhibits H3Lys9 acetylation in mice and recombinant PCAF mediated acetylation with near complete specificity in vitro. Further, using embelin, we have identified the PCAF regulated gene networks during muscle differentiation, further highlighting the broader regulatory functions of PCAF in muscle differentiation, apart from regulation through MyoD acetylation.
Project description:KAT2B (PCAF) belongs to GNAT family of lysine acetyltransferases (KAT), which specifically acetylates histone H3K9 residue and several nonhistone proteins. PCAF is also a transcription coactivator. Due to the lack of PCAF, KAT specific small molecule inhibitor, the exclusive role of the acetyltransferase activity of PCAF is not well understood. Here we report that a hydroxy benzoquinone class of natural compound, embelin, specifically inhibits H3Lys9 acetylation in mice and recombinant PCAF mediated acetylation with near complete specificity in vitro. Further, using embelin, we have identified the PCAF regulated gene networks during muscle differentiation, further highlighting the broader regulatory functions of PCAF in muscle differentiation, apart from regulation through MyoD acetylation.
Project description:Histone acetyltransferases (HATs) GCN5/PCAF and CBP/p300 are transcription coactivators. However, how these HATs regulate ligand-induced nuclear receptor target gene expression remains unclear. Here we show in mouse embryonic fibroblasts (MEFs), deletion of GCN5/PCAF specifically eliminates acetylation on H3K9 (H3K9Ac) while deletion of CBP/p300 selectively reduces acetylation on H3K18 and H3K27 (H3K18/27Ac). Treating MEFs with a specific ligand for nuclear receptor PPARdelta induces sequential increases of H3K18/27Ac and H3K9Ac on the promoter of PPARdelta target gene Angptl4, which correlates with a robust ligand-induced Angptl4 expression. Inhibiting transcription elongation blocks ligand-induced H3K9Ac but not H3K18/27Ac on Angptl4 promoter. Finally, we show CBP/p300 and their HAT activities are required, while GCN5/PCAF and H3K9Ac are dispensable, for ligand-induced PPARdelta target gene expression in MEFs. These results highlight the substrate and site specificities of HATs in cells, and suggest that GCN5/PCAF- and CBP/p300-mediated histone acetylations play distinct roles in regulating ligand-induced nuclear receptor target gene expression. PCAF and GCN5 have some redundant function. To identify PCAF/GCN5-regulated genes, immortalized MEFs with PCAF knockout and GCN5 conditional knockout were infected with retroviruses expressing either Cre recombinase or vector alone. We prepared duplicated RNAs from either vector or Cre infected cells (PCAF-/-;GCN5+/- or PCAF-/-;GCN5+/-) and RNAs from either Vector or Cre infected the other independently immortalized cells for 6 affymetrix microarray.
Project description:Histone acetyltransferases (HATs) GCN5/PCAF and CBP/p300 are transcription coactivators. However, how these HATs regulate ligand-induced nuclear receptor target gene expression remains unclear. Here we show in mouse embryonic fibroblasts (MEFs), deletion of GCN5/PCAF specifically eliminates acetylation on H3K9 (H3K9Ac) while deletion of CBP/p300 selectively reduces acetylation on H3K18 and H3K27 (H3K18/27Ac). Treating MEFs with a specific ligand for nuclear receptor PPARdelta induces sequential increases of H3K18/27Ac and H3K9Ac on the promoter of PPARdelta target gene Angptl4, which correlates with a robust ligand-induced Angptl4 expression. Inhibiting transcription elongation blocks ligand-induced H3K9Ac but not H3K18/27Ac on Angptl4 promoter. Finally, we show CBP/p300 and their HAT activities are required, while GCN5/PCAF and H3K9Ac are dispensable, for ligand-induced PPARdelta target gene expression in MEFs. These results highlight the substrate and site specificities of HATs in cells, and suggest that GCN5/PCAF- and CBP/p300-mediated histone acetylations play distinct roles in regulating ligand-induced nuclear receptor target gene expression.
Project description:We analyzed the transcriptional programs regulated by PCAF and p27 in the colon cancer cell line HCT116 by ChIP-seq. We identified 269 protein-encoding genes that contain both p27 and PCAF binding sites being the majority of these sites different for PCAF and p27
Project description:Gcn5/PCAF double knockout (dKO) leads to loss of the global H3K9ac. RNA-Seq was performed to define the changes of gene expression in response to Gcn5/PCAF deletion and H3K9ac loss
Project description:PArtner and Localizer of BRCA2 (PALB2) is essential to maintain genome stability in human cells. Upon DNA damage by double DNA strand breaks, PALB2 is required to repair DNA by homologous recombination. In undamaged conditions, PALB2 protects coding regions, by preventing DNA stress due to collisions between transcription and replication machineries. PALB2 associates with chromatin, which is essential to fulfill its function in genome stability maintenance, however molecular mechanisms regulating PALB2 chromatin association remain unknown. In our previous published study describing the KAT2A/B(GCN5/PCAF)-acetylome (Fournier et al., Nat.Comm. 2016, doi: 10.1038/ncomms13227) we have identified PALB2 as an acetylated protein target of the acetyltransferases KAT2A (GCN5) and KAT2B (PCAF) in vivo. KAT2A/B-acetylated sites of PALB2 were mapped within its DNA/Chromatin association domain. In this current study, we have conducted in vitro acetyltransferase (AT) assays, by mixing purified recombinant GST-tagged PALB2 full-length (FL) and PALB2 fragment P2.2 which associates with DNA/chromatin (from residues 295-610), with Flag-PCAF, Flag-GCN5 and Flag-GCN5 catalytic mutant (mut), followed by proteomics analysis to map PALB2 acetylated lysines by KAT2A(GCN5) and KAT2B(PCAF) in vitro. PALB2 FL and P2.2 alone, or PALB2 FL and P2.2 mixed with GCN5 catalytic mutant, were used as negative controls.
Project description:Gcn5/PCAF double knockout (dKO) leads to loss of the global H3K9ac. RNA-Seq was performed to define the changes of gene expression in response to Gcn5/PCAF deletion and H3K9ac loss PCAF-/-;Gcn5f/D MEFs were infected with retroviral Cre to delete Gcn5 to generate Gcn5/PCAF dKO cells, followed by RNA-Seq analysis using spike-in RNA as controls