Histone deacetylase 1 activates PU.1 gene transcription through regulating TAF9 deacetylation and transcription factor IID assembly

Abstract

Histone acetyltransferases and histone deacetylases (HDACs) are important epigenetic coregulators. It has been thought that HDACs associate with corepressor complexes and repress gene transcription; however, in this study, we have found that PU.1-a key master regulator for hematopoietic self-renewal and lineage specification-requires HDAC activity for gene activation. Deregulated PU.1 gene expression is linked to dysregulated hematopoiesis and the development of leukemia. In this study, we used erythroid differentiation as a model to analyze how the PU.1 gene is regulated. We found that active HDAC1 is directly recruited to active PU.1 promoter in progenitor cells, whereas acetylated HDAC1, which is inactive, is on the silenced PU.1 promoter in differentiated erythroid cells. We then studied the mechanism of HDAC1-mediated activation. We discovered that HDAC1 activates PU.1 gene transcription via deacetylation of TATA-binding protein-associated factor 9 (TAF9), a component in the transcription factor IID (TFIID) complex. Treatment with HDAC inhibitor results in an increase in TAF9 acetylation. Acetylated TAF9 does not bind to the PU.1 gene promoter and subsequently leads to the disassociation of the TFIID complex and transcription repression. Thus, these results demonstrate a key role for HDAC1 in PU.1 gene transcription and, more importantly, uncover a novel mechanism of TFIID recruitment and gene activation.-Jian, W., Yan, B., Huang, S., Qiu, Y. Histone deacetylase 1 activates PU.1 gene transcription through regulating TAF9 deacetylation and transcription factor IID assembly.

Keywords: HDAC1; HDACi; TFIID; acetylation; downstream promoter element.

Figure 1.

HDAC activity is required for PU.1 gene expression. A) MEL cells were treated with HDACi [100 nM TSA or 10 nM depsipeptide (Depsi)] for 8 h. PU.1 mRNA level was measured by using real-time PCR. B) K562 cells were treated with an increasing amount of TSA with or without 20 μm cycloheximide (CHX). PU.1 mRNA level was measured by using real-time PCR. C) K562 cells were treated with 10 nM Depsi for different time points. PU.1 mRNA level was measured by using real-time PCR. D) MEL cells were induced with 1.5% DMSO at different time points, and PU.1 mRNA level was measured by using real-time PCR. E) Recruitment of HDAC1 and acetyl-HDAC1 on mouse PU.1 promoter. ChIP assays with HDAC1 and acetylated HDAC1 Ab was performed in MEL cells induced with DMSO for 0 and 3 d. The resulting precipitated DNA was analyzed by using real-time PCR. F) Human CD34⁺ cells were induced for differentiation with Epo for 5 d, and CD36⁺ cells were collected via flow cytometry after differentiation. PU.1 mRNA level was measured by using real-time PCR. G) Human CD34⁺ cells were treated with TSA for 8 h. PU.1 mRNA level was measured by using real-time PCR. H) HDAC1 recruitment on human PU.1 promoter. ChIP assays with HDAC1 Ab were performed in CD34⁺ or CD36⁺ cells. The resulting precipitated DNA was analyzed by using real-time NT, no treatment; TSS, transcription start site; UTR, untranslated region. All data are represented as means ± sem.

Figure 2.

TAF9 is acetylated on PU.1 core promoter, interacts with HDAC1, and is deacetylated by HDAC1. A) The recruitment of acetyl-histone H3 and -4 on mouse PU.1 promoter. ChIP assay with acetyl-H3 (Ac-H3) and acetyl-H4 (Ac-H4) Abs was performed in MEL cells induced with DMSO for 0 and 3 d. The resulting precipitated DNA was analyzed by using real-time PCR. Data are represented as means ± sem. B) Schematic representation of biotin-labeled nucleosome pull-down assay (left). Reconstituted nucleosome and control DNA were resolved in 5% native gel (right). Asterisk indicates reconstituted nucleosome. C) Proteins that bound to in vitro reconstituted PU.1 nucleosome from MEL nuclear extract treated with or without TSA were Western blotted with anti–acetyl-lysine (Ac-K) and anti-TAF9 Abs. The acetylated band colocalized with the TAF9 band. D) Schematic representation of TAF9 protein. K5 and K108 are reported acetylation sites. E) GST, GST-TAF9, K5Q, and K108Q mutants were purified from Escheria coli and incubated with p300 and ³H-labeled acetyl-CoA. The reaction mix was then subjected to SDS-PAGE and autoradiography. F) GST pull-down assay was performed by incubating immobilized GST, GST-TAF9, or K5Q mutant with in vitro translated [³⁵S]-labeled HDAC1. The bond protein fraction was eluted and subjected to SDS-PAGE and autoradiography. G) K562 cell nuclear extract was immunoprecipitated with TAF9 Ab. The precipitated proteins were Western blotted with indicated Abs. H) Illustration of procedures of acetylation coupled deacetylation assay (top). In vitro acetylated TAF9 was incubated with or without HDAC1 (bottom). The reaction mix was Western blotted with acetyl-lysine Ab. CR, conserved region; DBD, DNA binding domain; HFD, histone fold domain; NT, no treatment; TSS, transcription start site; UTR, untranslated region.

Figure 3.

TAF9 recruitment correlates with HDAC1 deacetylase activity on human and mouse PU.1 promoter. A) K562 cells were treated with or without TSA for 8 h, and ChIP assay was performed with the indicated Abs. The resulting precipitated DNA was analyzed by using real-time PCR. B) ChIP assays with indicated Abs were performed in MEL cells that were treated with TSA or depsipeptide for 0 and 8 h. The resulting precipitated DNA was analyzed by using real-time PCR. C) MEL cells were induced with DMSO for 0 and 3 d. The recruitment of the TFIID complex on mouse PU.1 promoter was measured by ChIP assays with indicated Abs. The resulting precipitated DNA was analyzed by using real-time PCR. Depsi, depsipeptide; NT, no treatment; TSS, transcription start site; UTR, untranslated region. Data are represented as means ± sem.

Figure 4.

TAF9 is required for PU.1 gene transcription and TFIID recruitment. A, B) TAF9 was stably knocked down in K562 cells by using inducible lentiviral shRNA. TAF9 gene expression was measured by using real-time RT-PCR (A), and cell extracts were subjected to Western blot against TAF9 and β-actin Ab (B). C) PU.1 gene expression was measured by using real-time RT-PCR from cells with or without induction of TAF9 knockdown for 3 d with doxycycline (Dox). D) ChIP assay was performed in K562 cells with induced TAF9 knockdown with Ab as indicated. The resulting precipitated DNA was analyzed by using real-time PCR. CTL, control; TSS, transcription start site; UTR, untranslated region; WT, wild type. Data are represented as means ± sem.

Figure 5.

TAF9 acetylation results in the loss of DNA binding and transcription repression. A) Schematic representation of PU.1 core promoter. It consists of a conserved DPE site in both mice and humans. B, C) DPE sequence on pGL3-PU.1pro plasmid was mutated and transfected to K562 cells (B) or RAW 264.7 cells (C). The luciferase activity of cells was determined with or without TSA treatment. D) pGL3-PU.1pro or pGL3-PU.1proDPEmut plasmid was transiently transfected into K562 cells, and ChIP assay was performed with the indicated antibodies. The resulting precipitated DNA was analyzed by using real-time PCR with primers specific for mouse PU.1 promoter and the pGL3 plasmid. E) Schematic representation of biotin-labeled DNA pull-down assay. GST, GST-TAF9, and GST-TAF9K5Q were incubated with biotin-labeled PU.1pro or PU.1pro mut DNA fragment, and the bound proteins were Western blotted with anti-TAF9 Ab. NT, no treatment. Asterisk indicates input GST and GST TAF9 proteins.

Figure 6.

Acetylated TAF9 does not bind to chromatin and results in the disassociation of the entire TFIID complex from chromatin. A) Control and knockdown cells (shTAF9) were induced with or without doxycycline (Dox) for 3 d and transiently transfected with shTAF9-resistant TAF9 or TAF9 K5Q mutant (TAF9r or K5Qr). Cell extracts of treated cells were subjected to Western blot with indicated Abs. B, C) Knockdown cells (shTAF9) were induced with or without Dox for 3 d and transiently transfected with TAF9 or TAF9 K5Q mutant. Cells were subjected to ChIP assay with TAF9 and TAF6 (B); and TAF1, TAF5, and TBP (C). The resulting precipitated DNA was analyzed by using real-time PCR. CTL, control; TSS, transcription start site; UTR, untranslated region. Data are represented as means ± sem.

Figure 7.

Acetylation of TAF9 results in the disassembly of the TFIID complex. A) GST, GST-TAF9, or GST-TAF9 K5Q protein was immobilized on glutathione agarose beads and incubated with 3134 cell extracts. The associated protein was subjected to SDS-PAGE and was Western blotted with indicated Abs. B) Cell extracts from wild-type (WT) and TAF9-knockdown (KD) 3134 cells were immunoprecipitated (IP) with TBP Ab. The resulting immunoprecipitates were subjected to Western blot with indicated Abs. C) Model for HDAC1-dependent gene activation and acetylated HDAC1 mediated gene repression. TAF9 is deacetylated in the presence of HDAC1, and the TFIID complex is recruited to PU.1 promoter, which results in gene activation (left). HDAC1 is acetylated or inactivated by HDACi, which causes an inability to deacetylate TAF9 (right). Acetylated TAF9 is displaced from DNA. Acetylated TAF9 also causes the disassembly of the TFIID complex from the promoter, which results in gene repression. Inr, initiator. Asterisk indicates GST proteins.