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. 2005 Dec;25(23):10338-51.
doi: 10.1128/MCB.25.23.10338-10351.2005.

Gfi1 coordinates epigenetic repression of p21Cip/WAF1 by recruitment of histone lysine methyltransferase G9a and histone deacetylase 1

Zhijun Duan  1 , Adrian Zarebski, Diego Montoya-Durango, H Leighton Grimes, Marshall Horwitz
Affiliations

Gfi1 coordinates epigenetic repression of p21Cip/WAF1 by recruitment of histone lysine methyltransferase G9a and histone deacetylase 1

Zhijun Duan et al. Mol Cell Biol. 2005 Dec.

Abstract

The growth factor independent 1 (Gfi1) transcriptional regulator oncoprotein plays a crucial role in hematopoietic, inner ear, and pulmonary neuroendocrine cell development and governs cell processes as diverse as self-renewal of hematopoietic stem cells, proliferation, apoptosis, differentiation, cell fate specification, and oncogenesis. However, the molecular basis of its transcriptional functions has remained elusive. Here we show that Gfi1 recruits the histone lysine methyltransferase G9a and the histone deacetylase 1 (HDAC1) in order to modify the chromatin of genes targeted for repression by Gfi1. G9a and HDAC1 are both in a repressive complex assembled by Gfi1. Endogenous Gfi1 colocalizes with G9a, HDAC1, and K9-dimethylated histone H3. Gfi1 associates with G9a and HDAC1 on the promoter of the cell cycle regulator p21Cip/WAF1, resulting in an increase in K9 dimethylation at histone H3. Silencing of Gfi1 expression in myeloid cells reverses G9a and HDAC1 recruitment to p21Cip/WAF1 and elevates its expression. These findings highlight the role of epigenetics in the regulation of development and oncogenesis by Gfi1.

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Figures

FIG. 1.
FIG. 1.
Ectopic expression of Gfi1 in HeLa cells inhibits growth and retards cell cycle. (A) Growth curve of HeLa cells transiently transfected with vectors expressing Gfi1 or β-Gal, as a control. (B) Flow cytometric analysis of HeLa cells transiently cotransfected with either Gfi1 or β-Gal along with GFP expression vectors. Cells expressing or not expressing GFP (as a marker of transfection) were sorted and analyzed for DNA content by Hoechst 33342 staining.
FIG. 2.
FIG. 2.
Gfi1 transcriptionally represses its target genes via histone H3-K9 dimethylation in HeLa cells. (A) ChIP assay demonstrating target promoter occupancy with Gfi1 overexpression compared to β-Gal-expressing negative control in transfected HeLa cells. Antibodies to Gfi1, but not normal IgG, immunoprecipitate (IP) chromatin fragments from which specific sequences within each target promoter can be PCR amplified and resolved by agarose gel electrophoresis with ethidium staining. (B) Semiquantitative RT-PCR (products resolved by ethidium-stained agarose gels, left) and real-time RT-PCR (TaqMan) assays (graphed results, right) confirm that Gfi1 promoter occupancy in transfected HeLa cells coincides with transcriptional repression, compared to nontarget β-actin, PDE4D, and GAPDH negative controls. ddH2O, double-distilled water as PCR control. (C) Recruitment of Gfi1 to target genes increases dimethylated histone H3-K9 (diMeH3K9), as shown by ChIP assay in transfected HeLa cells.
FIG. 2.
FIG. 2.
Gfi1 transcriptionally represses its target genes via histone H3-K9 dimethylation in HeLa cells. (A) ChIP assay demonstrating target promoter occupancy with Gfi1 overexpression compared to β-Gal-expressing negative control in transfected HeLa cells. Antibodies to Gfi1, but not normal IgG, immunoprecipitate (IP) chromatin fragments from which specific sequences within each target promoter can be PCR amplified and resolved by agarose gel electrophoresis with ethidium staining. (B) Semiquantitative RT-PCR (products resolved by ethidium-stained agarose gels, left) and real-time RT-PCR (TaqMan) assays (graphed results, right) confirm that Gfi1 promoter occupancy in transfected HeLa cells coincides with transcriptional repression, compared to nontarget β-actin, PDE4D, and GAPDH negative controls. ddH2O, double-distilled water as PCR control. (C) Recruitment of Gfi1 to target genes increases dimethylated histone H3-K9 (diMeH3K9), as shown by ChIP assay in transfected HeLa cells.
FIG. 3.
FIG. 3.
Gfi1 associates with G9a in vivo. (A) Antibodies to Gfi1 coimmunoprecipitate Gfi1 with G9a, but not with Suv39H1, when plasmids expressing native Gfi1, HA epitope-tagged G9a, and Myc epitope-tagged Suv39H1 are cotransfected together into HeLa cells, as revealed by Western blots (WB) with detection by the indicated antibodies. (B) Gfi1 coimmunoprecipitates with HA-tagged G9a when both are transfected into HeLa cells, using either anti-HA or anti-Gfi1 antibodies for immunoprecipitation and anti-Gfi1 and anti-HA or anti-G9a antibodies (three left panels, respectively) for Western blot detection; however, Gfi1 does not coimmunoprecipitate with Myc-tagged Suv39H1 even when both are coexpressed in HeLa cells (as assayed by immunoprecipitation with anti-Gfi1 and Western blotting with anti-Myc, right panel). (C) Endogenous Gfi1 forms an immunoprecipitable complex with endogenous G9a in HL-60 cells, demonstrated by immunoprecipitation with either anti-Gfi1 or anti-G9a. Similar results were also observed in U937 promonocytes and Jurkat cells (not shown). (Dash denotes blank lane.) (D) Truncated forms of Gfi1 used to identify domains required for association with G9a. Myc-tagged Gfi1 mutants were cotransfected with HA-tagged G9a into HeLa cells; immunoprecipitation was performed with anti-HA, and Western blotting was performed with anti-Myc.
FIG. 4.
FIG. 4.
Gfi1 complex recruits G9a-type histone methyltransferase activity. (A) Immunoprecipitated Gfi1-containing complex possesses histone methyltransferase activity. Vectors expressing either Gfi1 or β-Gal, as a negative control, were transfected into HeLa cells, and cell extracts were immunoprecipitated with anti-Gfi1 or nonspecific antibody, then incubated with histone mixtures (H1 and core histones) and S-[3H]adenosylmethionine, and resolved by SDS-PAGE with Coomassie blue staining (bottom panel). Autoradiography (top panel) demonstrates transfer of tritiated methyl to histones. (B) Coexpression of G9a in HeLa cells, but not Suv39H1, increases histone methyltransferase activity of the immunoprecipitated Gfi1 complex. G9a was HA tagged, Suv39 was Myc tagged, and immunoprecipitation was performed with anti-Gfi1. (Western blotting with the indicated antibodies was performed on the cell extract to confirm expected expression [bottom three panels].) (C) Histone methyltransferase activity recruitment by Gfi1 requires interaction with G9a. Immunoprecipitates of full-length Gfi1, but not the Gfi1(N160) fragment (which does not interact with G9a), demonstrate histone methyltransferase activity. [Gfi1 was Myc tagged, and anti-Myc was used for immunoprecipitation, because the Gfi1 antibody does not recognize the Gfi1(N160) fragment.] (D) Endogenous Gfi1 complex immunoprecipitated from HL-60 cells possesses histone methyltransferase activity, and the Gfi1 immunoprecipitate was similarly assayed. (E) The Gfi1 complex predominantly methylates histone H3, as demonstrated by comparing a mixture of histone substrates to those enriched for H3. (F) The Gfi1 complex methylates H3 but not H4. (G) The Gfi1 complex methylates H3-K9, but not H3-K4. A synthetic peptide corresponding to the amino-terminal 20 residues of H3 and one in which the K4 was predimethylated are both methylated, but predimethylation of K9 diminishes its acceptance as a substrate. (H) Gfi1 complex demonstrates the same site specificity as purified G9a on a series of recombinant histone H3 substrates. The first 84 amino acids of H3, in which lysines were each mutated to an arginine, were purified as GST fusion proteins and tested as substrates. Immunoprecipitates were prepared from HeLa cells transfected with the indicated vector, and their activity was compared to that of purified G9a. Cotransfection with a G9a expression vector does not add significant activity to the Gfi1 immunoprecipitate. Asterisks mark positions corresponding between Coomassie blue-stained and autoradiogram-detected proteins on SDS-polyacrylamide gels.
FIG. 5.
FIG. 5.
G9a and HDAC1 are in the same complex with Gfi1. (A) Transfected Gfi1 coimmunoprecipitates with endogenous HDAC1 in HeLa cells. (B) Endogenous Gfi1 forms an immunoprecipitable complex with endogenous HDAC1 in HL-60 cells. Immunoprecipitation was carried out with either anti-Gfi1 or anti-HDAC1 antibody. Similar results were also observed with U937 and Jurkat cells (not shown). (C) Gfi1 domains required for association with endogenous HDAC1 in HeLa cells are identified with truncations of Gfi1 (same approach as shown in Fig. 3D). (D) Both HDAC1 and G9a form stable complexes with Gfi1 that are resistant to washing at increasing salt concentrations following immunoprecipitation. (E) G9a and HDAC1 exist in the same complex recruited by Gfi1. Vectors expressing native HDAC1, HA-tagged G9a, and Myc-tagged Gfi1 were transfected into HeLa cells; immunoprecipitation was performed with anti-HDAC1; and Western blots were detected with antibodies specific for each of the three proteins. The Gfi1 expression vector was replaced with one containing β-Gal to serve as a negative control. (Ten percent of input is shown, to normalize signals.)
FIG. 5.
FIG. 5.
G9a and HDAC1 are in the same complex with Gfi1. (A) Transfected Gfi1 coimmunoprecipitates with endogenous HDAC1 in HeLa cells. (B) Endogenous Gfi1 forms an immunoprecipitable complex with endogenous HDAC1 in HL-60 cells. Immunoprecipitation was carried out with either anti-Gfi1 or anti-HDAC1 antibody. Similar results were also observed with U937 and Jurkat cells (not shown). (C) Gfi1 domains required for association with endogenous HDAC1 in HeLa cells are identified with truncations of Gfi1 (same approach as shown in Fig. 3D). (D) Both HDAC1 and G9a form stable complexes with Gfi1 that are resistant to washing at increasing salt concentrations following immunoprecipitation. (E) G9a and HDAC1 exist in the same complex recruited by Gfi1. Vectors expressing native HDAC1, HA-tagged G9a, and Myc-tagged Gfi1 were transfected into HeLa cells; immunoprecipitation was performed with anti-HDAC1; and Western blots were detected with antibodies specific for each of the three proteins. The Gfi1 expression vector was replaced with one containing β-Gal to serve as a negative control. (Ten percent of input is shown, to normalize signals.)
FIG. 6.
FIG. 6.
Gfi1 recruits G9a and HDAC1 to the c-Myc and p21Cip/WAF1 promoters and increases H3-K9 methylation. HeLa cells were cotransfected with the noted combinations of plasmids and subjected to ChIP assays with antibodies against Gfi1, Myc tag, HA tag, dimethylated histone H3-K9, or control IgG. Each group of three lanes for the c-Myc promoter represents PCR analysis of threefold serial dilutions of the DNA templates. Analysis of the p21Cip/WAF1 promoter and the PDE4D promoter, included as a negative control, was performed at the highest of the three DNA concentrations.
FIG. 7.
FIG. 7.
Endogenous Gfi1 recruits G9a and HDAC1 to p21Cip/WAF1 promoter and transcriptionally represses the expression of p21Cip/WAF1 in HL-60 cells but does not have such effects on c-Myc. (A) Western blot demonstrating specificity of Gfi1, β-actin, and nonspecific control siRNAs in reducing protein levels in treated HL-60 cells. (B) Epifluorescence microscopy confirming uptake in HL-60 cells transfected with Alexa Fluor 488-labeled Gfi1 siRNA. Nuclei were counterstained with DAPI (4′,6′-diamidino-2-phenylindole). (C) Fluorescence-activated cell sorting of HL-60 cells electroporated with fluorescent Alexa Fluor 488-labeled siRNA to distinguish transfected from nontransfected populations. Left panel, side scatter profile for setting gates (pink); middle panel, cells not treated with siRNA; right panel, cells electroporated with Alexa Fluor 488-labeled Gfi1 siRNA with sorted populations identified. (D) Real-time RT-PCR (TaqMan) analysis of Gfi1 and p21Cip/WAF1 expression in control, untransfected, and transfected HL-60 cells sorted above. From left to right, HL-60 cells electroporated with unlabeled β-actin control siRNA (unsorted), electroporated with fluorescently labeled Gfi1 siRNA and sorted negatively for fluorescence (nontransfected), and electroporated with Gfi1 siRNA and sorted positively for fluorescence (transfected). (E) ChIP in HL-60 cells showing the presence of endogenous Gfi1, HDAC1, and G9a on p21Cip/WAF1 promoter and Gfi1 and HDAC1, but not G9a, on c-Myc promoter but neither Gfi1, HDAC1, nor G9a on control PDE4D promoter. (F) Specific silencing of Gfi1 by siRNA, but not nonspecific control siRNA (as shown by Western blotting, right panel), diminishes binding of G9a and HDAC1, diminishes incorporation of dimethylated histone H3-K9, and modestly increases incorporation of AcyH3, on the p21Cip/WAF1 promoter in HL-60 cells (as revealed by ChIP, left and middle panels). However, Gfi1 siRNA does not affect G9a or HDAC1 recruitment to the c-Myc promoter and correspondingly shows no changes in dimethylated histone H3-K9 and AcyH3.
FIG. 7.
FIG. 7.
Endogenous Gfi1 recruits G9a and HDAC1 to p21Cip/WAF1 promoter and transcriptionally represses the expression of p21Cip/WAF1 in HL-60 cells but does not have such effects on c-Myc. (A) Western blot demonstrating specificity of Gfi1, β-actin, and nonspecific control siRNAs in reducing protein levels in treated HL-60 cells. (B) Epifluorescence microscopy confirming uptake in HL-60 cells transfected with Alexa Fluor 488-labeled Gfi1 siRNA. Nuclei were counterstained with DAPI (4′,6′-diamidino-2-phenylindole). (C) Fluorescence-activated cell sorting of HL-60 cells electroporated with fluorescent Alexa Fluor 488-labeled siRNA to distinguish transfected from nontransfected populations. Left panel, side scatter profile for setting gates (pink); middle panel, cells not treated with siRNA; right panel, cells electroporated with Alexa Fluor 488-labeled Gfi1 siRNA with sorted populations identified. (D) Real-time RT-PCR (TaqMan) analysis of Gfi1 and p21Cip/WAF1 expression in control, untransfected, and transfected HL-60 cells sorted above. From left to right, HL-60 cells electroporated with unlabeled β-actin control siRNA (unsorted), electroporated with fluorescently labeled Gfi1 siRNA and sorted negatively for fluorescence (nontransfected), and electroporated with Gfi1 siRNA and sorted positively for fluorescence (transfected). (E) ChIP in HL-60 cells showing the presence of endogenous Gfi1, HDAC1, and G9a on p21Cip/WAF1 promoter and Gfi1 and HDAC1, but not G9a, on c-Myc promoter but neither Gfi1, HDAC1, nor G9a on control PDE4D promoter. (F) Specific silencing of Gfi1 by siRNA, but not nonspecific control siRNA (as shown by Western blotting, right panel), diminishes binding of G9a and HDAC1, diminishes incorporation of dimethylated histone H3-K9, and modestly increases incorporation of AcyH3, on the p21Cip/WAF1 promoter in HL-60 cells (as revealed by ChIP, left and middle panels). However, Gfi1 siRNA does not affect G9a or HDAC1 recruitment to the c-Myc promoter and correspondingly shows no changes in dimethylated histone H3-K9 and AcyH3.
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