Role of hPHF1 in H3K27 methylation and Hox gene silencing - PubMed (original) (raw)

Role of hPHF1 in H3K27 methylation and Hox gene silencing

Ru Cao et al. Mol Cell Biol. 2008 Mar.

Abstract

Polycomb group (PcG) proteins are required for maintaining the silent state of the homeotic genes and other important developmental regulators. The silencing function of the PcG proteins has been linked to their intrinsic histone modifying enzymatic activities. The EED-EZH2 complex, containing the core subunits EZH2, EED, SUZ12, and RbAp48, functions as a histone H3K27-specific methyltransferase. Here we describe the identification and characterization of a related EED-EZH2 protein complex which is distinguished from the previous complex by the presence of another PcG protein, hPHF1. Consistent with the ability of hPHF1 to stimulate the enzymatic activity of the core EED-EZH2 complex in vitro, manipulation of mPcl1, the mouse counterpart of hPHF1, in NIH 3T3 cells and cells of the mouse male germ cell line GC1spg results in global alteration of H3K27me2 and H3K27me3 levels and Hox gene expression. Small interfering RNA-mediated knockdown of mPcl1 affects association of the Eed-Ezh2 complex with certain Hox genes, such as HoxA10, as well as Hox gene expression concomitant with an alteration on the H3K27me2 levels of the corresponding promoters. Therefore, our results reveal hPHF1 as a component of a novel EED-EZH2 complex and demonstrate its important role in H3K27 methylation and Hox gene silencing.

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Figures

FIG. 1.

Purification and identification of hPHF1-containing EED-EZH2 complex. (A) Scheme used for purification of EED-EZH2 complexes. Numbers indicate the salt concentration (mM) at which the HMTase activity elutes from the respective columns. Nucleosomes were used as substrate in all of the HMTase assays. (B) HMTase activity assay of the fractions derived from the DEAE-5PW column. α-, anti-. (C) HMTase activity assay (top panel) and Western blot analysis (bottom panels) of fractions derived from the phenyl-Sepharose column. The antibodies used for Western blot analysis are indicated on the left side of the panel. (D) Silver staining of immunoprecipitated samples using antibodies against SUZ12. The positions of the protein size markers are indicated to the left of the panel. In, Ft, and IP represent input, flowthrough, and immunoprecipitates, respectively. The polypeptides copurified were identified by mass spectrometry, and their identities are indicated on the right. Representative peptides identified from mass spectrometry covering 41% of hPFH1 (GenBank accession no. BC008834) are shown in the box. (E) Phylogenetic tree of hPHF1 homologs from humans, mice, and flies. The relative positions of the conserved tudor domain and PHD domains are indicated. Numbers of amino acids for each protein are indicated.

FIG. 2.

Characterization of the hPHF1-containing EED-EZH2 complex in vitro. (A) Scheme for the steps carried out to reconstitute hPHF1-containing EED-EZH2 complex. a-Flag, anti-Flag. (B) Silver staining (top panel) and Western blotting (bottom panels) of the fractions derived from the Superose 6 gel-filtration column. The elution profile of the protein standards is indicated on top of the panel. The protein size markers are indicated to the left of the top panel. The antibodies (α-, anti-) used for Western blotting are indicated on the right. The six components of the reconstituted complex are indicated with asterisks. hPHF1 is stained weakly by silver. (C) Coomassie staining of a polyacrylamide-SDS gel containing the EED-EZH2 complexes in the presence or absence of hPHF1. Contaminating proteins from insect cells are indicated by asterisks. (D) Comparison of the substrate specificities of the two different recombinant EED-EZH2 complexes. Equal amounts of histone H3 alone or in octamer or mono- or oligonucleosome forms (bottom panel) were used as substrates for methylation by the two complexes shown in panel C (top two panels). (E) Time course experiment comparing the HMTase activities of the two complexes shown in panel C. A quantification of the top panel by scintillation counting is shown in the bottom panel.

FIG. 3.

Kinetic analysis of the EED-EZH2 complex in the presence or absence of hPHF1. Representative autoradiographs of HMTase assays containing different concentrations of 3H-SAM are shown in the top panels. Lineweaver-Burk plots (or double-reciprocal plots) of the reactions are shown in the middle panels. _V_max and Km were determined and are indicated on the plots. Michaelis-Menten plots were generated and are shown in the bottom panels. EED-EZH2 complex in the absence or presence of hPHF1 was used as the enzyme in panels A and B, respectively.

FIG. 4.

Knockdown of mPcl1 and Ezh2 in NIH 3T3 cells affects Hox gene expression. (A) Characterization of the stable mPcl1 knockdown cell lines by RT-PCR (left panel) and RT-qPCR (right panel). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as control (Ctrl) in both experiments. (B) Characterization of a stable Ezh2 knockdown cell lines by RT-PCR (left panel) and RT-qPCR (right panel). GAPDH was used as a control. (C) RT-PCR analysis of Hox gene expression pattern in response to knockdown of mPcl1 (lanes 2 and 3) or Ezh2 (lane 5). GAPDH serves as a control for equal input. The Hox genes affected by knockdown of mPcl1 or Ezh2 are underlined.

FIG. 5.

Knockdown of mPcl1 in GC1Spg cells affects Hox gene expression. (A) Characterization of the stable mPcl1 knockdown cell lines by RT-PCR (left panel) and RT-qPCR (right panel). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control (Ctrl) in both experiments. (B) RT-PCR analysis of Hox gene expression pattern in response to knockdown of mPcl1 relative to control. GAPDH serves as a control for equal input. The Hox genes whose expression is affected by mPcl1 knockdown are underlined. (C) RT-qPCR analysis of selected genes affected by mPcl1 knockdown shown in panel B. Results are normalized to GAPDH and are presented as means ± standard deviations from two independent experiments.

FIG. 6.

HoxA10 is a direct target of mPcl1. (A) Characterization of a stable Flag-mPcl1-rescued cell line by Western blotting. Equal loading was confirmed by Western blotting using α-tubulin antibody. α-Flag, anti-Flag antibody; Ctrl, control. (B) RT-PCR analysis of HoxA10 and mPcl1 expression in mock, mPcl1 knockdown, and Flag-mPcl1-rescued cell lines. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a control for equal input. (C) Western blot analysis of histone extracts from control, knockdown, and Flag-mPcl1-rescued cell lines. The antibodies used are indicated. Equal loading was verified by antibody against histone H3 (top panel). (D). Quantitative analysis of the changes of mono-, di-, and trimethylation shown in panel C by Licor image software. The data were normalized with total histone H3 and are presented as relative intensity from three independent experiments.

FIG. 7.

mPcl1 knockdown leads to a decreased H3K27me2 level at the promoter region, which can be rescued by expression of Flag-mPcl1. (A) Diagram of the HoxA10 gene in which the two exons are indicated by boxes labeled with 1 and 2. The regions analyzed are indicated with bars and are labeled from 1 to 8. Each region covers about 500 bp. (B) ChIP analysis of the HoxA10 gene using various antibodies (α-, anti-) indicated on the right. The cell lines used in ChIP are indicated at the bottom of each panel. The different amplicons analyzed are indicated on top of the panel. Ctrl, control. (C). ChIP-qPCR analysis of the relative levels of mono-, di-, and trimethylation of H3K27, Suz12, and Flag-mPcl1 on region 4 shown in panel A. Results are shown as percentages of enrichment relative to input. The data shown represent means ± standard deviations from two independent experiments.

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Role of hPHF1 in H3K27 methylation and Hox gene silencing - PubMed (original) (raw)