Relationship between histone H3 lysine 9 methylation, transcription repression, and heterochromatin protein 1 recruitment - PubMed (original) (raw)

Relationship between histone H3 lysine 9 methylation, transcription repression, and heterochromatin protein 1 recruitment

M David Stewart et al. Mol Cell Biol. 2005 Apr.

Abstract

Histone H3 lysine 9 (H3-K9) methylation has been shown to correlate with transcriptional repression and serve as a specific binding site for heterochromatin protein 1 (HP1). In this study, we investigated the relationship between H3-K9 methylation, transcriptional repression, and HP1 recruitment by comparing the effects of tethering two H3-K9-specific histone methyltransferases, SUV39H1 and G9a, to chromatin on transcription and HP1 recruitment. Although both SUV39H1 and G9a induced H3-K9 methylation and repressed transcription, only SUV39H1 was able to recruit HP1 to chromatin. Targeting HP1 to chromatin required not only K9 methylation but also a direct protein-protein interaction between SUV39H1 and HP1. Targeting methyl-K9 or a HP1-interacting region of SUV39H1 alone to chromatin was not sufficient to recruit HP1. We also demonstrate that methyl-K9 can suppress transcription independently of HP1 through a mechanism involving histone deacetylation. In an effort to understand how H3-K9 methylation led to histone deacetylation in both H3 and H4, we found that H3-K9 methylation inhibited histone acetylation by p300 but not its association with chromatin. Collectively, these data indicate that H3-K9 methylation alone can suppress transcription but is insufficient for HP1 recruitment in the context of chromatin exemplifying the importance of chromatin-associated factors in reading the histone code.

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Figures

FIG. 1.

FIG. 1.

Lysine 9 methylation alone is insufficient for recruitment of HP1 to chromatin. (A) Experimental design. mRNAs encoding Gal4 fusion proteins [Gal-G9a(SET) and Gal-SUV39H1] and HA-HP1 isoforms were injected into the oocyte cytoplasm. The single-stranded reporter DNA was 4xUAS-TRβA-CAT. The injected oocytes were incubated overnight (O/N) and processed for Western blot analysis and ChIP assay. (B) Western blots showing expression of the Gal4-G9a and Gal4-SUV39H1 fusion proteins and the three HA-tagged HP1 isoforms in Xenopus oocyte extracts. The positions of molecular mass markers (in kilodaltons) are shown to the left of the blots. Cx, no mRNA injection; α Gal4(DBD), anti-Gal4(DBD) antibody; α HA-Tag, anti-HA tag antibody. (C) ChIP assays to study modifications to histones associated with the promoter of the reporter DNA. Antibodies against H3 dimethyl-K9 (H3 dimeK9) and H3 dimethyl-K27 (H3 dimeK27) were used in this experiment. ChIP using a Gal4(DBD)-specific antibody was included to show that both fusion proteins bound the reporter. Both Gal-G9a(SET) and Gal-SUV39H1 induced methylation of H3-K9 and decreased H3-K9 acetylation (H3 acK9). The results of these and replicate experiments were analyzed by LS-ANOVA and subject to preplanned orthogonal contrasts. The fold difference between the LS mean for each group and the control group (Cx) is reported below the appropriate lane. Values that are significantly different (P ≤ 0.05) are indicated by different superscript letters. (D) SUV39H1, but not G9a(SET), was able to recruit all HP1 isoforms to chromatin as determined by ChIP assay using anti-HA tag antibody.

FIG. 2.

FIG. 2.

G9a(SET)-induced H3-K9 methylation is sufficient for transcriptional repression in the absence of HP1 recruitment. (A) Experimental design. The groups of oocytes were injected with the indicated mRNA or DNA, incubated overnight (O/N), and processed by three methods: Western blot analysis to measure protein expression, primer extension analysis to measure transcription, and ChIP assay to study histone modifications. The reporter DNA was 4xUAS-TK-CAT, 4xUAS-AdML-CAT, or 4xUAS-TRβA-CAT. (B) Expression of wild-type (wt) and mutant (mut) H1113K Gal-G9a(SET) proteins in Xenopus oocytes as revealed by Western blot analysis using a Gal4(DBD)-specific antibody. (C) ChIP assays showing that both G9a(SET) constructs bound the reporter but that only the wild type was able to methylate K9. Antibodies against H3 dimethyl-K9, -K4, and -K27 were used as indicated (H3 dimeK9, H3 dimethyl-K9). rIgG, normal rabbit IgG. cx, no mRNA injection. (D) The wild-type protein, but not mutant Gal-G9a(SET), suppressed transcription of all three reporter genes as determined by primer extension analysis. For the TRβA-CAT reporter, oocytes were divided into five groups. The first group received no mRNA (cx), and the other groups received one of two concentrations (undiluted or 1:3 dilution) of wild-type (wt) Gal-G9a(SET) or mutant (mut) H1113K mRNA. For the AdML- and TK-CAT reporters, mRNA was injected at a single concentration (undiluted). All the experiments in this figure were repeated three times to ensure reproducibility.

FIG. 3.

FIG. 3.

Transcriptional repression by lysine 9 methylation involves histone deacetylation. (A) ChIP assays comparing the effect of K9 methylation on acetylation of histones H3 (acH3) and H4 (acH4). Groups of oocytes were not injected with mRNA (cx) or were injected with wild-type (wt) Gal-G9a(SET) or mutant (mut) (H1113K) Gal-G9a(SET) mRNA as indicated and the single-stranded 4xUAS-TRβA-CAT reporter. After overnight incubation, ChIP assays were performed using the indicated antibodies. Fold differences between the LS mean for each group and the control group (cx) are reported to the right of the blots. H3 dimeK9, H3 dimethyl-K9; H3 acK9, H3 acetyl-H9. (B) Primer extension assays to determine the role of histone deacetylation on methyl-K9-induced transcriptional repression. The HDAC inhibitor TSA (1.65 μM) was added (+) immediately after injection of reporter DNA. RNA was prepared the following day. Note that TSA blocked repression by Gal-G9a(SET). (C) Effect of inhibiting deacetylase activity with TSA on the histone modifications induced by Gal-G9a(SET). Oocytes were injected as described above for panel B, and ChIP analyses were performed using groups of oocytes injected with the 4xUAS-TRβA-CAT reporter DNA. Note that H3-K9 methylation was almost completely blocked by TSA treatment and that TSA induced histone acetylation. Fold differences between the LS mean for each group and the control group (Cx) are reported to the right of the blots.

FIG. 4.

FIG. 4.

(A) Western blots showing expression of p300 and Gal-G9a(SET) proteins in Xenopus oocyte extracts. (B) ChIP assays to determine the effect of K9 methylation on the association of p300 with chromatin and its ability to acetylate histones. K9 methylation did not inhibit the association of p300 with chromatin [p300 versus p300 and G9a(SET), P = 0.19]; however, it did block p300-induced H3 acetylation. H3 dimeK9, H3 dimethyl-K9; acH3, acetyl-H3.

FIG. 5.

FIG. 5.

SUV39H1 suppresses transcription and recruits HP1 through multiple mechanisms. (A) Schematic diagrams of the various Gal-SUV39H1 fusion proteins. mut, mutant. (B) Expression of the various Gal-SUV39H1 fusion proteins in Xenopus oocytes the day after injection of their corresponding in vitro-synthesized mRNA. Western blots were performed using a Gal4(DBD)-specific antibody. The positions of molecular mass markers (in kilodaltons) are shown to the left of the blots. wt, wild type; mut, mutant. (C) Recruitment of HP1 to chromatin by the Gal-SUV39H1 fusion proteins. Groups of oocytes were injected with mRNAs encoding the various Gal-SUV39H1 fusion proteins as indicated, and ChIP assays were performed using anti-Gal4(DBD) or anti-HA tag antibody to determine binding of Gal4 fusions or HP1α to chromatin, respectively. Note that for the full-length protein, the recruitment of HP1α is not dependent on HMT activity, whereas the recruitment of HP1α by SUV(SET) is dependent on HMT activity. cx, no mRNA injected. (D) Effects of the Gal-SUV39H1 fusion proteins on transcription of the 4xUAS-TRβA-CAT reporter gene as determined by primer extension. Note that for the full-length protein, repression is not dependent on the HMT activity, whereas repression by SUV(SET) is HMT activity dependent.

FIG. 6.

FIG. 6.

Both lysine 9 methylation and a HP1-interacting protein must be targeted to chromatin for HP1 recruitment. (A) ChIP assay to compare di- and trimethylation of K9 induced by Gal-G9a(SET), Gal-SUV39H1, and Gal-SUV(SET). The fold difference between the LS mean for each group and the control group (Cx) is reported beneath each blot. Note that SUV(SET) mainly induced K9 dimethylation, while the full-length SUV39H1 primarily induced trimethylation of K9. H3 dimeK9, H3 trimeK9, H3 dimethyl-K9; H3 trimethyl-K9. (B) Each construct was tested for its ability to directly interact with HP1α by an in vitro GST pull-down assay. For a control, an equal amount of GST protein was used. SUV(SET)m, mutant SUV(SET). (C) The novel HP1 interaction site in the C terminus of SUV39H1 was further mapped to the C-terminal half of the SET domain excluding the post-SET region by in vitro pull-down assay. (D) Reciprocal coimmunoprecipitations to study the two HP1 interaction sites on SUV39H1. HA-HP1α easily coimmunoprecipitated with the N terminus of SUV39H1; however, the interaction between HP1α and the SUV39H1 SET domain was not detected, implying a weak protein-protein interaction. (E) Tethering the N-terminal region of SUV39H1 (SUVΔC) to chromatin resulted in increased H3-K9 methylation as revealed by ChIP assay. Cx, no mRNA injected; wt, wild type; mut, mutant.

FIG. 7.

FIG. 7.

Targeting a HP1-interacting protein to chromatin is not sufficient for HP1 recruitment in the absence of lysine 9 methylation. (A) Experimental design. Oocytes were divided into four groups. All groups received mRNA encoding HA-HP1α. Two groups received an additional injection of Gal-SUV39H1 mRNA, while the other two groups did not (Cx groups). After 3 h, all oocytes were injected with the 4xUAS-TRβA-CAT reporter and then immediately treated with TSA (1.65 μM) or not treated with TSA. After overnight (O/N) incubation, the oocytes were harvested for ChIP analysis using the indicated antibodies. (B) In the absence of TSA (−), Gal-SUV39H1 methylated K9 and recruited HP1 to chromatin; however, in the presence of TSA (+), K9 methylation was reduced and HP1 recruitment was impaired. acH3, acetyl-H3; H3 dimeK9, H3 dimethyl-K9; H3 trimeK9, H3 trimethyl-K9. (C) TSA did not affect the in vitro binding of SUV39H1 to HP1α as determined by GST pull-down assay. (D) TSA did not affect the in vivo binding of SUV39H1 to HP1α as determined by coimmunoprecipitation.

FIG. 8.

FIG. 8.

Two-interaction model for HP1 recruitment to chromatin. Based on the data presented here and the observations of others (see Discussion), K9 methylation by itself is insufficient to recruit HP1 to chromatin. Stable association of HP1 with chromatin involves multiple interactions. HP1 recruitment to chromatin by SUV39H1 requires both a direct interaction between HP1 and SUV39H1 and methyl-K9 (meK9) binding.

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