Differential histone H3 Lys-9 and Lys-27 methylation profiles on the X chromosome - PubMed (original) (raw)
Differential histone H3 Lys-9 and Lys-27 methylation profiles on the X chromosome
Claire Rougeulle et al. Mol Cell Biol. 2004 Jun.
Free PMC article
Abstract
Histone H3 tail modifications are among the earliest chromatin changes in the X-chromosome inactivation process. In this study we investigated the relative profiles of two important repressive marks on the X chromosome: methylation of H3 lysine 9 (K9) and 27 (K27). We found that both H3K9 dimethylation and K27 trimethylation characterize the inactive X in somatic cells and that their relative kinetics of enrichment on the X chromosome as it undergoes inactivation are similar. However, dynamic changes of H3K9 and H3K27 methylation on the inactivating X chromosome compared to the rest of the genome are distinct, suggesting that these two modifications play complementary and perhaps nonredundant roles in the establishment and/or maintenance of X inactivation. Furthermore, we show that a hotspot of H3K9 dimethylation 5' to Xist also displays high levels of H3 tri-meK27. However, analysis of this region in G9a mutant embryonic stem cells shows that these two methyl marks are dependent on different histone methyltransferases.
Figures
FIG. 1.
ChIP analysis of H3 lysines 9 and 27 methylation in female ES cells. Sonicated chromatin was immunoprecipitated using antibodies specific for dimethylated H3K9 (A to D) and di/trimethylated H3K27 (E to H). The immunoprecipitated DNA was analyzed by quantitative real-time PCR with primers distributed across a 350-kb region 5′ to Xist (A and E), across the Xist/Tsix region (B and F), in promoters (▪) and exons (□) of X-linked genes (C and G), and of autosomal genes (D and H). The graphs represent the percentage of immunoprecipitation obtained for each position tested. The differences in scale obtained by using the anti-dimethyl K9 and the anti-di/trimethyl K27 most likely correspond to differences in both the degree of enrichment for the modified histones, as well as in the efficiency of each antibody to immunoprecipitate chromatin. A partial map of the X inactivation center (Xic) is represented below the graphs to schematize the relative positions of the Xic primers used. The map ends after Chic1 and Cdx4, since the other genes tested map outside the Xic.
FIG. 2.
Simultaneous analysis of the H3K9 and H3K27 methylation enrichment in the vicinity of the Xist gene in ES cells. Representative nuclei of undifferentiated or differentiating ES cells hybridized with Xist RNA (green) and double stained with antibodies to H3 di-meK9, detected by using goat anti-rabbit Alexa 568 secondary antibody (Molecular Probes) (pseudocolored red, left), and H3 tri-meK27, detected by using goat anti-mouse Alexa 680 secondary antibody (Molecular Probes) (pseudocolored red, right). Arrowheads indicate the position of the unstable Xist/Tsix primary transcript and an accompanying hotspot of H3 di-meK9 but a lack of such a hotspot of H3 tri-meK27 in both undifferentiated and differentiating ES cells. The arrow indicates the accumulation of Xist RNA accompanied by enrichment of both H3 di-meK9 and H3 tri-meK27 in differentiating ES cells. It should be noted that the peptide competitions shown in Fig. S2 in the supplemental material are also applicable to the data shown here.
FIG. 3.
Analysis of H3K9, H3K27, and H3K4 methylation profiles in the region 5′ to Xist in G9a mutant ES cell lines. Three male cell lines were used in the present study, a double-null G9a (G9a−/−) mutant (31), a double-null G9a carrying a G9a transgene (G9a−/− Tg) (36), and the parental line (WT). The results of three experiments carried out on independent chromatin preparations are shown. Three different batches of H3 di-meK9 antibody with variable ChIP efficiencies were used. The low efficiency of some of the batches (batches 27271 and 23032) compared to others (batch 23424) explains the high standard deviations seen in the WT cell line. The same batches of H3 di/tri-meK27 (7B11) and H3 di-meK4 (24747) were used throughout. Six positions within the hotspot of H3K9 methylation were analyzed (░⃞), as well as the promoters of Xist (Xt1) and Hprt (Hprt-p).
FIG. 4.
Kinetics of histone H3K27 and H3K9 methylation on the Xist RNA-coated X chromosome during the differentiation of female ES cells. (A) Representative immunofluorescence for histone H3 trimethyl K27 (H3 di/tri-meK27, columns 2 and 3) and dimethyl K9 (H3 di-meK9, columns 4 and 5) combined with Xist RNA FISH. Immunodetections with goat anti-mouse Alexa 680- and goat anti-rabbit Alexa 568-conjugated secondary antibodies (in red, columns 2 and 4), were combined with Xist RNA FISH (Spectrum green-labeled probe, green, columns 3 and 5). The first row shows a typical example of a nucleus with an enrichment only for H3 di/tri-meK27 on the Xist RNA domain (column 3, arrowhead; yellow indicates the overlap of green and red signals). The second row shows an example of enrichment for both H3 di/tri-meK27 (arrowheads) and di-meK9 (arrows) on the Xist RNA domain (columns 3 and 5, yellow). DNA is stained with DAPI (blue, column 1). The bar represents 5 μm in each case. (B) Relative kinetics of H3K9 and H3K27 methylation on the X chromosome undergoing inactivation in female ES cells during differentiation (9 days of differentiation of female ES cells). Between 50 and 200 cells were counted for each time point.
FIG. 5.
ChIP analysis of H3 lysines 9 and 27 methylation in 13.5 dpc mouse embryonic fibroblasts. The levels of H3K9 dimethylation (A and B) and H3K27 di/trimethylation (C and D) within several X-linked (Chic1, G6pd, and Hprt) and autosomal (Myc and β-actin) genes were analyzed in both male (A and C) and female (B and D) fibroblasts. Two positions were analyzed for each gene: one in the promoter (▪) and one in an exon (□). Promoter-specific primers were designed to span the transcription initiation site.
FIG. 6.
Histone H3 dimethylation at K9 and di- and trimethylation at K27 on the inactive X chromosome in female fibroblasts. (A) Representative immunofluorescence for histone H3 modifications in mouse embryonic (13.5 dpc) fibroblasts. The histone H3 modifications (column 2), H3 di-meK9 (row 1), H3 di/tri-meK27 (row 2), and H3 di-meK27 (row 3) are shown combined with Xist RNA FISH data (column 3). Green coloration indicates the absence of the modification on the Xist RNA-coated X chromosome; yellow coloration (overlap of green and red signals) shows the enrichment of the modification on the Xist domain (arrowheads). DNA is stained with DAPI (blue, column 1). (B) Representative immunofluorescence for histone H3 modifications in ear fibroblasts from an 8-week-old female. Note the absence of H3K27 methylation enrichment on the Xist RNA-coated X chromosome (asterisk) and the variable nuclear levels of H3K27 methylation in some nuclei. The bar represents 5 μm in each case.
References
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