Mouse polycomb proteins bind differentially to methylated histone H3 and RNA and are enriched in facultative heterochromatin - PubMed (original) (raw)

Mouse polycomb proteins bind differentially to methylated histone H3 and RNA and are enriched in facultative heterochromatin

Emily Bernstein et al. Mol Cell Biol. 2006 Apr.

Abstract

The chromodomain (CD) of the Drosophila Polycomb protein exhibits preferential binding affinity for histone H3 when trimethylated at lysine 27. Here we have investigated the five mouse Polycomb homologs known as Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8. Despite a high degree of conservation, the Cbx chromodomains display significant differences in binding preferences. Not all CDs bind preferentially to K27me3; rather, some display affinity towards both histone H3 trimethylated at K9 and H3K27me3, and one CD prefers K9me3. Cbx7, in particular, displays strong affinity for both H3K9me3 and H3K27me3 and is developmentally regulated in its association with chromatin. Cbx7 associates with facultative heterochromatin and, more specifically, is enriched on the inactive X chromosome. Finally, we find that, in vitro, the chromodomain of Cbx7 can bind RNA and that, in vivo, the interaction of Cbx7 with chromatin, and the inactive X chromosome in particular, depends partly on its association with RNA. We propose that the capacity of this mouse Polycomb homolog to associate with the inactive X chromosome, or any other region of chromatin, depends not only on its chromodomain but also on the combination of histone modifications and RNA molecules present at its target sites.

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Figures

FIG. 1.

FIG. 1.

Analysis of Pc-like Cbx CD binding affinities for trimethylated H3K9 and H3K27. (A) ClustalW alignment of the five mouse Pc-like CDs (aa 1 to 62) and the Drosophila Pc CD (aa 16 to 78). The asterisks represent the caging aromatic residues that mediate the histone methyl-lysine interaction. Note the high degree of conservation among these family members. (B) Fluorescence polarization of Cbx7 (top) and Cbx4 (middle) to histone tail peptides, including the me1, me2, and me3 states on residues K9 and K27 of H3, and K20me1, K20me2, and K20me3 of H4, respectively. Full-length Cbx7 (bottom) was tested against all of the above except for the monomethylated forms of H3K9 and H3K27, H4K20me2 and H4K20me3, and behaves identically to its CD for peptides tested. Histone tail sequences are represented above; note the ARKS motifs of H3K9 and H3K27. See panel D for actual peptides used. (C) Fluorescence polarization of Cbx5 (mouse HP1α) to H3K9 and H3K27 histone tail peptides in the me1, me2, and me3 states. (D) Dissociation constants (Kd, in micromolars) for Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8, as well as Cbx5 (mHP1α), with each series of methylated peptides for each backbone shown. Low-micromolar binding constants are highlighted in red. The asterisk depicts weak binding of Cbx7 for H3K9me2 (a mark that is enriched on the Xi). Values represent averages ± standard deviations for at least three independent experiments in all cases (except for Cbx5 with certain peptides). ND, not determined. (E) Peptide pull-down assays. (Left) All CDs were examined for binding to unmodified and trimethylated peptides representing H3K9, H3K27, and H4K20. Results support those obtained by FP (D). (Right) Cbx7 CD, Cbx7 caging aromatic point mutant F11A, and human HP1β CD recombinant proteins were tested for the ability to bind unmodified and me1, me2, and me3 peptides of H3K9 and K27. Note the trimethyl specificity of Cbx7. (Right, bottom) GST and full-length Cbx7 were examined for binding to unmodified and trimethylated biotinylated peptides of H3K9 and H3K27. Full-length Cbx7 behaves the same as its CD alone. GST does not bind any peptide, as expected. un, unmodified.

FIG. 1.

FIG. 1.

Analysis of Pc-like Cbx CD binding affinities for trimethylated H3K9 and H3K27. (A) ClustalW alignment of the five mouse Pc-like CDs (aa 1 to 62) and the Drosophila Pc CD (aa 16 to 78). The asterisks represent the caging aromatic residues that mediate the histone methyl-lysine interaction. Note the high degree of conservation among these family members. (B) Fluorescence polarization of Cbx7 (top) and Cbx4 (middle) to histone tail peptides, including the me1, me2, and me3 states on residues K9 and K27 of H3, and K20me1, K20me2, and K20me3 of H4, respectively. Full-length Cbx7 (bottom) was tested against all of the above except for the monomethylated forms of H3K9 and H3K27, H4K20me2 and H4K20me3, and behaves identically to its CD for peptides tested. Histone tail sequences are represented above; note the ARKS motifs of H3K9 and H3K27. See panel D for actual peptides used. (C) Fluorescence polarization of Cbx5 (mouse HP1α) to H3K9 and H3K27 histone tail peptides in the me1, me2, and me3 states. (D) Dissociation constants (Kd, in micromolars) for Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8, as well as Cbx5 (mHP1α), with each series of methylated peptides for each backbone shown. Low-micromolar binding constants are highlighted in red. The asterisk depicts weak binding of Cbx7 for H3K9me2 (a mark that is enriched on the Xi). Values represent averages ± standard deviations for at least three independent experiments in all cases (except for Cbx5 with certain peptides). ND, not determined. (E) Peptide pull-down assays. (Left) All CDs were examined for binding to unmodified and trimethylated peptides representing H3K9, H3K27, and H4K20. Results support those obtained by FP (D). (Right) Cbx7 CD, Cbx7 caging aromatic point mutant F11A, and human HP1β CD recombinant proteins were tested for the ability to bind unmodified and me1, me2, and me3 peptides of H3K9 and K27. Note the trimethyl specificity of Cbx7. (Right, bottom) GST and full-length Cbx7 were examined for binding to unmodified and trimethylated biotinylated peptides of H3K9 and H3K27. Full-length Cbx7 behaves the same as its CD alone. GST does not bind any peptide, as expected. un, unmodified.

FIG. 2.

FIG. 2.

Cbx7-associated histone modifications. (A) Far Western blotting assay. The GST-Cbx7 CD binds to female ES cell (LF2) histone H3 through K9me3 and K27me3, demonstrated by peptide competitions (lanes 1 to 6); Cbx7 CD does not bind to recombinant H3 (lane 2); GST alone and caging aromatic mutants fail to associate (lanes 7 to 9); Ponceau for equal loading of total histones (bottom). (B) IPs of mononucleosomal extracts prepared from Cbx7-flag stable 293 cells versus control cells. Inputs in both cases show presence of all histone modifications; Cbx7-flag associated histones (IP) are marked predominantly by facultative heterochromatic modifications, including H3K9me2, H3K27me3, and H4K20me1. un, unmodified.

FIG. 3.

FIG. 3.

Cbx proteins associate with the inactive X. (A) All Cbx-EGFP fusion proteins, except Cbx4, localize to the Xi in 3-day-differentiated female ES cells. The Xi is visualized by K27me3 staining (red). Fluorescence intensity plots (white line from a to b) across the nucleus and including the K27me3 signal (red) on the Xi illustrate the enrichment for, or lack of, each of the Cbx-GFP fusion proteins (green). (B) Point mutations of the caging aromatic residues in the Cbx7 CD (F11A, W32A, and W35A) disrupt localization to the Xi. When the CD of Cbx7 is replaced by the CD of Cbx4 (mCbx7Cbx4CD-EGFP), the chimeric protein no longer strongly associates with the Xi, although it is not disrupted completely (bottom panel). Note that the K27me3 peak coincides with a Cbx7-GFP peak for the wild-type (WT) and Cbx7Cbx4CD proteins but not for the three mutated versions. (C) GST overlay assays were performed on 3-day-differentiated female mouse ES cells. Protein was detected on paraformaldehyde-fixed cells using α-GST antibody (green). The Xi was visualized by α-Eed antibody (red). GST protein does not associate with the Xi (top), while full-length GST-Cbx7 is enriched on the Xi (bottom). It should be noted that although these experiments support our EGFP-fusion results, they were somewhat variable in their efficiency. (D) Female ES cells were tested throughout differentiation (up to day 7) for Cbx7 association with chromatin (un, undifferentiated). Cbx7 and K27me3 are expressed throughout the differentiation process (top, whole-cell extracts); however, Cbx7 specifically associates with chromatin on day 6 and onward (compare to HP1β and K27me3 in chromatin extracts at the bottom of the panel).

FIG. 4.

FIG. 4.

Pc-like Cbx CDs bind RNA. Recombinant CDs were tested for RNA binding by gel shift assays. (A) The Cbx5 (mouse HP1α) CD was compared to Cbx7 (mPc) for RNA binding with a 500-nt single stranded RNA (ssRNA), and only the latter demonstrates a shift. (B) Cbx4, Cbx6, Cbx7, and Cbx8 CDs interact with RNA, while Cbx1 (mHP1β) and Cbx2 do not. (C) Cbx7 binds to both single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA). Cbx7 binds dsDNA with only minor affinity. (D) Gel shift analysis of the Cbx7 CD with ssRNA (500mer) was performed by extensive titration of protein in order to determine a dissociation constant, which is approximately 100 μM. (E) Heat denaturation of Cbx6, Cbx8, and dPc CD proteins disrupts the interaction with RNA (+, heat denaturation). (F) Point mutations in the caging aromatic residues of the Cbx7 CD do not abrogate RNA binding. Recombinant GST was used as a negative control.

FIG. 5.

FIG. 5.

Cbx7 chromatin association is RNA dependent. (A) RNase treatment of 3-day-differentiated ES cells strongly diminished the accumulation of Cbx7-EGFP on the Xi. The Xi is visualized by K27me3 staining (red), and the signal of Cbx7-EGFP was enhanced using α-GFP antibody (green). (B) Degradation of Cbx7 is not observed after RNase treatment (or DNase I treatment), as detected by Western blot with GFP antibody. (C) Cbx7 is depleted from 6-day-differentiated ES cell chromatin by RNase treatment (top), while WDR5 and HP1β are not affected. See histones for equal loading (bottom).

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