Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 13 (8), R68

Proteogenomic Characterization and Mapping of Nucleosomes Decoded by Brd and HP1 Proteins

Proteogenomic Characterization and Mapping of Nucleosomes Decoded by Brd and HP1 Proteins

Gary LeRoy et al. Genome Biol.

Abstract

Background: Histone post-translational modifications (PTMs) constitute a branch of epigenetic mechanisms that can control the expression of eukaryotic genes in a heritable manner. Recent studies have identified several PTM-binding proteins containing diverse specialized domains whose recognition of specific PTM sites leads to gene activation or repression. Here, we present a high-throughput proteogenomic platform designed to characterize the nucleosomal make-up of chromatin enriched with a set of histone PTM binding proteins known as histone PTM readers. We support our findings with gene expression data correlating to PTM distribution.

Results: We isolated human mononucleosomes bound by the bromodomain-containing proteins Brd2, Brd3 and Brd4, and by the chromodomain-containing heterochromatin proteins HP1β and HP1α. Histone PTMs were quantified by mass spectrometry (ChIP-qMS), and their associated DNAs were mapped using deep sequencing. Our results reveal that Brd- and HP1-bound nucleosomes are enriched in histone PTMs consistent with actively transcribed euchromatin and silent heterochromatin, respectively. Data collected using RNA-Seq show that Brd-bound sites correlate with highly expressed genes. In particular, Brd3 and Brd4 are most enriched on nucleosomes located within HOX gene clusters, whose expression is reduced upon Brd4 depletion by short hairpin RNA.

Conclusions: Proteogenomic mapping of histone PTM readers, alongside the characterization of their local chromatin environments and transcriptional information, should prove useful for determining how histone PTMs are bound by these readers and how they contribute to distinct transcriptional states.

Figures

Figure 1
Figure 1
Isolation of Brd- and HP1-bound mononucleosomes. (a) Illustration of the domain architecture of the histone code readers. (b) Schematic of protocol used to isolate nucleosomes bound to FLAG-histone code readers (HCRs). (c) Visualization of FLAG-Brd- and FLAG-HP1-bound nucleosome purifications. The proteins present in the input (I), the immunoprecipitation unbound (U), and FLAG-peptide-eluted (E), fractions were examined by SDS-PAGE and Coomassie blue staining. The asterisk denotes Brd3 breakdown products detected by liquid chromatography-tandem mass spectrometry.
Figure 2
Figure 2
Histone H3 and H4 residue-specific PTMs quantified by mass spectrometry. (a) Total ion chromatogram and full MS spectrum of propionylated histone peptides used for quantification from an HP1β ChIP; ChIP elution D0 labeled (black), and the control ChIP input D5 labeled (red). Full MS spectrum of peptides shown; H3K9me3K14un, H3K9me1K14un, H4K5unK8unK12acK16ac, H3K18unK23me1 and H3K79me3. (b) Quantification of the degree of acetylation of the histone H4 peptide (amino acids 4 to 17, GKGGKGLGKGGAKR). Values in the table are the total percentage of each acetylated form and the heatmap is a representation of the fold change of the specified ChIP/Input in (log2) scale. (c) Heatmap depicting all modifications quantified on the histone H3 and H4 proteins. Values used to generate the heatmap are found in Additional file 2. The heatmap was generated with the fold change values of each PTM for each specified ChIP/Input in (log2) scale.
Figure 3
Figure 3
Quantification of PTMs on histone H3 and H4 peptides isolated with HP1 and Brd proteins. (a) Heatmap depicting all modified forms of H3 peptides on HP1- and Brd-bound nucleosomes by qMS. Values used to generate the heatmap are in Additional files 2 and 6. The heatmap was generated with the fold change values for each modified H3 peptide quantified from each specified ChIP/Input in (log2) scale. (b) Heatmap depicting all modified forms of H4 peptides on HP1- and Brd-bound nucleosomes by mass spectrometry. Values used to generate the heatmap are in Additional files 2 and 3. The heatmap was generated with the fold change values for each modified H4 peptide quantified from each specified ChIP/Input in (log2) scale.
Figure 4
Figure 4
Mapping of Brd- and HP1-bound nucleosomes to their genomic locations. (a) Chromosome maps showing the density of Brd- and HP1-bound nucleosomes generated by Solexa Deep sequencing on chromosomes 17, × and 12. (b) Table denoting the enrichment of Brd and HP1 nucleosomes within the genome relative to the genes. (c-g) Graphs showing the relative enrichment of Brd and HP1 nucleosomes within genes, grouped by expression levels (high, red; medium, blue; silent, green). TES, transcription end site; TSS, transcription start site.
Figure 5
Figure 5
Promoters bound by Brd- and HP1-bound nucleosomes. (a) Heatmap of promoters with nucleosomes bound by the Brd and HP1 proteins. Heatmaps are arranged as clusters of promoters bound by the various ChIPs (lanes 1 to 5: bound, red; unbound, blue) and the expression of these genes from RNA-Seq is represented in lane 6 (high and medium, red; silent, blue). (b) Ranked order table of genes (top 35) whose promoters are bound by the Brd and HP1 proteins. Genes are ordered based on Brd/HP1 binding score within the promoter region of all annotated genes (top of the list, most bound). Genes belonging to the HOX clusters or ZNF clusters are color coded accordingly. (c) HP1α and HP1β binding clusters on chromosome 19. The ZNF clusters are labeled 1 to 6.
Figure 6
Figure 6
Brd4 and HP1β regulate gene expression. (a) Right panel: heatmap showing the log2 fold change in gene expression from 293 cells with depleted Brd4 compared to control 293 cells. Genes shown are the top 100 genes whose promoter regions were the most enriched with Brd4 bound nucleosomes. Left panel: heatmap showing the log2 fold change in gene expression from 293 cells with depleted HP1β compared to control 293 cells. Genes shown are the top 100 genes whose promoter regions were the most enriched with HP1β-bound nucleosomes. (b) Right panel: heatmap of the log2 fold change in expression of all HOX genes with promoters bound by Brd4 in the Brd4-depleted 293 cell line compared to control 293 cells. Left panel: heatmap of the log2 fold change in expression of all ZNF genes found on chromosome 19 with promoters bound by HP1β in the HP1β-depleted 293 cell line compared to control 293 cells.
Figure 7
Figure 7
Brd and HP1 protein nucleosomal binding patterns of Brd4 and HP1β regulated genes. (a) HOXA10 and HOXA11. (b) HOXB9. (c) HOXC10. (d) ACTB. (e) ZNF599. (f) ZNF560. Top panels for are the ChIP-seq results depicting the Brd and HP1 binding patterns. Bottom panel is the RNA-Seq results representing the expression level of each gene.

Similar articles

See all similar articles

Cited by 47 PubMed Central articles

See all "Cited by" articles

References

    1. Kouzarides T. Chromatin modifications and their function. Cell. 2007;128:693–705. doi: 10.1016/j.cell.2007.02.005. - DOI - PubMed
    1. Meissner A, Mikkelsen TS, Gu H, Wernig M, Hanna J, Sivachenko A, Zhang X, Bernstein BE, Nusbaum C, Jaffe DB, Jaenisch R, Lander ES. Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature. 2008;454:766–770. - PMC - PubMed
    1. Wang Z, Zang C, Rosenfeld JA, Schones DE, Barski A, Cuddapah S, Cui K, Roh TY, Peng W, Zhang MQ, Zhao K. Combinatorial patterns of histone acetylations and methylations in the human genome. Nat Genet. 2008;40:897–903. doi: 10.1038/ng.154. - DOI - PMC - PubMed
    1. Barski A, Cuddapah S, Cui K, Roh TY, Schones DE, Wang Z, Wei G, Chepelev I, Zhao K. High-resolution profiling of histone methylations in the human genome. Cell. 2007;129:823–837. doi: 10.1016/j.cell.2007.05.009. - DOI - PubMed
    1. Hebbes TR, Thorne AW, Crane-Robinson C. A direct link between core histone acetylation and transcriptionally active chromatin. EMBO J. 1988;7:1395–1402. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources

Feedback