Chromatin proteomics reveals novel combinatorial histone modification signatures that mark distinct subpopulations of macrophage enhancers

Abstract

The integrated activity of cis-regulatory elements fine-tunes transcriptional programs of mammalian cells by recruiting cell type–specific as well as ubiquitous transcription factors (TFs). Despite their key role in modulating transcription, enhancers are still poorly characterized at the molecular level, and their limited DNA sequence conservation in evolution and variable distance from target genes make their unbiased identification challenging. The coexistence of high mono-methylation and low tri-methylation levels of lysine 4 of histone H3 is considered a signature of enhancers, but a comprehensive view of histone modifications associated to enhancers is still lacking. By combining chromatin immunoprecipitation (ChIP) with mass spectrometry, we investigated cis-regulatory regions in macrophages to comprehensively identify histone marks specifically associated with enhancers, and to profile their dynamics after transcriptional activation elicited by an inflammatory stimulation. The intersection of the proteomics data with ChIP-seq and RNA-seq analyses revealed the existence of novel subpopulations of enhancers, marked by specific histone modification signatures: specifically, H3K4me1/K36me2 marks transcribed enhancers, while H3K4me1/K36me3 and H3K4me1/K79me2 combinations mark distinct classes of intronic enhancers. Thus, our MS analysis of functionally distinct genomic regions revealed the combinatorial code of histone modifications, highlighting the potential of proteomics in addressing fundamental questions in epigenetics.

Open chromatin landscape of rat microglia upon proinvasive or inflammatory polarization

“…Interestingly, LPS induced changes in openness with a preference for regions that were intronic or located within 10 kb immediately downstream of the transcription end site (Figure a). These findings are in agreement with the results of a recent chromatin proteomics study that identified a subpopulation of intronic enhancers involved in inflammatory gene expression (Soldi et al, ). In contrast, the GCM treatment preferentially induced openness changes in open chromatin regions distant from the genes' bodies.…”

Nucleosome Turnover Regulates Histone Methylation Patterns over the Genome

“…This is thought to be mediated by the deposition of H3.3, which results in the incorporation of newly synthesized histones (Goldberg et al, 2010). Further, AF9 has been shown to be localized sharply at the TSS of active genes (Figure 3A; Li et al, 2016b), despite the formation of broad H3K79 methylation domains (>20 kb) at these active regions (Soldi et al, 2017). This suggests that a propagation mechanism may contribute to the establishment of the H3K79 landscape.…”

“…(A) Distribution of H3K79me2 histone modification and the AF9 subunit in macrophages (Li et al, 2016b; Soldi et al, 2017). …”

Lysine-Specific Histone Demethylase 1A Regulates Macrophage Polarization and Checkpoint Molecules in the Tumor Microenvironment of Triple-Negative Breast Cancer

“…CpG content, GC content, histone marks, and accessibility of genes in the NanoString panel. (A) CpG and GC content, (B,D) histone levels and (C) accessibility of genes ± 1 kb from transcription start site (TSS) of NanoString genes up-regulated, downregulated (FDR < 0.15), or unchanged (FDR > 0.15) and genes from refSeq in RAW264.7 cells (47). (C) shows accessibility of Nanostring genes against gene sets from bone marrow derived macrophages (BMDMs) with or without 6 h of LPS stimulation (48).…”

“…Up-regulated gene promoters had significantly less H3K4me3 ( p < 0.05) but not H3K27ac and H3K4me1 than the down-regulated gene promoters, in resting RAW264.7 cells (Figure 6B) (47). Importantly the up-regulated gene promoters had less H3K27me3 than the unchanged genes, suggesting their non-maximal gene expression is not due to H3K27me3-mediated repression (Figure 6B).…”