Chromatin affinity purification and quantitative mass spectrometry defining the interactome of histone modification patterns

DNA and histone modifications direct the functional state of chromatin and thereby the readout of the genome. Candidate approaches and histone peptide affinity purification experiments have identified several proteins that bind to chromatin marks. However, the complement of factors that is recruited by individual and combinations of DNA and histone modifications has not yet been defined. Here, we present a strategy based on recombinant, uniformly modified chromatin templates used in affinity purification experiments in conjunction with SILAC-based quantitative mass spectrometry for this purpose. On the prototypic H3K4me3 and H3K9me3 histone modification marks we compare our method with a histone N-terminal peptide affinity purification approach. Our analysis shows that only some factors associate with both, chromatin and peptide matrices but that a surprisingly large number of proteins differ in their association with these templates. Global analysis of the proteins identified implies specific domains mediating recruitment to the chromatin marks. Our proof-of-principle studies show that chromatin templates with defined modification patterns can be used to decipher how the histone code is read and translated.     

Chromatin remodeling and bivalent histone modifications in embryonic stem cells

Pluripotent embryonic stem cells (ESCs) are characterized by distinct epigenetic features including a relative enrichment of histone modifications related to active chromatin. Among these is tri‐methylation of lysine 4 on histone H3 (H3K4me3). Several thousands of the H3K4me3‐enriched promoters in pluripotent cells also contain a repressive histone mark, namely H3K27me3, a situation referred to as “bivalency”. While bivalent promoters are not unique to pluripotent cells, they are relatively enriched in these cell types, largely marking developmental and lineage‐specific genes which are silent but poised for immediate action. The H3K4me3 and H3K27me3 modifications are catalyzed by lysine methyltransferases which are usually found within, although not entirely limited to, the Trithorax group (TrxG) and Polycomb group (PcG) protein complexes, respectively, but these do not provide selective bivalent specificity. Recent studies highlight the family of ATP‐dependent chromatin remodeling proteins as regulators of bivalent domains. Here, we discuss bivalency in general, describe the machineries that catalyze bivalent chromatin domains, and portray the emerging connection between bivalency and the action of different families of chromatin remodelers, namely INO80, esBAF, and NuRD, in pluripotent cells. We posit that chromatin remodeling proteins may enable “bivalent specificity”, often selectively acting on, or selectively depleted from, bivalent domains.     

Detailed specificity analysis of antibodies binding to modified histone tails with peptide arrays

Chromatin structure is greatly influenced by histone tail post-translational modifications (PTM), which also play a central role in epigenetic processes. Antibodies against modified histone tails are central research reagents in chromatin biology and molecular epigenetics. We applied Celluspots peptide arrays for the specificity analysis of 36 commercial antibodies from different suppliers, which are directed towards modified histone tails. The arrays contained 384 peptides from eight different regions of the N-terminal tails of histones, viz. H3 1–19, 7–26, 16–35 and 26–45, H4 1–19 and 11–30, H2A 1–19 and H2B 1–19, featuring 59 post-translational modifications in many different combinations. Using various controls we document the reliability of the method. Our analysis revealed previously undocumented details in the specificity profiles of the tested antibodies. Most of the antibodies bound well to the PTM they have been raised for, but some failed. In addition, some antibodies showed high cross-reactivity and most antibodies were inhibited by specific additional PTMs close to the primary one. Furthermore, specificity profiles for antibodies directed toward the same modification sometimes were very different. The specificity of antibodies used in epigenetic research is an important issue. We provide a catalog of antibody specificity profiles for 36 widely used commercial histone tail PTM antibodies. Better knowledge about the specificity profiles of antibodies will enable researchers to implement necessary control experiments in biological studies and allow more reliable interpretation of biological experiments using these antibodies.Key words: histone modification, histone methylation, histone acetylation, histone phosphorylation, chromatin, antibody, specificity, ChIP     

Detailed specificity analysis of antibodies binding to modified histone tails with peptide arrays

Chromatin structure is greatly influenced by histone tail post-translational modifications (PTM), which also play a central role in epigenetic processes. Antibodies against modified histone tails are central research reagents in chromatin biology and molecular epigenetics. We applied Celluspots peptide arrays for the specificity analysis of 36 commercial antibodies from different suppliers which are directed towards modified histone tails. The arrays contained 384 peptides from 8 different regions of the N-terminal tails of histones, viz. H3 1-19, 7-26, 16-35 and 26-45, H4 1-19 and 11-30, H2A 1-19 and H2B 1-19, featuring 59 post-translational modifications in many different combinations. Using various controls we document the reliability of the method. Our analysis revealed previously undocumented details in the specificity profile. Most of the antibodies bound well to the PTM they have been raised for, but some failed. In addition some antibodies showed high cross-reactivity and most antibodies were inhibited by specific additional PTMs close to the primary one. Furthermore, specificity profiles for antibodies directed towards the same modification sometimes were very different. The specificity of antibodies used in epigenetic research is an important issue. We provide a catalog of antibody specificity profiles for 36 widely used commercial histone tail PTM antibodies. Better knowledge about the specificity profiles of antibodies will enable researchers to implement necessary control experiments in biological studies and allow more reliable interpretation of biological experiments using these antibodies.     

In situ histone landscape of nephrogenesis

In the developing kidney, self-renewing progenitors respond to inductive signaling from the adjacent branching ureteric bud by undergoing mesenchyme-to-epithelium transition. Nascent nephrons subsequently undergo elongation, segmentation, and differentiation into a mature renal epithelium with diverse functions. Epigenetic mechanisms have been implicated in impacting cell fate decisions during nephrogenesis; however, the chromatin landscape of nephron progenitors and daughter differentiating cells are largely unknown. Here, we examined the spatiotemporal expression patterns of histone H3 methylation and histone methyltransferases in E15.5 mouse kidneys. Kidney sections were probed with antibodies against histone modifications, enzymes, and markers of progenitors and differentiation. The results revealed that: (1) nephron progenitor cells exhibit a broad histone methylation signature that comprises both “active” and “repressive” marks (H3K4me3/K9me3/K27me3/R2me2/R17me2); (2) nascent nephrons retain high H3K4me3 but show downregulation of H3K9/K27me3 and; (3) maturing epithelial tubules acquire high levels of H3K79me2/3. Consistent with respective histone marks, the H3K4 methyltransferase, Ash2l, is expressed in progenitors and nascent nephrons, whereas the H3K9/K27 methyltransferases, G9a/Ezh2, are more enriched in progenitors than nascent nephrons. We conclude that combinatorial histone signatures correlate with cell fate decisions during nephrogenesis.     

In situ histone landscape of nephrogenesis

In the developing kidney, self-renewing progenitors respond to inductive signaling from the adjacent branching ureteric bud by undergoing mesenchyme-to-epithelium transition. Nascent nephrons subsequently undergo elongation, segmentation, and differentiation into a mature renal epithelium with diverse functions. Epigenetic mechanisms have been implicated in impacting cell fate decisions during nephrogenesis; however, the chromatin landscape of nephron progenitors and daughter differentiating cells are largely unknown. Here, we examined the spatiotemporal expression patterns of histone H3 methylation and histone methyltransferases in E15.5 mouse kidneys. Kidney sections were probed with antibodies against histone modifications, enzymes, and markers of progenitors and differentiation. The results revealed that: (1) nephron progenitor cells exhibit a broad histone methylation signature that comprises both “active” and “repressive” marks (H3K4me3/K9me3/K27me3/R2me2/R17me2); (2) nascent nephrons retain high H3K4me3 but show downregulation of H3K9/K27me3 and; (3) maturing epithelial tubules acquire high levels of H3K79me2/3. Consistent with respective histone marks, the H3K4 methyltransferase, Ash2l, is expressed in progenitors and nascent nephrons, whereas the H3K9/K27 methyltransferases, G9a/Ezh2, are more enriched in progenitors than nascent nephrons. We conclude that combinatorial histone signatures correlate with cell fate decisions during nephrogenesis.     

The CW domain, a new histone recognition module in chromatin proteins

Post-translational modifications of the N-terminal histone tails, including lysine methylation, have key roles in regulation of chromatin and gene expression. A number of protein modules have been identified that recognize differentially modified histone tails and provide their proteins with the capacity to sense such modifications. Here, we identify the CW domain of plant and animal chromatin-related proteins as a novel module that recognizes different methylated states of lysine 4 on histone H3 (H3K4me). The solution structure of the CW domain of the Arabidopsis ASH1 HOMOLOG2 (ASHH2) histone methyltransferase provides insight into how different CW domains can distinguish different methylated histone tails. We provide evidence that ASHH2 is acting on H3K4me-marked genes, allowing for ASHH2-dependent H3K36 tri-methylation, which contributes to sustained expression of tissue-specific and developmentally regulated genes. This suggests that ASHH2 is a combined 'reader' and 'writer' of the histone code. We propose that different CW domains, dependent on their specificity for different H3K4 methylations, are important for epigenetic memory or participate in switching between permissive and repressive chromatin states.     

ChIPnorm: A Statistical Method for Normalizing and Identifying Differential Regions in Histone Modification ChIP-seq Libraries

The advent of high-throughput technologies such as ChIP-seq has made possible the study of histone modifications. A problem of particular interest is the identification of regions of the genome where different cell types from the same organism exhibit different patterns of histone enrichment. This problem turns out to be surprisingly difficult, even in simple pairwise comparisons, because of the significant level of noise in ChIP-seq data. In this paper we propose a two-stage statistical method, called ChIPnorm, to normalize ChIP-seq data, and to find differential regions in the genome, given two libraries of histone modifications of different cell types. We show that the ChIPnorm method removes most of the noise and bias in the data and outperforms other normalization methods. We correlate the histone marks with gene expression data and confirm that histone modifications H3K27me3 and H3K4me3 act as respectively a repressor and an activator of genes. Compared to what was previously reported in the literature, we find that a substantially higher fraction of bivalent marks in ES cells for H3K27me3 and H3K4me3 move into a K27-only state. We find that most of the promoter regions in protein-coding genes have differential histone-modification sites. The software for this work can be downloaded from http://lcbb.epfl.ch/software.html.