Enzymes catalyzing CpG methylation in DNA, including DNMT1 and DNMT3A/B, are indispensable for mammalian tissue development and homeostasis 1-4. They are also implicated in human developmental disorders and cancers 5-8 , supporting a critical role of DNA methylation during cell fate specification and maintenance. Recent studies suggest that histone posttranslational modifications (PTMs) are involved in specifying patterns of DNMT localization and DNA methylation at promoters and actively transcribed gene bodies 9-11. However, mechanisms governing the establishment and maintenance of intergenic DNA methylation remain poorly understood. Germline mutations in DNMT3A define Tatton-Brown-Rahman syndrome (TBRS), a

SUMMARY Histone post-translational modifications (PTMs) are important genomic regulators often studied by chromatin immunoprecipitation (ChIP), whereby their locations and relative abundance are inferred by antibody capture of nucleosomes and associated DNA. However, the specificity of antibodies within these experiments have not been systematically studied. Here, we use histone peptide arrays and internally calibrated ChIP (ICeChIP) to characterize 52 commercial antibodies purported to distinguish the H3K4 methylforms (me1, me2, and me3, with each ascribed distinct biological functions). We find that many widely-used antibodies poorly distinguish the methylforms and that high- and low-specificity reagents can yield dramatically different biological interpretations, resulting in substantial divergence from the literature for numerous H3K4 methylform paradigms. Using ICeChIP, we also discern quantitative relationships between enhancer H3K4 methylation and promoter transcriptional output and can measure global PTM abundance changes. Our results illustrate how poor antibody specificity contributes to the “reproducibility crisis,” demonstrating the need for rigorous, platform-appropriate validation.

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Highlights d SETD5 is an epigenetic driver of pancreatic cancer resistance to MEK1/2 inhibition d SETD5 has no intrinsic methylation activity on histones, including at H3 lysine 36 d A SETD5 co-repressor complex regulates a network of drug resistance pathways d Co-targeting of MEK1/2 and the SETD5 complex results in sustained tumor inhibition

Nuc-MS characterizes histone modifications and variants directly from intact endogenous nucleosomes. Preserving whole nucleosome particles enables precise interrogation of their protein content, as for H3.3-containing nucleosomes which had 6fold co-enrichment of variant H2A.Z over bulk chromatin. Nuc-MS, validated by ChIPseq, showed co-occurrence of oncogenic H3.3K27M with euchromatic marks (e.g., H4K16ac and >15-fold enrichment of H3K79me2). By capturing the entire epigenetic landscape, Nuc-MS provides a new, quantitative readout of nucleosome-level biology. MainThe post-translational modifications (PTMs) decorating the four core histones in an intact nucleosome encode information for nuclear effectors that trigger defined cellular events critical to health and disease. [1][2][3][4] For decades, affinity reagents, mass spectrometry and proteomics have played key roles in characterizing the many histone isoforms and PTMs involved in epigenetic regulation. [5][6][7] However, the standard practice of relying on digestion 8 and/or denaturation 5 removes the linkage between modifications and their nucleosomes of origin (Fig. 1a, at left). By digesting histone mixtures into small peptides, correlations among PTMs are forfeited, precluding strong assertions about the original composition of the intact histone. Similarly, when a nucleosome population is denatured, information about co-localization of histone isoforms and PTMs within the same nucleosome particle is lost. This inference problem diminishes our understanding of the organization and impact of co-occurring modifications, isoforms and mutations in epigenetics and disease pathogenesis. 9,10