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In nucleosomes, histone N-terminal tails exist in dynamic equilibrium between free/accessible and collapsed/DNA-bound states. The latter state is expected to impact histone N-termini availability to the epigenetic machinery. Notably, H3 tail acetylation (e.g., K9ac, K14ac, K18ac) is linked to increased H3K4me3 engagement by the BPTF PHD finger, but it is unknown if this mechanism has broader extension. Here we show that H3 tail acetylation promotes nucleosomal accessibility to other H3K4 methyl readers, and importantly, extends to H3K4 writers, notably methyltransferase MLL1. This regulation is not observed on peptide substrates yet occurs on thecisH3 tail, as determined with fully-defined heterotypic nucleosomes.In vivo, H3 tail acetylation is directly and dynamically coupled withcisH3K4 methylation levels. Together, these observations reveal an acetylation ‘chromatin switch’ on the H3 tail that modulates read-write accessibility in nucleosomes and resolve the long-standing question of why H3K4me3 levels are coupled with H3 acetylation.

Bulk chromatin encompasses complex sets of histone posttranslational modifications (PTMs) that recruit (or repel) the diverse reader domains of Chromatin-Associated Proteins (CAPs) to regulate genome processes (e.g., gene expression, DNA repair, mitotic transmission). The binding preference of reader domains for their PTMs mediates localization and functional output, and are often dysregulated in disease. As such, understanding chromatin interactions may lead to novel therapeutic strategies, However the immense chemical diversity of histone PTMs, combined with low-throughput, variable, and nonquantitative methods, has defied accurate CAP characterization. This chapter provides a detailed protocol for dCypher, a novel approach for the rapid, quantitative interrogation of CAPs (as mono- or multivalent Queries) against large panels (10s to 100s) of PTM-defined histone peptide and semisynthetic nucleosomes (the potential Targets). We describe key optimization steps and controls to generate robust binding data. Further, we compare the utility of histone peptide and nucleosome substrates in CAP studies, outlining important considerations in experimental design and data interpretation.

Histone post-translational modifications (PTMs) play a critical role in chromatin regulation. It has long been suspected that the poorly understood capability of ‘PTM-specific’ antibodies (i.e., their specificity and efficiency) is a prime driver of the reproducibility crisis in biomedical research. Here we confirm the validity of this concern as it applies to epigenomic mapping studies. Extensive spike-in panels of PTM-defined DNA-barcoded nucleosome standards show that >70% of >500 commercial antibodies (and >80% of the most highly cited) to >50 histone lysine methyl and acyl states have failing performance in ChIP (>20% cross reactivity/ Citation Format: Andrea L. Johnstone, Danielle N. Maryanski, Keli L. Rodriguez, Ellen N. Weinzapfel, Bryan J. Venters, Katherine Novitzky, Matthew R. Marunde, Carolina P. Lin, Michael-Christopher Keogh. A field survey of histone PTM antibodies in epigenomic mapping approaches reveals widespread liabilities: Best practices and resources for reliable epigenetic studies. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A022.