In nucleosomes, histone N-terminal tails exist in dynamic equilibrium between free/accessible and collapsed/DNA-bound states. The DNA-bound state is expected to impact histone N-termini availability to the epigenetic machinery. Notably, H3 tail acetylation (K9ac, K14ac, K18ac) is linked to increased engagement of H3K4me3 by the BPTF PHD finger, but it is unknown if this mechanism has broader extension. Here we show that cis H3 tail acetylation promotes nucleosomal accessibility to other H3K4 methyl readers, and further extends to H3K4 writers, notably methyltransferase MLL1. This regulation is nucleosome-dependent and also observed in vivo, where H3 acetylation correlates with increased levels of cis H3K4me. These observations reveal an acetylation "chromatin switch" on the H3 N-terminal tail that modulates the accessibility and function of H3K4 methylation in chromatin. Our findings also resolve the long-standing question of why H3K4me3 levels are linked with H3 acetylation.
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/<5% target recovery; www.ChromatinAntibodies.com). Variable lot behavior (of both polyclonals and monoclonals) shows the danger of focusing on catalog numbers without considering the inherent variability of biological reagents. Ultimately, these studies support the inclusion of in situ standards to control genomic mapping assays as an improved path out of this morass. Despite these advances, the application of ChIP-seq for cancer research has been hampered by its requirement for high cell inputs and low reliability. The recent development of immunotethering assays, such as Cleavage Under Targets and Release Using Nuclease (CUT&RUN), deliver high signal-to-noise mapping data using a fraction of the required cells compared to ChIP-seq. These innovations enable the application of epigenomics for cancer research, particularly for primary cells, sorted populations, and clinical samples where cell numbers are limited. By adapting our nucleosome spike-in control approach for CUT&RUN assays, we show that while the same antibody problems observed in ChIP-seq also pervade CUT&RUN, continuous use of spike-in controls improves assay rigor and reproducibility to realize the potential of CUT&RUN to advance cancer research. 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.
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