The relationships between the stability of the +1 nucleosome and DNA superhelicity

When the effect of various posttranslational histone tail modifications (PTMs) on nucleosome stability was compared in an in situ assay involving agarose-embedded nuclei, a subpopulation of the promoter proximal H3K4me3, H3K27ac positive nucleosomes exhibited relative sensitivity to intercalators as compared to bulk H3-GFP or nucleosomes carrying any of the following marks: H3K27me1, H3K27me2, H3K27me3, H3K9me1, H3K9me2, H3K9me3, H3K36me3, H3K4me0, H3K4me1, H3K4me2, H3K9ac, and H3K14ac. Nickase or DNase I treatment of the nuclei, or bleomycin treatment of live cells, did not affect the stability of nucleosomes carrying H3K4me3 or H3K27ac, while those of the second group were all destabilized upon treatment with intercalators. These observations support the possibility that the promoter proximal marks specifying dynamic nucleosomes are juxtaposed with relaxed DNA sequences due to DNA breaks generated in vivo . In line with this interpretation, endogeneous, 3'OH nicks were mapped within the nucleosome free region of promoters in human mononuclear cells as well as in mES. We also present evidence that the chromatin regions harboring the breaks are topologicaly separated from the domains containing superhelical chromatin. Regarding the mechanism eliciting the breaks, two alternative models were tested experimentally using kncok out cells, inhibitors and degrader systems, based on TOP2 activity or on histone demethylation induced oxydative DNA lesions and their repair. These observations lend support for a model where the role of DNA strand discontinuities in transcriptional regulation and in higher-order chromatin organization are integrated. Grants: OTKA/NKFIH 138524, ERA-Net NEURON 2024-1.2.2-ERA_NET-2024-00009 *PN and LI contributed equally