Nanasi, P. | bioRxiv Science

bioRxiv2023

KDM4-dependent DNA breaks at active promoters facilitate +1 nucleosome eviction

When the effect of various posttranslational histone tail modifications (PTMs) on nucleosome stability was compared in an in situ assay involving agarose-embedded nuclei, the promoter proximal H3K4me3, H3K27ac and H4K8ac 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 specify dynamic nucleosomes accomodating relaxed DNA sequences due to DNA breaks generated in vivo. In line with this interpretation, endogeneous, 3OH nicks were mapped within the nucleosome free region of promoters controlling genes active in human mononuclear cells, a conclusion supported by superresolution colocalization studies. The +1 nucleosomes were stabilized and the incidence of nicks was decreased at the promoters upon KDM4a,b,c KO induction (Pedersen et al, EMBO J, 2016) in mouse embryonic stem cells (mES). While etoposide did not further destabilize +1 nucleosomes in control mES, their stabilized state in the KO state was reversed by the drug. A significant fraction of the DNA breaks comprises TOP2-generated nicks according to the results of molecular combing experiments. The chromatin regions harboring nicks are topologicaly separated from the domains containing superhelical chromatin. 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.

genetics↗

bioRxiv2021

Fundamental Role Of The H2A.Z C-Terminal Tail In The Formation Of Constitutive Heterochromatin

H2A.Z-nucleosomes are present in both euchromatin and heterochromatin and it has proven difficult to interpret their disparate roles in the context of their stability features. Using an in situ assay of nucleosome stability and DT40 cells expressing engineered forms of the histone variant we show that native H2A.Z, but not C-terminally truncated H2A.Z (H2A.Z{Delta}C), is released from nucleosomes of peripheral heterochromatin at unusually high salt concentrations. H2A.Z and H3K9me3 landscapes are reorganized in H2A.Z{Delta}C-nuclei and overall sensitivity of chromatin to nucleases is increased. These tail-dependent differences are recapitulated upon treatment of HeLa nuclei with the H2A.Z-tail-peptide (C9), with MNase sensitivity being increased at specific regions including promoters. Introduced into live cells C9 elicits down-regulation of [~]560 genes with nonrandom chromosomal band-localization and pathway-spectrum. Thus, tail-dependent heterogeneity of H2A.Z-nucleosomes is revealed at all organization levels of chromatin and epigenetic modulation can be achieved by targeting molecular interactions involving its C-terminal tail.

cell biology↗