Preparation and Characterization of Chromatin Templates for Histone Methylation Assays

Liu, Cuifang; Zhao, Ji‐Cheng; Li, Guohong

doi:10.1007/978-1-0716-2481-4_4

Cited by 2 publications

(3 citation statements)

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“…For reconstitution of histone octamers, equimolar H2A, H2B, H3 and H4 histones were mixed and dialyzed against 2 L refolding buffer (2 M NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM 2-Mercaptoethanol) overnight at 4 °C with at least two times changes of refolding buffer. The histone octamers were purified through Superdex 200 column (GE) 59 .…”

Section: Methodsmentioning

confidence: 99%

“…Mono-nucleosomes and polynucleosomes were assembled using the salt-dialysis method 59 . Octamers and DNA were mixed in a buffer containing 10 mM Tris-HCl, pH 8.0, 1 mM EDTA and 2 M NaCl, and then the mixture was dialyzed against 450 ml of this buffer.…”

Section: Methodsmentioning

confidence: 99%

“…HMT assays were performed as reported previously with some modifications 59 . For details, the enzymatic reaction system includes 50 mM Tris-HCl (pH 8.5), 1 mM MgCl 2 , 4 mM DTT and 1 μg of nucleosome substrates.…”

Section: Methodsmentioning

confidence: 99%

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Histone H1 facilitates restoration of H3K27me3 during DNA replication by chromatin compaction

Liu

Song

et al. 2023

Nat Commun

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During cell renewal, epigenetic information needs to be precisely restored to maintain cell identity and genome integrity following DNA replication. The histone mark H3K27me3 is essential for the formation of facultative heterochromatin and the repression of developmental genes in embryonic stem cells. However, how the restoration of H3K27me3 is precisely achieved following DNA replication is still poorly understood. Here we employ ChOR-seq (Chromatin Occupancy after Replication) to monitor the dynamic re-establishment of H3K27me3 on nascent DNA during DNA replication. We find that the restoration rate of H3K27me3 is highly correlated with dense chromatin states. In addition, we reveal that the linker histone H1 facilitates the rapid post-replication restoration of H3K27me3 on repressed genes and the restoration rate of H3K27me3 on nascent DNA is greatly compromised after partial depletion of H1. Finally, our in vitro biochemical experiments demonstrate that H1 facilitates the propagation of H3K27me3 by PRC2 through compacting chromatin. Collectively, our results indicate that H1-mediated chromatin compaction facilitates the propagation and restoration of H3K27me3 after DNA replication.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%