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 3 publications

(3 citation statements)

References 22 publications

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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%

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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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H2AK119ub1 differentially fine-tunes gene expression by modulating canonical PRC1- and H1-dependent chromatin compaction

Zhao,

Lan,

Wang

et al. 2024

Molecular Cell

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Single amino acid mutations in histone H3.3 illuminate the functional significance of H3K4 methylation in plants

Xue,

Ma,

et al. 2024

Preprint

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Although numerous data have revealed correlations between histone modifications and chromatin activities such as transcription, proofs of their causal importance in gene expression regulation remain limited. Sequence variants within each histone family expand chromatin diversity and may carry specific modifications, further raising the question of how histone modifications coordinate with different variants. Here, we investigate the regulatory role of lysine 4 (K4) of Arabidopsis histone H3 by point mutating K4 in two major H3 variants, H3.1 and H3.3. K4 is essential for the function of H3.3 but not H3.1 in plant development. H3K4 methylation levels decrease drastically upon K4 mutation in H3.3, and the associated transcriptome changes are similar to those observed in a mutant lacking SDG2, a major enzyme responsible for depositing H3K4 trimethylation (H3K4me3). Moreover, H3.3K4 and SDG2 are required for de novo gene activation and RNA Pol II elongation. H3K4 methylation is preferentially accumulated on H3.3 compared to H3.1, likely due to the close association of the H3.3 deposition and H3K4 methylation machineries. Furthermore, we reveal the diverse impacts of K4 nearby residue mutations on H3K4 methylation and H3.3 function. Collectively, these findings suggest that H3.3 serves as a critical substrate for H3K4 methylation, which is important for gene expression regulation. In addition, our work highlights the potential of using plants as a platform to directly investigate the importance of histone amino acids and their modifications.

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Preparation and Characterization of Chromatin Templates for Histone Methylation Assays

Cited by 3 publications

References 22 publications

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

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

H2AK119ub1 differentially fine-tunes gene expression by modulating canonical PRC1- and H1-dependent chromatin compaction

Single amino acid mutations in histone H3.3 illuminate the functional significance of H3K4 methylation in plants

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