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Single amino acid mutations in histone H3.3 illuminate the functional significance of H3K4 methylation in plants [BS-seq]

ABSTRACT: Although numerous data have revealed correlations of histone modifications with 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 decreased 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.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE274523 | GEO | 2025/03/14

REPOSITORIES: GEO

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Project description:Epigenetic environment of histone H3.3 on promoters revealed by integration of imaging and genome-scale chromatin and methyl-DNA immunoprecipitation information. Chromatin regions with different transcriptional outputs are distinguished by the deposition of histone variants. Histone H3.3 is incorporated into chromatin in a replication-independent manner; yet the relationship between H3.3 deposition, chromatin environment is incompletely understood. We have integrated imaging and genome-scale chromatin and methyl-DNA immunoprecipitation approaches to investigate the genomic distribution of epitope-tagged H3.3 in relation to histone modifications, DNA methylation and transcription. Results: Imaging shows that H3.3, in contrast to replicative H3.1 or H2B, is enriched in chromatin marked by histone modifications of active genes. A genome-wide survey identifies 1,649 H3.3-enriched promoters, only a subset of which is co-enriched in H3K4me3, H3K9me3 and/or H3K27me3, with a preference for H3K4me3, corroborating imaging data. H3.3-enriched promoters are depleted of H3.3 at the TSS in a transcription-independent manner. H3.3 is found predominantly on CpG-rich unmethylated promoters, creating a condition favourable for transcription. In undifferentiated mesenchymal stem cells, H3.3 target genes are linked to signaling and mesodermal differentiation, suggesting that H3.3 may be a mark of lineage priming. Conclusions: A minor fraction of H3.3 is targeted to promoters, which are predominantly CpG-rich, DNA unmethylated and devoid of detectable trimethylated H3K4, K9 and K27. Among H3.3 target promoters co-marked by methylated H3, H4K4me3 is preferred, with or without H3K27me3, arguing that in mesenchymal stem cells H3.3 marks transcriptionally active or potentially active promoters. Key words: Imaging, ChIP-chip, MeDIP-chip, histone H3.3, mesenchymal stem cells ChIP-chip and MeDIP-chip experiments: Performed with two independent biological replicates. Gene expression profiling experiments: Total RNA obtained from H3.3-EGFP transfected or empty-EGFP transfected mesenchymal stem cells compared to untransfected mesenchymal stem cells. Raw expression data linked below as supplementary file (GSE17053_Illumina_non-normalized_data.txt).