Single amino acid mutations in histone H3.3 illuminate the functional significance of H3K4 methylation in plants [ChIP-seq_Pol II Ser5p & Pol II Ser2p]
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. Overall design: Examination of Pol II Ser5p and Pol II Ser2p enrichment in Col, h3.3ko;HTR5, h3.3ko;HTR5 K4A and sdg2 mutant seedlings