A plant histone H3.3-specific amino acid safeguards the deposition of H3K36 methylation for proper plant development and stress responses [H3K36me3_ChIPseq]

Histone variants are critical determinants for chromatin function with diverse regulatory mechanisms. The H3 variants, H3.1 and H3.3, evolved independently in animals and plants. A significant difference between H3.1 and H3.3 is the amino acid variation at position 31, with H3.1 carrying alanine (A), while H3.3 bearing serine (S) in animals or threonine (T) in plants. Both S and T can be phosphorylated, but why plants have selectively adopted T over S remains unclear. Here, we report the specific role of plant H3.3T31 in controlling plant development and stress responses by promoting the deposition of histone H3 lysine 36 trimethylation (H3K36me3) on H3.3. T31 prevents plant-specific H3K27 methyltransferases, ATXR5 and ATXR6, from depositing H3K27 monomethylation (H3K27me1), which inhibits the activity of H3K36 methyltransferase EFS. Substituting H3.3T31 with an S or A residue results in increased ATXR5/6 activity and elevated H3K27me1 levels, leading to a reduction in H3K36me3. Moreover, we show that unlike H3.3 T31S and T31A mutations, cancer-associated G34R and G34W mutations directly disrupt H3K36me3 deposition without affecting H3K27me1. These G34 mutations may also influence H3.3 function through mechanisms beyond the disruption of H3K36me3. Our data suggest a co-evolution of the plant-specific H3.3T31 residue and H3K27 methyltransferases ATXR5/6, which ensures the selective accumulation of H3K27me1 on H3.1 and H3K36me3 on H3.3, thereby enabling proper chromatin function. Overall design: Examination of H3K36me3 enrichment in seedlings

组蛋白变体（Histone variants）是染色质功能的关键决定因子，其调控机制多样。H3变体H3.1与H3.3在动物与植物中独立演化而来。H3.1与H3.3的显著差异在于第31位氨基酸的变异：H3.1携带丙氨酸（A）；在动物中，H3.3携带丝氨酸（S），而在植物中，H3.3则携带苏氨酸（T）。丝氨酸与苏氨酸均可被磷酸化，但植物为何选择性地保留苏氨酸而非丝氨酸，这一问题仍未明确。
本研究揭示了植物H3.3第31位苏氨酸（H3.3T31）通过促进组蛋白H3赖氨酸36三甲基化（H3K36me3）在H3.3上的沉积，进而调控植物发育与胁迫响应的具体作用。T31可阻止植物特异性组蛋白H3赖氨酸27甲基转移酶ATXR5与ATXR6催化组蛋白H3赖氨酸27单甲基化（H3K27me1）的沉积，而H3K27me1会抑制组蛋白H3赖氨酸36甲基转移酶EFS的活性。若将H3.3T31替换为丝氨酸（S）或丙氨酸（A）残基，则会增强ATXR5/6的活性并提升H3K27me1的水平，最终导致H3K36me3的含量降低。
此外，本研究发现，与H3.3 T31S和T31A突变不同，癌症相关的G34R与G34W突变可直接破坏H3K36me3的沉积，且不会影响H3K27me1的水平。这类G34突变还可能通过破坏H3K36me3以外的其他途径影响H3.3的功能。
本研究数据表明，植物特异性H3.3T31残基与组蛋白H3赖氨酸27甲基转移酶ATXR5/6存在协同演化关系，这一关系确保了H3K27me1选择性富集于H3.1，而H3K36me3选择性富集于H3.3，从而保障染色质功能正常行使。
实验整体设计：检测幼苗中H3K36me3的富集情况。