Dot1L recruits OGT target chromatin to regulate histones O-GlcNAcylation

OGT (O-GlcNAc transferase) is the distinctive enzyme responsible for catalyzing O-GlcNAc to the serine or threonine residues of thousands of cytoplasm and nuclear proteins that are involved in DNA damage, RNA splicing, and transcription preinitiation and initiation complex assembly. However, the molecular mechanism by OGT regulating gene transcription remains elusive. Using proximity labeling based mass spectrometry, we searched for functional partners of OGT and found that Dot1L, the conserved and unique histone methyltransferase mediated histone H3 lys79 methylation required for gene transcription, DNA damage repair, cell proliferation, and embryo development, interacts with OGT. Although this specific interaction does not regulate the enzymatic activity of Dot1L, it facilitates OGT-dependent histones O-GlcNAcylation. Moreover, OGT associates with Dot1L at transcription start sites, and depleting Dot1L decreased OGT associated with chromatin globally. Notably, downregulation of Dot1L reduces the levels of histone H2B S112 O-GlcNAcylation and histone H2B K120 ubiquitination in vivo, which are associated with gene transcription regulation. Taken together, these results reveal a Dot1L-dependent O-GlcNAcylation of chromatin. Cut&Tag dataset of OGT and Dot1L in HEK293T cells. Cut&Tag dataset of OGT in HEK293T cells treated with or without siRNA targeted Dot1L.Study the roles of DOT1L in histones O-GlcNAcyaltion regulation

O-GlcNAc转移酶（O-GlcNAc transferase，OGT）是一类独特的酶，负责催化O-GlcNAc修饰结合至数千种细胞质与核蛋白的丝氨酸或苏氨酸残基；这些蛋白参与DNA损伤修复、RNA剪接以及转录预起始与起始复合物组装等过程。然而，OGT调控基因转录的分子机制仍未阐明。本研究采用基于邻近标记的质谱分析技术，筛选OGT的功能互作伙伴，发现Dot1L——一种保守且独特的组蛋白甲基转移酶，其介导的组蛋白H3赖氨酸79甲基化是基因转录、DNA损伤修复、细胞增殖与胚胎发育所必需的——可与OGT发生特异性相互作用。尽管该互作并不会调控Dot1L的酶促活性，但它可促进依赖于OGT的组蛋白O-GlcNAc修饰。此外，OGT与Dot1L可共同结合至转录起始位点，且敲低Dot1L会全局降低染色质结合的OGT水平。值得注意的是，Dot1L的下调可在体内降低组蛋白H2B S112位点的O-GlcNAc修饰与组蛋白H2B K120位点的泛素化水平，这两者均与基因转录调控密切相关。综上，本研究结果揭示了一种依赖于Dot1L的染色质O-GlcNAc修饰调控机制。本研究包含两类数据集：一是HEK293T细胞中OGT与Dot1L的Cut&Tag测序数据集；二是经靶向Dot1L的小干扰RNA（siRNA）或对照处理的HEK293T细胞中OGT的Cut&Tag测序数据集，旨在探究DOT1L在组蛋白O-GlcNAc修饰调控中的作用。