组蛋白 3 赖氨酸 36 (H3K36me) 的甲基化已成为基因表达忠实调控的重要表观遗传成分。尽管它在发育、疾病和癌症中很重要，但分子因子如何共同塑造 H3K36me 景观尚不清楚。我们使用小鼠间充质干细胞模型来扰乱 H3K36me 沉积机制并推断五个最重要参与者的活动：SETD2、 NSD1、NSD2、NSD3 和 ASH1L。我们发现 H3K36me2 是三种甲基化状态中最丰富的，并且主要由 NSD1 沉积在基因间区域，部分由 NSD2 沉积在基因间区域。相比之下，H3K36me1/3 在外显子内最为丰富，并且与基因表达呈正相关。我们证明，虽然 SETD2 沉积了大部分 H3K36me3，但它也在转录基因内沉积了 H3K36me2。此外，SETD2 的缺失会导致外显子 H3K36me1 的增加，这表明其他 H3K36 甲基转移酶 (K36MT) 的主要基因体在转录前具有较低的甲基化状态。通过还原方法，我们揭示了 NSD3 和 ASH1L 催化的 H3K36me2 的分布模式。 NSD1/2 建立了广泛的基因间 H3K36me2 结构域，而 NSD3 将 H3K36me2 峰沉积在活性启动子和增强子上。同时，ASH1L 的活性仅限于发育相关基因的调控元件，我们的分析表明 PBX2 是一种潜在的招募因子。在基因内，SETD2 沉积 H3K36me2/3，而其他 K36MT 能够独立沉积 H3K36me1/2 SETD2 活动。对于 H3K36me1/2 的沉积，我们发现了 K36MT 活性的层次结构，其中 NSD1>NSD2>NSD3>ASH1L。虽然 NSD1 和 NSD2 负责 H3K36me2 的大部分全基因组传播，但 NSD3 和 ASH1L 的活性仅限于主动调控元件。

Methylation of histone 3 lysine 36 (H3K36me) has emerged as an essential epigenetic component for the faithful regulation of gene expression. Despite its importance in development, disease, and cancer, how the molecular agents collectively shape the H3K36me landscape is unclear.We use a mouse mesenchymal stem cell model to perturb the H3K36me deposition machinery and infer the activities of the five most prominent players: SETD2, NSD1, NSD2, NSD3, and ASH1L. We find that H3K36me2 is the most abundant of the three methylation states and is predominantly deposited at intergenic regions by NSD1, and partly by NSD2. In contrast, H3K36me1/3 are most abundant within exons and are positively correlated with gene expression. We demonstrate that while SETD2 deposits most H3K36me3, it also deposits H3K36me2 within transcribed genes. Additionally, loss of SETD2 results in an increase of exonic H3K36me1, suggesting other H3K36 methyltransferases (K36MTs) prime gene bodies with lower methylation states ahead of transcription. Through a reductive approach, we uncover the distribution patterns of NSD3- and ASH1L-catalyzed H3K36me2. While NSD1/2 establish broad intergenic H3K36me2 domains, NSD3 deposits H3K36me2 peaks on active promoters and enhancers. Meanwhile, the activity of ASH1L is restricted to the regulatory elements of developmentally relevant genes, and our analyses implicate PBX2 as a potential recruitment factor.Within genes, SETD2 deposits both H3K36me2/3, while the other K36MTs are capable of depositing H3K36me1/2 independently of SETD2 activity. For the deposition of H3K36me1/2, we find a hierarchy of K36MT activities where NSD1>NSD2>NSD3>ASH1L. While NSD1 and NSD2 are responsible for most genome-wide propagation of H3K36me2, the activities of NSD3 and ASH1L are confined to active regulatory elements.