Depletion of Nsd2-mediated histone H3K36 methylation impairs adipose tissue development and function

Histone tails are post-translationally modified at multiple sites, including Lys36 on histone H3 (H3K36). The H3K36 methylation has been shown to associate with the transcription of active euchromatin, alternative splicing, DNA repair and recombination. However, the role of H3K36 methylation during cell differentiation is still obscure.Previous investigations found that a site specific Lys-to-Met mutation on histones can serve as an inhibitor of this site specific methyltransferases to repress global methylation on that lysine of histones. In this study we usedH3K36 Lys-to-Met mutant (H3K36M) as a tool to investigaterole of H3K36 methylation in our cell differentiation system. Expression of H3K36M repressed global H3K36 methylation but increased H3K27me3 as previously reported. On our cell differentiation system, H3K36M suppressed adipogenesis and myogenesis. Our pioneer RNA-seqstudy further showed that H3K36M repressed the expression of master regulator genes. Here, we did ChIP-seq of several histone modifications to further explore the changes on the epigenome by expression of H3K36M. Interestingly, on some important master regulator genes loci, the repressive marker H3K27me3 increased significantly, correlated with the repression on their expression. The H3K36M decreases global H3K36 di- and tri-methylation. To clarify which H3K36 methyltransferase is important for cell differentiation, we knocked down H3K36 di-methyltransferases Nsd1 and Nsd2, H3K36 tri-methyltransferase Setd2 separately. Nsd2 knockdown, but not Nsd1 or Setd2,can phenocopythe adipogenesis defect in H3K36M expressed cells.Comparing in RNA-seq results, Nsd2 knockdown cells showed similar gene expression profile with H3K36M expressed cells in adipogenesis. These suggest that Nsd2-mediated H3K36me2 plays an important role in adipogenesis.To study the role of H3K36 methylation in vivo, we generated an aP2 promoter driven H3K36M expressed transgenic mouse (Tg), to express H3K36M specifically in adipose tissue. The Tg mice showedsignificant dysfunction in both white and brown adipose tissues. Their fat tissues gene expression profile changed significantly. All of these indicate that H3K36 methylation is important for adipose tissue developmentin vivo.Together, our comprehensive studies provide novel insights into dynamics of H3K36 methylation and its important role in transcriptional regulation of cell differentiation and mouse fat tissue development.

组蛋白尾巴（histone tails）可在多个位点发生翻译后修饰，包括组蛋白H3（histone H3）的第36位赖氨酸（H3K36）。H3K36甲基化已被证实与激活型常染色质的转录、可变剪接、DNA修复及重组相关，但H3K36甲基化在细胞分化过程中的作用仍尚不明确。既往研究发现，组蛋白上的位点特异性赖氨酸-甲硫氨酸突变可作为该位点特异性甲基转移酶的抑制剂，以抑制该赖氨酸位点上的组蛋白全局甲基化。本研究采用H3K36赖氨酸-甲硫氨酸突变体（H3K36M）作为工具，探究H3K36甲基化在本研究的细胞分化体系中的作用。正如既往报道，H3K36M的表达可抑制全局H3K36甲基化，但会升高H3K27me3水平。在本研究的细胞分化体系中，H3K36M可抑制脂肪生成与肌生成。我们的先导性RNA测序（RNA-seq）研究进一步显示，H3K36M可抑制主调控基因的表达。本研究通过染色质免疫共沉淀测序（ChIP-seq）检测了多种组蛋白修饰，以进一步探究H3K36M表达对表观基因组的影响。有趣的是，在部分关键主调控基因的基因座上，抑制性修饰标记H3K27me3水平显著升高，与其表达抑制呈显著相关。H3K36M可降低全局H3K36二甲基化与三甲基化水平。为明确哪种H3K36甲基转移酶对细胞分化至关重要，我们分别敲低了H3K36二甲基转移酶Nsd1、Nsd2以及H3K36三甲基转移酶Setd2。仅敲低Nsd2（而非Nsd1或Setd2）可模拟表达H3K36M的细胞中出现的脂肪生成缺陷。对比RNA测序结果发现，在脂肪生成过程中，Nsd2敲低细胞的基因表达谱与表达H3K36M的细胞高度相似。上述结果表明，Nsd2介导的H3K36me2在脂肪生成中发挥重要作用。为在体内研究H3K36甲基化的功能，我们构建了由aP2启动子驱动的H3K36M表达转基因小鼠（Tg），使其在脂肪组织中特异性表达H3K36M。该转基因小鼠的白色脂肪组织与棕色脂肪组织均出现明显功能异常，其脂肪组织的基因表达谱也发生显著改变。上述所有结果均表明，H3K36甲基化对体内脂肪组织发育至关重要。综上，我们的综合研究为H3K36甲基化的动态调控机制提供了新见解，并揭示了其在细胞分化的转录调控以及小鼠脂肪组织发育中的重要作用。