Role of the Histone Modifier KDM6A in Stem Cell Differentiation

描述（由申请人提供）：组蛋白赖氨酸(K)甲基化已成为与基因表达转录调控相关的关键表观遗传标记。特别是，组蛋白H3赖氨酸27 （H3K27me3）的三甲基化与基因沉默有关，而组蛋白H3赖氨酸4 （H3K4me3）的三甲基化与基因激活高度相关。H3K4me3和H3K27me3分别占据和影响胚胎干细胞中所有人类基因调控区域的68-75%和7%，并在包括干细胞分化在内的许多表观遗传过程中发挥关键作用。有趣的是，这两种具有相反作用的甲基化共同占据了许多启动子区域，形成了所谓的二价结构域，在细胞分化过程中可以分解为只含有H3K4me3的单价结构域。二价结构域在转录上不活跃，表明H3K27me3比H3K4me3起主导作用。最近，我们发现了H3K27去甲基化酶KDM6A（也称为UTX），这是一种长期寻找的组蛋白甲基化修饰因子，通过去甲基化H3K27me3来上调基因表达。我们的长期目标是确定KDM6A在干细胞分化中的表观遗传学作用。我们的KDM6A敲低实验表明，KDM6A是维甲酸诱导的人干细胞NT2/D1分化的关键表观遗传调控因子。重要的是，我们之前发现KDM6A在组蛋白修饰复合物中与H3K4甲基转移酶混合谱系白血病（MLL） 3/4相互作用。因此，KDM6A复合物能够催化H3K4甲基化和H3K27去甲基化的酶促过程，从而将二价结构域分解为单价结构域。与此一致的是，我们的额外初步数据表明，在NT2/D1和ES细胞系H9中，KDM6A参与了几个关键发育/分化特异性HOX基因启动子的二价结构域的解析。基于这些令人兴奋的初步结果，我们的中心假设是KDM6A通过与其相关蛋白合作修改关键的表观遗传特征（如二价结构域），在干细胞分化中起着至关重要的作用。本研究的重点是了解KDM6A及其相关蛋白在介导干细胞分化中的功能及其在修饰干细胞表观遗传特征中的作用。在此，我们提出以下三个具体目标：1)表征KDM6A在介导干细胞分化中的作用；2)确定KDM6A在修饰二价结构域中的作用；3)明确kdm6a相关蛋白在kdm6a介导的细胞分化中的作用。这些研究将揭示表观遗传修饰因子KDM6A及其辅助因子蛋白在干细胞分化中的前所未有的作用，并且是我们在分子水平上理解干细胞分化过程中如何修饰关键表观遗传特征（如二价结构域）的基础。

DESCRIPTION (provided by applicant): Histone lysine (K) methylation has emerged as a key epigenetic mark associated with transcriptional regulation of gene expression. In particular, trimethylation at histone H3 lysine 27 (H3K27me3) is linked with gene silencing, whereas trimethylation at histone H3 lysine 4 (H3K4me3) is highly correlated with gene activation. H3K4me3 and H3K27me3 occupy and affect 68-75% and 7%, respectively, of all human gene- regulatory regions in embryonic stem cells and play critical roles in numerous epigenetic processes, including stem cell differentiation. Interestingly, these two types of methylation with opposing roles co-occupy many promoter regions, forming so-called bivalent domains that can be resolved to monovalent domains containing only H3K4me3 during cellular differentiation. Bivalent domains are transcriptionally inactive, indicating a dominant role for H3K27me3 over H3K4me3. Recently, we identified the H3K27 demethylase KDM6A (also called UTX), a long-sought histone methylation modifier that up-regulates gene expression by demethylating H3K27me3. Our long-term objective is to define the epigenetic role of KDM6A in stem cell differentiation. Our KDM6A knockdown experiments indicate that KDM6A acts as a key epigenetic regulator for retinoic acid- induced differentiation of the human stem cell line NT2/D1. Importantly, we previously showed that KDM6A interacts with the H3K4 methyltransferases mixed-lineage leukemia (MLL) 3/4 in a histone modifier complex. Therefore, the KDM6A complex is able to catalyze enzymatic processes for both H3K4 methylation and H3K27 demethylation by which bivalent domains can be resolved to monovalent domains. Consistent with this, our additional preliminary data indicate that in both NT2/D1 and the ES cell line H9, KDM6A contributes to the resolution of bivalent domains at the promoters of several key development/differentiation-specific HOX genes. Based on these exciting preliminary results, our central hypothesis is that KDM6A plays an essential role in stem cell differentiation by modifying key epigenetic signatures, such as bivalent domains, in cooperation with its associated proteins. The focus of this proposed study is on understanding the functions of KDM6A and its associated proteins in mediating stem cell differentiation and their roles in modifying epigenetic signatures of stem cells. Here, we propose the following three specific aims: 1) Characterize the role of KDM6A in mediating stem cell differentiation; 2) Determine the role of KDM6A in modifying bivalent domains; and 3) Define the roles of the KDM6A-associated proteins in KDM6A-mediated cellular differentiation. These studies will uncover the unprecedented roles for the epigenetic modifier KDM6A and its cofactor proteins in stem cell differentiation and are fundamental to our understanding at the molecular level of how key epigenetic signatures, such as bivalent domains, are modified during stem cell differentiation.