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的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（H3 K27 me 3）的三甲基化与基因沉默有关，而组蛋白H3赖氨酸4（H3 K4 me 3）的三甲基化与基因激活高度相关。H3 K4 me 3和H3 K27 me 3分别占据和影响胚胎干细胞中所有人基因调控区的68-75%和7%，并且在许多表观遗传过程（包括干细胞分化）中起关键作用。有趣的是，这两种具有相反作用的甲基化共同占据许多启动子区域，形成所谓的二价结构域，其可以在细胞分化期间被解析为仅含有H3 K4 me 3的单价结构域。二价结构域是转录失活的，表明H3 K27 me 3在H3 K4 me 3上起主导作用。最近，我们发现了H3 K27去甲基化酶KDM 6A（也称为UTX），这是一种长期寻找的组蛋白甲基化修饰剂，通过使H3 K27 me 3去甲基化来上调基因表达。我们的长期目标是确定KDM 6A在干细胞分化中的表观遗传作用。我们的KDM 6A敲低实验表明，KDM 6A作为视黄酸诱导的人干细胞系NT 2/D1分化的关键表观遗传调节因子。重要的是，我们先前表明KDM 6A与H3 K4甲基转移酶混合谱系白血病（MLL）3/4在组蛋白修饰物复合物中相互作用。因此，KDM 6A复合物能够催化H3 K4甲基化和H3 K27去甲基化的酶促过程，通过该酶促过程，二价结构域可以被解析为单价结构域。与此一致，我们额外的初步数据表明，在NT 2/D1和ES细胞系H9中，KDM 6A有助于解析几个关键发育/分化特异性HOX基因启动子处的二价结构域。基于这些令人兴奋的初步结果，我们的中心假设是KDM 6A通过与其相关蛋白质合作修饰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。这项研究的重点是了解KDM 6A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。在这里，我们提出了以下三个具体目标：1）表征KDM 6A在介导干细胞分化中的作用; 2）确定KDM 6A在修饰二价结构域中的作用; 3）确定KDM 6A相关蛋白在KDM 6A介导的细胞分化中的作用。这些研究将揭示表观遗传修饰剂KDM 6A及其辅因子蛋白在干细胞分化中的前所未有的作用，并且对于我们在分子水平上理解关键的表观遗传标记（如二价结构域）在干细胞分化过程中如何被修饰是至关重要的。

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.