Role of UTX condensation in chromatin regulation

项目摘要 UTX/KDM6A是一种重要的肿瘤抑制和发育调节因子，经常 在人类癌症和包括歌舞伎在内的某些神经发育障碍中发生突变 综合症。虽然UTX是一种组蛋白H3K27去甲基酶，但去甲基酶的活性通常是 在介导肿瘤抑制和发育调节中不可或缺的关键分子 UTX在这些过程中的活动仍然难以捉摸。UTX被认为可以调节染色质的活性 通过联系和协调MLL3/KMT2C和MLL4/KMT2D的职能，主要 H3K4单甲基酶和p300组蛋白乙酰转移酶，所有这些都是 在人类癌症中也经常发生突变。在我们的初步研究中，我们发现肿瘤 UTX的抑制活性需要其核心所赋予的相分离特性 本质无序区域(CIDR)。内源性UTX在小鼠体内形成动态凝结物 胚胎干细胞(ESCs)及其CIDR对胚胎干细胞的分化具有重要意义。UTX和MLL4 形成增强H3K4单甲基化活性的共缩合物。我们还发现UTY， UTX的Y染色体同源物，具有较弱的肿瘤抑制活性，这与 形成动态程度较低的凝析油。这些结果让我们阐明了我们的中心假设 UTX需要在具有适当生物物理性质的凝析油中才能发挥作用 调控染色质基因表达调控肿瘤发生和干细胞分化。 我们提出了两个具体目标。 目的1.确定UTX缩合调节染色质的机制 活动。我们将通过以下方式确定UTX缩合如何调节染色质修饰活动 生化重建，也通过多水平染色质的综合分析 转导的癌细胞和内源性UTX编辑的ESCs的活性。 目的2.确定UTX冷凝物的生物物理性质如何调节其 生物活动。我们将研究UTX、WT和各种凝析油的生物物理性质 以及与之相关的共同激活剂。我们将确定凝析油的性质对生物的影响 在转导的癌细胞和内源性UTX编辑的ESCs中，都有不同水平的活性。 我们还将检查与疾病相关的UTX错义突变对凝集液的影响 属性。

PROJECT ABSTRACT UTX/KDM6A is an important tumor suppressor and developmental regulator, and is frequently mutated in human cancers and certain neurodevelopmental disorders including Kabuki syndrome. Though UTX is a histone H3K27 demethylase, the demethylase activity is often dispensable in mediating tumor suppression and developmental regulation, and the key molecular activity of UTX in these processes remains elusive. UTX is thought to regulate chromatin activity by associating with and coordinating the function of MLL3/KMT2C and MLL4/KMT2D, the major H3K4 mono-methylase at enhancers, and the p300 histone acetyltransferase, all of which are also frequently mutated in human cancers. In our preliminary studies, we found that the tumor suppressive activity of UTX requires its phase separation property conferred by its core Intrinsically Disordered Region (cIDR). Endogenous UTX forms dynamic condensates in mouse embryonic stem cells (ESCs) and its cIDR is important for ESC differentiation. UTX and MLL4 form co-condensates that enhance the H3K4 mono-methylation activity. We also found that UTY, the Y chromosome homolog of UTX, has weaker tumor suppressive activity that is associated with formation of less dynamic condensates. These results let us formulate our central hypothesis that UTX needs to be in condensates with appropriate biophysical properties to be active in regulating gene expression on chromatin in regulating tumorigenesis and stem cell differentiation. We proposal two Specific Aims. Aim 1. Determine the mechanisms by which UTX condensation regulates chromatin activity. We will determine how UTX condensation regulates chromatin modification activities by biochemical reconstitution, and also through integrative analyses of multiple levels of chromatin activity in both transduced cancer cells and endogenous Utx-edited ESCs. Aim 2. Determine how biophysical properties of the UTX condensates regulate its biological activities. We will study biophysical properties of UTX WT and variant condensates and its associated co-activators. We will determine impacts of condensate properties on biological activities at different levels in in both transduced cancer cells and endogenous Utx-edited ESCs. We will also examine effect of disease-associated missense mutations of UTX on condensate properties.