Epigenetic mechanisms of Ash1L in transcriptional activation

项目摘要 转录调控区域的表观遗传修饰在促进谱系特异性方面发挥着重要作用 干细胞分化过程中的基因表达。除了谱系特异的转录因子外， Trithroax-group(TXG)-group蛋白通过拮抗基因表达促进谱系特异性基因表达 多梳介导的转录抑制。在哺乳动物中，Mll1/Mll2(混合血统白血病)和Ash1 (不存在的、小的或类同胚盘的)复合体是两个TXG复合体，它们介导共价组蛋白 通过组蛋白甲基转移酶(HMTase)活性对组蛋白H3、赖氨酸4和 组蛋白H3赖氨酸分别为36。尽管先前的遗传学研究揭示了M111/ML12和Ash1 复合体在功能上参与了一个常见的表观遗传调控过程，目前尚不清楚这些过程是如何进行的 两个复合体在分子水平上连接在一起，实现它们在转录激活方面的功能。 此外，与研究良好的MLL1/MLL2复合体和组蛋白H3K4甲基化相反，MLI1/MLL2的功能 Ash1及其介导组蛋白H3K36在启动子上的甲基化在很大程度上是未知的。为了努力解决 这些基本问题，我们纯化了与Ash11相互作用的蛋白，并鉴定了Spindlin1(Spin1)，a 组蛋白H3K4me3特异性阅读器，物理上与Ash1结合。小鼠的Ash1或Spin1基因缺失 胚胎干细胞在诱导分化时会损害早期谱系特异性基因的表达， 这表明Spin1和Ash1在细胞中存在功能联系。基于这些结果，我们提出了一种 统一ML1/MLL2和Ash1转录激活功能的新模型。在这个模型中，Mll1/Mll2， Spin1和Ash1形成表观遗传调节轴，在该轴中，单个成分被顺序招募 在转录激活期间介导组蛋白修饰的谱系特异性基因启动子。 具体地说，Spin1通过将Ash1招募到 H3K4me3标记的谱系特异性基因启动子。了解各个组件的功能作用 在这个Mll1/Mll2-Spin1-Ash11表观遗传调节轴中，我们将结合生化分析、CRISPR/Cas9- 中介的基因组编辑和基于下一代测序的基因分析，以解剖个体 工作模式中提出的监管步骤。具体地说，我们将确定(1)Ash1及其 HMTase活性是其促进转录激活所必需的；(2)分子 ASH11招募到基因启动子的机制；(3)相互作用的结构基础 在Ash1、Spin1和组蛋白H3K4me3之间。这项研究的完成不仅将大大推进我们的 对调控谱系特异性基因激活的基本表观遗传机制的理解，也 揭示阻断癌症异常基因激活的新治疗靶点。

Project Abstract Epigenetic modifications at transcriptional regulatory regions play an important role in facilitating lineage-specific gene expression during stem cell differentiation. In addition to lineage-specific transcription factors, Trithroax-group (TxG)-group proteins promote lineage-specific gene expression through antagonizing the Polycomb-mediated transcriptional repression. In mammals, Mll1/Mll2 (Mixed Lineage Leukemia) and Ash1L (Absent, Small, or Homeotic discs 1-Like) complexes are two TxG complexes that mediate covalent histone modifications through their histone methyltransferase (HMTase) activities towards histone H3 lysine 4 and histone H3 lysine 36 respectively. Although previous genetic studies have revealed that Mll1/Mll2 and Ash1L complexes are functionally involved in a common epigenetic regulatory process, it remains unknown how these two complexes are connected at the molecular level to carry out their functions in transcriptional activation. Additionally, in contrast to the well-studied Mll1/Mll2 complexes and histone H3K4 methylation, the functions of Ash1L and its mediated histone H3K36 methylation at promoters are largely unknown. In an effort to address these fundamental questions, we purified the Ash1L-interacting proteins and identified Spindlin1 (Spin1), a histone H3K4me3-specific reader, physically binds to Ash1L. Deletion of either Ash1L or Spin1 in mouse embryonic stem cells impairs the expression of early lineage-specific genes upon induced differentiation, suggesting a functional connection between Spin1 and Ash1L in cells. Built upon these results, we propose a new model to unify the function of Mll1/Mll2 and Ash1L in transcriptional activation. In this model, Mll1/Mll2, Spin1, and Ash1L form an epigenetic regulatory axis, in which the individual component is sequentially recruited to the lineage-specific gene promoters to mediate histone modifications during transcriptional activation. Specifically, Spin1 plays a central role in connecting Mll1/Mll2 and Ash1L by recruiting Ash1L to the H3K4me3-marked lineage-specific gene promoters. To understand the functional role of individual components in this Mll1/Mll2-Spin1-Ash1L epigenetic regulatory axis, we will combine biochemical assays, CRISPR/Cas9- mediated genome editing, and next generation sequencing-based genetic analysis to dissect individual regulatory step proposed in the working model. Specifically, we will determine (1) whether Ash1L and its HMTase activity are required for its function in facilitating transcriptional activation; (2) the molecular mechanisms for the recruitment of Ash1L to gene promoters; (3) the structural basis underlying the interaction between Ash1L, Spin1 and histone H3K4me3. Completion of this study will not only significantly advance our understanding on the basic epigenetic mechanisms regulating the lineage-specific gene activation, but also reveal new therapeutic targets for blocking aberrant gene activation in cancers.