Epigenetic mechanisms of Ash1L in transcriptional activation

Ash1L 在转录激活中的表观遗传机制

基本信息

批准号：
9923726
负责人：
Jin He
金额：
$ 33.36万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-05-01 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9923726
关键词：
ASH1L gene Address Binding Biochemical Biological Assay CRISPR/Cas technology Cells Complex Cryoelectron Microscopy Development Disease Drosophila genus ES Cell Line Enzymes Epigenetic Process Event Gene Activation Gene Expression Genes Genetic Genetic Transcription Genetic study Histone H3 Histones Homologous Gene Impairment In Vitro Individual Lead Link Lysine MLL gene Malignant Neoplasms Mammalian Cell Mammals Mediating Methylation Mixed-Lineage Leukemia Modeling Modification Molecular Mus Mutate Nucleic Acid Regulatory Sequences Phenotype Play Polycomb Process Proteins Reader Research Institute Role Structure Tail Time Transcriptional Activation base design embryonic stem cell epigenetic regulation gene repression genetic analysis genome editing histone methylation histone methyltransferase histone modification inhibitor/antagonist mutant new therapeutic target next generation sequencing promoter recruit stem cell differentiation transcription factor

项目摘要

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.

项目摘要转录调控区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。除了谱系特异性转录因子外， Trithroax 组 (TxG) 组蛋白通过拮抗多梳介导的转录抑制。在哺乳动物中，Mll1/Mll2（混合谱系白血病）和 Ash1L （缺失、小或同源盘 1 样）复合物是介导共价组蛋白的两个 TxG 复合物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。尽管之前的遗传学研究表明Mll1/Mll2和Ash1L 复合物在功能上参与共同的表观遗传调控过程，但目前尚不清楚这些复合物如何两个复合物在分子水平上连接以在转录激活中发挥其功能。此外，与充分研究的 Mll1/Mll2 复合物和组蛋白 H3K4 甲基化相比， Ash1L 及其介导的启动子处组蛋白 H3K36 甲基化在很大程度上是未知的。为了努力解决针对这些基本问题，我们纯化了 Ash1L 相互作用蛋白并鉴定了 Spindlin1 (Spin1)，这是一种组蛋白 H3K4me3 特异性阅读器，与 Ash1L 物理结合。删除小鼠中的 Ash1L 或 Spin1 胚胎干细胞在诱导分化后损害早期谱系特异性基因的表达，表明细胞中 Spin1 和 Ash1L 之间存在功能联系。基于这些结果，我们提出了新模型统一了 Mll1/Mll2 和 Ash1L 在转录激活中的功能。在此模型中，Mll1/Mll2， Spin1 和 Ash1L 形成表观遗传调控轴，其中各个组件依次招募到谱系特异性基因启动子，以在转录激活过程中介导组蛋白修饰。具体来说，Spin1 通过将 Ash1L 募集到 Mll1/Mll2 和 Ash1L 中，在连接 Mll1/Mll2 和 Ash1L 方面发挥着核心作用。 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-Ash1L表观遗传调控轴中，我们将结合生化检测、CRISPR/Cas9- 介导的基因组编辑和基于下一代测序的遗传分析来剖析个体工作模型中提出的监管步骤。具体来说，我们将确定 (1) Ash1L 及其 HMTase 活性是其促进转录激活功能所必需的； (2)分子 Ash1L 招募至基因启动子的机制； (3)相互作用的结构基础 Ash1L、Spin1 和组蛋白 H3K4me3 之间。完成这项研究不仅将显着推进我们的了解调节谱系特异性基因激活的基本表观遗传机制，而且揭示了阻止癌症中异常基因激活的新治疗靶点。