Trithorax-related, MLL3 and MLL4 in enhancer-mediated cancer pathogenesis

描述(申请人提供)：果蝇三胸相关蛋白(TRR)，人类MLL3和MLL4是一个含有SET结构域的蛋白家族的同源物，这些蛋白构成了赖氨酸4(H3K4)上具有甲基转移酶活性的酶，并形成了高度保守的蛋白质复合体的催化活性枢纽，称为COMPASS。最近的全基因组研究表明，MLL3和MLL4在各种形式的癌症中经常发生突变。同样，TRR和UTX，一种果蝇TRR罗盘状复合体的复合体特异性成分，构成了组织生长的抑制因子。TRR是体内主要的H3K4单甲基转移酶，TRR和MLL3都在增强子介导的过程中发挥作用。越来越多的证据也表明，增强子活性的变化可能是致癌的驱动力之一。然而，尽管保持TRR/MLL3/MLL4的完整性对于适当的组织动态平衡很重要，但人们对依赖TRR/MLL3/MLL4介导肿瘤抑制的基本机制知之甚少。这项提议旨在为候选人提供一个培训平台，以提高他在与人类细胞合作(目标1-2)、在果蝇和人类细胞中设计和执行高通量RNAi筛查(目标2)的技能，熟练掌握流式细胞仪和生化技术(目标2)，并获得分析全基因组高通量测序数据的生物信息学技能(目标1)。基于TRR/MLL3/MLL4在增强子相关的H3K4单甲基化中的作用，候选假说是MLL3和/或MLL4突变癌症的增强子图谱的变化构成了肿瘤发生的驱动力。这项建议的所有研究目的都是为了获得对TRR/MLL3/MLL4在增强子介导的过程中的作用的机械性见解，特别是增强子活性的变化如何可能有助于肿瘤发生。实验将包括鉴定人类结肠癌细胞中具有致瘤潜力的依赖于MLL3/MLL4的增强子(目标1)。此外，我们希望通过在果蝇和人类细胞系中使用具有选择的增强子元件的全基因组RNAi筛选，并通过使用生化增强子下拉方法来识别介导TRR/ML3/MLL4依赖的增强子活性的因素(目标2)。最后，我们想要确定TRR/MLL3/MLL4及其H3K4甲基转移酶活性在体内增强子介导的肿瘤抑制中的作用(目标3)。用野生型、催化失活或高活性的TRR结构对TRR突变体进行的挽救实验，以及全基因组的研究，将有助于我们评估TRR的H3K4甲基转移酶活性是否对其在增强子介导的肿瘤抑制中的作用至关重要。总之，这些目标将使我们能够确定在携带MLL3/MLL4突变的癌症中至关重要的选定因素和增强子介导的过程。这将允许开发可用于治疗增强子介导的肿瘤形成的特定药物。

DESCRIPTION (provided by applicant): Drosophila Trithorax-related (Trr), human MLL3 and MLL4 are homologs of a family of SET domain-containing proteins that constitute enzymes with methyltransferase activity towards histone H3 on lysine 4 (H3K4) and form the catalytically active hub of highly conserved protein complexes termed COMPASS. Recent genome- wide studies have revealed that MLL3 and MLL4 are frequently mutated in various forms of cancer. Similarly, Trr and Utx, a complex-specific component of the Drosophila Trr COMPASS-like complex, constitute suppressors of tissue growth. Trr acts as a major H3K4 monomethyltransferase in vivo and both Trr and MLL3 play a role in enhancer-mediated processes. Accumulating evidence also suggests that changes in enhancer activity might be one of the driving forces of carcinogenesis. However, despite the importance to maintain Trr/MLL3/MLL4 integrity for proper tissue homeostasis, very little is known about the Trr/MLL3/MLL4- dependent basic mechanisms that mediate tumor suppression. This proposal is designed to provide a training platform for the candidate to enhance his skills in working with human cells (Aim 1-2), designing, and executing high-throughput RNAi screens in Drosophila and human cells (Aim 2), to become proficient in flow cytometry and biochemical techniques (both Aim 2) and to acquire bioinformatics skills to analyze genome-wide high-throughput sequencing data (Aim 1). Based on the role of Trr/MLL3/MLL4 in enhancer-associated H3K4 monomethylation, the candidate hypothesizes that changes in the enhancer landscape of MLL3 and/or MLL4 mutant cancers constitute a driving force of tumorigenesis. All research aims of this proposal are designed to gain mechanistic insights into the role of Trr/MLL3/MLL4 in enhancer-mediated processes in general, and particularly how changes in enhancer activity might contribute to tumorigenesis. Experiments will include the identification of MLL3/MLL4-dependent enhancers with tumorigenic potential in human colon cancer cells (Aim 1). Additionally, we want to identify factors that mediate Trr/MLL3/MLL4-dependent enhancer-activity using genome-wide RNAi screens with select enhancer elements in Drosophila and human cell lines, and by using biochemical enhancer pull down approaches (Aim 2). Finally, we want to define the role of Trr/MLL3/MLL4 and its H3K4 methyltransferase activity in enhancer-mediated tumor suppression in vivo (Aim 3). Rescue experiments of trr mutants with wild-type, catalytically inactive or hyperactive trr constructs, together with the genome-wide studies, will help us to assess whether the H3K4 methyltransferase activity of Trr is vital for its role in enhancer-mediated tumor suppression. In summary, these aims will enable us to identify select factors and enhancer-mediated processes that are crucial in cancers bearing mutations in MLL3/MLL4. This will permit the development of specific pharmaceuticals that can be used for the treatment of enhancer-mediated tumorigenesis.