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

描述（由申请人提供）：果蝇Trithorax相关（Trr）、人MLL 3和MLL 4是含SET结构域蛋白家族的同源物，其构成对赖氨酸4上的组蛋白H3（H3 K4）具有甲基转移酶活性的酶，并形成称为COMPASS的高度保守蛋白复合物的催化活性中心。最近的全基因组研究已经揭示，MLL 3和MLL 4在各种形式的癌症中频繁突变。类似地，Trr和Utx，果蝇Trr COMPASS样复合物的复合物特异性组分，构成组织生长的抑制因子。Trr在体内充当主要的H3 K4单甲基转移酶，并且Trr和MLL 3两者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，尽管维持Trr/MLL 3/MLL 4完整性对于适当的组织稳态很重要，但对介导肿瘤抑制的Trr/MLL 3/MLL 4依赖性基本机制知之甚少。该提案旨在为候选人提供一个培训平台，以提高其与人类细胞合作（目标1-2）、设计和执行果蝇和人类细胞高通量RNA干扰筛选（目标2）的技能，精通流式细胞术和生化技术（均为目标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/MLL 3/MLL 4在增强子相关H3 K4单甲基化中的作用，候选人假设MLL 3和/或MLL 4突变型癌症的增强子景观的变化构成了肿瘤发生的驱动力。该提案的所有研究目的都是为了获得对Trr/MLL 3/MLL 4在增强子介导的过程中的作用的机制见解，特别是增强子活性的变化如何可能有助于肿瘤发生。实验将包括在人结肠癌细胞中鉴定具有致瘤潜力的MLL 3/MLL 4依赖性增强子（Aim 1）。此外，我们希望通过在果蝇和人类细胞系中使用选择增强子元件的全基因组RNAi筛选以及通过使用生化增强子下拉方法来鉴定介导Trr/MLL 3/MLL 4依赖性增强子活性的因子（目的2）。最后，我们想要确定Trr/MLL 3/MLL 4及其H3 K4甲基转移酶活性在增强子介导的体内肿瘤抑制中的作用（目的3）。trr突变体与野生型，催化失活或过度活跃的trr构建体的拯救实验，以及全基因组研究，将帮助我们评估Trr的H3 K4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MLL 3/MLL 4突变的癌症中至关重要的选择因子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发生的特定药物。

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.