The COMPASS family of H3K4 methylases in development and cancer

描述（由申请人提供）： 在过去的十七年里，我的实验室的研究集中在Set 1/MLL蛋白家族的分子功能和生化特性的表征上。它们的嵌合体和突变与儿童白血病和其他形式的癌症有关。我们的希望是，我们的分子研究将通过这个蛋白质家族促进我们对重排和突变型癌症分子机制的理解。在目前的资助周期内，我的实验室在包括果蝇和哺乳动物细胞培养在内的多个模型系统中采用了遗传学和生物化学。我们证明，果蝇细胞具有三个Set 1相关的蛋白质：dSet 1，Trithorax（Trx），和Trithorax相关的（Trr），都发现在COMPASS样组合物能够甲基化组蛋白H3 K4。哺乳动物细胞具有果蝇中发现的三个子类中的每一个的两个代表，总共有六个COMPASS家族成员：SET 1A/SET 1B（与dSet 1相关）; MLL 1和MLL 2（与Trx相关）;以及MLL 3和MLL 4（与Trr相关）。此外，鉴于许多MLL易位伴侣之间几乎没有序列同源性，多年来，尚不清楚为什么MLL易位到如此多的不相关基因中导致白血病的发病机制。我们对MLL-嵌合体纯化的生物化学研究表明，许多MLL易位伴侣是我们命名为超延伸复合物（SEC）的相同大分子复合物的一部分。我们证明了MLL在SEC的任何亚基内的易位导致SEC向MLL靶基因的错误募集以及这些基因的转录检查点控制的扰动，从而触发白血病生长。此外，我们了解到，最近对癌症体细胞突变的编目已经在血液恶性肿瘤和实体瘤中的ML 1 -4和Set 1 A/B复合物的成分中发现了大量突变。事实上，MLL 1 -4和Set 1A/B在不同形式的癌症中似乎比p53携带更多的突变。然而，我们对MLL 1 -4和Set 1A/B家族的发展以及为什么它们的突变与癌症相关知之甚少。鉴于我们在过去的17年里已经开发了一套很棒的试剂和工具，用于在多个模型系统中研究这些因子及其相关蛋白质，我的实验室处于一个非常独特的位置，可以确定这些因子参与癌症发病机制的分子基础，以实现靶向治疗的目的。因此，本次更新申请的目标是MLL 1 -4和Set 1A-B及其复合物在基因表达和发育调控中的完整分子和生物化学表征，以及它们的突变如何促进人类癌症的发病机制。本次更新申请的目标将通过三个具体目标积极追求。具体目标1侧重于Trx/COMPASS家族成员（MLL 1和MLL 2）的分子特性的表征;鉴定其分子特性和特异性招募染色质;以及它们的易位如何有助于白血病发病机制。具体目标2将集中在定义Trr/COMPASS家族（MLL 3和MLL 4）在增强子单甲基化中的作用，以及如何通过该家族组分的特定突变导致癌症发病机制中的增强子功能障碍。具体目标3集中在Set 1/COMPASS家族（Set 1A和Set 1B）的分子表征和结构研究，以及这类酶实施的组蛋白H3 K4三甲基化如何参与整个发育过程中基因表达的调控。我们将在多个模型系统中利用各种生物化学，分子和遗传工具来实现所提出的目标。这些研究应该（i）有助于我们理解COMPASS家族在调节H3 K4甲基化模式中的不同作用以及它们如何调节发育和分化;（ii）对我们理解COMPASS家族内的突变如何导致癌症产生根本性影响。这些信息有可能证明有助于研究人员试图设计合理的方法来治疗癌症。

DESCRIPTION (provided by applicant): Project Summary Studies from my laboratory during the past seventeen years have focused on the characterization of the molecular functions and biochemical properties of the Set1/MLL family of proteins. Their chimeras and mutations are associated with childhood leukemia and other forms of cancers. Our hope is that our molecular studies will advance our understanding of the molecular mechanisms of rearrangement and mutation-based cancer through this family of proteins. During the current funding cycle of this grant, my laboratory has employed genetics and biochemistry in multiple model systems including Drosophila and mammalian cell cultures. We demonstrated that Drosophila cells possess three Set1-related proteins: dSet1, Trithorax (Trx), and Trithorax-related (Trr), all found within COMPASS-like compositions capable of methylating histone H3K4. Mammalian cells possess two representatives for each of the three subclasses found in Drosophila for a total of six COMPASS family members: SET1A/SET1B (related to dSet1); MLL1 and MLL2 (related to Trx); and MLL3 and MLL4 (related to Trr). Furthermore, given that there is almost no sequence homology between many of the MLL translocation partners, for many years, it was unclear why MLL translocations into so many unrelated genes result in the pathogenesis of leukemia. Our biochemical studies on the purification of the MLL-chimeras demonstrated that many of the MLL translocation partners are part of the same macromolecular complex we named the Super Elongation Complex (SEC). We demonstrated that the translocations of MLL within any of the subunits of SEC result in the misrecruitment of SEC to the MLL target genes and in the perturbation of the transcriptional checkpoint control of these genes, triggering leukemic growth. Additionally, what we have learned is that recent cataloging of somatic mutations in cancer have identified a large number of mutations in the components of the MLL1-4 and Set1A/B complexes in both hematological malignancies and solid tumors. As a matter of fact, collectively MLL1-4 and Set1A/B appear to bear more mutations in different forms of cancers than p53. However, we know very little about the MLL1-4 and Set1A/B families in development and why their mutations are associated with cancer. Given that we have developed a fantastic set of reagents and tools within the past seventeen years towards these factors and their associated proteins in multiple model systems, my laboratory is in a very unique position to define the molecular bases of these factors' involvement in cancer pathogenesis for the purpose of targeted therapeutics. Therefore, the goals of this renewal application are the full molecular and biochemical characterization of MLL1-4 and Set1A-B and their complexes in the regulation of gene expression and development and how their mutations contribute to the pathogenesis of human cancer. The goals of this renewal application will be aggressively pursued via three specific aims. Specific Aim 1 is focused on the characterization of the molecular properties of the Trx/COMPASS family members (MLL1 and MLL2); identification of their molecular properties and specific recruitment to chromatin; and how their translocations contribute to leukemic pathogenesis. Specific Aim 2 will be focused in defining the role of the Trr/COMPASS family (MLL3 and MLL4) in enhancer monomethylation and how enhancer malfunction through specific mutations of the components of this family result cancer in pathogenesis. Specific Aim 3 is focused on the molecular characterization and structural studies of the Set1/COMPASS family (Set1A and Set1B) and how histone H3K4 trimethylation implemented by this class of enzymes is involved in the regulation of gene expression throughout development. We will take advantage of a variety of biochemical, molecular, and genetic tools in multiple model systems to address the proposed aims. These studies should (i) be instrumental for our understanding of the diverse roles of the COMPASS family in the regulation of the pattern of H3K4 methylation and how they regulate development and differentiation; and (ii) have a fundamental impact on our understanding of how mutations within the COMPASS family result in cancer. This information has the potential of proving helpful to investigators attempting to design rational approaches for the treatment of cancer.