Biology of the Myb-MuvB Oncoprotein-Tumor Suppressor Protein Complex

描述（由申请人提供）：视网膜母细胞瘤（Rb）肿瘤抑制通路在大多数（如果不是全部）人类癌症中发生突变。该途径调节S期进展所需的基因以及细胞分化所必需的基因，并且还涉及异染色质的建立和维持。在脊椎动物中的Rb途径的生物化学研究一直受到阻碍的困难，从细胞核中提取Rb复合物在其天然状态。脊椎动物中Rb通路的遗传研究由于冗余、世代时间长和遗传筛选的高成本而变得复杂。幸运的是，Rb通路在后生动物进化过程中一直是保守的，允许对更简单的生物体进行信息分析。这项建议的重点是最近发现的Rb肿瘤抑制通路和Myb癌基因家族之间的一个非常意外的连接。从果蝇细胞核提取物的分馏揭示了一个大的，以前未知的多蛋白复合物称为Myb-MuvB。Myb DNA结合蛋白由脊椎动物Myb癌基因家族的唯一果蝇同源物编码，其在线虫C elegans中不存在。复合物中的其他蛋白质由synMuvB基因的果蝇同源物编码，所述synMuvB基因是在线虫中发现的，由于功能丧失突变体，其导致与RTK-RAS-MAP激酶途径中的功能获得突变体所引起的多叶表型相同的多叶表型。Rb和RAS通路都包含大多数人类癌症发展中的突变的关键靶点。此外，最近对人类乳腺癌分子标志物的筛选显示，B-MYB/MYBL 2（果蝇Myb的人类直向同源物）的表达增加是预后不良的强有力预测因子。因此，更好地了解Myb-MuvB复合物可能会改善人类癌症的诊断和治疗。我们建议使用遗传，细胞生物学和生物化学分析在果蝇中，以确定急性损失或获得的Myb-MuvB复合物的组件的功能的后果。我们的初步数据表明，在没有Myb的情况下，染色体凝聚的正常进展失败。此外，我们发现，沉积的着丝粒特异性组蛋白H3变体CID/CENP-A的Myb的情况下改变。我们的假设是，Myb-MuvB复合物的其他组分也是染色体生物学中这些关键步骤所必需的。因此，我们的具体目标是：（1）确定Myb-MuvB的哪些组分调节染色体凝聚;（2）描述Myb-MuvB在异染色质的建立、维持和扩散中的作用;（3）测试Myb-MuvB的组分是否调节组蛋白H3变体的交换。

DESCRIPTION (provided by applicant): The retinoblastoma (Rb) tumor suppressor pathway is mutated in most, if not all, human cancers. This pathway regulates genes required for S phase progression as well as genes essential for cellular differentiation, and has also been implicated in the establishment and maintenance of heterochromatin. Biochemical studies of the Rb pathway in vertebrates have been hampered by difficulties in extracting Rb complexes from cell nuclei in their native state. Genetic studies of the Rb pathway in vertebrates have been complicated by redundancy, long generation times, and the high cost of genetic screens. Fortunately, the Rb pathway has been conserved during metazoan evolution, permitting the informative analysis of simpler organisms. This proposal focuses on a recently discovered and quite unexpected connection between the Rb tumor suppressor pathway and the Myb oncogene family. Fractionation of nuclear extracts from Drosophila cells revealed a large, previously unknown multiprotein complex called Myb-MuvB. The Myb DNA-binding protein is encoded by the sole Drosophila homologue of the vertebrate Myb oncogene family which is absent in the nematode C elegans. The other proteins in the complex are encoded by Drosophila homologues of the synMuvB genes which were discovered in C elegans due to loss-of-funotion mutants that cause a multivulval phenotype identical to that caused by gain-of- function mutants in the RTK-RAS-MAP kinase pathway. Both the Rb and RAS pathways contain critical targets for mutation in the development of most human cancers. In addition, recent screens for molecular markers in human breast cancer have revealed that increased expression of B-MYB/MYBL2 (the human orthologue of Drosophila Myb) is a strong predictor of poor prognosis. Therefore, better understanding of the Myb-MuvB complex will likely lead to improvements in the diagnosis and treatment of human cancer. We propose to use genetic, cell biological, and biochemical analyses in Drosophila to determine the consequences of acute loss or gain of function of components of the Myb-MuvB complex. Our preliminary data show that a failure of the normal progression of chromosome condensation occurs in the absence of Myb. In addition, we found that the deposition of the centromere-specific histone H3 variant CID/CENP-A is altered in the absence of Myb. Our hypothesis is that other components of the Myb-MuvB complex are also required for these critical steps in chromosome biology. Therefore, our specific aims are: (1) To determine which components of Myb-MuvB regulate chromosome condensation; (2) To delineate the role of Myb-MuvB in the establishment, maintenance, and spread of heterochromatin; (3) To test whether components of Myb- MuvB regulate the exchange of histone H3 variants.