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。该通路调控S期进展所需的基因以及细胞分化所必需的基因，也与异染色质的建立和维持有关。由于难以从天然状态的细胞核中提取Rb复合物，阻碍了脊椎动物Rb途径的生化研究。脊椎动物Rb通路的遗传研究由于冗余、长世代和高成本的遗传筛选而变得复杂。幸运的是，Rb通路在后生动物进化过程中被保存下来，允许对更简单的生物进行信息分析。本提案的重点是最近发现的Rb肿瘤抑制通路和Myb癌基因家族之间的意想不到的联系。对果蝇细胞的核提取物进行分离，发现了一种以前未知的大型多蛋白复合物Myb-MuvB。Myb dna结合蛋白是由脊椎动物Myb致癌基因家族中唯一的果蝇同源基因编码的，而在线虫中是不存在的。该复合体中的其他蛋白由synMuvB基因的果蝇同源物编码，这些同源物是在秀丽隐杆线虫中发现的，由于功能丧失突变导致多外阴表型，与RTK-RAS-MAP激酶途径中功能获得突变引起的多外阴表型相同。Rb和RAS通路都包含大多数人类癌症发生过程中突变的关键靶点。此外，最近对人类乳腺癌分子标记的筛选表明，B-MYB/MYBL2（果蝇Myb的人类同源基因）的表达增加是不良预后的一个强有力的预测因子。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。我们建议在果蝇中使用遗传、细胞生物学和生化分析来确定Myb-MuvB复合物组分功能急性丧失或获得的后果。我们的初步数据表明，染色体凝聚正常进程的失败发生在Myb缺失的情况下。此外，我们发现在没有Myb的情况下，着丝粒特异性组蛋白H3变体CID/CENP-A的沉积发生了改变。我们的假设是，Myb-MuvB复合体的其他成分也需要染色体生物学的这些关键步骤。因此，我们的具体目标是：(1)确定Myb-MuvB的哪些成分调节染色体凝聚；(2)阐明Myb-MuvB在异染色质形成、维持和扩散中的作用；(3)检测Myb- MuvB组分是否调控组蛋白H3变异体的交换。

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.