Genetic and Molecular Studies of Drosophila Chromatin Remodeling Factors

描述（由申请人提供）：核小体和染色质的其他组分通过阻断转录因子和其他调节蛋白进入DNA，作为真核生物转录的有效抑制因子。染色质抑制通过两种一般机制调节：atp依赖性染色质重塑和核小体组蛋白的共价修饰。这些过程的破坏导致各种人类疾病，包括癌症。尽管在确定染色质重塑因子和组蛋白修饰酶的作用机制方面取得了巨大进展，但它们在真核生物转录和人类疾病中的作用仍有待了解。最近的研究表明，染色质重塑因子可以调节高阶染色质结构，但它们在这一过程中的作用尚不清楚。为了解决这些重要问题，本实验室以果蝇为模型系统，研究了三种不同的染色质重塑因子：BRM、kiss - l和ISWI。我们最近发现BRM和kiss - l在RNA聚合酶II的转录中发挥全局作用。为了确定BRM和kiss - l在转录中的确切作用，我们将描述它们功能丧失导致的缺陷，并绘制它们在体内与之相互作用的活性基因区域。我们还将研究组蛋白尾部的位点特异性甲基化是否会影响BRM或kiss - l与其染色质底物相互作用的能力。与BRM和kiss - l不同，ISWI在染色质压实和转录抑制中发挥全局作用，可能是通过促进连接蛋白H1与染色质的关联。为了验证这一假设，我们将描述ISWI功能丧失导致的染色体结构缺陷，并使用RNAi和工程显性负突变研究组蛋白H1功能降低的后果。为了确定参与高阶染色质结构调节的其他因素，我们将筛选导致类似于ISWI突变体中观察到的染色体缺陷的突变。通过同时研究单一生物体中三种不同的染色质重塑因子，我们将更好地了解它们在转录和其他过程中的作用。果蝇染色质重塑因子的对应物——包括BRM、kiss - l和ISWI——存在于人类中，它们在基因表达和发育中起着高度保守的作用。这些因子的突变与多种癌症有关，包括横纹肌样肿瘤、乳腺癌和白血病。因此，我们对遗传模式生物中染色质重塑因子的研究应该揭示这些人类癌症和其他疾病的分子机制。

DESCRIPTION (provided by applicant): Nucleosomes and other components of chromatin act as potent repressers of eukaryotic transcription by blocking the access of transcription factors and other regulatory proteins to DNA. Chromatin repression is regulated via two general mechanisms: ATP-dependent chromatin remodeling and the covalent modification of nucleosomal histones. The disruption of these processes leads to a variety of human diseases, including cancer. In spite of tremendous progress toward determining the mechanism of action of chromatin- remodeling factors and histone-modifying enzymes, much remains to be learned about their roles in eukaryotic transcription and human disease. Recent studies have suggested that chromatin-remodeling factors can regulate higher-order chromatin structure, but their role in this process remains poorly understood. To address these important issues, our laboratory uses Drosophila melanogaster as a model system to study three different chromatin-remodeling factors: BRM, KIS-L and ISWI. We recently found that BRM and KIS-L play global roles in transcription by RNA Polymerase II. To determine the precise roles of BRM and KIS-L in transcription, we will characterize defects resulting from their loss of function and map the regions of active genes with which they interact in vivo. We will also examine whether the site-specific methylation of histone tails affects the ability of BRM or KIS-L to interact with their chromatin substrates. Unlike BRM and KIS-L, ISWI plays a global role in chromatin compaction and transcriptional repression, possibly by promoting the association of the linker histone H1 with chromatin. To test this hypothesis, we will characterize defects in chromosome structure resulting from the loss of ISWI function and investigate the consequences of reducing histone H1 function using RNAi and engineered dominant-negative mutations. To identify other factors involved in the regulation of higher-order chromatin structure, we will screen for mutations that cause chromosome defects similar to those observed in ISWI mutants. By simultaneously studying three different chromatin-remodeling factors in a single organism, we will gain a much better understanding of their roles in transcription and other processes. Counterparts of Drosophila chromatin-remodeling factors - including BRM, KIS-L and ISWI - are present in humans, where they play highly conserved roles in gene expression and development. Mutations in these factors are associated with a variety of cancers, including rhabdoid tumors, breast cancer, and leukemias. Our studies of chromatin-remodeling factors in a genetic model organism should therefore shed light on the molecular mechanisms underlying these human cancers and other diseases.