Eukaryotic Chromatin Structure and Gene Regulation

我们小组继续研究染色质结构和真核基因表达的调控。今年，我们在理解核小体重塑因子(NURF)对ATP依赖的染色质重塑的生物学方面取得了重大进展。我们实验室以前的一项研究报告了Nurf301突变的分离，该基因编码唯一的NURF特异性亚基，并证明了体内许多基因转录激活的需要。然而，依赖于NURF的基因是有限的；而且，这些靶标都不能解释后期幼虫的致死表型。通过全基因组表达分析，我们现在已经发现了数百个额外的靶点，其中最引人注目的几乎是蜕皮激素反应基因的所有成员，蜕皮激素反应基因是幼虫到蛹发育的关键调节因子(从而解释了致命的表型)。我们进一步通过对显性负突变的分析证明了NURF与ECR在遗传上的相互作用，进一步地，NURF以激素依赖的方式与ECR物理结合。这些发现表明，NURF是蜕皮激素反应基因的主要转录辅助激活因子，并暗示ECR招募了更广泛的染色质调节剂。由于ECR与哺乳动物的RAR受体同源，NURF在哺乳动物中也是保守的，我们的发现应该对类固醇激素信号转导具有广泛的意义。我们对SWR1复合体的研究也取得了很好的进展，SWR1复合体是染色质重塑酶SWI2/SNF2超家族的新成员。我们在SWR1复合体和特异组蛋白变异体H_2AZ之间建立了新的联系。组蛋白变异体H2 AZ优先结合在真核细胞染色质中的特定位置，在那里它调节染色体的功能。在酿酒酵母中，组蛋白H_2AZ的沉积是由多蛋白SWR1复合体介导的，该复合体催化依赖于ATP的核小体组蛋白H_2A与H_2AZ的交换。为了阐明组蛋白交换中最早的事件，我们定义了SWR1组分与H2 AZ之间的相互作用，揭示了Swr1的ATPase结构域与H2 AZ结合所需的三个亚基之间的联系。我们发现Swc2，一个从酵母到人的保守的组蛋白伴侣样模块，直接与H_2AZ结合，并对H_2AZ的转移是必不可少的。两个组分Swc6和Arp6是将Swc2连接到Swr1所必需的，而额外的亚基Swc5和Yaf9是进行H2 AZ转移所必需的，但不需要与H2 AZ结合。最后，H_2AZ必需的C-末端？-螺旋对其被SWR1识别是至关重要的。这些发现为组蛋白变体交换的第一阶段提供了洞察力。

Our group has continued studies of chromatin structure and the regulation of eukaryotic gene expression. This year we have made significant progress towards understanding the biiology of ATP-dependent chromatin remodeling by NURF (Nucleosome Remodeling Factor). A previous study from our laboratory reported the isolation of mutants in Nurf301, the gene encoding the only NURF-specific subunit, and demonstration of a requirement for transcription activation of a number of genes in vivo. However, the NURF-dependent genes were limited; furthermore, none of those targets could explain the late larval lethal phenotype. By whole genome expression analysis, we have now uncovered several hundred additional targets, the most striking of which are virtually all members of the ecdysone response genes, key regulators of larval to pupal development (thus accounting for the lethal phenotype). We have gone on to show that NURF interacts genetically with EcR by analysis of dominant negative mutants, and further, that NURF binds physically to EcR in a hormone-dependent manner. These findings indicate that NURF is a major transcriptional coactivator of Ecdysone-response genes, and implicate recruitment by EcR of a wider cast of chromatin modulators. Because EcR is orthologous to mammalian RAR receptors, and NURF is conserved in mammals as well, our findings should have broad implications for steroid hormone signaling.We are also making excellent progress on studies of the SWR1 complex, which is a new member of the SWI2/SNF2 superfamily of chromatin remodeling enzymes. We established a new link between the SWR1 complex and a specific histone variant H2AZ. Histone variant H2AZ is incorporated preferentially at specific locations in eukaryotic chromatin, where it modulates chromosome functions. In S. cerevisiae, deposition of histone H2AZ is mediated by the multi-protein SWR1 complex, which catalyzes ATP-dependent exchange of nucleosomal histone H2A for H2AZ. To elucidate the earliest events in histone exchange, we defined interactions between SWR1 components and H2AZ, revealing a link between the ATPase domain of Swr1 and three subunits required for the binding of H2AZ. We discovered that Swc2, a histone chaperone-like module conserved from yeast to human binds directly to and is essential for transfer of H2AZ. Two components, Swc6 and Arp6, are necessary to tether Swc2 to Swr1, while additional subunits, Swc5 and Yaf9, are required for H2AZ transfer but not H2AZ binding. Finally, the essential C-terminal ?-helix of H2AZ is critical for its recognition by SWR1. These findings provide insight into the first stage of histone variant exchange.