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。我们已经取得了重大进展，了解ATP依赖的染色质重塑的机制，果蝇Nucleosome Remodeling Factor（NF）。要阐明的生物学意义的cDNAF诱导的核小体滑动，我们已经分离出果蝇nurf 301突变。我们证实，在体内转录激活所需的BMPF。在nurf 301突变体中，热休克转录因子与hsp 70和hsp 26基因的结合和转录受损。此外，同源异型基因的表达也需要cDNAF。与此相一致，nurf 301突变体概括了双胸增强子的表型，双胸复合体的正调节子先前定位于相同的遗传间隔。最后，突变体在BMPF亚基表现出肿瘤转化的幼虫血细胞，诱导黑色素瘤的形成。这些结果表明，BDNF不仅是参与应激反应和发育的基因的适当调节所必需的，而且也是肿瘤抑制所必需的。我们还在SWI 2/SNF 2超家族成员INO 80的研究方面取得了很大进展。我们已经完成了所有亚基的质谱鉴定，并正在进行遗传和生化实验，以阐明复合物的机制和生理功能。INO 80复合物含有化学计量的肌动蛋白和三种肌动蛋白相关蛋白（Arp 4、Arp 5和Arp 8）。Arp 4和肌动蛋白对细胞存活至关重要，但Arp 5和Arp 8的无效突变体的生存能力使我们能够进一步研究它们的功能作用。通过对无效突变体的生化分析，我们揭示了Arp 5和Arp 8在染色质重塑中的功能。从arp 5D和arp 8D细胞中纯化INO 80复合物表明，突变体复合物的DNA结合、核小体动员和Ino 80 ATP酶活性受到损害。与生化结果一致，arp 5D和arp 8D突变体显示出与ino 80 D相似的表型。这些结果为肌动蛋白和Arps在细胞核中的作用提供了新的见解。细胞核中肌动蛋白的长期报道。

Our group has continued studies of chromatin structure and the regulation of eukaryotic gene expression. We have made significant progress towards understanding the mechanism of ATP-dependent chromatin remodeling by Drosophila NURF (Nucleosome Remodeling Factor). To elucidate the biological significance of NURF-induced nucleosome sliding, we have isolated mutations for Drosophila nurf301. We confirm that NURF is required for transcription activation in vivo. In nurf301 mutants, heat-shock transcription factor binding to and transcription of the hsp70 and hsp26 genes are impaired. Additionally, NURF is required for homeotic gene expression. Consistent with this, nurf301 mutants recapitulate phenotypes of Enhancer of bithorax, a positive regulator of the Bithorax-Complex previously localized to the same genetic interval. Finally, mutants in NURF subunits exhibit neoplastic transformation of larval blood cells that induces melanotic tumor formation. These results reveal that NURF is required not only for proper regulation of genes involved in the stress response and development, but also for tumor suppression. We are also making excellent progress on studies of INO80, a member of the SWI2/SNF2 superfamily. We have completed identification of all subunits by mass spectrometry, and are conducting genetic and biochemical experiments to elucidate the mechanism and physiological functions of the complex. The INO80 complex contains stoichiometric amounts of actin and three actin related proteins (Arp4, Arp5, and Arp8). Arp4 and actin are essential for cell survival, but the viability of null mutants for Arp5 and Arp8 allowed us to further investigate their functional roles. We uncovered functions of Arp5 and Arp8 in chromatin remodeling by biochemical analysis of the null mutants. Purification of INO80 complexes from arp5D and arp8D cells showed that the mutant complexes are compromised for DNA binding, nucleosome mobilization, and Ino80 ATPase activity. Consistent with the biochemical findings, arp5D and arp8D mutants show phenotypes similar to ino80D. These results provide new insights on the role of actin and Arps in the cell nucleus. longstanding reports of actin in the cell nucleus.