Eukaryotic Chromatin Structure and Gene Regulation

我们继续研究染色质结构和真核基因的调控 通过分析几种ATP依赖性染色质重塑酶的表达。今年我们 已经进一步阐明了ATP依赖的染色质重塑的生物学由APNF（核小体 重塑因子）。我们完成了Bptf的分子和遗传研究，Bptf是Bptf的最大亚基。 Bptf在小鼠中的表达，表明Bptf是中胚层，内胚层， 外胚层组织谱系。我们发现Bptf是一个正或负调节因子 与小鼠早期发育有关的数百个基因的基因。这项工作发表在 PLoS Genetics.我们还参与了最近发表的合作实验， Drosophila cDNAF作为先天免疫相关基因的调节因子发挥作用， 果蝇中最大的cDNAF亚基的可变剪接形式产生了改变的 修饰的组蛋白结合特异性对精子发生至关重要。我们取得了 SWI 2/SNF 2成员之一的芽殖酵母多蛋白SWR 1复合物的研究进展 染色质重塑酶超家族，催化核小体的替换 组蛋白H2 A与组蛋白H2 AZ变体。我们发现14个SWR 1亚基中有3个 似乎具有辅助基本组蛋白置换活性的作用。n-末端 Swr 1 ATP酶亚基的区域含有第二个H2 AZ-H2 B特异性结合位点， 与之前鉴定的Swc 2亚基不同。这项工作发表在J. Biol. Chem. A 与Yawen Bais集团的合作项目导致了NMR结构的解决方案 H2 AZ-H2 B与Chz 1组蛋白伴侣结合。我们正在开发检测方法来确定 SWR 1复合物以启动子特异性方式被募集到染色质，并确定是否 核小体中的一个或两个组蛋白H2 A分子被H2 AZ取代。继续研究 芽殖酵母Scm 3非组蛋白，其特异性结合于着丝粒组蛋白 变体CenH 3，并且是CenH 3和内部动粒组装所需的，表明 Scm 3在体外与着丝粒DNA结合，并可能作为一种结构成分， 着丝粒染色质。Scm 3和CenH 3沉积的细胞周期分析显示Scm 3结合 在整个细胞周期中持续存在，而CenH 3在细胞周期中从着丝粒短暂丢失。 从G1期到S期的过渡。

We have continued studies of chromatin structure and the regulation of eukaryotic gene expression by analysis of several ATP-dependent chromatin remodeling enzymes. This year we have further elucidated the biology of ATP-dependent chromatin remodeling by NURF (Nucleosome Remodeling Factor). We completed a molecular and genetic study of Bptf, the largest subunit of NURF in the mouse, showing that Bptf is required for development of mesoderm, endoderm, ectoderm tissue lineages. We found that Bptf is required as a positive or negative regulator of several hundred genes implicated in early mouse development. This work was published in PLoS Genetics. We also participated in collaborative experiments, recently published, showing that the Drosophila NURF functions as a regulator of genes involved in innate immunity, and that an alternatively spliced form of the largest NURF subunit in Drosophila generates altered modified histone binding specificities that are critical for spermatogenesis. We have made progress on studies of the budding yeast multi-protein SWR1 complex, a member of the SWI2/SNF2 superfamily of chromatin remodeling enzymes that catalyzes the replacement of nucleosomal histone H2A with the histone H2AZ variant. We found that three out of fourteen SWR1 subunits appear to have roles auxiliary to the basic histone replacement activity. The N-terminal region of the Swr1 ATPase subunit contains a second H2AZ-H2B specific binding site, distinct from the previously identified Swc2 subunit. This work was published in J. Biol. Chem. A collaborative project with Yawen Bais group resulted in the solution of the NMR structure of H2AZ-H2B bound to the Chz1 histone chaperone. We are developing assays to determine how the SWR1 complex is recruited to chromatin in a promoter specific manner, and to define whether one or both histone H2A molecules in a nucleosome is replaced with H2AZ. Continuing studies of the budding yeast Scm3 nonhistone protein, which binds specifically to the centromeric histone variant CenH3, and is required for assembly of CenH3 and the inner kinetochore, indicate that Scm3 binds to centromeric DNA in vitro, and may function as a structural component of centromeric chromatin. Cell cycle analyses of Scm3 and CenH3 deposition show that Scm3 binding persists throughout the cell cycle, while CenH3 is transiently lost from centromeres in the transition from G1 to S phase.