Eukaryotic Chromatin Structure and Gene Regulation

通过分析atp依赖性染色质重塑酶，我们继续研究染色质结构和真核基因表达的调控。今年，我们进一步阐明了核小体重塑因子（NURF）对atp依赖性染色质重塑的生物学作用。我们正在完成一项关于小鼠Bptf （NURF的最大亚基）在胸腺发育中的作用的研究。具体来说，我们发现Bptf是胸腺细胞成熟所必需的，并将这种缺陷追溯到一些对胸腺细胞发育重要的基因的错误调节。我们还发现基因靶点染色质结构的bptf依赖性变化，以及序列特异性转录因子与染色质结合时需要NURF。这项工作目前正在准备出版。我们对组蛋白H2A的作用机制还在继续研究。SWR1染色质重塑复合体的Z替换。我们发现H2A启动子-近端核小体是高度异质的。在酿酒酵母中，具有含有一个、两个或不含H2A的核小体的大量代表。Z分子。SWR1-catalyzed H2A。Z在体外以逐步和单向的方式发生，一个H2A。Z-H2B二聚体，产生异型核小体作为中间体和同型H2A。作为最终产物的Z核小体。SWR1的atp酶活性被含有H2A的核小体特异性刺激，而不产生组蛋白H2A。值得注意的是，进一步添加了游离H2A。Z-H2B二聚体导致atp酶活性过度刺激，核小体H2A-H2B的排出和H2A.Z-H2B的沉积。这些结果表明含H2A的核小体与游离H2A结合。Z-H2B二聚体同时作为SWR1的效应物和底物，控制着替代反应的特异性和结果。这项工作正在审查中，准备出版。我们继续研究含有组蛋白变体CenH3的着丝粒特异性核小体的分子结构。我们发现DNA碱基组成影响含有酿酒酵母CenH3 （Cse4）的核小体的重建。对非着丝粒DNA的重构是稳健的，但对着丝粒DNA的重构是低效的，这是由于富含at的着丝粒元件阻碍了含cse4的组蛋白八聚体的结合。相比之下，Cse4/H4包涵非组蛋白Scm3允许在天然着丝粒序列上重建。Scm3具有一个偏爱富含at的着丝粒元件的DNA结合域和一个促进Cse4/H4装载的组蛋白伴侣样结构域。在体内，Scm3-GFP在细胞周期的所有阶段（包括有丝分裂）都定位于着丝粒簇。在整个细胞周期中，Scm3也可以与同步细胞中的着丝粒交联，即使Cse4/H4在早期S期暂时移位。这些发现提示着丝粒dna结合的Scm3有助于募集Cse4/H4来组装H2A/ h2b缺陷着丝粒核小体的模型。这部作品正准备出版。

We have continued studies of chromatin structure and the regulation of eukaryotic gene expression by analysis of ATP-dependent chromatin remodeling enzymes. This year we have further elucidated the biology of ATP-dependent chromatin remodeling by NURF (Nucleosome Remodeling Factor). We are completing a study of the role of mouse Bptf, the largest subunit of NURF, in the development of the thymus. Specifically, we found that Bptf is required for maturation of thymocytes, and traced this defect to the misregulation of a number of genes important for thymocyte development. We also found Bptf-dependent changes in chromatin structure at gene targets as well as a requirement for NURF in the binding of sequence-specific transcription factors to chromatin. This work is currently in preparation for publication. We have continued studies on the mechanism of histone H2A.Z replacement by the SWR1 chromatin remodeling complex. We found that promoter-proximal nucleosomes are highly heterogeneous for H2A.Z in Saccharomyces cerevisiae, with substantial representation of nucleosomes containing one, two, or no H2A.Z molecules. SWR1-catalyzed H2A.Z replacement in vitro occurs in a stepwise and unidirectional fashion, one H2A.Z-H2B dimer at a time, producing heterotypic nucleosomes as intermediates and homotypic H2A.Z nucleosomes as end products. The ATPase activity of SWR1 is specifically stimulated by H2A-containing nucleosomes without ensuing histone H2A eviction. Remarkably, further addition of free H2A.Z-H2B dimer leads to hyperstimulation of ATPase activity, eviction of nucleosomal H2A-H2B and deposition of H2A.Z-H2B. These results suggest that the combination of H2A-containing nucleosome and free H2A.Z-H2B dimer acting as both effector and substrate for SWR1 governs the specificity and outcome of the replacement reaction. This work is under review for publication. We have continued investigations on the molecular architecture of centromere-specific nucleosomes containing histone variant CenH3. We found that DNA base composition influences reconstitution of nucleosomes containing Saccharomyces cerevisiae CenH3 (Cse4). Reconstitution is robust for non-centromere DNA, but inefficient for centromere DNA, due to an AT-rich centromere element that hinders association of Cse4-containing histone octamers. By contrast, inclusion of nonhistone Scm3 with Cse4/H4 permits reconstitution on natural centromere sequences. Scm3 has a DNA binding domain that exhibits preference for the AT-rich centromere element, and a histone chaperone-like domain that promotes loading of Cse4/H4. In vivo, Scm3-GFP localizes to the centromere cluster in all phases of the cell cycle, including mitosis. Scm3 can also be cross-linked to centromeres in synchronized cells throughout the cell cycle, even when Cse4/H4 is temporarily dislodged in early S phase. These findings suggest a model in which centromere DNA-bound Scm3 aids recruitment of Cse4/H4 to assemble an H2A/H2B-deficient centromeric nucleosome. This work is in preparation for publication.