Eukaryotic Chromatin Structure and Gene Regulation

我们通过分析ATP依赖的染色质重塑酶，继续研究染色质结构和真核基因表达的调控。我们在小鼠中完成了对核小体重塑因子（Nucleosome Remodeling Factor，NF）的ATP依赖性染色质重塑的生物学研究。Bptf是胸腺细胞成熟所必需的BptF的最大亚基。我们将这种缺陷追溯到对胸腺细胞发育重要的一些基因的失调，并发现基因靶点处染色质结构的Bptf依赖性变化以及序列特异性转录因子SRF与染色质结合中对Bptf的要求。这项工作已提交，修订，接受出版，现在发表在基因和发展。我们还完成了SWR 1染色质重塑复合物替代组蛋白H2A.Z的机制研究。我们发现，启动子近端核小体是高度异质性的H2A.Z在酿酒酵母，与大量的核小体含有一个，两个，或没有H2A.Z分子的代表。SWR 1催化的H2A.Z体外置换以逐步和单向的方式发生，一次一个H2 A. Z-H2 B二聚体，产生异型核小体作为中间体和同型H2A.Z核小体作为终产物。SWR 1的ATP酶活性被含H2 A的核小体和游离的H2 A. Z-H2 B二聚体特异性地刺激，导致核小体H2 A-H2 B的驱逐和H2 A. Z-H2 B的沉积。这些结果表明，在体内底物支配的特异性和结果的替代反应。这项工作已提交，修订，接受出版，现在发表在细胞。我们继续研究含有组蛋白变体CenH 3的着丝粒特异性核小体的分子结构。我们发现，DNA碱基组成的影响重组的核小体含有酿酒酵母CenH 3（Cse 4）。重组是强大的非着丝粒DNA，但效率低下的着丝粒DNA，由于AT丰富的着丝粒元件，阻碍协会的Cse 4-含组蛋白八聚体。相比之下，包含非组蛋白Scm 3与Cse 4/H4允许重建天然着丝粒序列。Scm 3有一个DNA结合结构域，表现出对富含AT的着丝粒元件的偏好，和一个组蛋白伴侣样结构域，促进Cse 4/H4的加载。在体内，Scm 3-GFP定位于细胞周期的所有阶段的着丝粒簇，包括有丝分裂。Scm 3也可以在整个细胞周期中与同步化细胞中的着丝粒交联，即使Cse 4/H4在早期S期暂时移位。这些研究结果表明，着丝粒DNA结合Scm 3艾滋病招聘Cse 4/H4组装H2 A/H2 B缺陷的着丝粒核小体的模型。这项工作已提交，修订，接受出版，现在发表在分子细胞。

We have continued studies of chromatin structure and the regulation of eukaryotic gene expression by analysis of ATP-dependent chromatin remodeling enzymes. We completed studies on the biology of ATP-dependent chromatin remodeling by NURF (Nucleosome Remodeling Factor) in the mouse. Bptf, the largest subunit of NURF, is required for maturation of thymocytes. We traced this defect to the misregulation of a number of genes important for thymocyte development, and found Bptf-dependent changes in chromatin structure at gene targets as well as a requirement for NURF in the binding of the sequence-specific transcription factor SRF to chromatin. This work was submitted, revised, accepted for publication, and is now published in Genes and Development. We have also completed a study 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 and free H2A.Z-H2B dimers, leading to eviction of nucleosomal H2A-H2B and deposition of H2A.Z-H2B. These results suggest that the in vivo substrates govern the specificity and outcome of the replacement reaction. This work was submitted, revised, accepted for publication, and is now published in Cell. 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 was submitted, revised, accepted for publication, and is now published in Molecular Cell.