Eukaryotic Chromatin Structure and Gene Regulation

我们小组通过分析几种atp依赖性染色质重塑酶，继续研究染色质结构和真核基因表达的调控。今年，我们进一步阐明了小鼠核小体重塑因子（NURF）在atp依赖性染色质重塑中的生物学作用。我们通过研究缺乏该亚基的胚胎干细胞，扩展了对NURF最大亚基Bptf突变体的分析。分化早期的微阵列分析已经确定了bptf依赖的基因靶点，包括同源盒转录因子和外胚层、中胚层、终胚层和内脏胚层发育所必需的基因。Bptf-/-胚胎干细胞向胚状体的分化揭示了其对中胚层、内胚层和外胚层组织谱系发育的要求，并揭示了许多依赖Bptf激活或抑制的基因。一份描述这些发现的手稿正在修订中，准备出版。我们在出芽酵母多蛋白SWR1复合体的研究上也取得了进展，这是染色质重塑酶SWI2/SNF2超家族的成员。之前，我们在SWR1复合体和组蛋白变体H2AZ之间建立了一个新的联系，H2AZ优先结合在基因启动子区域。我们发现组蛋白H2AZ的沉积是由SWR1复合物介导的，SWR1复合物催化核小体组蛋白H2A与H2AZ的atp依赖性交换。我们正在通过一些生化分析分析组蛋白替代的早期步骤，这将为组蛋白交换机制提供深入的见解。我们还继续研究了Scm3非组蛋白，我们之前发现它与着丝粒组蛋白变体CenH3相互作用，并且是体内CenH3和内部着丝点组装所必需的。我们目前正在进行Scm3和CenH3在细胞周期中的沉积的体内研究，以及Scm3-CenH3- h4复合物与着丝粒DNA的体外重建。我们继续与校内和校外同事进行富有成效的合作。去年发表了几篇报道这些合作研究的手稿。

Our group has 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) in the mouse. We have extended analysis of mutants for Bptf, the largest subunit of NURF, by studying ES cells deficient for that subunit. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcription factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf-/- embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. A manuscript describing these findings is being revised for publication. We are also making progress on studies of the budding yeast multi-protein SWR1 complex, a member of the SWI2/SNF2 superfamily of chromatin remodeling enzymes. Previously, we established a new link between the SWR1 complex and the histone variant H2AZ, which is incorporated preferentially at gene promoter regions. We showed that deposition of histone H2AZ is mediated by the SWR1 complex, which catalyzes ATP-dependent exchange of nucleosomal histone H2A for H2AZ. We are in the process of analyzing the early steps of histone replacement by a number of biochemical assays, which should provide insight into histone exchange mechanism. We have also continued studies of the Scm3 nonhistone protein, which we previously found to interact with the centromeric histone variant CenH3, and to be required for assembly of CenH3 and the inner kinetochore in vivo. We are currently undertaking in vivo studies of Scm3 and CenH3 deposition in the cell cycle, and in vitro reconstitution of Scm3-CenH3-H4 complexes with centromeric DNA. We have continued fruitful collaborations with intramural and extramural colleagues. Several manuscripts reporting these collaborative studies were published in the past year.