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。今年，我们在理解转基因小鼠中由核小体重塑因子（Nucleosome Remodeling Factor，NF）引起的ATP依赖性染色质重塑的生物学方面取得了进一步的进展。除了作为TGF β-反应基因的调节剂的作用外，TGF β还参与调节胸腺中的T细胞发育和成熟。我们也取得了重大进展，我们的理解H2 AZ纳入转录活性染色质。通过建立在早期的观察参与的SWR 1复合物在这一过程中，我们能够发现一种新的核伴侣Chz 1和其活性和结构的特点。在多种H2 A-H2 B分子伴侣中，Chz 1显示出对H2 AZ变体的特异性，我们能够证明它作为H2 AZ-H2 B二聚体到SWR 1复合物的供体发挥作用，SWR 1复合物反过来催化二聚体掺入染色质。体外组装的Chz 1-H2 AZ-H2 B异源三聚体的分析揭示了一个保守的基序，这也是存在于其他物种，从而表明H2 AZ特异性伴侣可能是广泛保守的。在真核生物中，有丝分裂期间染色体的分离由着丝粒指定，其指导染色体通过动粒附着到纺锤体。利用生物化学、遗传学和分子细胞技术的组合，我们证明了酵母Scm 3非组蛋白蛋白对于CenH 3（组蛋白H3的着丝粒特异性变体）掺入着丝粒核小体是必需的，并且Scm 3实际上构成酵母着丝粒的稳定结构组分。Scm 3缺失导致着丝粒在结构和功能水平上的缺失。此外，使用纯化的重组蛋白在体外重构着丝粒核小体核心揭示了Scm 3与CenH 3-H4四聚体的缔合导致排除H2 A-H2 B二聚体。因此，我们提出了一个新的模型染色质在酵母着丝粒，涉及非组蛋白的核心着丝粒核小体。

Our group has continued studies of chromatin structure and the regulation of eukaryotic gene expression. This year we have made further progress towards understanding the biology of ATP-dependent chromatin remodeling by NURF (Nucleosome Remodeling Factor) in transgenic mice. In addition to NURF role as a regulator of TGF beta-responsive genes, NURF is also involved in the regulation of T-cell development and maturation in the thymus. We are also making significant progress in our understanding of the H2AZ incorporation into transcriptionally active chromatin. By building on earlier observations of the involvement of the SWR1 complex in that process, we were able to discover a novel nuclear chaperone Chz1 and characterized its activity and structure. Among multiple H2A-H2B chaperones, Chz1 displays specificity for H2AZ variant and we were able to demonstrate that it functions as a donor of the H2AZ-H2B dimer to SWR1 complex, which in turn catalyzes dimer's incorporation into chromatin. Analysis of in vitro assembled Chz1-H2AZ-H2B heterotrimers revealed the existence of a conserved motif which is also present in other species, thus suggesting that H2AZ-specific chaperones may be widely conserved. In eukaryotes, the segregation of chromosomes during mitosis is specified by centromere, which directs chromosome attachment, through kinetochore, to the spindle apparatus. Using a combination of biochemical, genetic and molecular cell techniques we demonstrated than yeast Scm3 nonhistone protein is necessary for the incorporation of CenH3 (centromere-specific variant of histone H3)into centromeric nuclosome and that Scm3 actually constitutes stable structural component of yeast centromere. Scm3 depletion causes loss of centromere at both structural and functional level. Moreover, reconstitution of centromerioc nucleosome core in vitro using purified recombinant protins revealed that Scm3 association with CenH3-H4 tetramer leads to the exclusion of H2A-H2B dimers. Therefore, we have proposed a new model for chromatin at yeast centromeres, involving a nonhistone protein at the core of the centromeric nucleosome.