Studies On Silencing

研究兴趣 我们实验室的研究致力于了解基因组的整个区域无法进行转录和重组的机制。我们采用遗传分析加上生化分馏和重建实验，以探讨基因组可及性的问题。 目前的研究 基因组结构域的沉默需要沉默子和阻遏蛋白之间的一系列复杂的相互作用。沉默子募集由Sir蛋白组成的阻遏蛋白复合物，其与核小体中的组蛋白相互作用以形成对各种细胞过程不可接近且惰性的染色质结构域。我们目前正在研究1）沉默结构域结构的生物化学和分子表征及其体外重建2）沉默结构域限制于基因组特定区域的机制和3）组蛋白变体在细胞周期进程中的作用。 我们一直在纯化Sir蛋白复合物，并纯化了由杆状病毒感染的昆虫细胞中表达的亚单位重建的复合物。我们已经开始研究使用这些复合物和组蛋白在核小体中重建沉默的染色质。这些研究将为我们目前对转录沉默的理解提供一个重要的指标，这取决于是否有可能重建沉默状态，因为机制很少通过遗传手段建立，通常需要生化测试。 除了这些研究，我们也有兴趣了解沉默的染色质结构域被限制在DNA纤维沿着特定区域的机制。我们已经证明，特定的元素作为沉默的染色质的连续传播的障碍。我们进行了一个基因组范围的筛选蛋白质，可以阻止沉默的染色质的传播和分析的分子机制，通过特定的天然屏障功能，以阻止沉默和我们的研究结果表明，两个重叠的机制功能，以限制沉默的传播，其中之一涉及的DNA结合元件，而第二个机制涉及特定的染色质修饰活动。我们也开始确定染色质折叠在限制沉默染色质扩散中的作用。 我们目前有兴趣了解组蛋白H2A变体（称为Htz1p）在细胞中的许多作用。经典的分子遗传学和生物化学实验正在进行中，以确定Htz1p在转录，DNA复制和细胞周期进程中的作用以及这些过程之间的相互关系。 总之，这些研究将使人们更好地了解沉默的过程，并对基因沉默的机制产生新的见解。

Research Interests Research in our laboratory is devoted to understanding the mechanisms by which entire regions of the genome are rendered inaccessible to transcription and recombination. We employ genetic analysis coupled with biochemical fractionation and reconstitution experiments to explore the issues of genome accessibility. Current Research Silencing of genomic domains requires a complex series of interactions between silencers and repressor proteins. The silencers recruit repressor protein complexes composed of the Sir proteins that interact with histones in nucleosomes to form a chromatin domain that is inaccessible and inert to various cellular processes. We are presently working on 1) The biochemical and molecular characterization of the structure of the silent domain and its reconstitution in vitro 2) The mechanism by which the silent domain is restricted to a specific region of the genome and 3) The role of histone variants in cell cycle progression. We have been purifying Sir protein complexes and have purified complexes reconstituted from sub-units expressed in baculovirus infected insect cells. We have begun studies on the reconstitution of silenced chromatin using these complexes and histones in nucleosomes. These studies will provide an important index of our current understanding of transcriptional silencing depending on whether or not it is possible to reconstitute the silenced state, since mechanisms are rarely established by genetic means and usually require biochemical tests. In addition to these studies we are also interested in understanding the mechanism by which the silenced chromatin domains are restricted to specific regions along the DNA fiber. We have demonstrated that specific elements act as barriers to the continuous spread of the silenced chromatin. We performed a genome wide screen for proteins that can block the spread of silenced chromatin and analyzed the molecular mechanism by which specific native barriers function to block silencing and our results suggest two overlapping mechanisms function to restrict the spread of silencing, one of which involves a DNA binding element, while the second mechanism involves specific chromatin modifying activities. We have also begun determining the role of folding of chromatin in restricting the spread of silenced chromatin. We are currently interested in understanding the many roles of a histone H2A variant called Htz1p in the cell. Classical molecular genetic and biochemical experiments are in progress to determine the role of Htz1p in transcription, DNA replication and cell cycle progression and the inter-relationships between these processes. Together, these studies will allow a better understanding of the process of silencing and yield new insight into the mechanism by which genes are silenced.