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转录和重组的机制。我们使用遗传分析结合生化分离和重组实验来探索基因组可及性问题。 当前研究进展 沉默基因组结构域需要在沉默因子和抑制蛋白之间进行一系列复杂的相互作用。沉默分子招募由SIR蛋白组成的阻遏蛋白复合体，与核小体中的组蛋白相互作用，形成一个染色质结构域，该结构域对各种细胞过程都是不可访问和惰性的。我们目前正致力于1)沉默区域结构的生化和分子特征及其在体外的重组，2)沉默区域被限制在基因组特定区域的机制，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蛋白复合体，并纯化了由杆状病毒感染的昆虫细胞中表达的亚基重组而成的复合体。我们已经开始研究利用这些复合体和核小体中的组蛋白来重建沉默的染色质。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，我们还有兴趣了解沉默的染色质结构域被限制在DNA纤维上的特定区域的机制。我们已经证明，特定的元素对沉默的染色质的持续传播起到了障碍作用。我们对可以阻止沉默染色质传播的蛋白质进行了全基因组筛选，并分析了特定天然屏障阻止沉默的分子机制，我们的结果表明有两种重叠的机制可以限制沉默的传播，其中一种涉及DNA结合元件，而第二种机制涉及特定的染色质修饰活动。我们还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一个名为Htz1p的组蛋白H2A变体在细胞中的许多角色。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复制和细胞周期进展中的作用以及这些过程之间的相互关系。 总之，这些研究将使我们能够更好地理解沉默的过程，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.