Studies On Silencing

沉默研究

• 批准号: 6813950
• 负责人: Rohinton T. Kamakaka
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6813950
• 关键词: chromatin; fungal genetics; fungal proteins; gene induction /repression; genetic recombination; genetic screening; genetic transcription; heterochromatin; histones; nucleosomes; protein protein interaction; protein purification; protein structure function

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 inactivation centers called silencers and numerous 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 currently focused on the Sir proteins and their interactions with the histones to understand in molecular detail the mechanism by which these proteins effect silencing. 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 silencing and cell cycle progression. We have been purifying Sir protein complexes and analyzing the composition of these complexes and their associated enzymatic activities to understand the mechanism by which these proteins function in silencing. We have also begun studies on the reconstitution of silenced chromatin using these purified 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 and our results suggest that barrier activity may arise from an underlying competition between chromatin remodeling and silencing activities at the interface of euchromatin and heterochromatin. We are currently performing a genome wide screen for proteins that can block the spread of silenced chromatin in order to determine the general principles that underlie the mechanism of barrier function. 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 domains of Htz1 involved in cell cycle progression, the response to drugs as well as the proteins that interact with Htz1 to function in the cell. 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.

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称为沉默子的失活中心和许多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。我们目前专注于爵士蛋白及其与组蛋白的相互作用，以了解这些蛋白质影响沉默的分子机制。 我们目前正在研究1）沉默结构域的结构及其体外重建的生物化学和分子表征2）沉默结构域限制于基因组特定区域的机制和3）组蛋白变体在沉默和细胞周期进程中的作用。 我们一直在纯化Sir蛋白复合物，并分析这些复合物的组成及其相关的酶活性，以了解这些蛋白质在沉默中发挥作用的机制。我们也开始研究使用这些纯化的复合物和核小体中的组蛋白重建沉默的染色质。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，特定的元素作为屏障沉默的染色质的连续传播，我们的研究结果表明，屏障活动可能会出现从染色质重塑和沉默活动之间的潜在竞争常染色质和异染色质的界面。我们目前正在进行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，以确定屏障功能的机制的一般原则。 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，以确定参与细胞周期进程的Htz1结构域，对药物的反应以及与Htz1相互作用以在细胞中发挥功能的蛋白质。 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。