Chromatin Remodeling and Lymphocyte Regulation

染色质重塑和淋巴细胞调节

• 批准号: 7132283
• 负责人: MICHAEL J PAZIN
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 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/7132283
• 关键词: Diptera; T lymphocyte; adenosine triphosphate; animal tissue; cell free system; cell line; chromatin; enzyme activity; gene expression; gene targeting; genetic regulation; histones; human tissue; immunoglobulin genes; tissue /cell culture; transcription factor

Chromatin structure is increasingly recognized as a key component in gene regulation. Chromatin structure is highly dynamic, and is determined by chromatin remodeling enzymes. There are three classes of remodeling enzymes; ATP-dependent remodeling enzymes use the energy of ATP hydrolysis to alter histone-DNA contacts, histone modifying enzymes covalently modify histone proteins, while DNA modifying enzymes add and remove methyl marks from the DNA itself. The importance of remodeling is especially appreciated in the immune system, where there are many examples of changes in chromatin structure associated with changes in gene expression. Moreover, it is possible to obtain defined populations of lymphocytes for experimentation. However, little is known about the enzymes mediating these changes. We use cell-based and cell free approaches to address this deficit by examining the role of remodeling in gene regulation, and the mechanism of remodeling. Cell-based studies We are investigating the role of ATP-dependent remodeling enzymes in T cell gene regulation using cultured cells. During this fiscal year, we developed conditions to transduce T cell lines with high efficiency (greater than 60%), and to enrich for transduced cells. We used this methodology to knock down expression of remodeling enzyme subunits. This allowed us to ask what genes required remodeling enzyme expression for proper regulation. Some of this work was reported at the NIA IRP retreat. We will expand these studies to include primary lymphocytes from mice or people. Cell-free studies We are investigating the mechanism of ATP-dependent remodeling enzymes using a cell-free biochemical system. We assemble chromatin with physiologic properties using either purified proteins or extracts from fly embryos. We then ask how chromatin structure changes occur, and what effect they have on chromatin function, using transcription or recombination as a readout. The current project uses an enhancer fragment from the immunoglobulin heavy-chain locus, and was initiated as a collaboration with Dr. Ranjan Sen while he was at Brandeis and I was at MGH/Harvard, and is related to work on the Ets family of transcription factors (Lu et al., 2004). Unexpectedly, we found that a transcription factor could prevent chromatin remodeling in an extract (Ishii et al., 2004). We have extended these findings using a purified system. This work has revealed that one transcription factor can reverse the remodeling directed by a first, and two different ATP-dependent remodeling enzymes mediate opposing effects.

染色质结构越来越被认为是基因调节中的关键成分。染色质结构是高度动态的，由染色质重塑酶确定。有三类的重塑酶；依赖ATP的重塑酶利用ATP水解的能量改变组蛋白DNA接触，组蛋白修饰酶共价修饰组蛋白蛋白，而DNA修饰酶则添加并从DNA本身中添加并去除甲基标记。重塑的重要性在免疫系统中尤其值得注意，其中有许多与基因表达变化相关的染色质结构变化的例子。此外，有可能获得定义的淋巴细胞种群进行实验。但是，关于介导这些变化的酶知之甚少。我们使用基于细胞的无细胞和无细胞方法来解决这种赤字，通过检查重塑在基因调节中的作用以及重塑的机制。 基于细胞的研究 我们正在研究使用培养细胞在T细胞基因调控中ATP依赖性重塑酶的作用。在这个财政年度，我们开发了效率高（大于60％）的T细胞系的条件，并富含转导的细胞。我们使用这种方法来击倒重塑酶亚基的表达。这使我们能够询问哪些基因需要重塑酶表达以进行适当调节。 NIA IRP撤退报告了这项工作中的一些。我们将扩大这些研究，包括来自小鼠或人的原发性淋巴细胞。 无细胞研究 我们正在研究使用无细胞生化系统的ATP依赖性重塑酶的机制。我们使用纯化的蛋白质或蝇胚提取物与生理特性组装染色质。然后，我们询问染色质结构的变化是如何发生的，以及它们对染色质功能的影响，使用转录或重组作为读数。当前的项目使用免疫球蛋白重链基因座的增强剂片段，并在Brandeis时与Ranjan Sen博士合作而启动，我当时在MGH/Harvard，与在ETS转录家族中工作有关因素（Lu等，2004）。出乎意料的是，我们发现转录因子可以防止提取物中的染色质重塑（Ishii等，2004）。我们已经使用纯化系统扩展了这些发现。这项工作表明，一个转录因子可以逆转由第一个转录因素引导的重塑，而两个不同的ATP依赖性重塑酶介导了相反的作用。