Transcriptional regulation by protein sumoylation

描述（由申请人提供）：拟议研究的目标是了解细胞如何使用sumoylation来调节RNA聚合酶II （RNAP II）的转录。分析将在酵母和哺乳动物系统中进行，并提出以下具体目标。1 .确定sumoylation在酵母基因失活中的作用。我们最近发现启动子结合因子的聚合化发生在基因激活过程中，但矛盾的是对转录有负面影响。在诱导的ARG1基因上，sumoylation促进了启动子结合因子的清除，使其被关闭。这至少部分是由于Gcn4的酰化，Gcn4是结合ARG1的激活剂。本文将详细描述这种改性及其效果。这一分析将扩展到其他基因，以确定sumo化是否在诱导基因失活中起普遍作用。2。确定酵母RNAP II的sumo化作用。我们最近确定酵母RNAP II的最大亚基Rpb1和Rpb2是被聚合的。Rpb1在靠近Rpb4亚基的Lys 1487位点被转录。Rpb1 K1487的sumoylation是否增强了核心RNAP II与Rpb4/7异源二聚体的相互作用，以及/或以其他方式，将被研究。我们将研究酵母RNAP II对SUMO蛋白酶Ulp2的不寻常要求。我们将探讨Rpb2聚合化的功能。3。确定在哺乳动物系统中转录是如何被聚合化调节的。ChIP分析和siRNA敲低实验将进行，以确定是否summoylation影响转录在人类细胞。sumo化对HeLa核提取物转录的影响也将被确定。实验最初将使用裸DNA模板，但随后将扩展到染色质模板。四、确定人RNAP II及一般转录因子的sumo化作用。人类RNAP II，以及来自酵母和人类细胞的gtf，将被纯化以鉴定受sumo化调节的成分。将产生不可聚合的突变体，以确定阻断聚合化是否会影响活性或对活性基因的招募。将对纯化的RNAP II进行直接聚合，以确定其对活性的影响。

DESCRIPTION (provided by applicant): The goal of the proposed research is to understand how cells use sumoylation to regulate transcription by RNA polymerase II (RNAP II). Analyses will be performed in both yeast and mammalian systems, and the following Specific Aims are proposed. I. Determine the role of sumoylation in gene deactivation in yeast. We recently found that sumoylation of promoter-bound factors takes place during gene activation, but paradoxically has a negative effect on transcription. At the induced ARG1 gene, sumoylation facilitates clearance of promoter-bound factors, enabling it to be shut off. This is due at least in part to sumoylation of Gcn4, the activator that binds ARG1. This modification and its effects will be characterized in detail. This analysis will be extended to other genes to establish whether sumoylation plays a general role in deactivation of induced genes. II. Determine the effect of sumoylation of yeast RNAP II. We recently determined that the largest subunits of yeast RNAP II, Rpb1 and Rpb2, are sumoylated. Rpb1 is sumoylated at Lys 1487, which is proximal to the Rpb4 subunit. Whether sumoylation at Rpb1 K1487 functions to enhance the interaction of core RNAP II with the Rpb4/7 heterodimer, and/or in some other way, will be investigated. The unusual requirement of yeast RNAP II sumoylation for Ulp2, a SUMO protease, will be examined. The function of Rpb2 sumoylation will be explored. III. Determine how transcription is regulated by sumoylation in mammalian systems. ChIP assays and siRNA knockdown experiments will be performed to determine whether sumoylation affects transcription in human cells. Effects of sumoylation on transcription in HeLa nuclear extracts will also be determined. Experiments will initially employ naked DNA templates, but will subsequently be extended to chromatin templates. IV. Determine the effect of sumoylation of human RNAP II and general transcription factors. Human RNAP II, as well as GTFs from both yeast and human cells, will be purified to identify components regulated by sumoylation. Non-sumoylatable mutants will be generated to determine whether blocking sumoylation affects activity or recruitment to active genes. Direct sumoylation of purified RNAP II will be performed to determine its effect on activity.