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。将在酵母和哺乳动物系统中进行分析，并提出以下具体目的。I.确定类小泛素化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类小泛素化在基因激活过程中发生，但矛盾的是对转录有负面影响。在被诱导的ARG 1基因中，类小泛素化促进启动子结合因子的清除，使其被关闭。这至少部分是由于Gcn 4（结合ARG 1的激活剂）的类小泛素化。将详细描述这种修改及其效果。这种分析将扩展到其他基因，以确定类小泛素化是否在诱导基因的失活中发挥普遍作用。二.确定酵母RNAP II的sumoylation的效果。我们最近确定，酵母RNAP II的最大亚基，Rpb 1和Rpb 2，是sumoylated。Rpb 1在靠近Rpb 4亚基的Lys 1487处被sumoylated。是否sumoylation在Rpb 1 K1487的功能，以增强核心RNAP II与Rpb 4/7异二聚体的相互作用，和/或以其他方式，将被调查。将研究酵母RNAP II类小泛素化对SUMO蛋白酶Ulp 2的不寻常要求。Rpb 2类小泛素化的功能将被探索。三.确定哺乳动物系统中类小泛素化是如何调节转录的。将进行ChIP测定和siRNA敲低实验以确定类小泛素化是否影响人细胞中的转录。还将确定SUMO化对HeLa核提取物中转录的影响。实验最初将采用裸DNA模板，但随后将扩展到染色质模板。四.确定人RNAP II和一般转录因子类小泛素化的作用。将纯化人RNAP II以及来自酵母和人细胞的GTF，以鉴定由sumoylation调节的组分。将产生不可小肽化的突变体，以确定阻断小肽化是否会影响活性基因的活性或募集。将进行纯化的RNAP II的直接类小泛素化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.