Transcriptional regulation by protein sumoylation

描述(申请人提供)：拟议研究的目标是了解细胞如何通过RNA聚合酶II(RNAP II)利用相加作用来调节转录。将在酵母和哺乳动物系统中进行分析，并提出以下具体目标。确定酵母中和甲基化在基因失活中的作用。我们最近发现，启动子结合因子在基因激活过程中发生总合作用，但矛盾的是，它对转录有负面影响。在诱导的ARG1基因中，和值化有助于清除启动子结合的因子，使其能够被关闭。这至少部分是由于与ARG1结合的激活剂Gcn4的苏木酸化。这一修改及其效果将得到详细描述。这一分析将扩展到其他基因，以确定相思作用是否在诱导基因失活中起到普遍作用。我们最近确定了酵母RNAP II中最大的亚基Rpb1和Rpb2是被总甲基化的。Rpb1在Rpb4亚基附近的Lys 1487处相加。Rpb1 K1487上的总甲基化是否能增强核心RNAP II与Rpb4/7异源二聚体的相互作用，和/或以其他方式，将被研究。我们将研究酵母RNAP II对相扑蛋白水解酶Ulp2的异常要求。我们将探讨Rpb2苏莫化的作用。确定哺乳动物系统中转录是如何通过相加作用来调节的。将进行芯片分析和siRNA敲除实验，以确定SUMO基化是否影响人类细胞的转录。对HeLa核提取液中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。IV.确定人RNAP II和一般转录因子的总合作用。人RNAP II，以及酵母和人类细胞中的GTF将被提纯，以鉴定受SUM化调节的成分。将产生不可相加的突变体，以确定阻断相加是否会影响活性或对活性基因的招募。将对纯化的RNAP II进行直接和甲基化，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.