Novel Mekk1-p53 mediated transcriptional regulation: mechanism and genome wide st

描述（由申请人提供）：MAPK（丝裂原活化蛋白激酶）级联参与多种细胞功能，包括增殖、分化和凋亡。在被多种机制激活后，Mekk（一种MAPK激酶）激活Mkk（一种MAPKK或Sek）， Mkk又激活SAPK（一种MAPK）。然后MAPK从细胞质转移到细胞核，在那里它直接调节转录因子的活性，控制许多基因的表达。最近，我们观察到Mekk1及其组成活性片段CA-Mekk1 （1174-1493 aa）在HEK293T、COS和HCT细胞中过表达时易位到细胞核，并通过抗肿瘤蛋白p53影响Pkd1（多囊肾病）基因和其他基因的转录。因此，我们已经开始揭示一种新的转录调控机制。因此，为了了解Mekk1-p53介导的Pkd1和其他基因转录调控的新机制，我们提出了以下四个具体目标：目标1：鉴定Mekk1易位到细胞核的片段并确定其核易位的机制。目标2：通过以下方式表征p53-Mekk1结合机制：(a)确定直接结合位点，或(b)确定间接结合的中间伙伴。目的3：探讨p53升高的机制。目的4：进行ChIP-Seq分析，确定Mekk1、p53及两者的全基因组结合位点。我们将使用多种生物化学和细胞/分子生物学方法以及最新的尖端技术ChIP-Seq。这些研究不仅将提高我们对p53和Mekk1转录调控的认识，还可能揭示Mekk1-p53通路的机制。因此，所寻求的答案既应具有根本意义，又应提供导致未来治疗方法的信息。这项研究将吸引西北密苏里州立大学（NWMSU）的本科生，以及西北校区密苏里科学、数学和计算学院（MASMC）有才华的高中生。在这个项目中接受的培训将为学生进入科学项目和/或科学专业生涯做好准备。因此，该项目将(i)为本科生和有才华的高中生提供接触和参与生物医学研究的机会，（ii）加强西北密歇根州立大学的研究环境，（iii）给PI一个继续有价值的研究的机会。此外，与一个研究密集型机构（堪萨斯大学医学中心）的研究人员合作，将确保将最新的方法和信息注入生物化学/细胞生物学课程。因此，该项目将促进发现和理解，同时也促进研究、培训和学习。

DESCRIPTION (provided by applicant): MAPK (mitogen-activated protein kinase) cascades are involved in a variety of cellular functions including proliferation, differentiation, and apoptosis. Upon activation by any of a number of mechanisms, Mekk (a MAPK kinase kinase) activates Mkk (a MAPKK or Sek), which in turn activates SAPK (a MAPK). The MAPK is then translocated from the cytoplasm to the nucleus, where it directly regulates the activity of transcription factors controlling the expression of a number of genes. Recently, we observed Mekk1 and its constitutively active fragment CA-Mekk1 (1174-1493 aa) were translocated to the nucleus when overexpressed in HEK293T, COS, and HCT cells and affected transcription of the Pkd1 (Polycystic kidney disease) gene and possibly other genes via the anti-tumor protein, p53. As such, we have begun to unravel a novel mechanism of transcriptional regulation. Thus, with a broad goal to understand the novel mechanism of Mekk1-p53 mediated transcriptional regulation of the Pkd1 and other genes, we propose to accomplish the following four Specific Aims: Aim 1: Identify fragment(s) of Mekk1 translocating to the nucleus and determine their mechanism of nuclear translocation. Aim 2: Characterize the p53-Mekk1 binding mechanism by: (a) identifying the binding site if it is direct, or (b) identifying the intermediate partners, if it is indirect. Aim 3: Investigate the mechanism of p53 elevation. Aim 4: Perform ChIP-Seq analysis to identify genome-wide binding sites for Mekk1, p53 and both. We will use a variety of biochemical and cellular/molecular biological approaches as well as the latest cutting edge technique, ChIP-Seq. These studies will not only improve our understanding of transcriptional regulation by both p53 and Mekk1, but may also uncover the mechanism of the Mekk1-p53 pathway. Thus, the answers sought should have both fundamental significances and should provide information leading to future therapeutic approaches. The research will engage undergraduates at Northwest Missouri State University (NWMSU), as well as talented high school students in the Missouri Academy of Science, Math, and Computing (MASMC) housed on the Northwest campus. Training received in this project will prepare students for entry into science programs and/or scientific professional careers. Thus, the project will (i) provide undergraduate and talented high school students benefits of exposure to and participation in biomedical research, (ii) strengthen the research environment at NWMSU, and (iii) give the PI an opportunity to continue meritorious research. Furthermore, collaborations with investigators at a research-intensive institution (University of Kansas Medical Center) will ensure the infusion of the latest methodology and information into the biochemistry/cell biology curricula. Thus, the project will advance discovery and understanding while also promoting research, training and learning. PUBLIC HEALTH RELEVANCE: Recently, we unraveled a novel mechanism in which nuclear translocation of Mekk1 and its interaction with p53 leads to transcriptional repression of PKD1, independent of the MAPK phosphorylation cascade. The mechanism was previously unknown. This proposal will study the mechanism in detail and will seek to determine how Mekk1, a membrane-associated protein, translocates to the nucleus and interacts with p53; and will utilize cutting-edge genome-wide approaches to identify other genes that might be under regulation of this Mekk1-p53 pathway.