YY1 functions in vascular smooth muscle cells

描述（由申请人提供）：平滑肌细胞（SMC）增殖在血管损伤反应中的激活是临床医学中广泛认识的血管问题，但其基础尚不清楚。我们的假设是，在血管损伤后，蛋白质的异常翻译后修饰对激活的平滑肌细胞增殖的启动起了重要作用，并与血管内膜形成相关。我们特别感兴趣的是YY1的o - glcn酰化，在新生儿平滑肌细胞中显著升高，调节YY1- rb相互作用和下游转录促进基因表达，从而触发平滑肌细胞增殖的可能性。我们感兴趣的可能性是，Rb在特定氨基酸上的磷酸化也会抑制YY1-Rb络合，从而促进平滑肌细胞增殖的激活。该项目的目标是确定YY1的o糖基化如何调节YY1- rb相互作用以及SMC从静止到活跃增殖转变过程中发生的转录变化。目的1：确定生长阻滞和生长刺激SMC培养中YY1 o - glcn酰化的具体位点。O-GlcNAc在馏分中附着于YY1的位置将通过应用自动三重三重和离子捕获串联质谱的组合来确定。目的2：分析大鼠颈动脉血管球囊损伤模型中YY1 o - glcn酰化反应。测定大鼠球囊损伤后第2天、第4天和第8天YY1 o -糖基化的速率和位置。目的3：基于结构的位点定向诱变，鉴定YY1-Rb结合表面及其通过Rb磷酸化调控。pRb上抑制YY1结合的磷酸化丝氨酸/苏氨酸残基将被识别。目的4：利用流式细胞术、基因表达调控和染色质免疫沉淀（ChIP）确定o - glcn酰化如何影响细胞YY1功能。

DESCRIPTION (provided by applicant): Activation of smooth muscle cell (SMC) proliferation in response to vascular injury is a widely appreciated vascular problem in clinical medicine, but its basis remains obscure. Our hypothesis is that abnormal posttranslational modification of proteins makes a major contribution to the initiation of activated smooth muscle cellular proliferation associated with vascular neointimal formation following vascular injury. We are particularly interested in the possibility that O-GlcNAcylation of YY1, significantly elevated in the neontimal smooth muscle cells, regulates the YY1-Rb interaction and downstream transcription promoting gene expression that triggers smooth muscle cellular proliferation. We are interested in the possibility that the phosphorylation of Rb at specific amino acids also inhibits the YY1-Rb complexation contributing to the activation of smooth muscle cellular proliferation. The goal of this project is to identify how O-glycosylation of YY1 regulates the YY1-Rb interaction and the transcriptional changes that occur in SMC transition from quiescence to active proliferation. Aim 1: Determine the specific sites of YY1 O-GlcNAcylation in growth arrested and in growth stimulated SMC cultures. The position of O-GlcNAc attachments to YY1 in the fractions will be determined by applying a combination of automated triple quadruple and ion trapping tandem mass spectroscopy. Aim 2: Analysis of YY1 O-GlcNAcylation in response to vascular balloon injury in a rat carotid artery model. The rate and the position of YY1 O-glycosylation 2 days, 4 days, and 8 days after rat balloon injury will be determined. Aim 3: Structure-based site-directed mutagenesis for the identification of the YY1-Rb binding surface and its regulation by Rb phosphorylation. The phosphorylated Ser/Thr residues on pRb that inhibit YY1 binding will be identified. Aim 4: Determine how O-GlcNAcylation influences cellular YY1 functions applying flow cytometry, gene expression regulation, and chromatin immunoprecipitation (ChIP).