YY1 functions in vascular smooth muscle cells

描述(由申请人提供)：血管损伤引起的平滑肌细胞(SMC)增殖激活是临床医学中广泛认识的血管问题，但其基础仍不清楚。我们的假设是，蛋白质的异常翻译后修饰在血管损伤后与血管新生内膜形成相关的激活的平滑肌细胞增殖的启动中起着重要作用。我们特别感兴趣的是，YY1的O-GlcN酰化可能调节YY1-Rb的相互作用和下游转录促进基因的表达，从而触发平滑肌细胞的增殖。我们感兴趣的是，Rb在特定氨基酸的磷酸化也可能抑制YY1-Rb的络合作用，从而激活平滑肌细胞的增殖。本项目的目标是确定YY1的O-糖基化如何调节YY1-Rb的相互作用，以及SMC从静止到活跃增殖过程中发生的转录变化。目的1：确定YY1O-GlcN酰化在生长停滞和生长刺激的SMC培养中的特异性位点。YY1上O-GlcNAc连接的位置将通过应用自动三重四重和离子捕获串联质谱学相结合的方法来确定。目的：在大鼠颈动脉球囊损伤模型上分析YY1O-GlcN酰化对血管球囊损伤的影响。测定大鼠球囊损伤后2天、4天、8天YY1 O-糖基化的速度和位置。目的3：基于结构的定点突变鉴定YY1-Rb结合表面及其Rb磷酸化调控。将确定抑制YY1结合的pRb上的磷酸化Ser/Thr残基。目的：应用流式细胞术、基因表达调控和染色质免疫沉淀(ChIP)技术研究O-GlcN酰化对细胞YY1功能的影响。

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).