MECHANISMS OF TRANSCRIPTIONAL COORDINATION AMONG PHOSPHORYLASE KINASE GENES

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。磷酸化酶激酶(PhK)是糖原分解途径中的关键调节酶，通过磷酸化来催化磷酸化酶的激活。这种具有亚基组成(阿尔法、贝塔、伽马、德尔塔)的十六聚体酶4，用于整合荷尔蒙、代谢和神经信号，导致肌肉和肝脏中糖原储存的分解。PhK的每个亚基都由一个单独的基因编码，所有的亚基都存在多种组织特异性亚型。虽然已经做了很多工作来阐明PhK催化的生物化学调节，但关于这种复杂低聚物的转录调节的信息很少。我们的工作旨在通过对所有人类α、β和伽马启动子的鉴定和功能分析来阐明那些负责PhK基因表达的时间和空间调节的元件。拟议的项目针对这一目标有四个目标。1)通过对人类基因组数据库的生物信息学分析，确定所有α、β和伽马亚型的可能的PhK启动子启动子，并通过聚合酶链式反应进行扩增。2)将为每个启动子确定表达空间和时间基因所需的最小序列。3)将为每个启动子确定关键的顺式和反式作用因子。4)将对PhK基因缺陷小鼠的RNA进行微阵列分析，以确定参与协调基因表达的新蛋白质。从这些目标中获得的知识将有助于确定如何协调PhK的表达，以确保代谢可塑性被调节以响应不断变化的能量需求。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Phosphorylase kinase (PhK) is a key regulatory enzyme involved in the glycogenolytic pathway, catalyzing the activation, by phosphorylation, of phosphorylase. The hexadecameric enzyme, with subunit composition (alpha,beta,gamma,delta)4, serves to integrate hormonal, metabolic and neural signals, leading to the breakdown of glycogen stores in muscle and liver. Each subunit of PhK is encoded by a separate gene, and multiple tissue specific isoforms exist for all of the subunits. While much work has been done to elucidate the biochemical regulation of PhK catalysis, little information regarding transcriptional regulation of this complex oligomer is available. Our work seeks to elucidate those elements responsible for the temporal and spatial regulation of PhK gene expression by providing for the identification and functional analysis of all human alpha, beta and gamma promoters. The proposed project has four aims directed at this goal. 1) The putative PhK promoter reg ions for all alpha, beta and gamma isoforms will be identified through bioinformatic analysis of the human genome database and amplified by PCR. 2) The minimal sequence necessary to convey spatial and temporal gene expression will be determined for each promoter. 3) Key regulatory cis- and trans-acting factors will be identified for each promoter. 4) Microarray analysis will be performed on RNA from PhK deficient mice to identify novel proteins involved in coordinated gene expression. Knowledge gained from these aims will help determine how PhK expression is orchestrated to ensure that metabolic plasticity is modulated in response to changing energy demands.