Transcription factor interactions at the GH promoter

描述(申请人提供)：生长激素是线性生长和代谢动态平衡的中心调节器。GH的合成在转录水平上仅限于脑下垂体前叶的细胞亚群。我们先前的研究表明，与进化相关的生长激素和催乳素(PRL)基因的转录被垂体特异性转录因子Pit-1和包括CCAAT/增强子结合蛋白α(C/EBPalpha)在内的其他转录因子协同激活。我们发现Pit-1、C/EBPalpha、共激活因子CBP以及基础因子TBP和TFIIB的分子相互作用参与了协同激活。我们还发现了一个泛基因组层来进行合作活性：C/EBPalpha通过与α卫星重复DNA结合，在着丝粒周围的异染色质中保持不活跃。PIT-1阻止C/EBPalpah与a-卫星DNA结合，从而将C/EBPalpha重新定位到细胞核转录活跃的亚室。这种功能区隔正在成为一种被忽视的表观遗传转录调节因子，因此，Pit-1既允许C/EBPalpha获得活性基因，又在GH和PRL启动子上直接与C/EBPalpha合作。 异染色质中C/EBPalpha的Pit-1释放和启动子的直接协同作用之间的生化和功能相互关系仍有待确定。我们的假设是，垂体特异的基因转录是由启动子上不同的分子相互作用引起的，这些相互作用的转录因子和辅助因子受到相互作用的转录因子和辅助因子的亚核区划的调节。我们的目标是确定启动子靶向和表观遗传层的分子基础以及它们对协同作用的相对贡献。 我们会： 目的1.比较特异性Pit-L和C/EBPalpha活性突变对转录协同作用、C/EBPalpha结合到α卫星DNA的释放以及C/EBPalpha到常染色质Pit-1重定位的影响。 目的2.明确Pit-1和C/EBPalpha突变体对C/EBPalpha、Pit-L及其相关辅助因子生化相互作用的影响， 目的3.检测Pit-L和C/EBPalpha的表达对Pit-1、C/EBPalpha的生化募集的影响以及相关的相关辅助因素，特别是在生长激素和催乳素启动子以及α卫星DNA上。

DESCRIPTION (provided by applicant): Growth hormone is a central regulator of linear growth and metabolic homeostasis. GH synthesis is restricted transcriptionally to a subpopulation of ceils in the anterior pituitary gland. Our prior studies showed transcription of the evolutionary-related GH and prolactin (PRL) genes to be cooperatively activated by the pituitary-specific transcription factor Pit-1 and other transcription factors including CCAAT/enhancer binding protein alpha (C/EBPalpha). We found molecular interactions of Pit-1, C/EBPalpha, the coactivator CBP and the basal factors TBP and TFIIB to participate in cooperative activation. We also uncovered a pan-genomic layer to cooperative activity: C/EBPalpha is held inactive at pericentromeric heterochromatin through C/EBPalpha binding to alpha-satellite repetitive DNA. Pit-1 prevents C/EBPalpah binding to a-satellite DNA and thereby relocates C/EBPalpha to the transcriptionally active subcompartment of the cell nucleus. Such functional compartmentalization is emerging as an overlooked, epigenetic regulator of transcription Thus, Pit-1 both grants C/EBPalpha access to active genes and directly cooperates with C/EBPalpha at the GH and PRL promoters. The biochemical and functional inter-relationships between Pit- 1 release of C/EBPalpha from heterochromatin and direct synergy at the promoters remain to be defined. Our hypothesis is that pituitary-specific gene transcription results from distinct molecular interactions at the promoter that are regulated by the subnuclear compartmentalization of the interacting transcription factors and co-factors. Our goal is to define the molecular basis of the promoter-targeted and epigenetic layers and their relative contributions to synergy. We will: Aim 1. Compare the effects of mutations in specific Pit- l and C/EBPalpha activities on transcriptional synergy, release of C/EBPalpha binding to alpha-satellite DNA, and Pit-1 re-location of C/EBPalpha to euchromatin, Aim 2. Define the effects of the Pit-1 and C/EBPalpha mutants on the biochemical interactions of C/EBPalpha, Pit-l, and their relevant co-factors, Aim 3. Determine the effects of Pit- l and C/EBPalpha expression on biochemical recruitment of Pit- 1, C/EBPalpha and relevant co-factors specifically at the GH and PRL promoters and at alpha-satellite DNA.