Transcription factor interactions at the GH promoter

描述（由申请人提供）：生长激素是线性生长和代谢稳态的中心调节剂。生长激素的合成在转录上仅限于垂体前叶细胞亚群。我们先前的研究表明，进化相关的GH和催乳素（PRL）基因的转录被垂体特异性转录因子Pit-1和其他转录因子，包括CCAAT/增强子结合蛋白α（C/EBPalpha）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的分子间相互作用参与协同激活。我们还发现了一个泛基因组层的合作活动：C/EBPalpha是通过C/EBPalpha结合α-卫星重复DNA在着丝粒周围异染色质保持无活性。Pit-1阻止C/EBPalpah与α-卫星DNA结合，从而将C/EBPalpha重新定位到细胞核的转录活性亚室。这种功能区室化作为一种被忽视的表观遗传转录调节因子而出现。因此，Pit-1既赠款C/EBPalpha接近活性基因，又在GH和PRL启动子处与C/EBPalpha直接合作。 Pit- 1从异染色质中释放C/EBPalpha与启动子直接协同作用之间的生物化学和功能相互关系仍有待确定。我们的假设是，垂体特异性基因转录的结果不同的分子相互作用的启动子，相互作用的转录因子和辅因子的亚核区室化的调节。我们的目标是确定启动子靶向和表观遗传层的分子基础及其对协同作用的相对贡献。 我们将： 目标1.比较特异性Pit-1和C/EBPalpha活性的突变对转录协同作用、C/EBPalpha与α-卫星DNA结合的释放以及C/EBPalpha向常染色质的Pit-1重新定位的影响， 目标2.确定Pit-1和C/EBPalpha突变体对C/EBPalpha、Pit-1及其相关辅因子的生物化学相互作用的影响， 目标3.确定Pit-1和C/EBPalpha表达对Pit- 1、C/EBPalpha和相关辅因子的生化募集的影响，特别是在GH和PRL启动子和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.