EPIGENETIC ACTIVATION OF THE HUMAN GROWTH HORMONE GENE

描述(由申请人提供)： 组织特异性基因座控制区(HGH LCR)调控人生长激素(HGH)基因簇。HGH LCR的组分位于hGH簇上游14.5至32kb之间，以DNase I超敏位点(HS)为标志。我们已经将-14.5kb的hSI定义为垂体促生长激素中hGH LCR的主要增强子成分。该决定簇在小鼠脑下垂体hGH-N转基因激活中起着必不可少的、非冗余的作用。HGH LCR和hGH-N基因包含在一个连续的32kb的生长激素特异性乙酰化染色质结构域中。HSI包含一个由三个结合位点组成的阵列，可与脑垂体特异的POU同源结构域反式因子Pit-1结合。HSI的这些核心决定因素对于广泛的乙酰化结构域的建立和hGH-N基因在发育过程中的激活至关重要。为了扩大对hGH LCR作用的机制理解，我们提出了以下三个目标。目的1.确定LCR介导的长程转录增强的顺式和反式效应因子。这一目标将集中在鉴定顺式和反式调节元件的完整补充，这些元件促进HSI介导的14.5kb距离的hGH-N启动子的远程激活，并定义修饰的染色质结构域的边界。目的II.明确乙酰化在hGH LCR中扩散的机制。这一目标将解决这些决定因素建立染色质修饰的广泛区域并触发目标hGH-N启动子的远程基因激活的机制。目的III.建立特定hGH LCR活性与hGH基因亚核定位之间的功能关系。这一目标将利用反映LCR活动矩阵的一系列转基因来建立LCR功能与hGH基因亚核定位之间的联系。值得注意的是，拟议的研究将为P.I.提供机会，充分利用一系列耗时但信息量大的小鼠转基因研究，拓宽和加强他的科学能力，为独立的学术研究生涯做准备

DESCRIPTION (provided by applicant): A tissue-specific locus control region (hGH LCR) regulates the human growth hormone (hGH) gene cluster. The components of the hGH LCR, marked by DNase I hypersensitive sites (HS), are located from 14.5 to 32 kb upstream of the hGH cluster. We have defined HSI, at -14.5 kb, as the major enhancer component of the hGH LCR in pituitary somatotropes. This determinant plays an essential and non-redundant role in hGH-N transgene activation in the mouse pituitary. The hGH LCR and hGH-N gene are encompassed within a continuous 32 kb somatotrope-specific acetylated chromatin domain. HSI contains an array of three binding sites for the pituitary-specific POU-homeodomain trans-factor, Pit-1. These core determinants of HSI are critical for establishment of the broad acetylated domain and for the activation of the hGH-N gene during development. To extend the mechanistic understanding of hGH LCR action we propose the following three aims. Aim 1. Define cis- and trans-effectors of LCR-mediated long-range transcriptional enhancement. This aim will focus on the identification of the full complement of cis- and trans-regulatory elements that facilitate HSI-mediated long-range activation of the hGH-N promoter over a distance of 14.5 kb and that define the borders of the modified chromatin domain. Aim II. Define the mechanism of acetylation spreading within the hGH LCR. This aim will address the mechanisms by which these determinants establish an extensive domain of chromatin modification and trigger long-range gene activation of the target hGH-N promoter. Aim III. Establish functional relationships between specific hGH LCR activities and the sub-nuclear localization of the hGH locus. This aim will utilize a series of transgenes that reflect a matrix of LCR activities to establish the linkages between LCR functions and sub-nuclear localization of the hGH locus. Significantly, the proposed studies will provide the P.I. the opportunity to fully exploit a set of time-intensive but highly informative mouse transgenic studies, broadening and strengthening his scientific capabilities in preparation for an independent career as an academic researcher