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）调节人生长激素（hGH）基因簇。由DNA酶I超敏位点（HS）标记的hGH LCR组分位于hGH簇上游14.5至32 kb处。我们已经将约14.5kb的HSI定义为垂体促生长素中hGH LCR的主要增强子组分。该决定簇在小鼠垂体中的hGH-N转基因激活中起着重要而非冗余的作用。hGH LCR和hGH-N基因包含在一个连续的32 kb促生长细胞特异性乙酰化染色质结构域中。HSI包含一系列的三个结合位点的垂体特异性POU-同源结构域反式因子，Pit-1。HSI的这些核心决定因素对于广泛乙酰化结构域的建立和发育期间hGH-N基因的激活至关重要。为了扩展对hGH LCR作用机制的理解，我们提出了以下三个目标。目标1.定义LCR介导的长距离转录增强的顺式和反式效应子。这一目标将集中在鉴定顺式和反式调节元件的完整互补物上，所述调节元件促进HSI介导的hGH-N启动子在14.5 kb的距离内的远程激活，并且限定经修饰的染色质结构域的边界。Aim II.定义hGH LCR内乙酰化扩散的机制。这一目标将解决这些决定因素建立一个广泛的染色质修饰域和触发目标hGH-N启动子的远程基因激活的机制。Aim III.建立特定hGH LCR活性与hGH基因座亚核定位之间的功能关系。该目标将利用一系列反映LCR活性矩阵的转基因来建立LCR功能与hGH基因座的亚核定位之间的联系。重要的是，拟议的研究将提供P.I.有机会充分利用一系列时间密集但信息量很大的小鼠转基因研究，扩大和加强他的科学能力，为独立从事学术研究做准备

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