LCR ACTIVATION OF THE HUMAN GROWTH HORMONE GENE

描述（申请人提供）：远端基因座控制区（LCR）调节人类生长激素（hGH）基因簇。hGH LCR的决定簇位于hGH-N基因5'端14.5 - 32 kb之间，由5个DNaseI超敏位点（HSI-HSV）标记。这些决定因素共同作用，以建立强大的和适当调节的垂体生长激素-N的表达。HSI是生长激素细胞中hGH-N转基因表达的主要决定因素。HSI含有三个必需的结合位点的垂体富集的转因子Pit-1。这些HSI核心元件似乎通过组蛋白乙酰转移酶活性的特异性募集发挥作用，导致建立32 kb的核心组蛋白超乙酰化结构域，其涵盖整个hGH LCR并延伸至远端hGH-N启动子。在HSI强大的Pit-1阵列发挥独特的，染色质为基础的功能，占主导地位的活性的Pit-1网站在hGH-N启动子。HSI活性的全部水平取决于与紧密相连的决定因素的协同作用。HSII是一种位于HSI 5'端约1 kb的垂体特异性HS，可能是这种协同作用的原因。32 kb hGH LCR结构域的末端边界对垂体细胞具有特异性，并且在转基因环境中用于将hGH基因座与整合位点效应和表达多样化隔离。目前的数据表明，在该结构域的5'边界处的边界和/或绝缘子活动可能是由HSV介导的。位于hGH-N基因3'端的边界元件可将胎盘hCS基因与垂体中的HSI介导的激活隔离。LCR在hGH基因座的初始激活中的作用之后可能是在染色质结构域内维持表观遗传修饰中的同样重要的作用，所述表观遗传修饰确保在整个成年生活中持续的hGH-N表达。本提案的具体目的侧重于hGH LCR功能的这些主要方面：目的I。表征HSI中启动hGH LCR活化的相互作用，目的II。通过LCR定义hGH-N的长程活化机制，目的III。描述HSII作为HSI促进者的角色，目标四。确定建立垂体特异性hGH LCR结构域的染色质边界，目的V.定义HSI在稳定维持hGH-N表达中的作用。值得注意的是，这些研究将解决参与LCR活性的基本表观遗传机制，并为理解正常和病理方面的hGH-N基因控制奠定基础。

DESCRIPTION (provided by applicant): A distal locus control region (LCR) regulates the human growth hormone (hGH) gene cluster. The determinants of the hGH LCR, located between 14.5 to 32 kb 5' to the hGH-N gene are marked by five DNaseI hypersensitive sites (HSI-HSV). These determinants act in concert to establish robust and appropriately regulated expression of hGH-N in pituitary somatotropes. HSI is the primary determinant of hGH-N transgene expression in somatotropes. HSI contains three essential binding sites for the pituitary-enriched transfactor Pit-1. These HSI core elements appear to function via specific recruitment of histone acetyltransferase activity, leading to the establishment of a 32 kb domain of core histone hyperacetylation that encompasses the entire hGH LCR and extends to the distal hGH-N promoter. The powerful Pit-1 array at HSI exerts unique, chromatin-based functions that are dominant over the activity of the Pit-1 sites at the hGH-N promoter. The full level of HSI activity is dependent on synergism with a closely linked determinant. HSII, a pituitary-specific HS located -1 kb 5' to HSI, may be responsible for this synergism. The terminal boundaries of the 32 kb hGH LCR domain are specific to pituitary cells and in a transgenic setting serve to insulate the hGH locus from site-of-integration effects and variegation of expression. Present data suggest that the boundary and/or insulator activities at the 5' border of this domain may be mediated by HSV. A boundary element located 3' to the hGH-N gene may insulate the placental hCS genes from HSI-mediated activation in the pituitary. The role of the LCR in the initial activation of the hGH locus may be followed by an equally important role in maintenance of epigenetic modifications within the chromatin domain that ensure continued hGH-N expression throughout adult life. The Specific Aims of this proposal focus on these major aspects of hGH LCR function: Aim I. Characterize interactions at HSI that initiate hGH LCR activation, Aim II. Define the mechanism(s) of long-range activation of hGH-N by its LCR, Aim III. Characterize the role of HSII as an HSI facilitator, Aim IV. Identify the chromatin boundaries that establish the pituitary-specific hGH LCR domain, and Aim V. Define the role of HSI in stable maintenance of hGH-N expression. Significantly, these studies will address fundamental epigenetic mechanisms involved in the activity of LCRs, and establish a foundation for understanding normal and pathological aspects of hGH-N gene control.