REMOTE CONTROL ELEMENTS IN SOMATOTROPE EXPRESSION

人类生长激素（hGH）基因簇包括垂体和胎盘特异性基因，这些基因对正常生长发育至关重要。一组组织特异性的，主要作用的远端调控元件，基因座控制区（LCR），被定位在14到34kb 5'之间，基于来自表达组织的染色质的DNaseI超敏性（HS）。这些元素参与垂体/胎盘中hGH基因簇表达的适当激活。然而，每个HS的具体功能作用，这些决定因素之间的冗余水平，以及它们各自的作用机制都没有定义。hGH基因簇中基因的组织结构、高水平表达和互斥的组织特异性，以及相应的LCR元件特异性集合，使其成为研究基因表达的一个有价值的模型。目的1：从功能上分析局限于垂体（HSI，II）或胎盘（HSIV）的LCR HS亚群。这些元素将在大（P1）人类转基因（hGH-P1）的背景下进行研究。这个87kb的hGH- p1转基因包含了整套LCR元件（HSI-V）和大部分连续的hGH基因簇，这些基因在宿主小鼠的适当组织中表达。hGH- p1将删除HSI II的一个小的、特征明确的功能片段，并将在转基因小鼠中研究其对hGH基因簇表达的影响。与此同时，同样的方法将应用于胎盘特异性的HSIV，以描述其在激活和/或限制胎盘表达中的功能。通过表征这些缺失对相对于天然hGH-P1的染色质表观遗传修饰的影响，我们将研究每种情况下HS功能的机制。Aim II将识别和表征小鼠中相应的LCR控制元件(m)。这个目的的一个主要目标将是确定LCR是否也指导mGH基因表达。具体来说，我们将确定HS的存在，并将其序列与相应的人类元素进行比较。这些数据将用于建立关键HSI，II决定因素的系统发育足迹，并作为Aim I中正在进行的功能/机制分析的基础，包括产生具有相应靶向缺失的小鼠。意义：这些研究将为LCR元件在hGH基因发育过程中建立和维持适当的基因表达谱和疾病相关表达模式中所起的作用提供新的见解。

The human growth hormone (hGH) gene cluster encompasses both pituitary- and placenta-specific genes that are essential for normal growth and development. A set of tissue-specific, dominantly acting distal regulatory elements, the locus control region (LCR), were mapped between 14 and 34kb 5' to the cluster on the basis of DNaseI hypersensitivity (HS) in chromatin derived from expressing tissues. These elements are involved in appropriate activation of hGH gene cluster expression in pituitary/placenta. However the specific functional role of each HS, the level of redundancy among these determinants, and their respective mechanism(s) of action are not defined. The organization of genes in the hGH gene cluster, their high-level expression and mutually exclusive tissue-specificities, and the corresponding specific sets of LCR elements make it a valuable model for studying gene expression. Aim I is to functionally analyze the subset of LCR HS restricted to either pituitary (HSI,II) or placenta (HSIV). These elements will be studied in the context of a large (P1) human transgene (hGH-P1). This 87kb hGH-P1 transgene encompasses the entire set of LCR elements (HSI-V) and most of the contiguous hGH gene cluster and the genes are expressed in appropriate tissues in the host mouse. A small, well-characterized functional segment of the HSI,II will be deleted from hGH-P1 and its impact on hGH gene cluster expression will be investigated in the transgenic mouse. In parallel the same approach will be applied to the placental-specific, HSIV to delineate its function in activating and/or restricting placental expression. The mechanism(s) of HS function will be studied in each case by characterizing the effects of these deletions on chromatin epigenetic modifications relative to the native hGH-P1. Aim II will identify and characterize corresponding LCR control elements in the mouse (m). A major goal of this aim will be to determine whether an LCR also directs mGH gene expression. Specifically we will identify the presence of HS and their sequences will be compared to the corresponding human elements. These data will be used to establish phylogenetic footprints of critical HSI,II determinants and serve as the foundation for the ongoing functional/mechanistic analyses in Aim I, including the generation of mice with corresponding targeted deletions. Significance: These studies will provide novel insight into the role(s) played by the LCR elements in the establishment and maintenance of appropriate gene expression profiles during development and in disease-related expression patterns of the hGH gene.