LCR activation of the human growth hormone gene

描述（由申请方提供）：远端基因座控制区（LCR）调节人生长激素（hGH）基因簇。位于垂体hGH-N基因启动子5'端14.5 - 32 kb之间的4个DNaseI超敏感位点HSI、II、III和V标记了该hGH LCR的决定簇。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。在-14.5到-15.5处的HSI和HSII是垂体染色质特异性的，并且构成生长激素细胞中hGH-N转基因表达的主要决定因素。HSI的核心元件包括垂体富集的转因子Pit-1的一系列结合位点，在hGH-N活化过程中整合了多组表观遗传调节修饰。这些HSI依赖性活动包括：1）建立包含hGH LCR并延伸至hGH-N启动子的32 kb核心组蛋白超乙酰化结构域，2）在LCR和邻近的CD 79 b基因内产生完整水平的hGH-N表达所必需的稳健PolII“转录结构域”，和3）在hGH-N转录增强过程中并置LCR/CD 79 b转录结构域和hGH-N启动子的高级染色质结构的组织。值得注意的是，Pit-1阵列在HSI处的强大的基于染色质的活性可以在功能上与Pit-1阵列在hGH-N启动子处的活性区分开。HSI Pit-1特异性部分由其独特的结合位点序列介导。HSI活性似乎与邻近的HSII协同作用。HSII可能负责通过表观遗传修饰的延伸或通过增强LCR内的PolII占用和非编码转录来增强HSI活性。LCR在激活和增强hGH-N表达中的作用可能是在整个成年期维持高水平的hGH-N表达中同样重要的作用。在本提案中，LCR作用的单个组分将使用体外、基于细胞和小鼠转基因方法的组合来表征。在hGH基因座的LCR依赖性改变的时间顺序将在一组新的组织培养系中确定，所述组织培养系在生长激素分化的限定阶段被阻止。选择性阻断hGH-N表达的人Pit-1蛋白的天然突变将用于进一步扩展我们对Pit-1作用于hGH LCR的特异性基础的理解。本提案的具体目的侧重于hGH LCR功能的这些主要方面，最终目标是扩展我们对hGH-N激活和表达的理解，并将我们的发现推广到真核基因组中远程转录激活的途径和机制。这些发现最终将与一系列遗传和获得性hGH表达的病理缺陷以及响应环境应激和衰老而发生的hGH表达的生理改变相关。 公共卫生相关性：从这些人类生长激素基因的研究中获得的知识将进一步确定参与人类垂体形成和功能的途径，扩展我们对在人群中观察到的生长激素基因表达的遗传和获得性缺陷的理解，并为这些和相关疾病的诊断和治疗提供新的途径。

DESCRIPTION (provided by applicant): A distal locus control region (LCR) regulates the human growth hormone (hGH) gene cluster. Four DNaseI hypersensitive sites, HSI, II, III, and V located between 14.5 to 32 kb 5' to the pituitary hGH-N gene promoter, mark the determinants of this hGH LCR. These determinants act in concert to establish robust and appropriately regulated expression of hGH-N in pituitary somatotropes. HSI and HSII, at -14.5 to -15.5, are specific to pituitary chromatin and constitute the primary determinants of hGH-N transgene expression in somatotropes. The core elements of HSI, comprising an array of binding sites for the pituitary-enriched transfactor Pit-1, integrate multiple sets of epigenetic regulatory modifications in the process of hGH-N activation. These HSI-dependent activities include: 1) establishing of a 32kb domain of core histone hyperacetylation encompassing the hGH LCR and extending to the hGH-N promoter, 2) generation of a robust PolII 'domain of transcription' within the LCR and adjacent CD79b gene that is necessary for full levels of hGH- N expression, and 3) organization of higher-order chromatin structure that juxtaposes the LCR/CD79b domain of transcription and the hGH-N promoter during the process of hGH-N transcriptional enhancement. Remarkably, the powerful chromatin-based activities of the Pit-1 array at HSI can be functionally distinguished from the activity of the Pit-1 array at the hGH-N promoter. The HSI Pit-1 specificity is mediated, in part, by its unique binding site sequence. HSI activity appears to act in synergy with the adjacent HSII. HSII may be responsible for augmenting HSI activity via extension of epigenetic modifications or by enhancing PolII occupancy and non-coding transcription within the LCR. The role of the LCR in the activation and enhancement of hGH-N expression may be followed by an equally important role in maintaining high levels of hGH-N expression throughout adult life. In this proposal, individual components of LCR action will be characterized using a combination of in vitro, cell-based, and mouse transgenic methodologies. The temporal order of LCR-dependent alterations at the hGH locus will be determined in a novel set of tissue culture lines that are arrested at defined stages of somatotrope differentiation. A naturally occurring mutation of the human Pit-1 protein that selectively blocks hGH-N expression will be used to further extend our understanding of the basis for the specificity of Pit-1 action at the hGH LCR. The Specific Aims of this proposal focus on these major aspects of hGH LCR function with the ultimate goal of extending our understanding of hGH-N activation and expression and in generalizing our findings to pathways and mechanisms of long-range transcriptional activation in the eukaryotic genome. These findings will eventually be correlated with an array of pathologic defects in hGH expression, both inherited and acquired, and with physiologic alterations in hGH expression that occur in response to environmental stress and ageing. PUBLIC HEALTH RELEVANCE: Knowledge gained from these studies of the human Growth Hormone gene will further define the pathways involved in the formation and function of the human pituitary, extend our understanding of genetic and acquired defects in Growth Hormone gene expression that are observed in human populations, and suggest novel avenues for diagnosis and therapy of these and associated disorders.