LCR activation of the human growth hormone gene

LCR 激活人类生长激素基因

• 批准号: 8055257
• 负责人: NANCY E. COOKE
• 金额: $ 1.14万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8055257
• 关键词: Adult; Age; Binding Sites; Cell Line; Cell Nucleus; Cells; Chromatin; Complement; Defect; Development; Diagnosis; Disease; Distal; Elements; Epigenetic Process; Functional RNA; Gene Cluster; Gene Expression; Generations; Genes; Genetic; Genetic Transcription; Genome; Goals; Higher Order Chromatin Structure; Histones; Human; In Vitro; Indium; Individual; Inherited; Knowledge; Life; Locus Control Region; Maps; Mediating; Methodology; Modeling; Modification; Mutation; Pathologic; Pathway interactions; Physiological; Pituitary Gland; Play; Population; Process; Proteins; Role; Site; Somatotropin; Somatropin; Specificity; Staging; Stress; Structure; Testing; Transcriptional Activation; Transgenic Mice; Transgenic Organisms; base; hGH Gene; macromolecular assembly; novel; pituitary gland development; promoter; public health relevance; response; tissue culture; transgene expression

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.

描述（由申请人提供）：远端基因座控制区（LCR）调节人类生长激素（hGH）基因簇。四个 DNaseI 超敏位点 HSI、II、III 和 V 位于垂体 hGH-N 基因启动子 5' 14.5 至 32 kb 之间，标记了该 hGH LCR 的决定因素。这些决定因素协同作用，在垂体生长激素中建立稳定且适当调节的 hGH-N 表达。 HSI 和 HSII（-14.5 至 -15.5）对垂体染色质具有特异性，构成生长激素中 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 激活过程中整合了多组表观遗传调控修饰。这些 HSI 依赖性活性包括：1) 建立包含 hGH LCR 并延伸至 hGH-N 启动子的核心组蛋白超乙酰化的 32kb 结构域，2) 在 LCR 和相邻 CD79b 基因内生成强大的 PolII“转录结构域”这对于完整水平的 hGH-N 表达是必需的，以及 3) 将转​​录的 LCR/CD79b 结构域并置的高阶染色质结构的组织hGH-N转录增强过程中的hGH-N启动子。值得注意的是，Pit-1 阵列在 HSI 处的基于染色质的强大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来。 HSI Pit-1 特异性部分是由其独特的结合位点序列介导的。 HSI 活性似乎与相邻的 HSII 具有协同作用。 HSII 可能通过扩展表观遗传修饰或通过增强 LCR 内的 PolII 占据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生活中维持高水平的 hGH-N 表达方面发挥同样重要的作用。在此提案中，将使用体外、基于细胞和小鼠转基因方法的组合来表征 LCR 作用的各个组成部分。 hGH 基因座上 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，这些组织培养系在生长激素分化的确定阶段被抑制。人类 Pit-1 蛋白天然发生的突变可选择性阻断 hGH-N 表达，将用于进一步扩展我们对 Pit-1 对 hGH LCR 作用特异性基础的理解。该提案的具体目标集中在 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 hGH 表达的一系列病理缺陷（遗传性和后天性）以及因环境压力和衰老而发生的 hGH 表达的生理改变相关。 公共健康相关性：从人类生长激素基因的这些研究中获得的知识将进一步定义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后天缺陷的理解，并提出诊断和治疗这些及相关疾病的新途径。