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。垂体HGH-N基因启动子的四个DNasei高度敏感性位点，HSI，II，III和V位于14.5至32 kb 5'之间，标志着该HGH LCR的决定因素。这些决定因素协同起作用，以建立垂体种子体中HGH-N的强大和适当调节的表达。 HSI和HSII为-14.5至-15.5，是垂体染色质的特异性，构成了hGH -N转基因表达的主要决定因素。 HSI的核心元素包括依赖垂体增强的转录器PIT-1的一系列结合位点，在HGH-N激活过程中整合了多个表观遗传调节修饰。这些依赖HSI的活性包括：1）建立一个核心组蛋白高乙酰化的32KB结构域，包括HGH LCR并延伸至HGH-N启动子，2）在LCR和相邻的CD79B基因内生成强大的polii polii“转录域”，该基因在CD79B基因中，是HGH-N的完整水平，并施加了HGH-N表现的完整水平。在HGH-N转录增强过程中，转录和HGH-N启动子的LCR/CD79B结构域。值得注意的是，在HSI上，PIT-1阵列的强大染色质活性可以与HGH-N启动子处的PIT-1阵列的活性区分开。 HSI PIT-1特异性部分通过其独特的结合位点序列介导。 HSI活性似乎与相邻的HSII协同作用。 HSII可能是通过扩展表观遗传修饰或增强LCR内的polii占用和非编码转录来增强HSI活性的原因。 LCR在HGH-N表达的激活和增强中的作用可能在整个成人生活中保持高水平的HGH-N表达方面同样重要。在此提案中，使用体外，基于细胞和小鼠转基因方法的组合来表征LCR作用的单个组成部分。 HGH基因座的LCR依赖性变化的时间顺序将在一组新型的组织培养基线中确定，该组织在定义的躯体分化阶段被捕。有选择地阻断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HGH LCR处PIT-1作用特异性的基础的理解。该提案的具体目的集中于HGH LCR功能的这些主要方面，其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表达中的一系列病理性缺陷相关，无论是遗传和获得的，以及HGH表达中的生理改变，这些变化是响应环境压力和衰老而发生的。 公共卫生相关性：从这些对人类生长激素基因的研究中获得的知识将进一步定义人类垂体的形成和功能所涉及的途径，扩展我们对人群中生长激素基因表达中遗传和获得的缺陷的理解，并在人类种群中观察到，并建议对这些和相关疾病的诊断和治疗进行新颖的途径。