Identifying pubertal regulators of Kiss1 expression

识别 Kiss1 表达的青春期调节因子

• 批准号: 7333069
• 负责人: Yee-Ming Chan
• 金额: $ 5.89万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7333069
• 关键词: A Mouse; Applications Grants; Area; Biological Process; Cells; Collection; Contraceptive methods; DNA; Delayed Puberty; Diagnostic tests; Disease; Elements; Endocrine; Gene Expression; Genes; Genetic; Genetic Transcription; Genomics; Goals; Gonadal Steroid Hormones; Green Fluorescent Proteins; Human; Human Biology; Human Identifications; Hybridization Array; Hypothalamic structure; Individual; Infertility; KISS1 gene; KISS1R gene; Kallmann Syndrome; Label; Lasers; Lead; Life; Malignant neoplasm of prostate; Microdissection; Molecular Profiling; Mus; Mutation; Neurons; Neurosecretory Systems; Nucleic Acid Regulatory Sequences; Patients; Puberty; RNA; Regulatory Element; Reporter; Rodent; Role; Sexual Maturation; Signaling Molecule; Stretching; System; Testing; Time; Trans-Activators; Work; in vivo; insight; malignant breast neoplasm; promoter; receptor; reproductive; research study; tumor

DESCRIPTION (provided by applicant): The neuroendocrine and genetic control of sexual maturation at puberty remains one of the great mysteries of human biology. Recently, the signaling molecule kisspeptin and its receptor GPR54 have been found to have key roles in regulating the reproductive endocrine axis. The experiments in this proposal seek to understand how kisspeptin function is regulated at the transcriptional level using three complementary approaches. 1) An in vivo mouse reporter system will be used to identify cis-acting DMA elements that regulate Kissl expression. DNA constructs containing stretches of the Kissl genomic region fused to GFP will be injected into the hypothalami of live mice. This will allow the identification of specific DNA elements required for Kissl expression. 2) Laser microdissection and gene array analysis will be used to identify trans-acting factors that regulate Kissl expression. Expression of GFP in Kissl-expressing neurons will allow them to be isolated using laser microdissection. RNA will be prepared from neurons that express or do not express Kissl and used for gene array hybridization to create gene expression profiles. Genes expressed in Kissl-expressing neurons but not in non-Kiss1-expressing neurons are likely to be involved in Kissl expression. 3) Human patients with idiopathic hypogonadotropic hypogonadism (IHH) will be screened for mutations in KISS1 regulatory regions. Once key regulatory regions of Kissl are identified in mouse, the analogous regions in human KISS1 region will be screened in our collection of DNA from over 500 patients with IHH. If a likely mutation is identified, the mouse reporter system will be used to test the effect of this mutation on Kissl expression. In this way, identification of human mutations can contribute to the analysis of the mouse Kissl promoter. By using these complementary approaches, I hope to discover how Kissl expression is regulated at the pubertal transition and thereby gain insight into the mechanisms that determine the timing of puberty. These experiments will further our understanding of the precise mechanisms that govern puberty. In addition to providing insight into a fundamental biological process, this work may lead to genetic diagnostic tests for individuals with reproductive disorders. Modulation of the reproductive endocrine axis has important applications for the treatment of precocious and delayed puberty, and also for contraception, infertility, endpmetriosis, and the treatment of sex-hormone dependent tumors such as breast cancer and prostate cancer.

描述（由申请人提供）：青春期性成熟的神经内分泌和遗传控制仍然是人类生物学的最大奥秘之一。近年来，研究发现信号分子kisspeptin及其受体GPR54在调节生殖内分泌轴中发挥重要作用。在这个提议中的实验试图了解kisspeptin功能是如何在转录水平上使用三种互补的方法进行调节。1)将使用体内小鼠报告系统来鉴定调节Kissl表达的顺式作用DMA元件。将含有与GFP融合的Kissl基因组区域的延伸的DNA构建体注射到活小鼠的下丘脑中。这将允许鉴定Kissl表达所需的特异性DNA元件。2)激光显微切割和基因阵列分析将用于确定调节Kissl表达的反式作用因子。GFP在表达Kissl的神经元中的表达将允许使用激光显微切割分离它们。将从表达或不表达Kissl的神经元制备RNA，并用于基因阵列杂交以创建基因表达谱。在Kissl表达神经元中表达而在非Kissl表达神经元中不表达的基因可能参与Kissl表达。3)将对患有特发性低促性腺激素性性腺功能减退症（IHH）的人类患者进行KISS 1调控区突变筛查。一旦在小鼠中鉴定出Kissl的关键调控区域，将在我们收集的来自500多名IHH患者的DNA中筛选人Kissl区域中的类似区域。如果识别出可能的突变，则将使用小鼠报告系统来测试该突变对Kissl表达的影响。以这种方式，人突变的鉴定可以有助于小鼠Kissl启动子的分析。通过使用这些互补的方法，我希望发现Kissl表达是如何在青春期过渡期进行调节，从而深入了解决定青春期时间的机制。这些实验将进一步加深我们对支配青春期的精确机制的理解。除了提供对基本生物学过程的深入了解外，这项工作还可能导致对患有生殖障碍的个体进行遗传诊断测试。生殖内分泌轴的调节对于治疗性早熟和青春期延迟，以及避孕、不孕、子宫内膜异位症以及治疗性激素依赖性肿瘤（例如乳腺癌和前列腺癌）具有重要应用。