Identification of target genes for Lhx3 in gonadotropes

描述(申请人提供)：R21资金申请寻求使用探索性研究，以了解调节垂体前叶促性腺细胞的发育和功能的基因表达网络。特别是，这些研究试图确定受LIM同源结构域转录因子LHX3调控的目标基因。在小鼠或人类中，发现使LHX3失活的突变会显著减少促性腺激素、黄体生成素(LH)和卵泡刺激素(FSH)的产生和分泌。发育研究表明，激素分泌的丧失是由于产生黄体生成素和卵泡刺激素的适当分化细胞的丧失。有证据表明，LHX3对编码糖蛋白激素亚基、FSH亚基和促性腺激素释放激素受体的基因具有调节作用。因此，LHX3在促性腺激素的发育和成熟的、分化的促性腺激素的功能中都起着至关重要的作用。鉴于LHX3的发育作用，LHX3很可能与促性腺激素中一些重要的靶基因结合并调节其活性。拟议的研究试图使用新开发的大规模并行DNA测序来确定促性腺激素中受LHX3调控的靶基因。1)利用染色质免疫沉淀和大规模并行高通量DNA测序，在L T2和T3-1促性腺激素细胞系中寻找LHX3的体内结合位点。2)通过鉴定shRNA敲除LHX3后减少的特定mRNAs，确定受LHX3功能调控的转录本。信使核糖核酸种群的变化将通过大规模并行、高通量的RNA测序来评估。将LHX3与体内结合位点的基因列表与受LHX3调控的基因列表进行比较，可以确定受LHX3直接结合调控的靶基因。我们相信，通过提供有关对促性腺激素的发育和功能至关重要的基因调控网络的新信息，该结果有可能产生巨大的影响。 公共卫生相关性：拟议的研究应该与非常重要的一般性问题相关，即了解基因组中的信息如何用于确定身体的所有不同细胞类型。这些发现还应该对了解脑垂体发育和基因表达具有重要意义，并增加对突变如何导致促性腺激素产生缺陷和人类疾病的理解。

DESCRIPTION (provided by applicant): This request for R21 funding seeks to use exploratory studies to understand the gene expression networks that regulate the development and function of the gonadotrope cells of the anterior pituitary. In particular, the studies seek to identify target genes that are regulated by the LIM homeodomain transcription factor, LHX3. Mutations in mice or humans that inactivate LHX3 have been found to drastically decrease production and secretion of the gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH). Developmental studies have shown that loss of hormone secretion is due to loss of the appropriate, differentiated cells that produce LH and FSH. There is evidence that LHX3 plays a role in regulating the genes encoding the glycoprotein hormone -subunit, the FSH -subunit, and the gonadotropin releasing hormone receptor. Thus LHX3 plays an essential role in both the development of gonadotropes and the function of mature, differentiated gonadotropes. In view of the development role of LHX3, it seems likely that LHX3 binds to and regulates the activity of a number of important target genes in gonadotropes. The proposed studies seek to use newly developed massively parallel DNA sequencing to identify target genes regulated by LHX3 in gonadotropes. The specific aims include 1) Use chromatin immunoprecipitation and massively parallel, high throughput DNA sequencing to identify in vivo binding sites for LHX3 in the L T2 and T3-1 gonadotrope cell line. 2) Identify transcripts that are functionally regulated by LHX3 by identifying specific mRNAs that are decreased after shRNA knockdown of LHX3. Changes in mRNA populations will be assessed by massively parallel, high throughput sequencing of RNA. Comparing the list of genes with in vivo binding sites for LHX3 with the list of genes regulated by LHX3 should identify target genes that are functionally regulated by direct binding of LHX3. We believe that the results have the potential for high impact by providing new information about the gene regulatory network important for development and function of gonadotropes. PUBLIC HEALTH RELEVANCE: The proposed studies should be relevant to the very important general issue of understanding how the information in the genome is used to specify all of the different cell types of the body. The findings should also have significance for the understanding of pituitary development and gene expression and increase understanding of how mutations lead to deficiencies in gonadotropin production and human disease.