Identification of target genes for Lhx3 in gonadotropes

描述（由申请人提供）：本R21基金申请旨在通过探索性研究了解调节垂体前叶促性腺细胞发育和功能的基因表达网络。特别是，这些研究试图确定由LIM同源结构域转录因子LHX3调节的靶基因。研究发现，小鼠或人类体内的LHX3基因突变会使促性腺激素、促黄体生成素（LH）和促卵泡激素（FSH）的产生和分泌急剧减少。发育研究表明，激素分泌的减少是由于产生黄体生成素和卵泡刺激素的适当分化细胞的减少。有证据表明LHX3在调节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中起作用。因此，LHX3在促性腺激素的发育和成熟分化的促性腺激素的功能中都起着至关重要的作用。从LHX3的发育作用来看，LHX3可能结合并调节促性腺激素中许多重要靶基因的活性。本研究旨在利用新开发的大规模平行DNA测序技术鉴定促性腺激素中LHX3调控的靶基因。具体目的包括：1)利用染色质免疫沉淀和大规模平行、高通量DNA测序技术鉴定LHX3在L T2和T3-1促性腺激素细胞系中的体内结合位点。2)通过鉴定LHX3 shRNA敲低后特异性减少的mrna，鉴定受LHX3功能调控的转录本。mRNA种群的变化将通过大规模平行、高通量的RNA测序来评估。将体内有LHX3结合位点的基因列表与受LHX3调控的基因列表进行比较，应该找出受LHX3直接结合功能调控的靶基因。我们相信，通过提供有关促性腺激素发育和功能重要的基因调控网络的新信息，该结果具有潜在的高影响。

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.