Identification of target genes for Lhx3 in gonadotropes

描述（由申请人提供）：这项R21资金申请旨在利用探索性研究来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LHX 3调节的靶基因。已经发现小鼠或人类中的LHX 3突变会显著降低促性腺激素、促黄体激素（LH）和促卵泡激素（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LH和FSH的适当的分化细胞的丧失。有证据表明LHX 3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，LHX 3在促性腺激素的发育和成熟、分化的促性腺激素的功能中起重要作用。鉴于LHX 3的发育作用，LHX 3似乎可能与促性腺激素中的许多重要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测序来识别促性腺激素中受LHX 3调节的靶基因。具体目的包括1）使用染色质免疫沉淀和大规模平行、高通量DNA测序来鉴定LHX 3在L T2和T3-1促性腺细胞系中的体内结合位点。2)通过鉴定LHX 3的shRNA敲低后减少的特定mRNA，鉴定LHX 3功能调节的转录物。将通过大规模平行、高通量RNA测序评估mRNA群体的变化。将具有LHX 3的体内结合位点的基因列表与由LHX 3调节的基因列表进行比较，应当鉴定出在功能上由LHX 3的直接结合调节的靶基因。我们认为，这些结果有可能通过提供有关促性腺激素发育和功能的基因调控网络的新信息产生重大影响。 公共卫生相关性：拟议的研究应该与理解基因组中的信息如何用于指定身体的所有不同细胞类型这一非常重要的一般性问题有关。这些发现对于理解垂体发育和基因表达也具有重要意义，并增加了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.