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 i nclude 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.
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.