Genetic Analysis of Pituitary Thyrotrope Development

Abstract

The pituitary gland produces polypeptide hormones that regulate many functions including growth, lactation, reproduction, metabolism, and the stress response. Pituitary thyrotrope cells produce the heterodimeric glycoprotein hormone thyrotropin, which is critical for stimulating thyroid gland development and production of thyroid hormone. Less is known about the drivers of thyrotrope cell fate than the other specialized cells in this organ. The transcription factor POU1F1 is critical for generation of thyrotropes, somatotropes and lactotropes, and GATA2 is critical for both thyrotropes and gonadotropes. Additional factors are likely involved in driving thyrotrope fate. SV40-immortalized cell lines have been invaluable for studying the regulation of pituitary hormone production. Here I use two established immortalized cell lines to identify epigenomic and gene expression changes that are associated with adoption of the thyrotrope fate. GHF-T1 cells represent a POU1F1-expressing progenitor which does not produce hormones, and TaT1 cells represent a thyrotrope-like line that expresses POU1F1, GATA2 and thyrotropin (TSH). I also developed a novel, genetically engineered mouse line that expresses SV40 in response to cre recombinase, and I used this line to develop novel pituitary cell lines. These cell lines can be used for transcriptome and epigenome studies to understand the development and function of the pituitary gland. I identified the transcription factors and epigenomic changes in chromatin that are associated with thyrotrope differentiation. I generated and integrated genome-wide information about DNA accessibility, histone modifications, POU1F1 binding and RNA expression data to identify regulatory elements and candidate transcriptional regulators. I identified POU1F1 binding sites that are unique to each cell line. POU1F1 binding sites are commonly associated with bZIP factor motifs in GHF-T1 cells and Helix-Turn-Helix or basic Helix-Loop-Helix motifs in TαT1 cells, suggesting classes of transcription factors that may recruit POU1F1 to unique sites. I validated enhancer function of novel elements we mapped near Cga, Pitx1, Gata2, and Tshb by transfection in TαT1 cells. Finally, I confirmed that an enhancer element near Tshb can drive expression in thyrotropes of transgenic mice and demonstrated that GATA2 enhances Tshb expression via this element. These data extend the ENCODE analysis to an organ that is critical for growth and metabolism. This information could be valuable for understanding pituitary development and disease pathogenesis. Targeted oncogenesis is the process of driving tumor formation by engineering transgenic mice that express an oncogene under the control of a cell-type specific promoter. Using CRISPR/Cas9 we inserted a cassette with coding sequences for SV40 T antigens and IRES-GFP into the Rosa26 locus, downstream from a stop sequence flanked by loxP sites: Rosa26 LSL-SV40-GFP . These mice were mated with previously established Prop1-cre and Tshb-cre transgenic lines. The majority of Rosa26 LSL-SV40-GFP/+ ; Prop1-cre and all Rosa26 LSL-SV40-GFP/+ ; Tshb-cre mice developed dwarfism and large tumors by 4 weeks. Prop1-cre-mediated activation of SV40 expression affected cell specification, reducing thyrotrope differentiation and increasing gonadotrope cell fate selection. GFP-positive cells from flow-sorted Rosa26 LSL-SV40GFP/+ LSL-SV40-GFP/+; Prop1-cre and Rosa26 ; Tshb-cre mice express PROP1 and TSH, respectively. Tumors from both of these mouse lines were adapted to growth in cell culture. I established a progenitor-like cell line (PIT-P1) that expresses Sox2 and Pitx1, and a thyrotrope-like cell line (PIT-T1) that expresses Cga and Pou1f1. These studies demonstrate the utility of the novel, Rosa26 LSL-SV40-GFP mouse line for targeted oncogenesis and development of cell lines.PHDGenetics and Genomics PhDUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/162910/1/azdaly_1.pd