Identification of pituitary thyrotrope signature genes and regulatory elements

Abstract

Pituitary thyrotropes are specialized cells that produce thyroid stimulating hormone (TSH), a critical factor for growth and maintenance of metabolism. The transcription factors POU1F1 and GATA2 have been implicated in thyrotrope fate and transcriptional regulation of the beta subunit of TSH, Tshb, but no transcriptomic or epigenomic analyses of these cells has been undertaken. The goal of this work was to discover key transcriptional regulatory elements that drive thyrotrope fate. We identified the transcription factors and epigenomic changes in chromatin that are associated with differentiation of POU1F1-expressing progenitors into thyrotropes, a process modeled by two cell lines: one that represents an early, undifferentiated Pou1f1 lineage progenitor (GHF-T1) and one that is a committed thyrotrope that produces TSH (TT1). We generated and compared RNA-seq, ATAC-seq, histone modification (including H3K27Ac, H3K4Me1, and H3K27Me3), and transcription factor (POU1F1) binding in these two cell lines to identify regulatory elements and candidate transcriptional regulators. We identified POU1F1 binding sites that were unique to each cell line. POU1F1 binding sites are commonly associated with bZIP transcription factor consensus binding sites in GHF-T1 cells and Helix-Turn-Helix (HTH) or basic Helix-Loop-Helix (bHLH) factors in TT1 cells, suggesting that these classes of transcription factors may recruit or cooperate with POU1F1 binding to unique sites. We validated enhancer function of novel elements we mapped near Cga, Pitx1, Gata2, and Tshb by transfection in TT1 cells. Finally, we confirmed that an enhancer element near Tshb can drive expression in thyrotropes of transgenic mice, and we demonstrate that GATA2 enhances Tshb expression through this element. These results extend the ENCODE multi-omic profiling approach to an organ that is critical for growth and metabolism, which should be valuable for understanding pituitary development and disease pathogenesis.