Genome-wide map of FOXO1-bound chromatin in pituitary somatotropes
Pituitary gland function is regulated by the activity of various transcription factors which control cell fate decisions leading to cellular differentiation and hormone production. FOXO1 is necessary for the proper timing of somatotrope differentiation and for somatotrope function, but the exact mechanism of action has yet to be elucidated. Recent data implicate FOXO1 in the regulation of genes important for somatotrope differentiation including Gh1, Neurod4, and Pou1f1. Previously, a mouse model with conditional deletion of Foxo1 from the developing pituitary gland displayed reduced Gh1 and Neurod4 transcripts as early as embryonic day 18.5. Additional data from adult animals with conditional deletion of both Foxo1 and Foxo3 from the pituitary gland have a similar reduction in Neurod4 and Gh1, as well as Pou1f1. To investigate the mechanism by which FOXO1 regulates pituitary gland gene expression and confirm in vivo findings, the somatotrope-like cell line, GH3, was treated with the FOXO1 inhibitor, AS1842856, for 24 hours at various concentrations. Neurod4 was the most severely affected genes with a dose-dependent reduction in transcript at inhibitor concentrations as low as 30 nM. Gh1 transcripts were significantly reduced at 300 nM. Pou1f1 expression was trending down at 3 microM inhibitor (p=0.066). Consistent with these findings, CRISPR/Cas9-mediated deletion of Foxo1 in GH3 cells significantly reduced expression of Gh1, Neurod4, but not Pou1f1. To elucidate the molecular mechanisms underlying the role of FOXO1 in somatotropes, ChIPseq was performed for FOXO1 in the GH3 cell line. This study identified novel FOXO1 binding sites associated with the Neurod4, Gh1, and Pou1f1 genes. The FOXO1 binding site in the Neurod4 gene exhibits enhancer activity in somatotrope-like cells, but not in gonadotrope-like or heterologous cells. These data strongly suggest FOXO1 directly contributes to the transcriptional control of genes important for somatotrope differentiation. These novel findings contribute to the much-needed understanding of pituitary cell fate decisions. Overall design: ChIPseq was performed using GH3 cells and anti-FOXO1 (FKHR H-128 sc-11350, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at Active Motif (Carlsbad, CA, USA). Three separate passages were pooled. Active Motif performed the ChIPseq and generated approximately 33 x 107 reads. These reads were aligned to rat genome rn6 and compared to input control. 1 sample with FOXO1 Antibody and 1 input sample were sequenced