MON-483 FOXO1 Contributes to Somatotrope Differentiation through Regulation of Key Transcription Factors

Abstract

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. In order to investigate the mechanism by which FOXO1 regulates pituitary gland gene expression and confirm in vivo findings, the somatotrope-like cell line MtT/S was treated with FOXO1 inhibitor, AS184256, for 24 hours at various concentrations. Neurod4 and Pou1f1 were the most severely affected genes with a dose-dependent reduction in transcript at inhibitor concentrations as low as 30 nM (p < 0.05). Gh1 transcripts were significantly reduced at 3 μM (p < 0.01). To ascertain whether FOXO1 binds to regulatory regions of key somatotrope genes, ChIP-seq was performed using the MtT/S cell line and anti-FOXO1 antibody. This study identified FOXO1 interaction with genomic regions associated with Neurod4, Pou1f1, and Gh1. Interestingly, the FOXO1 binding intervals associated with Neurod4 and Pou1f1 are highly conserved between rat and human, indicating these sequences are important for survival. These data strongly suggest FOXO1 directly contributes to the transcriptional control of genes important for somatotrope differentiation. Investigation is also underway to identify proteins within the FOXO1 transcriptional complex. Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) was performed using the MtT/S cell line for this purpose. Proteins important for signal transduction and transcriptional regulation, including PKC, LRPPRC, and YBX1, were enriched in chromatin containing FOXO1, suggesting that they contribute to transcriptional control of FOXO1 targets. Overall, the emerging mechanism for FOXO1 control of somatotrope differentiation likely involves direct regulation of Neurod4, Pou1f1, and Gh1. These novel findings contribute to the much-needed understanding of pituitary cell fate decisions.