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.垂体腺的功能受多种转录因子（transcription factors）的活性调控，这些转录因子控制细胞命运决定（cell fate decisions），进而调控细胞分化（cellular differentiation）与激素生成（hormone production）。叉头框蛋白O1（FOXO1）对于生长激素细胞（somatotrope）分化的正常时序及生长激素细胞功能至关重要，但其确切的作用机制尚未阐明。近期研究数据表明，FOXO1可调控与生长激素细胞分化相关的关键基因，包括Gh1、Neurod4及Pou1f1。此前有研究显示，在发育阶段的垂体腺中条件性敲除（conditional deletion）Foxo1的小鼠模型，早在胚胎第18.5天就表现出Gh1与Neurod4转录本（transcript）水平的降低。另有针对成年动物的研究数据表明，在垂体腺中同时条件性敲除Foxo1与Foxo3，可导致Neurod4、Gh1以及Pou1f1的转录水平出现类似下降。为探究FOXO1调控垂体腺基因表达的分子机制并验证体内实验结果，研究人员以不同浓度的FOXO1抑制剂AS1842856处理生长激素细胞样细胞系GH3，处理时长为24小时。在最低浓度为30 nM的抑制剂处理下，Neurod4的转录本水平便呈现出剂量依赖性（dose-dependent）的显著降低，是受影响最严重的基因。Gh1的转录本水平在300 nM浓度处理时出现显著下调；Pou1f1的表达则在3 μM抑制剂处理下呈现下降趋势（p=0.066）。与上述结果一致，在GH3细胞中通过CRISPR/Cas9介导的Foxo1敲除，可显著降低Gh1与Neurod4的表达，但对Pou1f1的表达无明显影响。为阐明FOXO1在生长激素细胞中发挥作用的分子机制，研究人员对GH3细胞系中的FOXO1进行了染色质免疫共沉淀测序（ChIP-seq）。本研究鉴定出了与Neurod4、Gh1及Pou1f1基因相关的新型FOXO1结合位点。Neurod4基因区域的FOXO1结合位点在生长激素细胞样细胞中表现出增强子活性（enhancer activity），但在促性腺激素细胞（gonadotrope）样细胞或异源细胞（heterologous cells）中未观察到此活性。上述数据有力表明，FOXO1可直接参与调控与生长激素细胞分化相关的关键基因的转录过程。这些全新的研究发现，为亟需的垂体细胞命运决定机制研究提供了重要支撑。