Identification of a pituitary ER\u03b1-activated enhancer triggering the expression of Nr5a1, the earliest gonadotrope lineage-specific transcription factor

Vincent Pacini,David L’Hôte,Bruno Querat,Florence Petit,Joëlle Cohen-Tannoudji,Jean-Noël Laverriere

doi:10.1186/s13072-019-0291-8

Abstract

BackgroundGonadotrope lineage differentiation is a stepwise process taking place during pituitary development. The early step of gonadotrope lineage specification is characterized by the expression of the Nr5a1 transcription factor, a crucial factor for gonadotrope cell fate determination. Abnormalities affecting Nr5a1 expression lead to hypogonadotropic hypogonadism and infertility. Although significant knowledge has been gained on the signaling and transcriptional events controlling gonadotrope differentiation, epigenetic mechanisms regulating Nr5a1 expression during early gonadotrope lineage specification are still poorly understood.ResultsUsing ATAC chromatin accessibility analyses on three cell lines recapitulating gradual stages of gonadotrope differentiation and in vivo on developing pituitaries, we demonstrate that a yet undescribed enhancer is transiently recruited during gonadotrope specification. Using CRISPR/Cas9, we show that this enhancer is mandatory for the emergence of Nr5a1 during gonadotrope specification. Furthermore, we identify a highly conserved estrogen-binding element and demonstrate that the enhancer activation is dependent upon estrogen acting through ERα. Lastly, we provide evidence that binding of ERα is crucial for chromatin remodeling of Nr5a1 enhancer and promoter, leading to RNA polymerase recruitment and transcription.ConclusionThis study identifies the earliest regulatory sequence involved in gonadotrope lineage specification and highlights the key epigenetic role played by ERα in this differentiation process.

Highlights

Gonadotrope lineage differentiation is a stepwise process taking place during pituitary development
In this work, using functional genomic in vitro and in vivo approaches, we demonstrated that Nr5a1 expression is triggered by another early activated enhancer at the emergence of gonadotrope lineage
Differential chromatin accessibility in Nr5a1 locus during gonadotrope specification We performed an assay for transposase-accessible chromatin with high-throughput sequencing analysis (ATACseq) in αT1–1, αT3–1 and LβT2 cell lines, allowing the identification of new potential cis-regulatory sequences (Fig. 1a and Additional file 1)

Summary

Introduction

Gonadotrope lineage differentiation is a stepwise process taking place during pituitary development. The early step of gonadotrope lineage specification is characterized by the expression of the Nr5a1 transcription fac‐ tor, a crucial factor for gonadotrope cell fate determination. Significant knowledge has been gained on the signaling and transcriptional events controlling gonadotrope differentiation, epigenetic mechanisms regulating Nr5a1 expression during early gonadotrope lineage specification are still poorly understood. The αT1–1 cells are likely derived from E12.5 common precursor cells from which originate thyrotrope and gonadotrope lineages These cells do not express Nr5a1 yet. The αT3–1 cells are likely derived from cells engaged in a gonadotrope cell fate at E13.5 and express some of the gonadotrope-specific genes including Nr5a1. We observed that Nr5a1 is already expressed in αT3–1 cells, the epigenetic marks on the gonadotrope enhancer indicate that it is repressed, suggesting that it does not regulate Nr5a1 expression at early steps of gonadotrope specification

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Discussion

Conclusion