Chicken ovalbumin upstream promoter transcription factor type II interacts and functionally cooperates with GATA4 to regulate anti‐Müllerian hormone receptor type 2 transcription in mouse MA‐10 Leydig cells

Abstract

Background: Leydig cells produce testosterone and insulin-like 3, two hormones essential for male sex differentiation and reproductive function. The orphan nuclear receptor, chicken ovalbumin upstream promoter transcription factor type II (COUP-TFII), and the zinc finger factor GATA4 are two transcription factors involved in Leydig cell differentiation, gene expression, and function. Objectives: Several Leydig cell gene promoters contain binding motifs for both GATA factors and nuclear receptors. The goal of the present study is to determine whether GATA4 and COUP-TFII cooperate to regulate gene expression in Leydig cells. Materials and methods: The transcriptomes from GATA4-and COUP-TFII-depleted MA-10 Leydig cells were analyzed using bioinformatic tools. Functional cooperation between GATA4 and COUP-TFII, and other related family members, was assessed by transient transfections in Leydig (MA-10 and MLTC-1) and fibroblast (CV-1) cell lines on several gene promoters. Recruitment of GATA4 and COUP-TFII to gene promoters was investigated by chromatin immunoprecipitation. Co-immunoprecipitation was used to determine whether GATA4 and COUP-TFII interact in MA-10 Leydig cells.Results: Transcriptomic analyses of GATA4-and COUP-TFII-depleted MA-10 Leydig cells revealed 44 commonly regulated genes including the anti-Müllerian hormone receptor type (Amhr2) gene. GATA4 and COUP-TFII independently activated the Amhr2 promoter, and their combination led to a stronger activation. A GC-rich element, located in the proximal Amhr2 promoter was found to be essential for GATA4-and COUP-TFII-dependent activation as well as for the COUP-TFII/GATA4 cooperation.COUP-TFII and GATA4 directly interacted in MA-10 Leydig cell extracts. Chromatin immunoprecipitation revealed that GATA4 and COUP-TFII are recruited to the proximal Amhr2 promoter, which contains binding sites for both factors in addition to the GC-rich element. Cooperation between COUP-TFII and GATA6, but not GATA1 and GATA3, was also observed.

NR2F2 is required in the embryonic testis for Fetal Leydig Cell development

“…NR2F2 regulates the transition from the adult progenitor Leydig cell (characterized by their elongated shape, their ability to proliferate and a low level of testosterone synthesis) to the immature ALC (characterized by their round shape, low mitotic activity and increased testosterone production) (50,63). In agreement with its in vivo role promoting maturation along the ALC lineage, NR2F2 co-operates with NR5A1 and GATA4 to activate the transcription of Insl3, Star (encoding the cholesterol transporter) and Amhr2 (46,(67)(68)(69)(70) in MA-10 cells, an in vitro model for immature ALC. Here we found that the steroidogenic cells that differentiate in Nr2f2 mutant fetal testes exhibit a small size, an elongated shape and reduced steroidogenic gene expression, features of the earliest stages of FLC differentiation (19,48,63).…”