The nuclear receptors SF1 and COUP-TFII cooperate on the Insl3 promoter in Leydig cells

Abstract

Insulin-like 3 (INSL3), a hormone abundantly produced by Leydig cells, is essential for testis descent during foetal life and bone metabolism in adults. The mechanisms regulating Insl3 expression in Leydig cells have been studied in several species but remain poorly understood. To date, only a handful of transcription factors are known to activate the Insl3 promoter and include the nuclear receptors AR, NUR77, COUP-TFII, and SF1, as well as the Krüppel-like factor KLF6. Some of these transcription factors are known to transcriptionally cooperate on the Insl3 promoter but the mechanisms at play remain unknown. Here we report that COUP-TFII and SF1 functionally cooperate on the Insl3 promoter from various species, but not on the Inha, Akr1c14, Cyp17a1, Hsd3b1, Star, Gsta3, and Amhr2 promoters that are known to be regulated by COUP-TFII and/or SF1. The Insl3 promoter contains species-conserved binding sites for COUP-TFII (−91 bp) and SF1 (−134 bp). Mutation of either the COUP-TFII or the SF1 sequence had no impact on the COUP-TFII/SF1 cooperation but mutation of both binding sites abolished the cooperation. In agreement with this, we found that COUP-TFII and SF1 physically interact in Leydig cells. Finally, we report that the transcriptional cooperation is not limited to COUP-TFII and SF1 as it also occurred between all NR2F and NR5A family members. Our data provide new mechanistic insights into the cooperation between the orphan nuclear receptors COUP-TFII and SF1 in the regulation of Insl3 gene expression in Leydig cells.

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

“…Expression plasmids for mouse COUP‐TFI, COUP‐TFII, GATA1, GATA3, and rat GATA4 and GATA6 have been previously described 4,8,24,45 . To measure promoter activation, MA‐10, MLTC‐1, and CV‐1 cells were transiently transfected using polyethylenimine hydrochloride (Sigma‐Aldrich Canada, Ontario, Canada) as previously described 8,42 or using the calcium phosphate co‐precipitation method as described in 46–48 . Briefly, the cells were plated in 24‐well plates and co‐transfected with 400 ng of reporter vector along with 100 ng of expression vectors (empty expression vector pcDNA3.1 as control, COUP‐TFs, GATAs, or COUP‐TFs+GATAs).…”

“…After eluate collection, the remaining beads were boiled in 20 μl of 1x Laemmli buffer at 95°C for 10 min. The immunoprecipitated samples were separated by SDS‐PAGE and analyzed by Western blot using a monoclonal antibody against human COUP‐TFII made in the mouse (R&D Systems Cat# PP‐H7147‐00, ; dilution 1:1000) as described previously 4,6,8,9,48 . To detect GATA4, the same membrane was stripped with 0.2 N NaOH and re‐probed with a monoclonal antibody against human GATA4 made in the mouse (Santa Cruz Biotechnology Cat# sc‐25310, ; dilution 1:50).…”

“…4,8,24,45 To measure promoter activation, MA-10, MLTC-1, and CV-1 cells were transiently transfected using polyethylenimine hydrochloride (Sigma-Aldrich Canada, Ontario, Canada) as previously described 8,42 or using the calcium phosphate co-precipitation method as described in. [46][47][48] Briefly, the cells were plated in 24-well plates and co-transfected with 400 ng of reporter vector along with 100 ng of expression vectors (empty expression vector pcDNA3.1 as control, COUP-TFs, GATAs, or COUP-TFs+GATAs). For the calcium phosphate co-precipitation method, 1 μg of SP64 plasmid was also added as a carrier.…”

“…49 dilution 1:1000) as described previously. 4,6,8,9,48 To detect GATA4, the same membrane was stripped with 0.2 N NaOH and re-probed with a monoclonal antibody against human GATA4 made in the mouse (Santa Cruz Biotechnology Cat# sc-25310, RRID:AB_627667; dilution 1:50).…”

A 35-bp Conserved Region Is Crucial for Insl3 Promoter Activity in Mouse MA-10 Leydig Cells

“…These include the Krüppel-like factor KLF6 [ 18 ] as well as the nuclear receptors SF1 (Ad4BP, NR5A1) [ 13 , 17 , 19 , 20 ], LRH1 (NR5A2) [ 21 ], NUR77 (NGFI-B, NR4A1) [ 21 , 22 ], testosterone-activated androgen receptor (AR, NR3C4) [ 23 , 24 ], COUP-TFII (NR2F2) [ 25 ], and DAX1 (NR0B1) [ 19 ]. Transcriptional cooperation between COUP-TFII and SF1 [ 25 , 26 ], and between KLF6 and NUR77 and SF1 [ 18 ] on the Insl3 promoter has also been reported. However, most of these transcription factors are also found in cell types that do not express the Insl3 gene, such as Sertoli and adrenal cells, indicating that additional transcription factors are likely involved in directing Insl3 expression in Leydig cells.…”