Regulation of follicle-stimulating hormone β subunit transcription by newly discovered enhancers

Yangfan Jin¹, Carlos Agustin Isidro Alonso¹, Luisina Ongaro Gambino¹, Gauthier Schang¹, Xiang Zhou¹, Michel Zamojski², German Nudelman², Natalia Mendelev², Corrine K. Welt³, Louise M. Bilezikjian⁴, Stuart C. Sealfon², Frederique Ruf-Zamojski^2,5, Daniel J. Bernard¹

1. Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec

2. Neurology Department, Icahn School of Medicine at Mount Sinai, New York, NY

3. Division of Endocrinology and Metabolism, University of Utah, Salt Lake City, UT

4. Salk Institute for Biological Studies, La Jolla, CA

5. Department of Medicine, Cedars-Sinai, Los Angeles, CA

Abstract Text:

Follicle-stimulating hormone (FSH) is an essential regulator of gonadal function. FSH is a dimeric glycoprotein produced by pituitary gonadotrope cells. Expression of the FSHβ subunit (Fshb) is rate-limiting in the production of the mature hormone. TGFβ ligands in the activin class, including myostatin, stimulate Fshb transcription via complexes of SMADs and FOXL2, which bind to the Fshb promoter. Single nucleus ATAC-seq analysis of wild-type murine pituitary demonstrated the presence of four regions of co-accessibility upstream of the Fshb gene exclusively in gonadotropes. We refer to these as enhancers (Enh) 1-4, with Enh 1 most proximal to the gene. In gonadotrope-specific activin type II receptor knockout mice, which are FSH-deficient, the Fshb gene and three out of four enhancers are compacted (closed) in these animals. Enh 3 remains partly accessible. Enh 2 was recently described as an enhancer that increased activin A-stimulated murine Fshb promoter-reporter activity in gonadotrope-like LβT2 cells. In our hands, however, Enh 2 does not alter murine Fshb promoter activity in response to activin A. Moreover, we observe normal FSH synthesis and secretion in mice in which Enh 2 was knocked out using CRISPR-Cas9. To decrease enhancer redundancy, we knocked out Enh 2, 3 and 4 together, and these mice have decreased FSH production. The knockout females have reduced ovarian weights and ovulate fewer eggs in natural cycles than wild-type controls. Furthermore, Enh 3 and 4 markedly increase basal and activin A-stimulated murine Fshb promoter-reporter activity in LβT2 cells. Enh 3 and 4 are accessible, enriched for the enhancer mark H3K27Ac, and bind SMAD3 and FOXL2 in these cells. SMAD3/4 binding to Enh 3 and FOXL2 binding to Enh 4 are required for full enhancer activity in reporter assays in vitro. Collectively, the data indicate that there may be four upstream enhancers that regulate murine Fshb transcription in gonadotropes. At least two of these enhancers bind SMADs and FOXL2, which may facilitate their association with the Fshb promoter to regulate transcription.