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

Area of Science:

• Endocrinology
• Molecular Biology
• Genetics

Background:

• Activin-class ligands stimulate follicle-stimulating hormone (FSH) production in pituitary gonadotrope cells.
• Transcription factors SMAD3, SMAD4, and FOXL2 are known to regulate the FSHβ subunit (Fshb) promoter.
• Previous identification of an Fshb enhancer in vitro did not confirm its in vivo function.

Purpose of the Study:

• To identify and characterize novel regulatory elements controlling murine Fshb transcription.
• To investigate the role of activin signaling in regulating chromatin accessibility and enhancer activity at the Fshb locus.
• To elucidate the specific transcription factors involved in activin-mediated Fshb regulation.

Main Methods:

• Single-nucleus ATAC-seq was employed to map chromatin accessibility in murine pituitary gonadotropes.
• CRISPR-Cas9 was used to delete a previously identified enhancer region.
• Reporter assays in LβT2 cells were performed to assess enhancer activity and transcription factor binding (SMAD2/3, SMAD4, FOXL2) using gel shift assays and site-directed mutagenesis.

Main Results:

• Three novel open chromatin regions upstream of Fshb were identified exclusively in gonadotropes.
• These regions, along with the Fshb gene, showed reduced accessibility in FSH-deficient mice lacking functional activin type II receptors.
• Two distal enhancers significantly enhanced basal and activin A-stimulated Fshb promoter activity, exhibiting H3K27ac enrichment and binding by SMAD2/3, SMAD4, and FOXL2.

Conclusions:

• Multiple activin-sensitive enhancers, in addition to the previously identified one, regulate murine Fshb transcription.
• Activin signaling modulates chromatin accessibility and enhancer activity at the Fshb locus via transcription factors SMAD2/3, SMAD4, and FOXL2.
• These findings reveal a complex regulatory network controlling FSH production in the pituitary.