Abstract
Forkhead box L2 (Foxl2), a member of the forkhead transcription factor family, plays important roles in pituitary follicle-stimulating hormone synthesis and in ovarian maintenance and function. Mutations in the human FOXL2 gene cause eyelid malformations and premature ovarian failure. FOXL2/Foxl2 is expressed in pituitary gonadotrope and thyrotrope cells, the perioptic mesenchyme of the developing eyelid, and ovarian granulosa cells. The mechanisms governing this cell-restricted expression have not been described. We mapped the Foxl2 transcriptional start site in immortalized murine gonadotrope-like cells, LβT2, by 5’ rapid amplification of cDNA ends and then PCR amplified approximately 1 kb of 5’ flanking sequence from murine genomic DNA. When ligated into a reporter plasmid, the proximal promoter conferred luciferase activity in both homologous (LβT2) and, unexpectedly, heterologous (NIH3T3) cells. In silico analyses identified a CpG island in the proximal promoter and 5’ untranslated region, suggesting that Foxl2 transcription might be regulated epigenetically. Indeed, pyrosequencing and quantitative analysis of DNA methylation using real-time PCR revealed Foxl2 proximal promoter hypomethylation in homologous compared to some, though not all, heterologous cell lines. The promoter was also hypomethylated in purified murine gonadotropes. In vitro promoter methylation completely silenced reporter activity in heterologous and homologous cells. Collectively, the data suggest that differential proximal promoter DNA methylation may contribute to cell-specific Foxl2 expression in some cellular contexts. However, gonadotrope-specific expression of the gene cannot be explained by promoter hypomethylation alone.
Highlights
Forkhead transcription factors regulate diverse biological processes including embryogenesis, cellular differentiation, cell cycle control, and immune function [1,2]
As the caprine promoter was reportedly induced by FOXL2 and oxidative stress [29,31], we examined the effects of FOXL2 overexpression and knockdown as well as H2O2 exposure on murine forkhead box L2 (Foxl2) reporter activity
For the qAMP analyses in these additional cell lines, we focused on the proximal promoter and 5’ untranslated region (UTR) (-81/+43), assuming that the data for this region would reflect the pattern of methylation more distally (-523/-350), as we saw in LβT2 and NIH3T3 cells (Figure 3A)
Summary
Forkhead transcription factors regulate diverse biological processes including embryogenesis, cellular differentiation, cell cycle control, and immune function [1,2]. Global or gonadotropespecific ablation of Foxl causes impaired pituitary folliclestimulating hormone (FSH) β subunit transcription and FSH synthesis [22,23]. These phenotypes are consistent with Foxl2’s restricted pattern of expression in the perioptic mesenchyme of the developing eyelid, ovarian granulosa cells, and pituitary gonadotrope and thyrotrope cells [3,24]. The mechanisms controlling this cell-specific expression have not been described
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