Forkhead L2 (FOXL2) is a member of the forkhead/hepatocyte nuclear factor 3 (FKH/HNF3) gene family of transcription factors and acts as a transcriptional repressor of the Steroidogenic Acute Regulatory (StAR) gene, a marker of granulosa cell differentiation. FOXL2 may play a role in ovarian follicle maturation and prevent premature follicle depletion leading to premature ovarian failure. In this study, we found that FOXL2 interacts with Ubc9, an E2-conjugating enzyme that mediates sumoylation, a key mechanism in transcriptional regulation. FOXL2 and Ubc9 are co-expressed in granulosa cells of small and medium ovarian follicles. FOXL2 is sumoylated by Ubc9, and this Ubc9-mediated sumoylation is essential to transcription activity of FOXL2 on the StAR promoter. As FOXL2 is endogenous to granulosa cells, we generated a stable cell line expressing FOXL2 and found that activity of the StAR promoter in this cell line is greatly decreased in the presence of Ubc9. The sumoylation site was identified at lysine 25 of FOXL2. Mutation of lysine 25 to arginine leads to loss of transcriptional repressor activity of FOXL2. Taken together, we propose that Ubc9-mediated sumoylation at lysine 25 of FOXL2 is required for transcriptional repression of the StAR gene and may be responsible for controlling the development of ovarian follicles.

FOXL2 is expressed in granulosa cells of small and medium ovarian follicles, functions as a repressor of the human StAR gene, a marker of a granulosa cell differentiation, and its mutation is associated with premature ovarian failure in women with BPES type I. We now report that FOXL2 also represses the transcription of aromatase, P450scc, and cyclin D2, three other key genes involved in granulosa cell proliferation, differentiation, and steroidogenesis, and that a FOXL2 mutation found in BPES type I patients also fails to repress aromatase transcription, further supporting a role for FOXL2 in follicle maturation. KeywordsFOXL2; Premature ovarian failure; Granulosa cells; Aromatase; P450scc; Cyclin D2 (CCND2)The duration of a woman's reproductive life is determined before birth by a limited numbers of primordial follicles. Primordial follicles develop to primary, secondary (medium) and then to antral (large, mature) follicles. Most antral follicles go through atresia, however, under proper stimulation with gonadotropins during the reproductive period, a few survive and develop further to the preovulatory stage. Every ovulation cycle uses many primordial follicles and eventually the pool of resting primordial follicles is exhausted, resulting in menopause (1). Although the average age of menopause is 51 years of age, 1% of women are reported to undergo menopause before the age of 40 (2), known as premature ovarian failure (POF). Extensive research has identified genes involved in POF (3,4), which include the gene encoding FOXL2 (5,6). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Capsule:Human FOXL2 is a repressor of aromatase, P450scc, and cyclin D2 promoters. It may inhibit the expression of these genes in granulosa cells of small follicles. FOXL2 is a member of the winged helix/forkhead transcription family. FOX (forkhead box) family members are expressed in all eukaryotes and play important roles in the establishment of the body axis and in the development of embryonic tissue. Forkhead transcription factors bind to a common consensus DNA sequence, 5' [(G/A)(T/C)(C/A) A A (C/T) A] 3' in the promoters of target genes (7). FOXL2 is mainly expressed in the ovary and in the developing eyelid. In the mouse, FOXL2 expression starts at 12.5 dpc in female gonads, but not in developing male gonads, and is found in ovarian somatic cells in newborns (8). In the adult female, granulosa cells of early follicles express high levels of FOXL2 (8). Female FOXL2 −/− mice exhibit hypoplastic uterine tubes and ovaries and are infertile (8,9). In humans, mutations in the FOXL2 gen...

is expressed in the ovary and acts as a transcriptional repressor of the steroidogenic acute regulatory (StAR) gene, a marker of granulosa cell differentiation. Human FOXL2 mutations that produce truncated proteins lacking the COOH terminus result in blepharophimosis/ptosis/epicanthus inversus (BPES) syndrome type I, which is associated with premature ovarian failure (POF). In this study, we investigated whether FOXL2's activity as a transcriptional repressor is regulated by phosphorylation. We found that FOXL2 is phosphorylated at a serine residue and, using yeast two-hybrid screening, identified LATS1 as a potential FOXL2-interacting protein.LATS1 is a serine/threonine kinase whose deletion in mice results in an ovarian phenotype similar to POF. Using coimmunoprecipitation and kinase assays, we confirmed that LATS1 binds to FOXL2 and demonstrated that LATS1 phosphorylates FOXL2 at a serine residue. Moreover, we found that FOXL2 and LATS1 are coexpressed in developing mouse gonads and in granulosa cells of small and medium follicles in the mouse ovary. Last, we demonstrated that coexpression with LATS1 enhances FOXL2's activity as a repressor of the StAR promoter, and this results from the kinase activity of LATS1. These results provide novel evidence that FOXL2 is phosphorylated by LATS1 and that this phosphorylation enhances the transcriptional repression of the StAR gene, a marker of granulosa cell differentiation. These data support our hypothesis that phosphorylation of FOXL2 may be a control mechanism regulating the rate of granulosa cell differentiation and hence, follicle maturation, and its dysregulation may contribute to accelerated follicular development and POF in BPES type I. large tumor suppressor gene 1 phosphorylation; transcriptional regulation; forkhead L2; granulosa cell; premature ovarian failure PREMATURE OVARIAN FAILURE (POF) is defined as a condition causing amenorrhea, hypoestrogenism, and elevated gonadotropins in women under 40 yr of age (1) and can be associated with failure to endow the follicle pool or an early loss of the fixed follicle pool following excess follicle recruitment and/or atresia. A genetic basis for selective cases of POF has been determined. Patients with blepharophimosis-ptosis-epicanthus inversus (BPES) syndrome type I exhibit POF in association with characteristic eyelid dysplasia, blepharophimosis, ptosis, and epicanthus inversus (60). Ovaries from BPES type I patients are histologically variable, ranging from the presence of some primordial follicles with atretic follicles to complete absence of follicles and scarring of the ovaries (23). The gene encoding the transcription factor forkhead L2 (FOXL2) (15) maps to the BPES locus on chromosome 3q22-3q23. FOXL2 is a member of the forkhead/hepatocyte nuclear factor 3 family of transcription factors (15), which is characterized by the presence of a conserved winged helix domain that is essential for DNA binding as well as more divergent transactivation or transrepression domains (12,29,33). FOXL2 mutations in indivi...

Premature ovarian failure in the autosomal dominant disorder blepharophimosis-ptosis-epicanthus inversus is due to mutations in the gene encoding Forkhead L2 (FOXL2), producing putative truncated proteins. We previously demonstrated that FOXL2 is a transcriptional repressor of the steroidogenic acute regulatory (StAR), P450SCC (CYP11A), P450aromatase (CYP19), and cyclin D2 (CCND2) genes, markers of ovarian follicle proliferation and differentiation. Furthermore, we found that mutations of FOXL2 may regulate wild-type FOXL2, leading to loss of transcriptional repression of CYP19, similar to StAR. However, the regulatory mechanisms underlying these premature ovarian failure-associated mutations remain largely unknown. Therefore, we examined the effects of a FOXL2 mutant protein on the transcriptional repression of the CYP19 promoter by the full-length protein. We found that mutant FOXL2 exerts a dominant-negative effect on the repression of CYP19 by wild-type FOXL2. Both wild-type and mutant FOXL2 and can form homo- and heterodimers. We identified a minimal -57-bp human CYP19 promoter containing two potential FOXL2-binding regions and found that both wild-type and mutant FOXL2 can bind to either of these regions. Mutational analysis revealed that either site is sufficient for transcriptional repression by wild-type FOXL2, and the dominant-negative effect of mutant FOXL2, but these are eliminated when both sites are mutated. These findings confirm that mutant FOXL2 exerts a dominant-negative effect on wild-type FOXL2's activity as a transcriptional repressor of key genes in ovarian follicle differentiation and suggest that this is likely due to heterodimer formation and possibly also competition for DNA binding.

The forkhead transcription factor forkhead box L2 (FOXL2) is expressed in granulosa cells of small and medium follicles in the mouse ovary. Foxl2 female knockout mice exhibit primordial follicle depletion and primary ovarian failure, but evidence from adult female conditional Foxl2 knockout mice suggests that FOXL2 may also play a significant role in maintenance of ovarian differentiation at stages beyond the primordial follicle and initial wave of folliculogenesis. We previously showed that human FOXL2 functions as a transcriptional repressor of several key genes involved in granulosa cell proliferation and differentiation, including steroidogenic acute regulatory protein (STAR), P450aromatase (CYP19A1 (CYP19)), P450scc (CYP11A1 (CYP11A)), and cyclin D2 (CCND2). To elucidate the role of mouse FOXL2, we determined its role in transcriptional regulation in Chinese hamster ovary (CHO) cells and then confirmed our findings in mouse granulosa cells. We found that mouse FOXL2 represses the activities of the mouse Star, Cyp19a1, Cyp11a1 promoters in CHO cells, but may not repress the Ccnd2 promoter, and identified the minimal mouse Star, Cyp19a1, and Cyp11a1 promoter regions responsive to FOXL2 regulation. We then knocked down Foxl2 in mouse granulosa cells using siRNA, which resulted in significantly increased expression levels of mouse Star, Cyp19a1, and Cyp11a1 but not Ccnd2. To increase Foxl2 expression levels, we generated a mouse Foxl2 lentiviral construct and used it to infect mouse granulosa cells. Following lentiviral infection, the expression levels of mouse Star, Cyp19a1, and Cyp11a1, but not Ccnd2, decreased significantly. These data confirm that mouse FOXL2 functions as a transcriptional repressor of key granulosa cell genes that influence ovarian development.

Purpose To determine the expression of SMAD transcripts in human granulosa cells. Methods Luteinized mural granulosa cells were harvested from forty women undergoing oocyte retrieval, and RNAs were isolated. SMAD expression levels were determined by polymerase chain reaction (PCR) and quantitative real-time PCR (q-RTPCR). Results SMAD1-7 and 9 are expressed in human granulosa cells, with SMAD2, 3 and 4 showing the highest expression levels. Peak estradiol (E2) levels correlated with the number of oocytes retrieved during IVF. Oocyte number showed no correlation with SMAD expression levels or ratios. Fertilization rates also did not correlate with the expression levels of individual SMADs, but did correlate with higher SMAD4:SMAD3 ratios (p=0.0062) and trended with SMAD4:SMAD2 (p=0.0698). Conclusions SMAD transcripts are differently expressed in human granulosa cells, where they may mediate TGF-beta superfamily signaling during folliculogenesis and ovulation. Further, the relative expression ratios of SMAD2, 3 and 4 may differentially affect fertilization rate.

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