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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-0890
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 12 6596-6602
Copyright © 2005 by The Endocrine Society

Increased Transcription and Increased Messenger Ribonucleic Acid (mRNA) Stability Contribute to Increased GATA6 mRNA Abundance in Polycystic Ovary Syndrome Theca Cells

Clement K. M. Ho1, Jennifer R. Wood1, Douglas R. Stewart, Kathryn Ewens, Wendy Ankener, Jessica Wickenheisser, Velen Nelson-Degrave, Zhibing Zhang, Richard S. Legro, Andrea Dunaif, Jan M. McAllister, Richard Spielman and Jerome F. Strauss, III

Center for Research on Reproduction and Women’s Health (C.K.M.H., J.R.W., Z.Z., J.F.S.) and Department of Genetics (D.R.S., K.E., W.A., R.S.), University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; Departments of Cellular and Molecular Physiology (J.W., V.N.-D., J.M.M.) and Obstetrics and Gynecology (R.S.L.), Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033; and Division of Endocrinology, Metabolism, and Molecular Medicine (A.D.), Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611

Address all correspondence and requests for reprints to: Jerome F. Strauss III, M.D., Ph.D., Dean, School of Medicine, Virginia Commonwealth University, Sanger Hall, 1101 East Marshall Street, Room 1-071, P.O. Box 980565, Richmond, Virginia 23298. E-mail: jfstrauss{at}vcu.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Polycystic ovary syndrome (PCOS) theca cells secrete increased levels of androgens. The mRNA and protein levels of the transcription factor GATA6, which regulates expression of several steroidogenic enzymes, are increased in PCOS theca cells. Thus, GATA6 is a PCOS candidate gene.

Objective: The objective of the study was to explore mechanisms by which GATA6 mRNA levels are increased in PCOS theca cells.

Design: Theca cell cDNA and genomic DNA from normal individuals and PCOS patients were subjected to quantitative RT-PCR and sequence analysis, respectively.

Setting: The experiments were performed in a university laboratory.

Participants: Four hundred sixty-nine families that contain at least one PCOS patient were ascertained for genetic studies. Theca cells were obtained from four normal individuals and four PCOS patients.

Results: Nascent GATA6 transcript levels, which reflect GATA6 gene transcription, were significantly increased in PCOS theca cells. In normal theca cells, GATA6 mRNA has a short half-life, which was attributed to an AU-rich 3'-untranslated region sequence. The half-life of GATA6 transcripts was also significantly longer in the PCOS theca cells. However, no sequence variations in the GATA6 gene locus were associated with PCOS.

Conclusions: In PCOS theca cells, GATA6 gene transcription and the stability of the GATA6 mRNA are increased. Because there is no sequence variation in the GATA6 gene locus, which is associated with PCOS, it is likely that the increased gene transcription and mRNA stability are due to intrinsic differences in PCOS theca cells.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
POLYCYSTIC OVARY SYNDROME (PCOS) is a common endocrine and metabolic disease. The hallmark characteristic of PCOS is hyperandrogenemia, which clusters in families and appears to be inherited as a "modified autosomal-dominant trait" (1, 2, 3, 4). Theca cells from PCOS ovaries synthesize increased levels of androgens compared with normal theca cells (5, 6). This increased steroidogenic activity has been correlated to increased expression and/or activity of cytochrome P450 cholesterol side chain cleavage enzyme (CYP11A1), cytochrome P450 17{alpha}-hydroxylase, 17–20 lyase (CYP17), and 3ß-hydroxysteroid dehydrogenase type 2 (5, 7). Microarray analysis of theca cells from PCOS patients and normal individuals (NL) have also defined distinct differences between the normal and PCOS transcriptomes (8). Together, these data suggest that the PCOS theca cell has a stable biochemical and molecular phenotype that is distinct from the normal theca cell.

GATA6, which belongs to the GATA family of zinc finger transcription factors (9), exhibits increased mRNA and protein levels in PCOS theca cells (8). Recent reports indicate that GATA6, which is expressed in the gonads and adrenal cortex, regulates the expression of steroidogenic genes (9, 10). For example, GATA6 activates the StAR promoter in luteinized granulosa cells (11). Transient transfection assays demonstrate that the promoter activity of the CYP17, the CYP11A1, and the steroid sulfotransferase 2A1 genes are increased by GATA6 (8, 12, 13, 14). In addition to regulating the expression of these steroidogenic enzymes, GATA6 also increases the promoter activity of cytochrome b5, which is an allosteric modulator of CYP17 augmenting the 17,20 lyase activity of the enzyme (15). Interestingly, cytochrome b5 mRNA abundance is increased in PCOS theca cells (8), suggesting a functional link between increased GATA6 mRNA abundance and increased androgen synthesis in the PCOS theca cell and raising the possibility that GATA6 is a PCOS candidate gene.

Although microarray analysis identified increased levels of GATA6 mRNA in the PCOS theca cells, the mechanism for increased GATA6 expression remains unknown. To shed light on how GATA6 mRNA abundance is regulated, we examined the levels of newly transcribed GATA6 mRNAs, alternative mRNA transcript expression, and GATA6 mRNA stability. To determine whether GATA6 plays a role in the genetic etiology of PCOS, the sequence of the GATA6 gene locus was analyzed for variations associated with PCOS.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Experimental subjects

Four hundred sixty-nine families were ascertained and phenotyped as described previously (16). All patients gave informed consent before inclusion. Approval was obtained from the Institutional Review Boards at the University of Pennsylvania, the Pennsylvania State University, and Northwestern University.

Plasmids

The expression plasmids hG6-MALT, hG6-MYQ, and hG6-M147L have been described previously (13, 17). To generate the expression vector hG6-MYQ-R, the EcoRI/XhoI fragment of hG6-MALT was cloned into the hG6-MYQ plasmid. To construct pcDNA-hG6-Ia and pcDNA-hG6-Ib, exon 1a or 1b, respectively, of the GATA6 gene were PCR amplified (supplemental Table 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org/) and cloned into the BamHI/BstXI restriction site of hG6-MALT. To generate the plasmid pCR2.1-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the coding sequence of GAPDH was amplified (supplemental Table 1) and cloned into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA).

Cell culture and transient transfections

Theca cells were isolated from 3- to 5-mm follicles from the ovaries of four normal women and four PCOS patients, and independent cultures were established using the isolated cells as described previously (5, 18). The diagnosis of PCOS and the steroidogenic capacity of each sample were determined as described previously (5, 18, 19). HeLa cells were maintained in DMEM (Invitrogen) supplemented with 5% fetal bovine serum. Twenty-four hours before transfection, cells were seeded in 12-well plates at a density of 45,000 cells per well. Triplicate wells were transfected with 80 ng pcDNA3, hG6-MALT, hG6-MYQ-R, hG6-M147L, pcDNA-hG6-Ia, or pcDNA-hG6-Ib using FuGENE 6 (Roche Diagnostics, Indianapolis, IN)

Western analysis

Nuclear extracts (NEs) were prepared from the transfected HeLa cells. The NEs were separated by SDS-PAGE, transferred to Immobilon P (Millipore, Billerica, MA), and probed with GATA6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). GATA6 protein was detected using the SuperSignal West Pico Sensitivity reagent (Pierce, Rockford, IL).

Actinomyocin D (ActD) and cyclohexamide treatment of cells

Theca cells from three NL and three PCOS patients were plated in duplicate 60-mm dishes and cultured in serum-free medium (5) for 24 h. Cells were subsequently treated with ActD (10 µg/ml) and harvested 0, 20, 40, 60, 120, or 240 min later. Likewise, three NL and three PCOS theca cell samples were plated in duplicate 60 mm dishes, cultured in serum-free medium for 24 h, treated with 10 µg/ml cyclohexamide, and harvested 0, 1, 2, 4, 8, or 12 h later.

RT-quantitative PCR (QPCR)

Total RNA from untreated, ActD-treated, or cyclohexamide-treated normal and PCOS theca cells was isolated using Tri Reagent (Sigma, St. Louis, MO) and reverse transcribed using random hexamers (Roche Diagnostics) and Moloney murine-leukemia virus reverse transcriptase (Promega, Madison, WI) as described previously (8). To measure nascent GATA6 transcript levels, total RNA from normal and PCOS theca cells was reversed transcribed using 6 pmol of a GATA6-specific primer (5'-GGCTGGAATTGATAGGATAAACAAAA-3') and 100 fmol of a GAPDH-specific primer (5'-GTTCTCAGCCTTGACGGTGC-3').

Equivalent dilutions of the resulting cDNA was used to perform QPCR amplification of total GATA6 mRNA, nascent GATA6 RNA, exon Ia-containing GATA6 mRNA, or exon Ib-containing GATA6 mRNA as described previously (8). QPCR primers were designed using the Primer Express 2.0 software (Applied Biosystems, Foster City, CA) (supplemental Table 1). To account for differences in starting material, QPCR was also performed for each cDNA sample using the Applied Biosystems human GAPDH 20x primer and probe reagent (Applied Biosystems). The relative abundance of total GATA6 mRNA and nascent GATA6 transcripts was determined as described previously (8). To define the number of GATA6 Ia and GATA6 Ib copies in the normal and PCOS theca cell samples, the threshold cycle for GATA6 Ia and GATA6 Ib in each cDNA sample was converted to a copy number using a standard curve generated from serial dilutions of the hG6-Ia or the hG6-Ib plasmids, respectively. Likewise, the number of GAPDH copies in each sample was determined using a standard curve generated from serial dilutions of pCR2.1-GAPDH. The number of GATA6-Ia and -Ib copies per 1000 GAPDH copies was determined for each sample.

Genetic polymorphism studies

Single nucleotide polymorphism (SNP) genotyping of the GATA6 gene locus was performed using 6.75 ng of genomic DNA, the TaqMan SNP Genotyping Assays (hCV7490431 and hCV1892216) or Custom TaqMan SNP Genotyping Assay (rs1941084), and the ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Genotypes were auto called by the SDS 2.2 software with a quality value parameter of 0.95. Error checking of genotypes was performed by PedCheck software (20). Linkage disequilibrium between SNPs and PCOS in the families was tested using the transmission/disequilibrium test (TDT) (16, 21).

The CAC trinucleotide repeat within exon II of the GATA6 gene (Fig. 1AGo) was amplified by PCR using the fluorescently labeled primers 5'-FAM-CGAGCCCCAGTACAGCTC-3' and 5'-CTGAGGCGCGACCCTTAC-3' (Applied Biosystems). PCR products were electrophoresed on an Applied Biosystems 377 DNA Sequencer and analyzed using Genescan and Genotyper programs as detailed previously (16).



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  		FIG. 1. Schematic representation of the GATA6 gene and the mature mRNA species. A, The GATA6 gene is organized into seven exons with two alternative exon 1 sequences (Ia and Ib). The two translational start sites are indicated (+1710 and +2148). Previously described SNPs and a CAC trinucleotide repeat are indicated. The regions of the 5' promoter, 5'-UTR (exons Ia and Ib), and 3'-UTR (exon VII), which were sequenced in the present study, are emphasized with black dots. Distances are not to scale. B, The GATA6 gene is alternatively spliced into two mature GATA6 mRNA species that contain exons II–VII (hatched bar) and either exon 1a (white bar) or exon 1b (black bar). The two translational start sites (MALT or MYQ) are indicated.

 
To sequence the GATA6 promoter, exon Ia, and exon Ib, genomic DNA from 15 PCOS and five normal women was amplified (supplemental Table 1). The resultant PCR products were purified and sequenced. To sequence the GATA6 3'-untranslated region (UTR), cDNA from four NL and four PCOS patients was PCR amplified, and the resulting PCR products were sequenced (supplemental Table 1).

Rapid amplification of cDNA ends (RACE)

5' and 3' RACE of the GATA6 gene was performed using the Human Ovary Marathon-Ready cDNA kit (Clontech, Palo Alto, CA) as indicated by the directions of the manufacturer. 5' RACE was performed using two different gene-specific antisense primers (supplemental Table 1). 3' RACE reactions were performed using one gene-specific primer (supplemental Table 1) and an antisense primer supplied by the manufacturer. PCR products from the 5' and 3' RACE reactions were cloned into pCR2.1-TOPO (Invitrogen), and 20 clones from the 5'RACE reaction and seven clones from the 3'RACE reaction were sequenced and compared with published sequence of the GATA6 gene.

Statistical analysis. All statistical analyses were performed using the GraphPad Prism 4 software (GraphPad Software, San Diego, CA). Specifically, QPCR data were log transformed, and the unpaired Student’s t test was used to detect statistically significant differences (P < 0.05) in GATA6 mRNA abundance between normal and PCOS theca cells. To determine the half-life of the GATA6 and GATA4 mRNAs, the decay constant (K) for each mRNA was determined using the formula N = N0 x e–Kt for single-phase exponential decay, where N and N0 are mRNA abundance at time t and t0, respectively. The half-life of each mRNA was subsequently determined using the formula t1/2 = ln 2/K. Statistically significant differences in the half-life (t1/2) were determined by nonlinear regression analysis.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
QPCR suggests that GATA6 gene transcription is increased in PCOS theca cells

To determine whether the regulation of GATA6 gene transcription is altered in PCOS theca cells, the levels of nascent GATA6 transcripts in the normal and PCOS theca cells was measured using QPCR. Total RNA from four normal and four PCOS theca cell samples was reverse transcribed using a primer specific to intron 6 of the GATA6 gene, and QPCR was performed using primers to exon 6 and intron 6 (Fig. 1AGo). Sequence analysis of the QPCR product demonstrated that the exon 6/intron 6 primer pair specifically amplified nascent GATA6 transcript without amplification of mature mRNA species (data not shown). Nascent GATA6 transcript levels were increased 3.5-fold in the PCOS theca cells (Fig. 2AGo), which was consistent with our previously described differences in total GATA6 mRNA abundance between normal and PCOS theca cells (8) (Fig. 2BGo). Interestingly, treatment of the theca cells with 20 µM forskolin did not alter the levels of nascent or total GATA6 transcript levels in the normal or PCOS theca cells (Fig. 2Go). QPCR of the no-RT control samples revealed that genomic DNA was not detected during PCR amplification (data not shown).



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  		FIG. 2. QPCR analysis of the nascent and mature GATA6 mRNAs in human theca cells. Nascent GATA6 transcript levels (A) and total GATA6 mRNA levels (B) were determined by QPCR in four NL and four PCOS theca cell samples that were cultured in the absence (untreated) or presence of 20 µM forskolin. The relative abundance of GATA6 mRNA was expressed relative to GAPDH levels. Statistically significant differences in mRNA abundance are indicated (*, P < 0.05).

 
The GATA6 transcript is processed into two mRNA splice variants and translated into two protein isoforms in both normal and PCOS theca cells

The GATA6 gene contains two different exon 1 sequences (Ia and Ib) (22) (Fig. 1AGo). Alternative splicing of the nascent GATA6 transcript gives rise to two different mature mRNAs, which contain either exon Ia or Ib, and exons II–VII and two protein isoforms are generated from two alternative translational start sites (22) (Fig. 1BGo). To detect possible differences in the patterns of GATA6 mRNA splice variant expression in PCOS and normal theca cells, QPCR was performed using primers specific for exon Ia or exon Ib. The abundance of exon Ia-containing GATA6 transcripts was approximately 30-fold higher compared with the exon Ib-containing GATA6 mRNAs in both normal and PCOS theca cells. Furthermore, the levels of GATA6-Ia were 4.5-fold higher in PCOS theca cells, whereas the levels of GATA6-Ib were 2.5-fold higher in PCOS theca cells (Fig. 3AGo). These data were consistent with the QPCR results obtained using primers to the 3' end of the GATA6 mRNA (Fig. 2Go).



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  		FIG. 3. Expression of GATA6 alternative splice variants and identification of transcriptional start sites. A, Steady-state levels of the Ia- and Ib-containing GATA6 mRNAs were determined by QPCR in four NL and four PCOS theca cell samples. The copy number of the GATA6-Ia and GATA6-Ib transcripts was expressed relative to 1000 copies of GAPDH. Statistically significant differences in mRNA abundance are indicated (*, P < 0.05). B, The transcriptional start sites of the GATA6 gene were defined by 5' RACE. The position of the nested PCR primer and the nucleotide position of exons Ia, Ib, and II are indicated. The transcriptional start sites for 20 clones generated by the 5' RACE are indicated. None of the clones sequenced contain exon Ib. The organization of the nucleotide sequence was derived from Ref.22 .

 
To determine whether there were additional GATA6 transcriptional start site(s), 5' RACE was performed using a human ovary cDNA library. A gene-specific, nested primer was designed downstream of the second translational start site to amplify any mRNA that might contain only this 3' start codon (Fig. 3BGo). PCR products were cloned into pCR2.1-TOPO vector and sequenced. Of the 20 clones sequenced, there were no exon Ib-containing transcripts detected, which was consistent with the QPCR data and indicates that variants with a frequency of less than 5% could not be reliably detected in this screen. Four of the clones included genomic DNA immediately upstream of exon Ia, indicating transcriptional start sites immediately adjacent to exon Ia (Fig. 3BGo). Conversely, 16 clones contained sequence indicative of a transcriptional start site within the exon Ia sequence. These results together with the QPCR data demonstrated that the predominant GATA6 splice variant in the human ovary has a transcriptional start site within exon Ia.

In addition to the two splice variants of the GATA6 mRNA, there are two translational start sites in the GATA6 transcript (Fig. 1BGo) that give rise to two protein species in theca cells (8). To determine whether the specific GATA6 5'-UTR sequence influences the expression of the two GATA6 protein isoforms, plasmids that contain exon Ia–VII (hG6-Ia), exon Ib–VII (hG6-Ib), or exons II–VII (hG6-MALT, hG6-M147L, and hG6-MYQ-R) were transfected into HeLa cells, which do not express endogenous GATA6 protein (Fig. 4AGo). NEs from HeLa cells transfected with an empty vector (pcDNA3) or one of the GATA6-encoding plasmids were separated by SDS-PAGE, and the long or short form of the GATA6 protein was detected by Western blot analysis. When cells were transfected with hG6-M147L or hG6-MYQ-R, the long (64 kDa) or short (52 kDa) form of the GATA6 protein was detected, respectively. Conversely, both GATA6 protein isoforms were expressed when cells were transfected with hG6-Ia, hG6-Ib, or hG6-MALT (Fig. 4BGo), which was consistent with the protein expression profile of GATA6 in human theca cells and in in vitro translation studies (8, 22). Transient transfection studies also indicated that both protein isoforms of GATA6 increased the promoter activity of CYP17, CYP11A1, and 3ß-hydroxysteroid dehydrogenase type 2 (supplemental data), which was consistent with previous studies (8, 12, 13). These collective data suggest that alternative splicing is not a mechanism of increased GATA6 mRNA or protein abundance in PCOS theca cells.



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  		FIG. 4. Expression profile of the GATA6 protein isoforms. A, Schematic representation of the five plasmid constructs used to study protein isoform expression. The hatched rectangle indicates exons II to VII, and arrows designate translational start sites. Noncoding exons are in white (Ia) or black (Ib). B, NEs of HeLa cells transfected with pcDNA3, hG6-Ia, hG6-Ib, hG6-MALT, hG6-MYQ-R, or hG6-M147L were separated by SDS-PAGE, and the 64- and/or 52-kDa GATA6 isoforms were detected with a specific antibody.

 
GATA6 mRNA is more stable in PCOS theca cells

Increased GATA6 mRNA abundance in PCOS theca cells may also be due in part to increased message stability. To address this hypothesis, the t1/2 of the GATA6 transcript in PCOS and normal theca cells was compared. Cells were treated with ActD, which blocks mRNA transcription but does not alter intracellular mRNA decay for 0–240 min. QPCR was performed to determine the levels of GATA6 mRNA in ActD-treated cells, and the t1/2 and decay constant for GATA6 in the normal and PCOS theca cells were defined. In normal theca cells, the GATA6 mRNA had a relatively short t1/2 of 55.22 min (Fig. 5AGo). In PCOS theca cells, the t1/2 (81.24 min) was 38% longer. Likewise, the decay constant of the GATA6 transcript in normal and PCOS theca cells was also different (Fig. 5BGo). Nonlinear regression indicated that these differences were statistically significant. Using cDNA from NL and PCOS theca cells that were treated with cyclohexamide, a translational inhibitor that indirectly increases the intracellular levels of unstable mRNA by inhibiting mRNA turnover, confirmed that GATA6 mRNA stability is increased in PCOS compared to NL theca cells (data not shown). Conversely, the half-life and decay constant of GATA4, which does not exhibit altered total mRNA abundance in PCOS theca cells (data not shown), and the endogenous control GAPDH were not different in normal and PCOS theca cells (data not shown).



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  		FIG. 5. Determination of the t1/2 of the GATA6 mRNA in normal and PCOS theca cells. A, Three normal (black squares) and three PCOS (white circles) theca cell samples were cultured in the absence or presence of 10 µg/ml ActD for 0, 15, 30, 60, 90, 120, or 240 min. RNA was collected, and the relative abundance of GATA6 mRNA at each time point was determined by QPCR. Single-phase exponential decay analysis was used to determine the t1/2 and decay constant (K) for GATA6. B, Tabular results of the t1/2 and K of GATA6 mRNA in the NL and PCOS theca cells. Two-way ANOVA analysis demonstrated a statistically significant difference (P = 0.04) between the curves for NL and PCOS theca cells.

 
Cis-acting elements in 3'-UTRs are known to regulate mRNA stability (23). Furthermore, AU-rich domains in mRNAs often confer instability to the transcript (24). Given that the t1/2 of the GATA6 mRNA was short, the AU content of the GATA6 3'-UTR was defined using 3' RACE. Seven clones were obtained from the 3' RACE reaction and sequenced. All seven clones had a length of 1153–1158 nucleotides. The full-length 3'-UTR had an overall AU content of 61%, which was increased to 68% when the first 290 nucleotides of the region were excluded (Fig. 6Go). Thus, it is plausible that the GATA6 3'-UTR sequence regulates GATA6 mRNA stability.



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  		FIG. 6. Sequence analysis of the GATA6 3'-UTR. The GATA6 3'-UTR (1158 nucleotides) sequence was determined by 3'-RACE using ovarian cDNA as template. The translational stop codon (uga) is shown in lowercase. Adenosine (A) and uridine (U) nucleotides are indicated in bold.

 
There is no genetic variation in the GATA6 gene locus, which accounts for altered GATA6 mRNA abundance in the PCOS theca cells

Genetic predisposition is thought to play an important role in the etiology of PCOS. Therefore, we sought to determine whether genetic variation in the GATA6 gene was associated with increased transcription and/or increased stability of the GATA6 mRNA. There are three documented SNPs in the GATA6 gene (hCV7490431, hCV1892216, and rs1941084). hCV7490431 is approximately 5 kb upstream of the GATA6 gene, whereas hCV1892216 and rs1941084 are located within intron 6 and exon 7 (3'-UTR), respectively (Fig. 1AGo). To determine whether there is an association between a specific SNP and PCOS, genomic DNA from 401 families (hCV7490431) or 469 families (hCV1892216 and rs1941084) that had at least one PCOS female subject was assayed (2). When the SNP data were analyzed using the TDT (21), there was no association detected between PCOS and any of the three SNPs (hCV7490431, TDT {chi}2 = 0.013, nominal P value = 0.9; hCV1892216, TDT {chi}2 = 3.551, nominal P value = 0.06; and rs1941084, TDT {chi}2 = 0.385, nominal P value = 0.53).

Within exon II of the GATA6 gene, a CAC trinucleotide repeat gives rise to a polyhistidine tract (Fig. 1AGo). Genomic DNA from 96 individuals (PCOS and normal) was amplified to determine whether there was any polymorphic variation in this CAC trinucleotide repeat. PCR products from all 96 individuals contained 249 bp. Although there appeared to be additional bands in a few individuals (239 or 259 bp), the extra bands were not consistently transmitted in 47 simplex families, suggesting that they were the result of a PCR artifact.

Variations in promoter sequences are associated with differential rates of gene transcription. To determine whether genetic variation might contribute to increased transcription of GATA6 mRNA in PCOS theca cells, the promoter region and exons Ia and Ib of the GATA6 gene were sequenced (Fig. 1AGo). However, when genomic DNA from five normal women and 15 PCOS patients was sequenced, there was no genetic variation detected 1 kb upstream of exon Ia, within exon 1a, or within exon Ib. Likewise, when the 1158 bp of the GATA6 3'-UTR were sequenced using cDNA from four normal and four PCOS theca cell samples, there was no sequence variation associated with PCOS. Thus, the difference in GATA6 mRNA stability observed in the ActD experiments cannot be attributed to sequence variation in the 3'-UTRs of the GATA6 transcripts in normal and PCOS theca cells. Collectively, these data indicate that differences between the normal and PCOS theca cell environment are likely responsible for the differences in GATA6 gene transcription and GATA6 mRNA stability.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Our previous microarray analysis demonstrated that PCOS theca cells have a unique transcriptome compared with normal theca cells and identified several potential PCOS candidate genes, including the zinc finger transcription factor GATA6, which exhibited increased mRNA and protein levels in PCOS compared with normal theca cells (8). In the present study, we have demonstrated that both transcriptional and posttranscriptional mechanisms likely contribute to increased GATA6 mRNA abundance in the PCOS theca cell. Conversely, alternative splicing of the GATA6 transcript did not appear to be an important mechanism for the regulation of GATA6 mRNA abundance or protein isoform expression. Furthermore, when the GATA6 gene locus was subjected to genetic analysis, no polymorphism was identified that was associated with PCOS, making it unlikely that the GATA6 gene makes a significant contribution to any genetic predisposition to PCOS.

Increased gene transcription is a well-described mechanism for increased levels of a specific mRNA. Increased levels of newly transcribed GATA6 transcripts in PCOS compared with normal theca cells suggests that transcription of the GATA6 gene is increased in the PCOS theca cells and contributes to the increased steady-state levels of GATA6 mRNA. In the porcine ovary, GATA6 mRNA levels are regulated temporally, with a sharp increase in GATA6 levels detected in the granulosa cells of the preovulatory follicle (11), suggesting that gonadotropin-induced production of cAMP may regulate transcription of the GATA6 gene. However, the adenylate cyclase activator forskolin did not increase GATA6 mRNA levels in either normal or PCOS theca cells maintained in long-term culture (Fig. 2Go). In silico analysis of the GATA6 promoter revealed consensus binding sites for the basic helix-loop-helix/PAS family of transcription factors, including aryl hydrocarbon receptor, hypoxia-induced factor-{alpha} (HIF{alpha}), and aryl hydrocarbon receptor nuclear translocator (ARNT) (25). Interestingly, microarray analysis indicated that both ARNT and HIF{alpha} are increased in PCOS compared with normal theca cells (8). However, future studies will be required to determine whether HIF{alpha} and ARNT bind to the putative sites in the GATA6 promoter and differentially regulate transcription of the GATA6 gene in normal and PCOS theca cells.

Although transcriptional regulation of gene expression is often associated with increased mRNA abundance, the rate of mRNA decay also plays an important role in the regulation of mRNA steady-state levels and therefore gene expression (23). GATA6 mRNA in PCOS theca cells decayed at a slower rate than in normal cells. Given that there was no genetic variation in the GATA6 3'-UTR associated with PCOS, it is likely that the intracellular environment in the PCOS theca cells has a stabilizing effect on mRNA turnover. GATA6 mRNA is characterized by AU-rich elements in its 3'-UTR, which are known to be cis-acting factors that can regulate the rate of mRNA turnover (24). This study represents the first demonstration that the GATA6 transcript is relatively unstable and that differences in GATA6 mRNA turnover can contribute to altered steady-state GATA6 mRNA levels. Future studies will be required to address the trans-factors that bind to the GATA6 AU-rich element and regulate its stability.

In summary, increased levels of nascent transcript indicate that GATA6 gene transcription is increased in the PCOS theca cell. Likewise, an increase in the GATA6 t1/2 in PCOS cells suggests that posttranscriptional regulation of GATA6 mRNA stability is altered in PCOS. Together, these two mechanisms result in increased GATA6 transcript abundance and lead to increased GATA6 protein, which can stimulate the expression of steroidogenic enzymes involved in androgen synthesis and contribute to the hyperandrogenic phenotype of PCOS.


   Footnotes
 
This work was supported by National Institutes of Health Grants U54-34449 and HD06274 and the Mellon Foundation (to J.R.W.).

Present address for D.R.S.: National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892

First Published Online September 13, 2005

1 C.K.M.H. and J.R.W. contributed equally to this manuscript. Back

Abbreviations: ActD, Actinomyocin D; ARNT, aryl hydrocarbon receptor nuclear translocator; CYP11A1, cytochrome P450 cholesterol side chain cleavage enzyme; CYP17, cytochrome P450 17{alpha}-hydroxylase, 17–20 lyase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HIF{alpha}, hypoxia-induced factor-{alpha}; NE, nuclear extract; NL, normal individuals; PCOS, polycystic ovary syndrome; QPCR, quantitative PCR; RACE, rapid amplification of cDNA ends; SNP, single nucleotide polymorphism; TDT, transmission/disequilibrium test; UTR, untranslated region.

Received April 25, 2005.

Accepted September 1, 2005.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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