Proximal GATA-binding sites are essential for human HSD3B1 gene transcription in the placenta

The enzyme 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) is involved in the synthesis of active steroid hormones. Two human 3β-HSD isoforms are expressed in a tissue-specific pattern. HSD3B1 (type I) expression is essential to produce progesterone for pregnancy maintenance. To understand the mechanisms of human HSD3B1 activation in the placenta, 2.2 kb of 5′-flanking sequence and 5′-deletions were fused to the luciferase reporter gene and transfected into human JEG-3 cells. The proximal −238/+337 sequence had the highest promoter activity. Two GATA elements were identified at −106/−99 and −52/−45. Mutations of either sites greatly reduced promoter activity in JEG-3 cells, demonstrating the importance of GATA sites. EMSA revealed the specific binding of GATA2 and GATA3 to the GATA sequences at −106/−99 and −52/−45. ChIP assays demonstrated the association of GATA2 but not GATA3 with the GATA-binding regions of the HSD3B1 promoter in JEG-3 cells. GATA2 knockdown significantly reduced HSD3B1 expression in JEG-3 cells; however, GATA3 knockdown increased HSD3B1 expression. Western blot analysis revealed high levels of GATA2 but not GATA3 in human placental tissues. This study identified GATA motifs as essential control elements for HSD3B1 transcription and GATA2 as a novel transcriptional regulator of HSD3B1 expression in the human placenta.

Enhancement of HSD3B1 promoter activity by GATA2 and GATA3. A database search of the −238/+337 promoter region identified two putative GATA-binding sites at −106/−99 and −52/−45, which are referred to as Gu and Gp, respectively. To determine the role of these GATA-binding sites in the regulation of HSD3B1 promoter activity, mutated GATA-binding sequences were introduced into the −238/+337 promoter construct ( Fig. 2A). As shown in Fig. 2B, mutations of Gu and Gp significantly reduced promoter activity by 87% and 58%, respectively, in JEG-3 cells. In addition, the simultaneous mutation of both GATA sites resulted in a 93% reduction in promoter activity that was significantly lower than the Gu mutation (P < 0.05). These results suggested that both the Gu and Gp sites are involved in the regulation of HSD3B1 promoter activity.
The GATA transcription factor family consists of six members, GATA1-GATA6. GATA2 and GATA3 have previously been reported to regulate the expression of a number of genes in trophoblast cells and placental tissues 10 . To determine whether GATA proteins affect HSD3B1 promoter activity, the −238/Luc construct was co-transfected with GATA expression plasmids in HeLa cells. As shown in Fig. 3A, co-transfection of the −238/ Luc construct with GATA2 or GATA3 significantly increased promoter activity, whereas co-transfection with GATA4 did not affect reporter activity. Mutation of the Gu or Gp GATA-binding site resulted in the loss of GATA2-and GATA3-stimulated promoter activity ( Fig. 3C and D).
Expression of 3β-HSD, GATA 2, and GATA3 in the placenta. As shown in Fig. 4A, we confirmed the expression of 3β-HSD protein in JEG-3 cells by western blot analysis. The 3β-HSD type 2 is the isoform expressed −2226/+337) and HSD3B2 (B2: −1859/+556) promoter regions (shown on the top) were fused to luciferase reporter to generate promoter-luciferase constructs. "+1" is the transcription start site indicated by the arrow. The HSD3B1, HSD3B2 or empty (v) promoter-luciferase construct was transiently transfected into JEG-3 cells. (B) A series of 5′-deleted HSD3B1 promoter-luciferase constructs were transiently transfected into JEG-3 cells. Cells were harvested after 24 h of transfection and assayed for luciferase activity. Results are presented as the activity relative to the empty vector. Data are means ± SD of five (A) or three (B) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with vector control. # P < 0.05. in human adrenals 3 ; thus, a high amount of 3β-HSD protein was detected in H295R human adrenocortical carcinoma cells (Fig. 4A). In addition, Fig. 4A shows that GATA2 and GATA3 proteins were present at high levels in JEG-3 cells but at low to undetectable levels in H295R, HepG2, HeLa, or HEK293T cells. This is consistent with earlier studies showing the high expression of GATA2 and GATA3 in JEG-3 cells but not in H295R cells 11,12 . Cellular fractionation experiments demonstrated that GATA2 and GATA3 proteins were exclusively present in the nuclear fraction of JEG-3 cells (Fig. 4B).
The expressions of 3β-HSD, GATA 2, and GATA3 were examined in placental tissues by western blot analysis. Mouse 3β-HSD 6 is orthologous to human 3β-HSD 1, which is the only isoform expressed in the placenta and skin 13 . Consistent with previous studies, we found that 3β-HSD protein was highly expressed in mouse placenta on E10.5 and gradually decreased from E11.5-E12.5 (Fig. 4C). Similar to 3β-HSD, protein levels of GATA2 and GATA3 were high in mouse placenta on E10.5 and declined thereafter. However, GATA3 protein level was markedly decreased on E11.5. The protein expressions of 3β-HSD and GATA2 were also high in human placental tissues; conversely, GATA3 protein was detected at a very low level (Fig. 4C).
Analysis of GATA2 and GATA3 binding to the GATA elements. We performed electrophoretic mobility shift assay (EMSA) to examine the potential nuclear proteins involved in binding to the GATA-binding sites. Synthetic oligonucleotides containing the GATA site Gu (−115/−86) and Gp (−65/−36) were used as probes. Nuclear extracts from JEG-3 cells formed DNA-protein complexes with Gu probe (Fig. 5A, lane 2) that were competed by a 100-fold excess of unlabelled Gu (lane 3) or Gp (lane 4) probe. Similarly, protein complexes formed from JEG-3 nuclear extracts and Gp probe were competed by an excess of unlabelled Gu (lane 10) or Gp (lane 11) probe. Probes with mutation of the GATA-binding sequences (mu and mp) shown in Fig. 2 did not compete with the complexes (lane 5, 6, 12, and 13), indicating that the GATA sequence is required for protein binding. With Gu or Gp as the probe, a supershift was observed by the antiserum to GATA2 or GATA3, but not by the control serum (Fig. 5B). This suggests the involvement of GATA2 and GATA3 in the DNA-protein complex observed in EMSA. Furthermore, we used ChIP assays to confirm the presence of GATA2 or GATA3 at the endogenous promoter in JEG-3. As shown in Fig. 5C, GATA2 occupancy was observed in the GATA-binding region of the HDS3B1 promoter, whereas GATA3 was not found to be associated with this region.
Effects of GATA2 or GATA3 knockdown on HSD3B1 expression. To determine the roles of GATA2 and GATA3 in regulating HSD3B1 expression, we used shRNA to knock down GATA2 or GATA3 in JEG-3 cells. As shown in Fig. 6A and B, knockdown of GATA2 markedly reduced HSD3B1 mRNA levels by ~80% and protein levels by ~70% in JEG-3 cells. In contrast, we observed that GATA3 knockdown resulted in a 2-fold increase in with the −238/+337 HSD3B1 promoter-luciferase construct containing wild-type or mutated GATA sites (either or both sites). Cells were harvested after 24 h of transfection and assayed for luciferase activity. Results are presented as the activity relative to the wild-type construct. Data are means ± SD of five independent experiments. ***P < 0.001 compared with wild-type construct. # P < 0.05. the mRNA and protein levels of HSD3B1 ( Fig. 6C and D). The results suggest that GATA2 and GATA3 may play distinct functional roles in HSD3B1 transcription.

Discussion
Human HSD3B1 is involved in the production of placental progesterone for maintenance of pregnancy. Although several cis-acting elements have been implicated in the transcription of HSD3B1, the transcriptional mechanisms that regulate its basal expression in the placenta remain unknown. Here, we found that the proximal −238/+337 sequence of HSD3B1 exhibited high promoter activity in human choriocarcinoma JEG-3 cells. In this region, two GATA elements were identified at −106/−99 (Gu) and −52/−45 (Gp). Mutation of the GATA-binding sites at Gu or Gp greatly reduced promoter activity in JEG-3 cells. Our findings in this study strongly suggest that the proximal GATA motifs are important for trophoblast-specific expression of the HSD3B1 gene, and GATA2 and GATA3 play a role in the regulation of HSD3B1.
GATA transcription factors have conserved zinc finger domains that bind to the consensus DNA motif [(A/T) GATA(A/G)] to regulate the transcription of downstream genes. In mammals, six GATA factors are expressed in a tissue-specific manner and involved in the regulation of cell-specific gene expression. Earlier studies have indicated that GATA2 and GATA3 are the isoforms predominantly expressed in placental trophoblast cells, regulating the expression of a number of genes such as placental lactogen I, proliferin, and syncytin 10,14,15 . Our study showed that both GATA2 and GATA3 proteins were abundantly expressed in JEG-3 trophoblast cells and placental tissues (Fig. 4). GATA2 and GATA3 exhibited similar temporal expression patterns to HSD3B in mouse placenta. Furthermore, overexpression of GATA2 or GATA3 significantly stimulated HSD3B1 promoter activity in non-trophoblast cells (HeLa cells), whereas GATA4 had no effect on HSD3B1 promoter activity (Fig. 3). Mutations of GATA-binding sites significantly impaired GATA2-or GATA3-induced HSD3B1 promoter activity.
In addition, EMSA experiments demonstrated the specific binding of GATA2 and GATA3 proteins to the GATA sequences at −106/−99 and −52/−45. These data suggest that GATA2 and GATA3 can regulate the transcription of HSD3B1 through the GATA-binding sequences.
In EMSA, we observed that the two GATA probes (Gu and Gp) seemed to bind with a stronger affinity to GATA2 than to GATA3 (Fig. 5B). ChIP-qPCR assays further verified the binding of GATA2 at the GATA-binding regions of the HSD3B1 promoter in JEG-3 cells. GATA2 knockdown in JEG-3 cells resulted in a sharp reduction in the expression of HSD3B. Together, these findings reveal that GATA2 is the predominant protein recruited to the GATA motifs at −106/−45 to stimulate HSD3B1 gene expression in JEG-3 cells. In addition to the placenta, the expression of HSD3B1 has been identified in peripheral tissues including the mammary gland, prostate, and several human cancer cell lines [3][4][5] . The LNCaP human prostate cancer cells have been shown to express low levels of endogenous HSD3B1 5,16 . GATA2 ChIP-seq data derived from LNCaP cells are available in the GEO (Gene Expression Omnibus) database under accession number GSE69043 17 . Analysis of the ChIP-seq datasets for LNCaP cells revealed GATA2 ChIP enrichment in the regulatory regions of the HSD3B1 locus between −267 and +104 ( Supplementary Fig. S1), covering the two GATA-binding sites identified in this study. This is consistent with our ChIP-qPCR results of GATA2 in JEG-3, strongly indicating that GATA2 may be necessary for the basal transcription of the HSD3B1 gene.
In contrast to GATA2, the levels of HSD3B1 mRNA and protein were increased by GATA3 knockdown in JEG-3 cells, indicating that GATA3 may negatively regulate the expression of HSD3B1. EMSA experiments showed that both GATA2 and GATA3 were able to bind to the GATA elements at −106/−45 in vitro (Fig. 5B), suggesting that GATA2 and GATA3 may occupy identical GATA-binding regions of the HSD3B1 locus but exert distinct activities in HSD3B1 expression. Similar findings demonstrating that GATA factors exhibit different functions through shared chromatin sites were also observed in the transcriptional regulation of the Gata2   [18][19][20] . GATA2 is transcriptionally active in these regions, indicating that it positively autoregulates transcription. The process of "GATA switch" was also found in the development of the placenta. GATA3 was reported to repress the Gata2 gene by occupying the −3.9 kb and +9.5 kb regions of the Gata2 locus in undifferentiated trophoblast cells 21 . The displacement of GATA3 by GATA2 at these regions induces Gata2 transcription during trophoblast differentiation 21 . If a similar GATA switch occurs in the HSD3B1 locus, the concentration of GATA2 and GATA3 may be an important determinant in HSD3B1 gene regulation. In the present study, we showed that GATA3 protein was scarce in human placental tissues (Fig. 4C). In contrast, GATA2 protein was present at high levels, suggesting the binding of GATA2 to the GATA sites in the promoter to induce HSD3B1 transcription.
Although both GATA3 and GATA2 were abundant in JEG-3 cells, GATA3 binding to the GATA sites at −106/−45 was not observed in ChIP assays, indicating that this region may not be involved in GATA3-mediated HSD3B1 repression. Piao et al. 22 identified a GATA3-binding motif in a silencer element that represses the transcriptional activity of the HSD17B1 promoter in choriocarcinoma cells. By using a transcription factor binding site search program, we identified another putative GATA element present between −558 and −547 of the HSD3B1 promoter (Supplementary Fig. S2). Mutation of the GATA sequence in this region increased the promoter activity in JEG-3 cells, indicating that this motif is a potential silencer of the HSD3B1 gene. However, the specific association of GATA proteins to this GATA element was not detectable by EMSA analysis. Whether other GATA3-binding sites are present in the HSD3B1 locus and participate in the regulation of HSD3B1 transcription remain to be determined.
The expressions of CYP11A1 and HSD3B1 are required for the synthesis of progesterone in the human placenta. A previous study identified a GATA element at −475/−470 that is required for the activation of the Cyp11a1 promoter in mouse trophoblast giant cells 23 . The study demonstrated via EMSA that GATA2 is the predominant protein bound to this GATA site 23 . In this study, we found that two GATA-binding motifs located at −106/−45 are essential for the activation of the HSD3B1 promoter in human trophoblast cells. Our data demonstrate that GATA2 binds to the GATA motifs and acts as the critical transcriptional activator of the HSD3B1 gene in trophoblast cells. In addition, GATA3 seems to exert suppressive effects on HSD3B1 gene transcription. Together, GATA2 and GATA3 may play a critical role in the regulation of placental progesterone production.
Human tissues and animals. Human placental tissues were collected at term from the Department of Obstetrics and Gynecology, National Taiwan University Hospital (NTUH). The study protocol was approved by the institutional review board (IRB: 201406055RINC) at NTUH. The informed consent was obtained from all subjects. Animal experiments were approved by the National Taiwan University College of Medicine and College of Public Health Institutional Animal Care and Use Committee. All methods were performed in accordance with the relevant guidelines and regulations. C57BL/6 J mice were mated and checked for vaginal plugs the next morning. Noon on the day the vaginal plug was observed was considered embryonic day 0.5 (E0.5). Pregnant female mice were killed by CO 2 at the indicated times in the figure. Uterine horns were removed, and the placenta was separated from the embryo. Luciferase assay. Cells were subcultured 24 h before transfection in 24-well plates at a density of 10 5 cells/ well. Cells were transfected with 100 ng of reporter plasmid and 10 ng of the control reporter phRLuc. After 24 h, cells were harvested, and luciferase activity was determined using the Dual-Glo Luciferase Assay System (Promega). The results were normalised to internal Renilla luciferase activity. Data were obtained from at least three independent experiments. The significance of differences between group means was determined using one-way ANOVA followed by Newman-Keuls post-test. The statistical analysis were performed with GraphPad Prism 6 software (GraphPad Software).
shRNA knockdown. The shRNA-expressing lentiviral plasmids (pLKO.1-shRNA) were obtained from the National RNAi Core Facility (Academia Sinica, Taipei, Taiwan). GATA2 was efficiently targeted with the construct TRCN0000355775 or TRCN0000355776, and GATA3 was targeted with the construct TRCN0000273942 or TRCN0000273991. The shRNA targeting LacZ (TRCN0000072223) was used as a control. Lentiviral particles were prepared as described previously 24 .
RNA extraction and quantitative reverse transcription (qRT-PCR). Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific) and then reverse transcribed into cDNA by First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). qRT-PCR analyses were performed using SYBR ® Green PCR Master Mix (Thermo Fisher Scientific) in an ABI StepOnePlus TM machine (Thermo Fisher Scientific). Primer sequences used were as follows: HSD3B1 (forward primer 5′-CGGCTAACGGGTGGAATCTG-3′; reverse primer 5′-CCCCATAGATATACATGGGTCGTAAG-3′) and β-actin (forward primer 5′-GGGAAATCGTGCGTGAC-3′; reverse primer 5′-CAAGAAGGAAGGCTGGAA-3′). The relative quantification of mRNA levels was determined by the 2 −ΔΔCt method and normalised to β-actin in accordance with the manufacturer's instructions (Thermo Fisher Scientific). Triplicate qPCR amplifications were performed for each sample. Data were obtained from three independent experiments. The significance of differences between means of two groups was determined using Student's t-test.
Chromatin immunoprecipitation (ChIP). JEG-3 cells were incubated with 1% formaldehyde in growth medium for 10 min at room temperature, followed by adding glycine to a final concentration 125 mM for 5 min to stop the crosslinking reaction. Cells were then washed and scraped in ice-cold PBS and centrifuged, and the cell pellet was resuspended in ChIP lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 5 mM DTT, 2 mM PMSF, and 10 µg/ml leupeptin) on ice for 10 min. The cell lysate was sonicated with an ultrasonic processor (Sonics & Materials), diluted 1:3 with dilution buffer (50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2 mM PMSF, and 10 µg/ml leupeptin), and incubated with anti-GATA2 or anti-GATA3 antibody. Rabbit or mouse IgG isotype was used as a negative control. After incubation of the immunoprecipitate with Protein G Mag Sepharose Xtra (GE Healthcare) for 4 h, the beads were washed with high salt buffer (20 mM Tris pH 8.1, 2 mM EDTA, 0.1% SDS, 1% Triton X-100, and 500 mM NaCl) and eluted with elution buffer (1% SDS in TE buffer). RNase was added, and the crosslinks were reversed by incubation at 65 °C for 4 h. After proteinase K treatment at 55 °C for 1 h, the DNA was purified using QIAquick Gel Extraction Kit (Qiagen). The purified DNAs were subjected to qPCR using the forward primer 5′-TCCCATAGGAGGAGAGAGCA-3′ and reverse primer 5′-CCTCATTTCCTGTGGCAAGT-3′.