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. Author manuscript; available in PMC: 2012 Feb 1.
Published in final edited form as: Gen Comp Endocrinol. 2010 Dec 2;170(3):514–521. doi: 10.1016/j.ygcen.2010.11.023

Tissue-specific expression of squirrel monkey chorionic gonadotropin

Audrey A Vasauskas a,b, Tina R Hubler c, Lori Boston c, Jonathan G Scammell a,b,*
PMCID: PMC3022102  NIHMSID: NIHMS261234  PMID: 21130091

Abstract

Pituitary gonadotropins LH and FSH play central roles in reproductive function. In Old World primates, LH stimulates ovulation in females and testosterone production in males. Recent studies have found that squirrel monkeys and other New World primates lack expression of LH in the pituitary. Instead, chorionic gonadotropin (CG), which is normally only expressed in the placenta of Old World primates, is the active luteotropic pituitary hormone in these animals. The goal of this study was to investigate the tissue-specific regulation of squirrel monkey CG. We isolated the squirrel monkey CGβ gene and promoter from genomic DNA from squirrel monkey B-lymphoblasts and compared the promoter sequence to that of the common marmoset, another New World primate, and human CGβ and LHβ. Using reporter gene assays, we found that a squirrel monkey CGβ promoter fragment (−1898/+9) is active in both mouse pituitary LβT2 and human placenta JEG3 cells, but not in rat adrenal PC12 cells. Furthermore, within this construct separate cis-elements are responsible for pituitary- and placenta-specific expression. Pituitary-specific expression is governed by Egr-1 binding sites in the proximal 250 bp of the promoter, whereas placenta-specific expression is controlled by AP-2 sites further upstream. Thus, selective expression of the squirrel monkey CGβ promoter in pituitary and placental cells is governed by distinct cis-elements that exhibit homology with human LHβ and marmoset CGβ promoters, respectively.

Keywords: New World primate, luteinizing hormone, marmoset, placenta

1 Introduction

LH and chorionic gonadotropin (CG) are glycopeptide hormones made up of an identical α-subunit and different β-subunits. The β-subunits of these heterodimeric glycopeptides confer their biological specificities [20, 23]. Although LH and CG activate the same LH/CG receptor (LHR), they are differentially regulated in pituitary gland and placenta of humans and other Old World primates, consistent with their respective roles during the reproductive cycle and during pregnancy. Reproductive function is dependent on the proper expression and release of LH from gonadotrophs of the anterior pituitary gland. The upregulation and secretion of LH is stimulated by the pulsatile release of GnRH from the hypothalamus. In females, LH stimulates granulosa cell differentiation, cumulus expansion, and ovulation, as a mid-cycle surge of LH causes rupture of the mature follicle [17]. In males, LH activates testosterone biosynthesis in Leydig cells of the testes [11]. On the other hand, CG is expressed by syncytiotrophoblasts of the placenta of humans and other Old World primates during the first ten weeks of pregnancy and supports implantation and placental development by maintaining progesterone production by the corpus luteum [8].

Recent work from this laboratory and others has shown that New World primates, such as marmoset and squirrel monkey, do not express LH in the pituitary gland, but rather CG is the relevant luteotropic hormone [19, 22]. Müller et al. showed that the LHβ gene is present in marmoset. However, LHβ mRNA is absent from both pituitary and placental tissue, whereas CGβ mRNA is expressed in both tissues [19]. It is likely that pituitary expression of CG in New World primates arose secondary to expression of LHR that lack exon 10. The absence of amino acids encoded by exon 10 in LHR impairs LH, but not CG activity [5, 18, 30]. Therefore, unlike Old World primates, New World primates express CG in both pituitary and placenta, a finding that raises several questions. For example, what is the molecular organization of the CG gene in New World primates? Second, how is CG expressed in different tissues, specifically the pituitary gland and placenta? Answers to these questions are emerging.

Work in the common marmoset suggests that the CGβ gene is a single copy gene in New World primates [19]. Furthermore, differently sized mRNA transcripts of CGβ were detected in pituitary and placenta of the marmoset. These transcripts most likely arise from the tissue-specific utilization of different promoters in the CGβ gene. More recently, Henke and colleagues identified sites in the putative pituitary-specific promoter of the marmoset CGβ gene to which transcription factors steroidogenic factor 1 (SF-1) and early growth response protein 1 (Egr-1) bind [10]. These factors are important for the GnRH-responsiveness of the rat and bovine LHβ genes [3, 6, 27]. Here, in studying regulation of CGβ expression in squirrel monkey, another species of New World primate, we confirm and expand these previous studies in marmoset. We identified independent promoter regions in the squirrel monkey CGβ promoter that mediate pituitary- and placenta-specific expression of the gene. Comparisons are made to these regulatory regions in the marmoset and human CGβ promoters and the human LHβ promoter. Within these regions, we showed that specific cis-elements contribute to the tissue-specific expression of squirrel monkey CGβ.

2 Materials and Methods

2.1 Cell Culture

LβT2 mouse pituitary gonadotroph cells [1] were kindly provided by Dr. Pamela Mellon (University of California at San Diego, La Jolla, CA). JEG-3 human choriocarcinoma cells were obtained from American Type Culture Collection (Manassas, VA). PC12 rat pheochromocytoma cells were kindly provided by Dr. Edward Hawrot (Brown University, Providence, RI). Monolayers of LβT2, JEG-3, and PC12 were grown in DMEM (Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (FBS, Hyclone Logan, UT), 50 U/mL penicillin G, and 0.05 mg/mL streptomycin. Epstein-Barr virus-transformed squirrel monkey lymphoblast cells (SML) [21] were grown in RPMI 1640, 10% FBS, 4 mM L-glutamine, and antibiotics as above. Cells were grown at 37°C in a humidified atmosphere of 95% air-5% CO2.

2.2 Isolation of the squirrel monkey CGβ gene and promoter

Total genomic DNA was isolated from SML using the QIAGEN DNeasy Blood and Tissue Kit or the Flexigene Kit (QIAGEN, Valencia, CA). A DNA fragment containing the squirrel monkey CGβ gene was obtained by PCR using the Taq PCR Core Kit (QIAGEN) and upstream primer 5′-GAC GCA CCA AGG ATG GAG-3′ and downstream primer 5′-GCG GAT TGA GAA GCC TTT A-3′ corresponding to the human CGβ cDNA sequences 354–371 and 879–861 (NM_000737). The squirrel monkey CGβ promoter was obtained with the GenomeWalker Universal and Advantage-GC 2 PCR Kits (Clontech, Mountain View, CA), using an outer adaptor primer included in the kit and a reverse primer, 5′-TTG GAT CCC CAT GGC CCA CCT GTG CTC A-3′, corresponding to sequences 403-376 in the squirrel monkey CGβ gene. The squirrel monkey CGβ promoter and gene sequences were submitted to GenBank with accession number GU117708. Sequences were confirmed in several, independently generated DNA fragments.

2.3 Construction of luciferase plasmids and mutagenesis

A DNA fragment containing the squirrel monkey CGβ promoter (−1898 to +9) was amplified using a forward primer containing a linker sequence for MluI, TGT ACG CGT TCT AGC GAT TCT CTT GCC TCA ACC TC, corresponding to position −1898 to −1872 and a reverse primer containing a BglII site, CAC AGA TCT CCT TGG TGC CTC CCC TGC CTC GT, corresponding to position −16 to +9, (MluI and BglII sites underlined). After digestion with MluI and BglII, the amplified DNA fragment was subcloned into the MluI- and BglII-cut vector pGL3-Basic (Promega Corp, Madison, WI).

To study cis-elements responsible for cell type-specific activity of the squirrel monkey CGβ promoter, deletion constructs of the promoter were made. The 5′ deletion construct (−253 to +9) was generated by amplifying the region of interest using a forward primer containing a MluI site (underlined); TGT ACG CGT GGC GAG GCC TTT CTG TAC CCT ACT T, corresponding to position −253 to −229 and the reverse primer containing a BglII site described above. The product was cut with MluI and BglII and inserted into pGL3-Basic. To generate a construct in which 248 bp were deleted from the 3′ end of the squirrel monkey CGβ promoter, a HindIII restriction site was inserted at position −249 using site-directed mutagenesis. The mutagenesis was performed using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA), using forward and reverse primers as follows: (HindIII site underlined); 5′-CAG GAA GAG ACA GGG CGA AGC TTT TCT GTA CCC TAC TTC-3′ and 5′-GAA GTA GGG TAC AGA AAA GCT TCG CCC TGT CTC TTC CTG-3′, corresponding to position −227 to −266. The promoter fragment was digested with MluI and HindIII and subcloned into pGL3-Basic.

Mutations were made in the proximal and/or distal Egr-1, SF-1, or AP-2 binding sites of the squirrel monkey CGβ promoter (−1898 to +9) using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) (Table 1). PCR was performed according to the manufacturer’s instructions except that DMSO (5%) was added to the reactions. PCR products were digested to remove parental DNA according to the kit protocol, grown up in One Shot® Top 10 competent cells (Invitrogen), isolated, and sequenced.

TABLE 1.

Oligonucleotide primers used for mutagenesis studies

Site Sequence and position Primer sequence
pEgr-1 5′-CGC CCC CAC-3′ (−48/−40) 5′-AAC ACC ACC TGG TGG CCT TGT GAT TCT TAT AAC CCC GAG GTA T-3′
dEgr-1 5′-CG CCC CCG G-3′ (−110/−102) 5′-CTT GTC CGC CTC CTA GTA CTC AGG GGA TTA GTG TCC AGG TTA C-3′
pSF-1 5′-T GGC CTT G-3′ (−57/−50) 5′-ATG CTA ACA CCA CCT GGT GGA ATT CTC GCC CCC A CAA CCC CGA-3′
dSF-1 5′-TG GCC TTG-3′ (−127/−120) 5′-CTG AGC CCC TGC TGC GTC TCC CTG ATC ATG ATG TCC GCC TCC CG-3′
pAP-2 5′-GG CTG AGA G-3′ (−526/−518) 5′-ACT GCT CTG GGC TTC TTG ATG ATA GTG GTG TGG GAG AAG-3′
mAP-2 5′-CCT GCG GGT G-3′ (−599/−591) 5′-GCA GGC ACG CCC CCT GGA GAT TTT TTA AAT AAT GAG TTA AAT-3′
dAP-2 5′-GCC CCG AGG GC-3′ (−620/−610) 5′-ATG TTC ATG CAG GCT GTG GAT CCG AGA TCA GGC ACG CCC CCT G-3′
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pEgr-1, pSF-1, pAP-2 denote proximal Egr-1, SF-1, AP2 binding sites, respectively. mAP-2 denotes middle AP-2 site. dEgr-1, dSF-1, dAP-2 denote distal Egr-1, SF-1, AP-2 binding sites, respectively

2.4 Luciferase reporter gene assays

Cells were plated at a density of 3 × 105 cells/well in 6-well dishes. The next day, cells were transfected with 2 μg DNA/well of either promoterless luciferase plasmid (pGL3-Basic or pLuc-Link) or luciferase plasmid containing various promoter fragments using Superfect Transfection Reagent (QIAGEN) in complete medium for 3 h. Cells were washed, and fresh medium was added. The following day, after treatment with 100 nM GnRH (Sigma, St. Louis, MO) for 6 h, cells were harvested in 300 μL ice-cold Monolight lysis buffer (BD Biosciences, San Jose, CA), and luciferase activity measured in a Monolight 2010 luminometer (Analytical Luminesence Laboratory, San Diego, CA). Results were normalized to the promoterless luciferase plasmid control. Experiments were performed at least three times.

2.5 Statistical analysis

Statistical analysis was performed by using GraphPad Prism version 4.0 software (San Diego, CA). Comparisons between multiple groups were performed by using a one-way ANOVA and the Newman-Keuls posthoc test. Values were considered significantly different when the P value was less than 0.05.

3 Results

3.1 Isolation and analysis of the squirrel monkey CGβ promoter

A DNA fragment containing the squirrel monkey CGβ gene was amplified by PCR from genomic DNA and found to contain three exons and two introns with a similar organization to the human CGβ5 gene. Using a genome walking strategy, additional 5′ upstream DNA was isolated and approximately 1.9 kb were sequenced. This sequence was compared to the proximal marmoset CGβ promoter (Callithrix jacchus trace archive ti 1063492599) and rhesus macaque CGβ promoter (Macaca mulatta trace archive ti 542139165) and found to be highly similar (88% identity and 76% identity, respectively) (Figure 1). Sequences shown to bind the distal SF-1 and distal Egr-1 cis-elements in the marmoset promoter are conserved in the squirrel monkey CGβ promoter (Figure 2). Additionally, the squirrel monkey CGβ promoter was compared to the human CGβ, human LHβ, and rhesus macaque LHβ promoters (Macaca mulatta trace archive ti 349792889) and was found to contain regions of similarity with these promoters (79% and 86% identity with the human CGβ and LHβ promoters, respectively, and 74% identity with the rhesus macaque promoter) (Figures 1 and 2, respectively). Cis-elements known to be important for expression of human LHβ and CGβ genes are largely conserved in the squirrel monkey CGβ promoter. Taken together, these data suggest that the squirrel monkey CGβ promoter contains likely sequence important for both pituitary- and placenta-specific expression.

Figure 1.

Figure 1

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Nucleotide sequence alignment of the squirrel monkey CGβ promoter to the common marmoset CGβ (cmCGβ), human CGβ (huCGβ), and rhesus macaque CGβ (rmCGβ) promoters. Cis-elements known important for human CGβ promoter activity are conserved in the squirrel monkey CGβ promoter (underlined and in bold), the common marmoset CGβ, and the rhesus macaque CGβ promoters. The putative initiator element, based on the hCGβ initiator element, is underlined and in bold. Dashes indicate nucleotides bases that are identical to the squirrel monkey sequences. Asterisks indicate nucleotide deletions. The sequence is numbered according to the putative transcription start site for pituitary expression of squirrel monkey CGβ, labeled as +1, based on the transcription start site for human LHβ.

Figure 2.

Figure 2

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Nucleotide sequence alignment of the squirrel monkey CGβ (smCGβ) promoter to the common marmoset (cmCGβ), human LHβ (huLHβ), and rhesus macaque LHβ (rmLHβ) promoters. Cis-elements known to be important for human LHβ promoter activity are conserved in the squirrel monkey CGβ promoter (underlined and in bold), the common marmoset CGβ and rhesus macaque LHβ promoters. Putative transcription start sites for pituitary-specific expression are indicated as +1, based on the start site for human LHβ. Dashes indicate nucleotides bases that are identical to the squirrel monkey sequences. Asterisks indicate nucleotide deletions.

Thus, based on the sequence comparison of squirrel monkey CGβ promoter with marmoset CGβ, human CGβ and human LHβ promoters, we propose a model of the squirrel monkey CGβ gene and promoter wherein the expression of the gene is governed by two promoters: expression of the squirrel monkey CGβ gene in the pituitary is driven by a promoter that is found immediately upstream of exon 1, and contains a TATA-box, and cis-elements important for pituitary-specific expression; on the other hand, expression of the gene in the placenta is governed by an upstream TATA-less promoter that contains cis-elements similar to regions in the human CGβ promoter shown to be important for placenta-specific expression (Figure 3A).

Figure 3.

Figure 3

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Squirrel monkey CGβ is regulated in a tissue-specific manner by distinct promoter elements. A, Schematic depicting the putative structure of the squirrel monkey CGβ gene and promoter (not drawn to scale). The squirrel monkey CGβ gene is made up of three exons (solid bars) and two introns. The putative transcription start sites for pituitary- and placenta-specific expression are indicated by arrows. Pituitary-specific expression is governed by a promoter immediately upstream of the gene, whereas placenta-specific expression is governed by elements approximately 300 bp upstream of the pituitary start site. Luciferase promoter constructs are shown below the gene schematic, and sizes of each promoter fragment are indicated by position relative to the putative pituitary transcription start site: −1898/+9, −253/+9, and −1898/−249. B, LβT2, JEG-3, or PC12 cells were transiently transfected with pGL3-Basic promoter luciferase reporter plasmids driven by the −1898/+9 squirrel monkey CGβ promoter. Cells were harvested and assayed for luciferase activity, and the data are shown as fold-induction over that achieved with pGL3-Basic vector. Each bar represents the mean ± SEM of at least three independent experiments. *, significantly different from promoter activity in LβT2 and JEG3 cells.

3.2 Pituitary-specific regulation of the squirrel monkey CGβ promoter

To determine whether the squirrel monkey CGβ promoter confers pituitary- and placenta-specific expression, we generated a construct in which 1907 bp (−1898/+9) of 5′-flanking sequence of the squirrel monkey CGβ gene was placed upstream of the luciferase reporter gene in pGL3-Basic (Figure 3A). This construct was transiently transfected into either mouse pituitary gonadotroph LβT2 cells, human placental JEG-3 cells, or rat pheochromocytoma PC12 cells that do not express CGβ. For each experiment, a separate set of cells was transfected with empty pGL3-Basic vector to account for background and experiment-to-experiment variability. The squirrel monkey CGβ promoter fragment exhibited cell-type specific expression in both pituitary and placental cells (Figure 3B). Less than three-fold activity over the promoterless control was observed when the promoter fragment was transfected into PC12 cells. These data suggest that the −1898/+9 promoter fragment contains sequences that are sufficient for pituitary- and placenta-specific expression.

The LHβ gene promoter exhibits GnRH-responsiveness in pituitary cells [3, 27]. Additionally, Henke et al. showed that the marmoset CGβ promoter is also GnRH-responsive [10]. To examine whether the squirrel monkey CGβ promoter behaves similarly, we tested the GnRH-responsiveness of the squirrel monkey CGβ promoter in LβT2 gonadotroph cells. The −1898/+9 construct, and two deletion constructs, in which 5′ upstream (−253/+9) or 3′ downstream sequences (−1898/−249) were deleted (Figure 3A), were tested for GnRH-responsiveness. Treatment with 100 nM GnRH resulted in stimulation of reporter gene activity in cells transfected with both the −1898/+9 and −253/+9 constructs, whereas the −1898/−249 construct showed no GnRH-responsiveness (Figure 4A). These results suggest that the squirrel monkey CGβ promoter is regulated by GnRH in pituitary cells and expression is conferred by sequences within the most proximal ~250 bp of the promoter.

Figure 4.

Figure 4

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GnRH-responsiveness of squirrel monkey CGβ promoter in pituitary cells. A, LβT2 cells were transiently transfected with luciferase-reporter plasmids driven by either the −1898/+9, −253/+9, or −1898/−249 squirrel monkey CGβ promoter constructs. After 24 h, cells were incubated in the absence or presence of 100 nM GnRH for 6 h. Cells were harvested and assayed for luciferase activity, and data are shown as percent change in luciferase activity over that achieved in unstimulated cells, set at 100 percent. Each bar represents the mean ± SEM of at least three independent experiments. *, significantly different than activity in control cells. B, Schematic depicting relative positions of Egr-1, SF-1, and Pitx-1 binding sites of the squirrel monkey CGβ promoter. Mutated elements are denoted by “MUT.” The luciferase reporter plasmid containing mutations in the proximal Egr-1 binding site is designated as pEgr1m, the distal Egr-1 site as dEgr1m, and the construct containing mutations of both sites as pdEgr1m. C, LβT2 cells were transiently transfected with either the luciferase-reporter plasmid driven by the −1898/+9 squirrel monkey CGβ promoter (WT) or the same plasmid in which mutations were made in the proximal (pEgr1m), the distal (dEgr1m), or both Egr-1 binding sites (pdEgr1m). After 24 h, the cells were incubated in the absence or presence of 100 nM GnRH for 6 h. Cells were harvested and assayed for luciferase activity, and data are shown as percent change in luciferase activity over that achieved in unstimulated cells, set at 100 percent. Each bar represents the mean ± SEM of at least three independent experiments. *, significantly different from activity in control cells. †, significantly different from WT activity. D, Mutated SF-1 elements are denoted by “MUT.” The luciferase reporter plasmid containing mutations in the proximal SF-1 binding site is designated as pSF1m, the distal SF-1 site as dSF1m, and the construct containing mutations of both sites as pdSF1m. E, LβT2 cells were transiently transfected with either the luciferase-reporter plasmid driven by the −1898/+9 squirrel monkey CGβ promoter (WT) or the same plasmid in which mutations were made in the proximal (pSF1m), the distal (dSF1m), or both SF-1 binding sites (pdSF1m). After 24 h, the cells were incubated in the absence or presence of 100 nM GnRH for 6 h. Cells were harvested and assayed for luciferase activity, and data are shown as percent change in luciferase activity over that achieved in unstimulated cells, set at 100 percent. Each bar represents the mean ± SEM of at least three independent experiments.

Within this ~250 bp region of the squirrel monkey CGβ promoter are sequences homologous to known cis-elements in the LHβ promoter to which Egr-1, SF-1 and Pitx-1 bind (Figure 2). These factors are conserved among several species, and have been shown in the rat, mouse, and bovine LHβ promoters to act in concert to regulate basal and GnRH-induced expression in pituitary gonadotropes [3, 6, 27, 29]. To determine whether these sites are similarly involved in pituitary-specific expression and GnRH-responsiveness of the squirrel monkey CGβ promoter, we employed site-directed mutagenesis to mutate both the proximal and distal Egr-1 or SF-1 sites (Figure 4B). LβT2 pituitary gonadotroph cells were transiently transfected with the wild type (WT) promoter fragment (−1898/+9) or constructs containing mutations of Egr-1 and SF-1 binding sites (Table 1) that are known to affect GnRH-responsiveness of the LHβ promoter [3]. A mutation within the distal Egr-1 binding site (dEgr1m) resulted in a 60% reduction in GnRH-stimulated promoter activity, whereas a mutation within the proximal Egr-1 binding site (pEgr1m) reduced the GnRH response by 90% (Figure 4C). The construct containing mutations in both the proximal and distal Egr-1 (pdEgr1m) sites completely abrogated the effect of GnRH on the promoter. Thus, as reported for the LHβ promoter in other species, Egr-1 binding sites play a critical role in the regulation by GnRH of the pituitary-specific squirrel monkey CGβ promoter. On the other hand, mutation of either or both of the SF-1 binding sites within the squirrel monkey CGβ promoter (Figure 4D) had no effect on GnRH-responsiveness (Figure 4E).

3.3 Placenta-specific expression of the squirrel monkey CGβ promoter

Having identified regions in the squirrel monkey CGβ promoter that mediate pituitary-specific regulation, placenta-specific expression of the squirrel monkey CGβ gene was studied. Reporter constructs containing the squirrel monkey CGβ promoter were transiently transfected into JEG-3 cells, a human choriocarcinoma cell line used to study placenta-specific expression of reporter genes [4, 16]. The activity of the reporter construct containing −1898/+9 of the squirrel monkey CGβ promoter was dramatically higher than that of the promoterless vector in JEG-3 (Figure 5A). When upstream elements were deleted (−253/+9), promoter activity was eliminated. On the other hand, the construct containing −1898 to −249 of the promoter had similar activity to the −1898/+9 construct. These results suggest placenta-specific expression is governed by cis-elements between −1898 and ~−250 bp of the squirrel monkey CGβ promoter.

Figure 5.

Figure 5

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Placenta-specific expression of the squirrel monkey promoter. A, JEG3 cells were transiently transfected with luciferase-reporter plasmids driven by either the −1898/+9, −253/+9 or −1898/−249 squirrel monkey CGβ promoter constructs. After 24 h, cells were harvested and assayed for luciferase activity, and the data are shown as fold-induction over that achieved with pGL3-Basic vector. *, significantly different than activity in −1898/+9 and −1898/−249. B, Schematic depicting relative positions of AP-2 binding sites of the squirrel monkey CGβ promoter. Mutated elements are denoted by “MUT.” The luciferase reporter plasmid containing mutations in the proximal AP-2 binding site is designated as pAP2m, the middle AP-2 site as mAP2m, and the distal AP-2 site as dAP2m. C, JEG-3 cells were transiently transfected with either the luciferase-reporter plasmid driven by the −1900/+7 squirrel monkey CGβ promoter (WT) or the same plasmid in which mutations were made in either the proximal (pAP2m), middle (mAP2m), or distal (dAP2m) AP-2 binding sites. After 24 h, cells were harvested and assayed for luciferase activity, the data are shown as fold-induction over that achieved with pGL3-Basic vector. Each bar represents the mean ± SEM of at least three independent experiments. *, significantly different than activity in WT.

Previous studies have shown that three AP-2 binding sites within the human CGβ promoter are important for placenta-specific expression of the gene [12, 16]. These cis-elements are well conserved within the squirrel monkey CGβ promoter (Figure 1). We asked whether these binding sites play a role in the expression of the squirrel monkey CGβ promoter in placental cells. Site-directed mutagenesis was employed to mutate the proximal, middle, and distal AP-2 sites within the −1898/+9 construct (Figure 5B), and these reporter constructs were transiently transfected into JEG-3 placental cells. These mutations (Table 1) have been shown to affect the expression of the human CGβ promoter [12, 13]. Mutations in each of the AP-2 sites reduced the activity of the promoter by at least two-fold (Figure 5C). Similar results were observed in the JAR choriocarcinoma cell line, another cell line used to study placenta-specific gene expression (data not shown). These results suggest that like the human CGβ promoter, AP-2 sites play a role in placenta-specific expression of the squirrel monkey CGβ promoter.

4 Discussion

Recently, it has been found that New World primates lack expression of LH, and instead express CG as the luteotropic hormone secreted from the pituitary gland. Therefore, New World primates express CG in both the pituitary gland and the placenta. In this study, we isolated the squirrel monkey CGβ gene and demonstrated that its promoter is active in both pituitary and placental cells. Furthermore, distinct regions of the promoter appear to be important for this cell-type specific activity. Egr-1 sites play a critical role in pituitary-specific expression, whereas AP-2 sires are important for placenta-specific expression. This work confirms and expands work on the marmoset promoter by Gromoll and colleagues, who introduced the notion that New World primates express CG, and not LH, in the pituitary gland using cis-elements with homology to LHβ [10, 19].

Like the marmoset CGβ promoter, the squirrel monkey CGβ promoter shows high sequence identity to both the human CGβ and human LHβ promoters. The squirrel monkey CGβ promoter contains sequences known to confer pituitary-specific LHβ expression and GnRH-responsiveness. These include proximal and distal Egr-1 sites, which are paired with adjacent SF-1 sites [3, 6, 7]. A Pitx-1 site resides between the two SF-1/Egr-1 pairs [27]. Egr-1 appears to be a critical effector of GnRH stimulation, as Egr-1 −/− mice exhibit female infertility and male fertility dysfunction caused by lack of LHβ transcriptional activity in pituitary gonadotrophs [15, 26]. Egr-1 also appears to be a critical factor in the regulation of the squirrel monkey CGβ promoter, as mutations of the two Egr-1 sites eliminated GnRH-responsiveness. Henke et al. showed that basal and GnRH-responsive elements are located in the proximal 264 bp of the marmoset CGβ promoter, and that Egr-1 and SF-1 bind to a region of the marmoset CGβ promoter (−130/−92 in Figure 1) containing the distal Egr-1 and SF-1 sequences [10]. Additionally, specific point mutations in the distal Egr-1 binding site, which convert the marmoset CGβ promoter to corresponding sequences in the human CGβ promoter, reduced GnRH-responsiveness approximately two-fold [10]. Here, we conclude that both Egr-1 binding sequences in the squirrel monkey CGβ promoter are sufficient for its GnRH-responsiveness in pituitary gonadotrophs.

On the other hand, we did not observe significant changes in GnRH-responsiveness of the CGβ promoter when SF-1 elements were mutated. While the synergy of Egr-1 with SF-1 is necessary for GnRH-responsiveness of the LHβ promoter [3], SF-1 may play a lesser role in the regulation of the squirrel monkey CGβ promoter. Although SF-1 appears to bind to the distal SF-1 binding site of the marmoset CGβ promoter, it is not known whether SF-1 contributes to GnRH-responsiveness of the marmoset CGβ promoter [10]. SF-1 does not appear to be necessary for the GnRH-responsiveness of the squirrel monkey CGβ promoter. Collectively, these data add to the larger evolutionary picture of pituitary CGβ expression in New World primates.

Less is understood regarding the complex transcriptional regulation of the CGβ promoter in placenta. There are two major DNA footprint regions of the human CGβ5 promoter which bind several transcription factors. The most important cis-elements within the CGβ5 promoter, the most active promoter in humans [2], appear to be three AP-2, two Sp1, and five Ets-2 binding sites [2, 4, 12, 14, 16]. Of these, binding of AP-2 family members to the three AP-2 cis-elements is responsible for basal expression of the CGβ promoter, and Sp1 binding may contribute to this activity [12, 14, 16]. The three AP-2 sites are largely conserved within the squirrel monkey CGβ promoter, suggesting that they also play a role in the basal expression of this promoter. We found that the squirrel monkey CGβ promoter is very active in placental cells and that mutations in the AP-2 sites lead to a significant decrease in basal promoter activity. These data suggest that the squirrel monkey CGβ promoter is regulated similarly to that of the human CGβ promoter and highlights the role of AP-2-binding sites in basal expression of the gene in placenta.

Collectively, the work of a number of groups have highlighted the important role of CG in the regulation of reproductive function in New World primates [9, 10, 19, 24, 25]. The novel finding of pituitary expression of CG in New World primates raises a number of questions regarding its storage and secretion. In other species, such as humans and rodents, the luteotropic hormone LH is tightly regulated with the granin, secretogranin II (SgII). A question is whether a similar relationship is found between SgII and CG. This question is answered in a companion publication [28].

Research Highlights.

  • Squirrel monkey CGβ promoter is regulated in a tissue-specific manner

  • Egr-1 cis-elements necessary for GnRH-responsiveness of squirrel monkey CGβ

  • AP-2 cis-elements contribute to placenta-specific activity of CGβ promoter

Acknowledgments

This study was supported by Grant number 13200 from the National Center for Research Resources (NCRR), a component of the National Institutes of Heath (NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NCRR or NIH. Ms. Lori Boston and Dr. Tina Hubler were supported by REU: Structure and Function of Proteins National Science Foundation Research Experiences for Undergraduates (NSF #0751684) and an intramural grant from the University of North Alabama, respectively. We are grateful to Ms. Subha Pyakurel for her technical assistance.

Footnotes

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