285 A novel two-promoter-one-gene system of the chorionic gonadotropin b gene enables tissue-specific expression

Christian Adams*, Alexander Henke* and Jo¨ rg Gromoll Institute of Reproductive and Regenerative Biology, Centre of Reproduction and Andrology, University Hospital Mu¨nster, Domagkstrasse 11, 48129 Mu¨nster, Germany

(Correspondence should be addressed to J Gromoll; Email: [email protected])

*(C Adams and A Henke contributed equally to this work)

(A Henke is now at Medical Research Council, Centre for Reproductive Health, The Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, Scotland, UK)

Abstract

The New World monkey (NWM), Callithrix jacchus, a preferred model in medical research, displays an interesting endocrine regulation of reproduction: LH, the heterodimeric glycoprotein hormone, is functionally replaced by the chorionic gonadotropin (CG), a hormone indispensable for establishment of pregnancy in humans and normally expressed in the placenta. In the marmoset pituitary, the expression of the b-subunit (CGB) gene is regulated similar to human LH b-subunit, but its placental regulation is unknown. This study intended to decipher the underlying mechanism of tissue-specific expression of CGB in the marmoset placenta. We identified a new placental transcriptional start site, described a new, previously undiscovered exon, and define a novel placental core promoter in the marmoset CGB gene. This promoter contains a TATA box and binding sites for activating protein 2 and selective promoter factor 1, the latter acting synergistically by forming a regulation cassette. Differential first exon usage directed the tissue-specific expression. Methylation analyses revealed a tissue-specific pattern in the placental promoter indicating additional epigenetic regulation of gene expression. Our findings point toward a hitherto unknown evolutionary plasticity in the LH/CG hormonal system in NWM, which could be used as a model to study human CGB regulation in clinical pathologies. Journal of Molecular Endocrinology (2011) 47, 285–298

Introduction in OWM and humans. These result in the presence of six CGB genes and one LHB gene in humans, all Mammalian reproduction is controlled by the hypo- expressed at different levels, with CGB5 being the most thalamic GNRH that stimulates the pituitary to release abundant (Talmadge et al. 1983, Jameson et al. 1984, FSH, which triggers folliculogenesis in the female and Bo & Boime 1992, Miller-Lindholm et al. 1997). Notably, spermatogenesis in the male, and LH, which directs the CGB variants are arranged in a gene cluster together ovulation in the female and testosterone production in with the single-copy LHB gene (Maston & Ruvolo 2002, the male. A third glycoprotein hormone, the chorionic Hallast et al. 2005), suggesting rapid evolution in gonadotropin (CG), is essential for establishing parallel to the LHB gene itself (Wallis 2001). However, pregnancy as it is the first biochemical signal from the the ongoing evolution of the CGB/LHB genes in the embryo and ‘rescues’ the corpus luteum (Bousfield & NWM lineage is underlined by the absence of the LHB Ward 2006). Moreover, in humans, CG triggers fetal gene, while CGB is a single-copy gene in this taxon testosterone production and is hence crucial for (Maston & Ruvolo 2002, Muller et al. 2004a, Scammell male sexual development (Gromoll et al. 2000). The et al. 2008). It is noteworthy that equine species also gonadotropins are heterodimeric with identical possess a gonadotropin with CG-like properties that a-subunits but hormone-specific b-subunits that confer has evolved differently from primate CG via convergent specificity (Bousfield & Ward 2006). While LHB and evolution (Sherman et al. 1992, Chopineau et al. 1999). FSHB genes are present in nearly all mammals, the CGB Understanding the mechanism of CGB gene gene is exclusively present in Anthropoidea, such as regulation is relevant for clinical pathologies, e.g. New World monkeys (NWM), Old World monkeys reduced serum levels of CG are a risk factor for (OWM), great apes, and humans. Produced mainly by miscarriage, and association of specific CGB poly- placental trophoblasts (Maston & Ruvolo 2002), CGB is morphisms and recurrent miscarriage (Rull et al. derived from the LHB by gene duplication and a 2008b). Furthermore, increased serum CGB can serve frameshift mutation. Thus, this leads to an extension of as a marker for trisomy 21 and malignancies of the the protein, named the carboxy-terminal peptide reproductive system (Iles et al. 1987, Cuckle et al. 1994, (Talmadge et al. 1984), followed by several duplications Hotakainen et al. 2007).

Journal of Molecular Endocrinology (2011) 47, 285–298 DOI: 10.1530/JME-11-0026 0952–5041/11/047–285 q 2011 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org

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The CGB gene is also important from an evolutionary squirrel monkey (Saimiri sciureus) were obtained from point of view for primates and humans, as it is only our center’s own tissue bank. present in that particular taxa (Fiddes & Talmadge 1984, Henke & Gromoll 2008). The marmoset is the most common non-human primate animal model in DNA isolation reproductive sciences and preclinical toxicology Genomic DNA was isolated using the Qiagen ‘Whole (Zuhlke & Weinbauer 2003). Our previous study DNA Tissue Kit’ (Qiagen) following the manufacturer’s showed that the regulation of human LHB and instructions. marmoset CGB expression shows similarities in the pituitary. Both have a very short 50-untranslated region (50-UTR) of only 9 and 7 bp respectively. Furthermore, Sequences and in silico tools human LHB and marmoset CGB promoters show similar GNRH responsiveness and identical transcrip- Marmoset DNA sequence data were obtained from the tion factor binding sites (TFBS) in the core promoter of Ensembl website (www.ensembl.org). Sequence align- the pituitary (Henke et al. 2007). Findings were similar ments were performed with ClustalW (Chenna et al. to that of other NWM species as well (Scammell et al. 2003) and translation of DNA sequences was conducted 2008, Vasauskas et al. 2011). In contrast, human by EMBOSS transeq (Rice et al. 2000). Prediction of placental CGB transcription starts 365 bp upstream of potential TFBS in the CGB promoter was performed by exon 1 so that the human CGB 50-UTR encompasses the MatInspector (Cartharius et al. 2005) and protein region that would serve as a regulatory region in cleavage sites were predicted by SignalIP (Emanuelsson paralogous human LHB and orthologous marmoset et al. 2007). CGB. Thus, the regulation of CGB gene expression in the placenta of NWM deserves to be explored in detail. Cloning experiments The fact that the marmoset has only one CGB gene expressed in two different tissues (Muller et al. 2004b), Primers (outlined in Supplementary Table 1, see as well as other distinct biochemical properties, hints at section on supplementary data given at the end of a mechanism for tissue-specific regulation. In the last this article) were synthesized by Eurofins (Ebersburg, years, the role of epigenetic processes got more focus in Germany). Segments of the marmoset placental CGB research, which is also true for CGB. Very recently, gene promoter were amplified by PCR, cloned into Uuskula et al. (2010) demonstrated epigenetic pCR2.1 (Invitrogen, Karlsruhe, Germany), and ver- mechanisms regulating the human CGB genes during ified by sequencing. Constructs were then subcloned pregnancy. Meanwhile, the need to consider protein– into the luciferase vector pGL3-basic via XhoI and DNA interaction as well as epigenetic aspects of gene KpnI restriction sites (Promega, Mannhein, Germnay). regulation was highlighted for placental trophoblast differentiation and function in a recent review (Maltepe et al. 2010). Therefore, this study aimed to determine RNA isolation, rapid amplification of cDNA ends, and the placental regulation of the CGB gene in the reverse transcriptase PCR marmoset, taking into account classical transcriptional Total RNA was isolated with Ultraspec (AMS Bio- regulation as well as additional epigenetic regulation. technology, Frankfurt, Germany) according to the manufacturer’s instructions. Reverse transcription of RNA was performed using Superscript II Reverse Transcriptase (Invitrogen), random hexamer primers Materials and methods (Promega), and RNasin (Promega) according to the manufacturer’s instructions. 50-Rapid amplification of Animals and tissues cDNA ends (RACE) was performed using the GeneR- Animals were maintained in the center’s animal house acer kit with SuperScript III (both Invitrogen) accor- and kept under constant conditions. All animal ding to the product instructions. Reverse transcriptase procedures were approved by an ethics committee PCR (RT-PCR) and RACE primer sequences are given (G64/01) and were carried out in accordance with the in Supplementary Table 1, see section on supple- German Animal Protection Law. Pituitaries and pla- mentary data given at the end of this article. The first cental tissues of the common marmoset (Callithrix round of nested RACE PCR was performed with 1 ml jacchus) for RNA and DNA isolation were collected in a cDNA. The mix was run on a Robocycler (Stratagene, previous study (Henke et al. 2007), while genomic DNA La Jolla, CA, USA). For the second round of nested of the brown capuchin monkey (Cebus apella), the RACE-PCR, 500 ng DNA from round 1 was used. cotton-top tamarin (Saguinus oedipus), the brown- The products were purified and cloned into the headed tamarin (Saguinus fuscicollis), and the common pCR4TOPO vector (Invitrogen) according to the

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Downloaded from Bioscientifica.com at 09/29/2021 03:34:27AM via free access Regulation of CGB gene in the marmoset . C ADAMS, A HENKE and others 287 manual’s recommendations. Escherichia coli XL1Blue Luciferase assay was transformed with these plasmids and eight clones Luciferase activity was measured with the Dual-Lucifer- from the resulting colonies were DNA sequenced. ase Reporter Assay (Promega) according to the The expression of an alternative exon 1 of CGB was manufacturer’s protocol, using 20 ml lysate and 50 ml determined by semi-quantitative RT-PCR with exon- reaction buffers I and II. Firefly and Renilla luciferase specific primers and normalized to b-actin expression. activity was measured using the 96-well plate reader The reaction mix was the same as for the second of MicroLumat Plus LB96 (Berthold Technologies, Bad . m nested RACE PCRs in all aliquots except 1 0 lof Wildbad, Germany). Each DNA construct was measured . m tenfold diluted cDNA was added instead of 0 5 l in duplicates with two to five repetitions. Experiments undiluted cDNA. The program used was 94 8C for were normalized to the negative control (empty vector, 2 min initial denaturation, followed by 30 cycles of pGL3 basic), which was set arbitrarily as 1 relative light 30 s at 94 8C, 30 s at 56 8C, and 30 s at 72 8C unit (RLU). performed in a Hybaid Express thermal cycler. The PCR products were separated on an agarose gel, imaged in a MultiImage Light Cabinet (INTAS, In vitro mutagenesis Go¨ttingen, Germany) and analyzed using the accom- Mutagenesis of promoter sequences was performed panying software. The expression values of both using the QuickChangeII Site-directed mutagenesis kit transcript variants were each normalized to b-actin (Stratagene) according to the manufacturer’s instruc- expression and set as 100%. tions with an annealing temperature of 55 8C for 30 s. The primers used for mutagenesis are given in Supplementary Table 1, see section on supplementary Cell culture data given at the end of this article. Correct mutagen- The monkey kidney cell line COS7 was kept at 37 8C esis was verified by sequencing, the correct clone K 8 and 5% CO2 (v/v). Cells were cultured in DMEM/F12 amplified; DNA was isolated and stored at 20 C. 1:1 containing 10% (v/v) FCS and 1% (w/v) antibiotics. The human placental choriocarcinoma Immunohistochemistry cell line BeWo was purchased from Cell Line Service (CLS, Eppelheim, Germany). BeWo was established in We obtained tissue sections of formalin-fixed and 1968 and is an accepted model cell line for paraffin-embedded human full-term placenta from cytotrophoblasts (Pattillo et al. 1968, Sullivan 2004). two individuals from the Edinburgh Reproductive Tissue Bio Bank (ethical approval no. 09/S0704/3). These cells were cultured at 37 8C, 5% (v/v) CO2 in Ham’s F12 medium with 10% (v/v) FCS. These sections served as positive controls for the immunohistochemistry detection of SP1 and AP2g. Tissue sections of marmoset placenta from day 80 of Transient transfections gestation were retrieved from the tissue bank of the Medical Research Council, Centre for Reproductive . ! 5 . ! 4 BeWo (1 5 10 ) and COS7 cells (2 5 10 ) per well Health. Monoclonal mouse anti-SP1 antibody (Abcam, were seeded in 12-well plates. After 24 h, cells were Cambridge, UK, cat. no. Ab58199) was used at a covered with 400 ml serum-free medium (Ham’s F12 concentration of w1 mg/ml, while the monoclonal for BeWo and DMEM/F12 for COS7, Gibco), which g . mouse anti-AP2 antibody (sc-12762) and the mouse contained 1 mg promoter construct DNA, 0 05 mg anti-hCGB (sc-51605; Santa Cruz Biotechnology, Santa pRLSV40 Renilla vector (for normalization of transfec- Cruz, CA, USA) were used at w3and2mg/ml tion efficiency), and 2 ml Lipofectamine (Invitrogen). respectively. The peptides against which SP1 and Each DNA construct was transfected in duplicates. After AP2g antibodies were raised showed w95% homology 18 h, the medium was replaced with a medium between human and marmoset. Immunohistochemistry containing 10% (v/v) FCS (heat inactivated and (IHC) was performed as follows: tissue sections were charcoal stripped). After 24 h, the cells were washed dewaxed and rehydrated in a xylene/ethanol/water with PBS, then lysed with 300 ml PLB, and assessed by series, and antigens were retrieved by pressure-cooking luciferase assay. Overexpression experiments were in 0.01 M citrate buffer (pH 6). The antigen detection similarly performed, using 3 mgtransfectedDNA procedure for AP2g and CGB was automated on a (promoter, expression plasmid, and vehicle). Bond-Max machine (Leica Microsystems, Milton Expression plasmids used were activating protein 2 Keynes, UK) with the Bond Polymer Refine Detection (AP2), human AP2g isoform cloned in pBKCMV, and Kit (cat. no. DS9800; Vision BioSystems bond, selective promoter factor 1 (SP1), human isoform Milton Keynes, UK) according to the manufacturer’s cloned in pFCMV. Expression vectors were provided instructions and slides were mounted in Pertex by P Mellon, UCSD, La Jolla, USA. (Histolab, Go¨teborg, Sweden) and coverslips. Antigen www.endocrinology-journals.org Journal of Molecular Endocrinology (2011) 47, 285–298

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detection for SP1 was carried out manually: dewaxin- a region assumed to regulate placental CGB expression. g/rehydration in xylene/ethanol/water series, antigen A total of 2 mg DNA were bisulfite converted in a volume retrieval via pressure-cooking, blocking in methanol of 50 ml, according to the protocol of Hayatsu et al. with 3% (v/v) H2O2, blocking with streptavidin and (1970). Converted DNA was desalted and purified using biotin blocking solutions (Vector Laboratories, the DNA Purification Kit (Promega). PCR was per- Burlingame, CA, USA, cat. no. SP-202), and blocking formed using the primer listed in Supplementary with a mixture of 20% normal rabbit serum (NRS), Table 1, see section on supplementary data given at TBS, and 5% (w/v) BSA (NRS/TBS/BSA). Slides were the end of this article. 10! Buffer, 10 mM dNTPs, incubated with the primary antibody in NRS/TBS/BSA forward primer (20–100 pmol/ml), reverse primer overnight in a humidity chamber at 4 8C. The following (20–100 pmol/ml), 0.3 ml Taq Polymerase (Qiagen), day, the slides were incubated at room temperature with ddH2O, and 2 mg bisulfite DNA were added to a total first 1:500 dilutions of a secondary rabbit anti-mouse volume of 25 ml. Thermocycling program used was biotinylated antibody (Dako, Glostrup, Denmark; cat. an initial denaturation at 95 8C for 120 s, 95 8C for 30 s, no. E464) for 2 h in NRS/TBS/BSA and then 1:1000 48–54 8C for 30 s, and 72 8C for 30 s, final extension dilutions of tertiary streptavidin–HRP conjugate in TBS 72 8C for 10 min respectively. PCR product was checked (Vector Laboratories) for 2 h. ImmPACT DAB kit via gel electrophoresis, purified, and cloned into the (Vector Laboratories, SK-4105) was used to visualize pGemT-easy vector (Promega) in E. coli XL-1Blue. Ten antibody binding. Afterwards slides were mounted and resulting clones were sequenced. tissue sections were photographed with an Axiocam CGB promoter fragments were cloned into a CpG- HRc (Zeiss, Welwyn Garden City, UK) mounted to a Provis free backbone luciferase vector (Klug & Rehli 2006) AX70 microscope from Olympus (Southend-on-Sea, UK). followed by in vitro methylation by SssI Methylase (New Images were white balanced, composed, and annotated England Biolabs, Frankfurt, Germany) according to the with Photoshop. Omission of the primary antibody was manufacturer’s instructions. Briefly, 1 mg vector DNA, considered to be a sufficient negative control. 2 ml10! NEB-Buffer, 2 ml1! S-adenosylmethionine, and 1 ml SssI methylase (4 U/ml) were incubated for 1 h at 37 8C before the reaction was stopped by heating Chromatin immunoprecipitation with transiently at 65 8C for 20 min and vectors subjected to the transfected cells reporter assay. BeWo cells were seeded in 10 cm dishes at 80% confluence for 24 h. The next day, the cells were transfected with 15 mg of the promoter construct with Statistical analysis or without mutation using 66 ml Lipofectamine trans- Statistical analysis was performed using GraphPad fection reagent (Invitrogen). Plates were incubated for Prism (GraphPad Software, La Jolla, CA, USA), paired 24 h at 37 8C after which cells were cross-linked with t-test, one-way ANOVA with the Bonferroni post-test, formaldehyde, harvested, and chromatin immuno- and the Pearson test where appropriate. precipitation (ChIP) was performed. Cells (5!106) were used per immunoprecipitation reaction, using the ChampionChIP One-Day Kit (SABiosciences, Frederick, MD, USA) according to the previously published Results protocols (Wells & Farnham 2002) for AP2. For SP1, the Cell Signalling Simple ChIP Kit (Qiagen, Hilden, CGB mRNA from marmoset placenta contained Germany) was used, according to the manufacturer’s a novel exon recommendations. The resulting DNA was analyzed by We initially determined the transcriptional start site PCR with primers flanking the AP2/SP1 binding (TSS) for placental CGB expression in the marmoset via cassette (see Supplementary Table 1, see section on 50-RACE and sequenced the resulting clones. From supplementary data given at the end of this article). these, we identified a previously unknown TSS of CGB Antibodies used in the ChIP procedure included AP2 and a novel exon 1 (Fig. 1A). The conventional exon 1, (sc-12762X), SP1 (sc-17824X), Pol II (sc-9001X; all present in all known LHB and CGB transcripts from Santa Cruz Biotechnology), and anti-mouse IgG OWM, is absent with the novel exon 1 directly spliced to (included in the ChampionChIP One-Day Kit). exon 2. This novel exon contains 212G11 bp 50-UTR and a 15 bp protein-coding region after the first ATG Bisulfite sequencing codon (Fig. 1A). Thus, both exon 1 sequences, the novel as well as the conventional, encode the first five MethPrimer software (Li & Dahiya 2002) was used to amino acids (aa) of a signal peptide, while exon 2 predict in silico CpG islands in the marmoset CGB encodes the remaining 15 aa of the signal peptide and promoter, and primers designed accordingly to amplify 41 aa of the mature peptide. The in silico translated

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A

Placental promoter Pituitary promoter

376 bp352 bp 235 bp Exon 2 Exon 3 ′ 5 UTR 5′ UTR 168 bp 312 bp 3′ UTR 212 bp 7 bp 18 bp Novel exon 1 Conventional exon 1 15 bp 15 bp

B Cjac Ssci Cape Sfus Soed Mmul CG1 Mmul CG2 Mmul CG3 Ptro CG3 Ptro CG7 Hsap LH Hsap CG7 Hsap CG8 Hsap CG5 Hsap CG3 ****** ** * **** *** * ** ***** **** ** * ** C

Cjac CGB–N Ssci CGB–N Soed CGB–N Sfus CGB–N Cape CGB–N Anan CGB–C Sbol CGB–C Cape CGB–C Cjac CGB–C Soed CGB–C Ssci CGB–C Sfus CGB–C Hsap CGB3–5–8 Abbreviations : Hsap CGB7 Cjac, Callithrix jacchus, marmoset, NWM Hsap LHB Ssci, Saimiri sciureus, squirrel monkey, NWM Ptro CGB3 Soed, Saimiri oedipus, tamarin, NWM Ptro CGB7 Sfus, saguinus fuscicollis, tamarin, NWM Mfas CGB1 Cape, Cebus apella, brown capuchin, NWM Mmul CGB8 Anan, Aotus nancymaae, Ma's night monkey, NWM Mfas CGB2 Sbol, Saimiri boliviensis, brown squirrel monkey, NWM Hsap, Homo sapiens, human, Panu CGB Ptro, Pan troglodytes, chimp, OWM Ecab CGB Mfas, Macaca fasicularis, macaque, OWM Mmus LHB Mmul, Macaca mulatta, macaque, OWM *******. ::** .. Ecab, Equus caballus, horse Mmus, Mus musculus, mouse Figure 1 A novel exon 1 in NWM and its tissue-specific use in the marmoset CGB gene. (A) The gene structure of marmoset CGB. Arrows indicate the two different transcription start sites; the first exons are differentially labeled (white, novel exon 1; black, conventional exon 1), gray areas encode exons 2 and 3 whereas light gray boxes stand for UTRs. Promoter regions are also indicated. Numbering starts with the first nucleotide of start codon ATG of the conventional exon (conventional exon 1ZC1). The ATG of the novel exon 1 is K381 bp. (B) Sequence alignment displaying the novel exon 1 (bold, with ATG in light gray) present in all NWM, adjacent to a splicing donor site GT (dark gray). Lower light gray shading: initiator element of the human CGB genes with transcriptional start sites (underlined). The numbering is the same as in A, identical nucleotides compared to the Callithrix sequence are presented as dashes, ‘*’ indicates conserved sites and ‘:’ defines same charge of the amino acids, whereas ‘.’ marks amino acids belonging to the same group (pyridene or purine). (C) Sequence alignment representing LHb (ZLHB)orCGB (Z CGB), with gene variant number given or whether the signal peptide included the novel (N) or conventional (C) exon 1. Nine of 20 aa of the novel exon 1 were completely conserved across all species and gene variants. Human CGB3, 5, and 8 have been summarized due to identical signal peptides. Identical nucleotides to the sequence of the marmoset novel exon 1 are indicated by dashes and all other symbols are the same as in B. Species abbreviations are given on the right. peptide sequence of the marmoset CGB new exon 1 was anionic glutamate. Analysis of the peptide sequence via changed from the previously known pituitary CGB SignalP showed no altered cleavage site of the signal protein sequence MEMLQ – which is also conserved in peptide from the mature peptide. human CGB peptide sequences – to MALVE. This 4 aa substitution alters the polarity and charge of the signal Presence of the novel exon 1 in NWM peptide. A negatively charged glutamic acid is exchanged for an uncharged alanine, positively The surprising findings of a novel exon 1 in the charged methionine for an uncharged leucine, while marmoset raised the question of whether other primate in position K17 a lipophilic character is maintained species, especially the NWM, also possess the novel and in position K16 a polar glutamine is changed to exon 1. To address this, we retrieved CGB gene and www.endocrinology-journals.org Journal of Molecular Endocrinology (2011) 47, 285–298

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upstream sequences from public databases. In addition, 100 * the marmoset CGB gene and its upstream sequence 90 80 * were built in silico from raw data provided from the 70 marmoset genome project. This sequence served for 60 50 designing primers for cloning of the CGB region from 40 genomic DNA. We were able to clone the CGB genes 30 and their upstream promoter sequences from the 20 10 Conventional exon Novel exon common marmoset as well as four other NWM species. splice variant Percentage of RNA 0 The sequences were deposited in public databases (see Pituitary (n = 8) Placenta (n =6) Supplementary Table 2, see section on supplementary Figure 2 Tissue-specific usage of the novel exon 1. Pituitary and data given at the end of this article, for accession placental usage of the first two exons 1 are highly significantly ! . Z C numbers) and used for sequence alignments. different (*P 0 005, n 5). The mean S.E.M. percentage of splice variants in each tissue is displayed. Black columns Prerequisites for a functional exon 1 are generally represent the conventional exon 1 whereas blank columns a start codon and a donor splice site. These two indicate the novel exon 1. Percentage of mRNA was quantified conditions for a potential novel exon 1 were only densitometrically after RT-PCR. fulfilled in the marmoset and three other NWM (S. oedipus, S. fuscicollis, and C. apella). All three CGB study from marmoset pituitaries and placentas of genes of the rhesus macaque, chimpanzee, and human pregnant (8–12 weeks of gestation) and non-pregnant CGB7 showed an ATG codon for a potential novel exon female marmosets (Henke et al. 2007). 1 but not the consensus sequence for donor splice sites. In the pituitaries, a significantly elevated expression The other human and chimpanzee sequences fulfilled of the CGB isoform containing the conventional exon 1 was evident in both pregnant (nZ4) and non-pregnant neither criterion, suggesting that the presence and Z use of the novel exon 1 of CGB is limited to the females (n 4) (Supplementary Table 3, see section NWM (Fig. 1B). The signal peptide sequences were on supplementary data given at the end of this article). also aligned with known LHB/CGB signal peptide As there was no statistically significant difference between these two groups (PZ0.678), they were sequences from OWM (Macaca mulatta, Macaca Z fascicularis and Pan triglycerides), human, mouse (Mus combined (n 8) for subsequent comparison with the prevalence of the isoforms in the placenta. In the musculus), and horse (Equus caballus)asoutliers Z (Fig. 1C). The conventional consensus sequence placenta (n 6), the novel exon 1 was the predominant MEM(L/F)Q was found in all primates but not in transcript isoform. A significant difference of exon 1 usage between pituitary and placenta was detected mouse or horse. Different amino acids were restricted ! . to the third or fourth position in the human, (P 0 0001; Fig. 2, Supplementary Tables 3 and 4, chimpanzee, and olive baboon sequences (Fig. 1B and see section on supplementary data given at the end of Supplementary Figure 2, see section on supplementary this article). data given at the end of this article). The horse and mouse showed a single aa difference as well (Fig. 1C). Identification of the placental core promoter region The cladogram (Supplementary Figure 2, see section for marmoset CGB on supplementary data given at the end of this Due to the findings of a new TSS, a novel exon 1, and a article), showing the relationship between the tissue-specific expression of exon 1, we concluded that different primates as well as horse and mouse, the preferential use of the novel exon 1 in the placenta revealed similarities in the NWM group. The protein must be accompanied by a new promoter region, which consensus sequence for the novel exon 1 appeared to we wanted to identify. In the following, the positions are be MALVE and was found to be exclusively present given in relation to the ATG start codon of conventional only in the NWM. Only the brown capuchin C. apella exon 1. In the human, the TSS for CGB3/5/7/8 is showed one different aa, with a lysine residue located 365 bp upstream of the start codon of the replacing glutamine at the fifth position. conventional exon 1 and the TSS for CGB1/2 is 175 bp upstream (Henke & Gromoll 2008). However, this 0 Preferential use of novel exon 1 in the placenta region is part of the 5 -UTR in the placental transcript of the orthologous marmoset CGB gene. Promoter Having identified a novel exon 1, we next addressed regions of different lengths were used in a reporter the question of whether the marmoset uses the assay. Luciferase activity was measured after transient two different exons in a tissue-specific manner. transfection into the human choriocarcinoma cell line We examined the distribution of the two CGB transcript BeWo. The marmoset CGB promoter constructs were forms in pituitary and placenta by semi-quantitative successively truncated, leading to a subsequent loss of RT-PCR from cDNA samples generated in a previous TFBS (see Supplementary Figure 1, see section on

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Relative promoter activity (in RLU) 50 100 150 200 luc pGL3 basic Hs CGB 5

–1411/–381

–1167/–381

–1024/–381

–1000/–381

–837/–381

–750/–381

–735/–381 –691/–381 * EGR1 –668/–381

GCMa –645/–381 SP1 –624/–381 AP2 –264/–1 Figure 3 Identification of the placental CGB core promoter of marmoset. The placental core promoter resides within K837 to K624 bp upstream, displaying significant differences between these two promoter constructs. Empty vector pGL3 basic served as negative control whereas the human CGB5 promoter served as positive control. The construct (K264/K1) represents the CGB pituitary promoter. All activities are relative to pGL3 basic, for which the activity was set to 1. The different symbols indicate binding sites for transcription factors AP2, EGR1, GCMa, and SP1 on the left side. RLU, relative light unit. Given are the meansCS.E.M. of three independent transfections of BeWo cells per experiment. One-way ANOVA; *P!0.001. supplementary data given at the end of this article, for TFAP2C, ERF1, and hAP2-g) (K659/K650 and 50-ends of truncated promoter constructs). A reporter K641/K631) (see Supplementary Figure 1, see section construct containing the core promoter for marmoset on supplementary data given at the end of this article, CGB expression in the pituitary (K264/K1) showed for position). In addition, within an AT-rich region, minimal activity in BeWo cells. In contrast, distinct a TATA box was predicted, whose position is in activity was detected from the constructs containing accordance with the average distance between a TATA regions suspected of involvement in placental CGB box and a TSS. Finally, a binding site for the chorion- K K regulation. The shortest construct ( 624/ 381) specific transcription factor GCMa (K639/K633) was stretched to the beginning of the novel exon 1 also predicted, which is absent in the human CGB K (at position 381) and had a very low promoter promoter. K K activity, while longer constructs ( 1167/ 381 and We mapped the AP2 and SP1 sites on the promoter K K 1024/ 381) displayed the highest promoter activity constructs. Hence, only two EGR1, two SP1 sites, and of all constructs generated (Fig. 3). A construct located both AP2 sites are located within the core promoter further than 1176 nucleotide (nt) upstream led to a sequence (Fig. 3). Promoter sequences with both AP2 decrease in promoter activity. Thus, we identified the and SP1 trans elements displayed higher activity (up to essential placental CGB core promoter sequence 50-fold), compared with constructs without AP2 or SP1 (between K837 and K624) due to a significant loss of sites (120 RLU versus 2 RLU; Fig. 3). This correlates promoter activity after removal of this 200 bp and with studies showing placental transcriptional subjected this sequence to TFBS analysis. regulation by AP2 and SP1 in the human hCGB5 gene (Johnson & Jameson 1999) and by AP2 in the squirrel In silico prediction of TFBS within the placenta- monkey (Vasauskas et al. 2011). The GCMa factor was specific CGB core promoter excluded from further studies due to the minimal activity of the promoter constructs containing only the After identification of the placental core promoter, GCMa variant (K645/K381 vs K624/K381). we wanted to address its regulation via transcription. Within the placental CGB core promoter, several TFBS were predicted in silico by MatInspector, including Localization of AP2g, SP1, and CGB in the placenta three trans binding sites for early growth response protein 1 (EGR1; K875/K883, K744/K735, and Having excluded several possible candidate factors, K645/K639), five binding sites for SP1 (K885/ we examined whether the two remaining factors K879, K856/K849, K845/K838, K805/K798, and AP2 and SP1 are expressed together with CGB. We K655/K649), and two binding sites for AP2 (aliases found AP2g expression exclusively in the nuclei of www.endocrinology-journals.org Journal of Molecular Endocrinology (2011) 47, 285–298

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syncytiotrophoblasts and cytotrophoblasts of both CGB is expressed in syncytiotrophoblasts in the marmoset and human placenta (Fig. 4A, a–c). SP1 marmoset placenta. However, the strength of the signal expression in the marmoset placenta was detected in is weak compared with human tissue (Fig. 4A, g–i). varying levels in the nuclei of nearly all syncytiotropho- blasts, most cytotrophoblasts, but also in endothelial Synergistic effects of AP2 and SP1 on promoter and stromal cells. In the human placenta, SP1 activity expression was similarly detectable in most cell types but to a smaller extent than in the marmoset, Since it was previously demonstrated for human CGB particularly in syncytiotrophoblasts (Fig. 4A,d–f). that AP2 binding stimulates CGB expression, and a

A B (a) (b)me (c) Relative promoter activity c is 50 100 150 is st luc Empty vector fe v st v Wild type

AP2 M1

(d) (e)st (f) AP2 M2 me AP2 M1+2 v me is SP1 M1 v * st SP1 M2

is SP1 M1+2

AP2 M1+2 SP1 M1 (g) (h) (i) st AP2 M1+2 SP1 M2 is AP2 M1+2 SP1 M1+2 st is SP1 AP2

C 100 Wild type + SP1 and AP2 SP1 mutant + SP1 or AP2 80 Wild type + AP2 AP2 mutant + AP2 or SP1 Wild type + SP1 Wild type + SP1 (AP2 const·) 60 * * 40 20 D 0 Input IgG AP2 Pol II Input IgG SP1 Pol II –20 WT

Relative change in Relative –40 Input IgG AP2 Pol II Input IgG SP1 Pol II promoter activity (in %) promoter activity –60 MUT –80 * SP1 AP2 * –100 –837/–381 –837/–381 –837/–381 Mut Mut –837/–381

0·0 – 0·75 0·0 – 0·5 0·0 – 0·5 0·0 – 0·5 0·0 – 0·5 0·0 – 0·5 SP1 SP1/AP2 AP2 SP1 SP1 AP2 0·25 AP2 0·25 SP1 0·25 AP2 Figure 4 Immunohistochemistry, mutation analyses, and chromatin immunoprecipitation (ChIP) indicating importance of AP2 and SP1 for promoter activation. (A) Immunohistochemical detection of AP2g, SP1 and CGB in syncytiotrophoblasts of the marmoset placenta. (a–c) Staining for AP2g of marmoset placenta showed exclusive AP2g expression in syncytiotrophoblasts and cytotrophoblasts (small inset: negative control). Human term placenta showed a staining pattern identical to the marmoset. (d–f) Identical staining was also seen for marmoset and human SP1, with SP1 expression mainly in trophoblastic cells but also in endothelial cells and stromal cells. Human placenta has a similar SP1 pattern. (g–i) Marmoset placenta showing positive trophoblasts after staining for CGB. As control human placental trophoblasts were used that are also CGB positive. Second column showing magnification of the first one and the third column displays the human tissue as control. is, intervillous space; st, syncytiotrophoblasts; red asterisks (*), cytotrophoblast; c, fetal capillary; v, fetal vein; fe/me, fetal/maternal erythrocyte(s). Scale bar: 200 mm in a, d, and g and 50 mm in the other panels. (B) Promoter activity of mutated construct and untreated constructs in BeWo cells. The SP1 mutants and the AP2 double mutant cause a significant decrease in promoter activity. Values from three independent experiments (meanCS.E.M.) are normalized to pGL3 basic in each cell group (one-way ANOVA, *P!0.005). (C) Addition of AP2 increases promoter activity whereas SP1 decreases it. Activity of the promoter constructs in the presence of the transcription factors AP2 and SP1. Amounts of cotransfected expression plasmids are 0, 125, 250, and 500 ng (750 ng for AP2 and SP1 transfection). Every transfection was carried out with 3 mg of total DNA transfected (vehicle vector was added accordingly). Black bar: wild type with addition of both factors; checked gray bar: wild type with AP2 addition; striped bar: wild type with SP1 addition; small squared bar: SP1 mutant with SP1 or AP2 addition; gray bar: AP2 mutant with AP2 or SP1 addition; checked bars: wild type with constant AP2 level and SP1 addition. Data were normalized and are given as percentage compared to vehicle only transfections (exceptionK837/K381C250 ng AP2: 250 ng AP2 are already included) (D) ChIP revealed importance of AP2 and SP1 binding sites. The created constructs for ChIP are given on the left side, containing either a normal AP2/SP1 binding cassette (black box) or a mutated variant (AP2 only), where AP2 binding is abolished. On the right side, the PCR products of immunoprecipitation of transfected BeWo cells are presented. One ChIP contains the input probe (2% of total chromatin), the negative control (IgG), the target antibody (AP2 or SP1), and a positive control (RNA-Pol II).

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Downloaded from Bioscientifica.com at 09/29/2021 03:34:27AM via free access Regulation of CGB gene in the marmoset . C ADAMS, A HENKE and others 293 mutation of AP2 binding sites that eliminates AP2 Methylation analysis of the marmoset CGB promoter binding leads to decreased CGB activity (LiCalsi et al. The finding of different promoters linked to 2000), we mutated the two predicted AP2 binding sites different exons suggested the presence of an additional (K659/K650 and K641/K631) within the core tissue-specific regulatory mechanism. As mentioned in promoter sequence, according to LiCalsi et al. (2000). the introduction, there are several hints that epigenetic The constructs bearing both AP2 sites (K837 to regulation is important for CGB expression. Therefore, K381 bp) displayed higher activity, up to fivefold in we studied the role of methylation on CGB activity. the reporter assay, than constructs without AP2 sites (120 RLU versus 25 RLU; Fig. 3). Mutating each AP2 A Analyzed sequence site alone decreased the activity by 50% (60 RLU; M2) A BC or 33% (80 RLU; M1) compared with the wild-type K K construct (WT, construct 837/ 381) in transfected –1434 –1307–1208 –661 –512–411 +773 +995 Novel Pituitary Conv. Exon 2-3 BeWo cells (Fig. 4B). LiCalsi et al. (2000) hypothesized exon 1 promoter exon 1 Mean methylation throughout the analyzed sequence (in %) a possible interaction of SP1 with AP2 activation. B 10 20 30 40 50 60 70 80 90 100 Mutations of the two SP1 core promoter binding sites K K K K Placental promoter core (B) ** ( 805/ 798 and 655/ 649) according to LiCalsi ** et al. (2000) showed similar effects, which SP1 M1 Placental promoter all (A) *** decreased activity to 25% (35 RLU), whereas SP1 M2 * mutation showed 20% (23 RLU) remaining activity.

The SP1 M1C2 double mutant showed 15% activity Proximal placental promoter (C) **

(20 RLU), which was similar to the activity of the Blood Pituitary Placenta construct bearing no AP2 and SP1 sites (K645/K381, Relative promoter activity C 25 50 250 5002500 5000 7500 Fig. 4B). Additional mutations of AP2 and SP1 sites Empty reporter vector (AP2 M1C2 and SP1 M1 or AP2 M1C2 and SP1 M2) as Empty reporter vector meth Constitutive promoter well as the mutation of all four binding sites further Constitutive promoter meth NS decreased promoter activity to 15% (15–20 RLU). 126 bp core fragment 126 bp core fragment meth * Mutation of the distal EGR1 binding site, however, 67 bp core fragment showed no effect on placental promoter activity (data 67 bp core fragment meth * not shown). In COS7 cells, these effects were absent, 18 bp core fragment 18 bp core fragment meth * indicating a cell-specific regulatory mechanism only 10 bp core fragment present in the placenta and displayed by the tropho- 10 bp core fragment meth * blastic cell line BeWo (data not shown). Co-transfection Empty vector CMV prom. 126 bp 67 bp 18 bp 10 bp of the placental CGB wild-type core promoter construct Figure 5 Methylation analyses indicating CGB promoter regulation. (A) In silico prediction of CpG islands in the with either AP2 or SP1 overexpression constructs alone marmoset CGB promoter and gene. The placenta-specific displayed a moderate stimulative effect or no significant promoter, the novel exon, the pituitary promoter, and the CGB upregulation of the reporter respectively. However, gene are shown. Predicted CpG islands are given in gray with simultaneous addition of both factors even at lower starting and end point relative to the conventional exon 1. The arrows above indicate the analyzed sequence and the dashed box concentrations resulted in an increase yet a decrease represents the placental core promoter. (B) Hypomethylation of at high concentrations. Transfecting the promoter the placental core promoter. Mean methylation of the CpG island constructs that were mutated either at the AP2 or at (gray boxes in A) within the placental promoter in the placenta the SP1 binding site, in the presence of a vehicle only, (white bars), pituitary (gray bars), and blood (black bars). Values are given for the placental promoter in its entirety (K961 to K624), led to a slight non-significant decrease in activity the placental proximal promoter (K624 to K381), and the (Fig. 4C). Interestingly, transfecting cells with the placental core promoter (K837 to K624, dashed box in A). wild-type promoter construct and 0.25 mg AP2 with The placental core promoter (K837 to K624) was significantly increasing levels of SP1 showed an inhibitory effect hypomethylated in the placenta as well as the placental promoter full, whereas it was hypermethylated in the pituitary. Analysis of after activation with AP2 alone. genomic DNA from marmoset placenta (nZ3) and pituitary tissue In order to confirm AP2 and SP1 binding to the CGB (nZ4) and EDTA blood (nZ4). One-way ANOVA; *P!0.05, promoter, we performed ChIP analyses. Enrichment of **P!0.01, ***P!0.001. (C) In vitro methylation inhibits promoter AP2 and SP1 at the promoter could be demonstrated activity. CGB core promoter constructs of different length with pCpG luciferase reporter vector backbone transfected in BeWo by PCR (Fig. 4D). Mutation of the respective binding cells with (labeled: -meth) and without in vitro methylation. White sequences resulted in signal loss for AP2 and a bar with inlet: empty vector; light gray bars: CMV promoter; gray signal reduction for SP1. For SP1, these conclusions bars: 128 bp fragment; black bars: 67 bp fragment; white bars: 18 bp fragment; dark gray bars: 10 bp fragment. Values were were drawn from the SP1 band intensities being above normalized to pCpG basic, pCpG CMV served as positive control. or, in case of the mutated form, below the surprisingly RLU, relative luciferase units. Values are meanCS.E.M. One-way high IgG signal. ANOVA; *P!0.005. www.endocrinology-journals.org Journal of Molecular Endocrinology (2011) 47, 285–298

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We first determined the CG base content within the biomedicine in general and in reproductive research placental promoter sequence, as high CG content is specifically (Moore et al. 1985, Hearn 1986, Saunders indicative of possible regions for methylation. The high et al. 1987, Smith et al. 1987, Summers et al. 1987, Hearn CG bases number in the placental CGB promoter et al. 1988), we previously found that CG is strongly region (75% compared with 60% in the pituitary and expressed in the marmoset pituitary instead of LH gene) points to the potential of CGB regulation via (Muller et al. 2004b). This finding was confirmed when epigenetic mechanisms. MethPrimer software (Li & only CGB but not LHB gene expression was found in the Dahiya 2002) was used to predict four CpG islands NWM Ma’s night monkey (Aotus nancymaae) and the in silico in the marmoset CGB. Two are located in the brown squirrel monkey (Saimiri boliviensis; Scammell placental promoter (K1434/K1307 and K1208/ et al. 2008). An investigation of CGB gene copy numbers K661), one in the novel exon 1 (K512/K411), and in different primate taxa revealed that CGB is a single- one in exon 3 (C773/C955), indicated in Fig. 5A. copy gene in NWM (Maston & Ruvolo 2002), which As the second CpG-island within the placental raises the question ‘how is the marmoset able to promoter is the longest, spanning nearly the complete differentially regulate CGB expression in the pituitary promoter, it was chosen for further analysis by bisulfite and placenta?’ We have previously shown that the sequencing. Interestingly, no CpG islands were iden- regulation of CGB expression is similar to that of tified in the pituitary promoter (Fig. 5A). human LHB in the marmoset pituitary (Henke et al. A genomic DNA fragment containing the placental 2007). Both genes have a similar short 50-UTR of the promoter region (K964/K381) was isolated, bisulfite mRNA, are GNRH responsive, and have identical treated, and sequenced (nZ3 for placenta, nZ4 for binding sites for the transcription factor pituitary- blood and pituitary). The analysis showed that the induced factor (PITX1), stimulating factor (SF1), and mean CGB methylation level is drastically lowered in early growth receptor (EGR1) in the pituitary-specific the placenta compared with the other tissues (Fig. 5B). core promoter region (Henke et al. 2007). In the In the placenta, the mean methylation level in the current study, we investigated placental CGB regulation promoter (K961 to K624) throughout the analyzed in the marmoset and identified a new exon 1, termed G sequence was 10.8 3.7%, significantly lower than ‘novel exon 1 (nExon 1)’, to distinguish it from the G pituitary tissue (27.5 6.1%) and blood leukocytes conventional exon 1, which is present only in OWM G (45 4.5%). Interestingly, the placental core promoter CGB genes. It has its own TSS, located more than 200 bp K K ( 837 to 624) showed a significant hypermethy- upstream of the human CGB TSS, with the region G lation, which was much stricter (28.1 0.2% vs 65.4 involved in transcriptional regulation in orthologous G . . G . 0 9 8% vs 62 5 5 4%). However, mean methylation in human CGB5 being now part of the marmoset’s 5 -UTR the placental CpG island increased toward the novel of placental CGB. Thus, we discovered tissue-specific exon 1 in placental tissue. CGB isoforms between the marmoset monkey pituitary We next examined whether the methylation does and the placenta. The detection of tissue-specific indeed downregulate CGB transcription. The CGB transcripts is not exclusive to the marmoset. Several core promoter sequences were cloned into the genes share the phenomenon of multiple variable first methylation insensitive vector pCpG (Klug & Rehli exons, e.g. the novel spermatogenesis regulator in 2006) and in vitro DNA methylation was performed. Drosophila (Ding et al. 2010). Typically, tissue-specific Promoter activity after in vitro methylation was expression is predominantly found in 70–80% of dramatically decreased compared with the activity of mammalian regulatory genes (Lareau et al. 2004, non-methylated plasmids (Fig. 5C). Compared to the Ben-Dov et al. 2008). results of unmethylated plasmid constructs, activity Very recently, the CGB1/2 gene expression was declined significantly to 5–10% in all methylated confirmed in the human testis in alternative splice plasmids. As shown in Fig. 5C, CMV promoter activity forms, one of which could potentially give rise to a was not affected by in vitro methylation, proving that normal CGB protein. So far, no evidence exists for the the vector backbone was insensible to the in vitro functionality of CGB1 or CGB2 (Parrott et al. 2011). In methylation procedure. contrast, the marmoset CGB is actually the only known glycoprotein hormone gene that encodes two different peptides due to alternative promoter/exon 1 usage. Discussion This situation could potentially affect other processes, e.g. mRNA nuclear export, cytoplasmic localization, In anthropoid primates, including humans, CGB translational efficiency, and stability and regulatory expression has been limited to the trophoblast and motifs (Hughes 2006). placental syncytiotrophoblast, with minimal amounts in We also investigated the prevalence of the novel exon the pituitary (Dirnhofer et al. 1996). For the common 1 within the primate taxon. Interestingly, the putative marmoset, which serves as a preferred primate model in novel exon 1 was found in all NWM analyzed but not

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Downloaded from Bioscientifica.com at 09/29/2021 03:34:27AM via free access Regulation of CGB gene in the marmoset . C ADAMS, A HENKE and others 295 other primate species. Despite the high-sequence to that described for human CGB (Johnson et al. 1997): homology among LHB/CGB genes (85–99%), there hereby, SP1 binding is the prerequisite for AP2 are signs of functional differentiation among the gene binding, confirmed by Vasauskas et al. (2011), who copies (Hallast et al. 2008). It seems that in the NWM, independently identified a functional AP2 binding site the exon gains function during evolution, whereas in in the CGB promoter of the NWM squirrel monkey the OWM and humans no such function evolves. The (Vasauskas et al. 2011). Therefore, we propose a differential use of alternative first exons often indicates concurring model for AP2 and SP1 regulation of a two-promoter system. In a previous study, we placental CGB (Fig. 6A) in which one SP1 site acts as demonstrated that marmoset CGB expression in the a linker between AP2 and the other as an enhancer of pituitary is GNRH inducible and driven by SF1, EGR1, the AP2 and SP1 cassette. This model combines our and PITX1 (Henke et al.2007). Conversely, the findings with those of Johnson et al. (1997) and LiCalsi placental core promoter seemed to be regulated et al. (2000), for control of the human CGB promoter by differently. In silico predictions underlined the import- SP1, SP3, and AP2 (Fig. 6B). In this case, two clusters ance of two transcription factors, SP1 and AP2, in the exist. At the first site, SP1 and AP2 binding sites overlap, activation of placental CGB expression. We showed the and binding is mutually exclusive. The second site presence of SP1 and AP2g in the nuclei of syncytio- shows similarity to our findings in the marmoset. At this trophoblasts in marmoset placenta, which also express site, comparative SP1 and AP2 binding exists. The CGB. Previous investigations showed that CG is solely expressed in syncytiotrophoblasts in placentas (Gauster A & Huppertz 2008), leading us to conclude that AP2g Placenta (marmoset) SP1 and SP1 protein is present in CGB-expressing cells. This CGB mRNA SP1 premise was confirmed by the detection of CGB in AP2 AP2 nEx1 cEx1 syncytiotrophoblasts. In addition, our promoter mutagenesis and ChIP Binding cassette experiments confirmed AP2 and SP1 as transcription factors binding to the CGB promoter. Compared to IgG Pituitary (marmoset) and polymerase, which display similar signal strength, AP2 and SP1 the signal of the SP1 fraction is enhanced in the wild CGB mRNA type, suggesting an enrichment of SP1. The weaker SF1 EGR signal in the SP1 fraction with mutated DNA sequence Placental promoter Pituitary promoter is due to a loss of SP1 binding to the SP1 binding site. In B Placenta (human) order to successfully perform the SP1 ChIP, the method cAMP has been slightly modified. This could be the reason why we observed a higher background in the IgG AP2 SP3 fraction. Nevertheless, the loss of SP1 binding could be SP1AP2 SP1 AP2 clearly identified by the signal strength above back- ground in the wild type and the weaker signal in the Binding cassette mutated fraction. SP3 The presence of AP2 and SP1 binding sites within the Figure 6 Current model of placental CGB activation by SP1 and placental core promoter correlates with previous AP2. (A) Model for CGB regulation in the marmoset placenta and pituitary. Placental expression is controlled by SP1, in human CGB studies that show the existence of two combination with AP2 binding to specific regions within the minimal clusters of TFBS in the human CGB5 promoter (Bo & CG core promoter (binding cassette, white box). The distal SP1 Boime 1992, Johnson et al. 1997). Within these clusters, acts as an enhancer on the proximal SP1/AP2 binding cassette. AP2 and SP1 were the most prominent factors (Steger SP1 serves as a linker between the two AP2 sites (black circles). The loss of one of these components leads to a decrease in CG et al. 1993, 1994, LiCalsi et al. 2000, Thackray et al. 2010). expression. The promoter sequence in the placental tissue is Johnson et al. (1997) also showed that AP2 and SP1 activated by hypomethylation (small open circles). This leads to cooperate to induce gene activation, a finding con- expression of the placental CGB variant. In the pituitary, the binding firmed in our overexpression experiments. It appears of AP2 and SP1 is disturbed, because of methylated sequences that the model postulated by Johnson et al. (1997) and (small filled black circles) within the binding cassette. In the pituitary, the activation is triggered by EGR1 and SF1 (black LiCalsi et al. (2000) for the activation of human CGB hexagonal boxes). Therefore, stimulation of placental CGB expression in general also holds true for the marmoset, expression is not that strong. The novel exon 1 is represented by albeit with some differences. First is the enhancer the black box between the two TSS (angled black arrows) sites and function of the SP1 site (K805/K796), which had been the conventional exon is given by the gray box. (B) Model for human placental CGB regulation. In the human, placental CGB regulation postulated (Johnson et al.1997). Additionally, our is controlled by a similar binding cassette, an SP1 site is flanked by results indicate that the proximal SP1 site functions as two AP2 sites. The AP2 binding is stimulated by cAMP and SP1 a linker between the AP2 sites, forming a cassette, akin itself interacts with SP3. Adapted from Johnson et al. (1997). www.endocrinology-journals.org Journal of Molecular Endocrinology (2011) 47, 285–298

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different effects of AP2 and SP1 (activation versus promoter region, leading to increased binding by AP2 inhibition) seem to be present in the marmoset as well, and SP1 (Nowak-Markwitz et al. 2004a,b, Rull et al. as indicated by our overexpression experiments. 2008a). However, further studies addressing the Adding low levels of equimolar concentrations of AP2 influence of methylation on CGB promoter regulation and SP1 led to an increase in promoter activity but this are necessary. decreased when high concentrations were added. This In summary, this study gives new insights into the might be due to the action of SP1 that at high levels placental regulation of marmoset CGB via a novel two- may reverse the activating effect of AP2, but more likely promoter, one-gene system. Thus, our findings rep- the high concentrations of these factors are far beyond resent a hitherto unknown scenario of evolutionary their normal physiological range. This proposition is in plasticity in the CG hormonal system in NWM, which agreement with the finding that AP2 alone increases involves regulation by transcription factors as well as promoter activity while SP1 alone neither stimulates epigenetic mechanisms to address tissue-specific nor inhibits promoter activity. Our findings that the expression. addition of increasing amounts of SP1 to constant AP2 levels, however, do display an inhibitory effect, add evidence to the concept of a balanced interaction of Supplementary data AP2 and SP1. Hence, more AP2 leads to activation, This is linked to the online version of the paper at http://dx.doi.org/ more SP1 to inactivation of the mCGB promoter. 10.1530/JME-11-0026. This ability of SP1 to inhibit promoter activity was also shown by Novikov & Kamps (2001) who found it acting on peroxisomal multifunctional enzyme type 2 Declaration of interest (perMFE2), indicating that the regulation (activation or repression) may be dependent on the level of AP2 The authors declare that there is no conflict of interest that could be and SP1 in the marmoset trophoblasts. perceived as prejudicing the impartiality of the research reported. In addition to regulation by transcription factors, several genes have been described that are regulated via differential promoter methylation at the epigenetic Funding level. DNA methylation is associated with 56% of the This study was supported by the Deutsche Forschungsgemeinschaft promoters of mammalian genes (Ben-Dov et al. 2008). (DFG, grant nos GR1574-6/8-1), the funding of Innovative Medizi- Recently, a study demonstrated different methylation nische Forschung (IMF grant SI520601) of the University of Mu¨nster, patterns in CGB5 and -8 depending on whether and the Alexander-von-Humboldt Foundation. paternally or maternally inherited (Uuskula et al. 2010). There is an inverse relationship between CpG methylation and transcriptional activity (Bou et al. Acknowledgements 1987), as shown previously for the placental CGB The authors thank Prof. Pamela Mellon (UCSD, La Jolla, USA) for locus in the human placenta, i.e. significant hypo- donating the AP2 expression plasmid, Reinhild Sandhowe-Klaver- methylation is accompanied by high promoter activity kamp for technical assistance in cell culture, Elisabeth Lahrmann for (Campain et al. 1993). Our results strongly suggest an assistance in molecular biology, Dr Simon Riley for advice, and Dr epigenetic regulation of the placental CGB promoter James L Hutchinson and Dr Con Mallidis for language editing and with a different methylation in pituitary than in the comments. placenta and a hypomethylation in the placenta itself. This finding was confirmed by our in vitro methylation studies, in which methylation led to a dramatic loss of References placental CGB promoter activity compared with the unmodified promoter. The link between methylation Ben-Dov C, Hartmann B, Lundgren J & Valcarcel J 2008 Genome-wide analysis of alternative pre-mRNA splicing. Journal of Biological and transcription factor regulation has been shown by Chemistry 283 1229–1233. (doi:10.1074/jbc.R700033200) several groups before, leading to speculation concern- Bo M & Boime I 1992 Identification of the transcriptionally active ing the inhibitory effect of methylation by altered genes of the chorionic gonadotropin beta gene cluster in vivo. transcription factor binding (Watt & Molloy 1988, Journal of Biological Chemistry 267 3179–3184. Bou M, Hoshina M & Mochizuki M 1987 A study on the role of DNA Comb & Goodman 1990, Robertson et al. 1995, van methylation within hCG and hPL genes in trophoblastic disease. der Gun et al. 2011). Presumably, methylation leads to Nippon Sanka Fujinka Gakkai Zasshi 39 2144–2150. a block of AP2 or SP1 binding to the promoter Bousfield GR & Ward DN 2006 Gonadotropins: chemistry and sequence, resulting in a decrease in promoter activity. physiology. In Knobil and Neill’s Physiology of Reproduction, pp 1581– 1634. Ed JD Neill. St. Luis, MO, USA: Elsevier Academic Press. Interestingly, this might explain the observed high Campain JA, Gutkin DW & Cox GS 1993 Differential DNA methylation levels of hCGB expression in some tumors, which of the chorionic gonadotropin beta-subunit multigene family. may be linked to complete demethylation of the Molecular Endocrinology 7 1331–1346. (doi:10.1210/me.7.10.1331)

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(doi:10.1210/me.6.6.951) Made available online as an Accepted Preprint 5 August 2011

Journal of Molecular Endocrinology (2011) 47, 285–298 www.endocrinology-journals.org

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