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HLA-G Transactivation by cAMP-response Element-binding Protein (CREB)

2002; Elsevier BV; Volume: 277; Issue: 42 Linguagem: Inglês

10.1074/jbc.m112273200

1083-351X

Sam J. P. Gobin, Paula J. Biesta, Jurriaan E.M. de Steenwinkel, Gert Datema, Peter J. van den Elsen,

Reproductive Biology and Fertility

The expression of HLA-G in extravillous cytotrophoblast cells coincides with a general lack of classical major histocompatibility complex (MHC) class I expression in this tissue. This differential expression of HLA-G and classical HLA class I molecules in trophoblasts suggests a tight transcriptional control of MHC class I genes. Transactivation of the classical MHC class I genes is mediated by two groups of juxtaposed cis-acting elements that can be viewed as regulatory modules. Both modules are divergent in HLA-G, rendering this gene unresponsive to NF-κB, IRF1, and class II transactivator (CIITA)-mediated induction pathways. In this study, we searched for alternative regulatory elements in the 1438-bp HLA-G promoter region.HLA-G was not responsive to interferon-α (IFNα), IFNβ, or IFNγ, despite the presence of an upstream ISRE binding IRF1 in vitro. However, the HLA-G promoter contains three CRE/TRE elements with binding affinity for CREB/ATF and Fos/Jun proteins both in vitro and in vivo. In transient transfection assays, it was shown that HLA-Gtransactivation is regulated by CREB, CREB-binding protein (CBP), and p300. Moreover, immunohistochemical analysis demonstrated that HLA-G is co-expressed with CREB and CBP in extravillous cytotrophoblasts, revealing the in vivo relevance of this transactivation pathway. This implies a unique regulation of HLA-Gtranscription among the MHC class I genes. The expression of HLA-G in extravillous cytotrophoblast cells coincides with a general lack of classical major histocompatibility complex (MHC) class I expression in this tissue. This differential expression of HLA-G and classical HLA class I molecules in trophoblasts suggests a tight transcriptional control of MHC class I genes. Transactivation of the classical MHC class I genes is mediated by two groups of juxtaposed cis-acting elements that can be viewed as regulatory modules. Both modules are divergent in HLA-G, rendering this gene unresponsive to NF-κB, IRF1, and class II transactivator (CIITA)-mediated induction pathways. In this study, we searched for alternative regulatory elements in the 1438-bp HLA-G promoter region.HLA-G was not responsive to interferon-α (IFNα), IFNβ, or IFNγ, despite the presence of an upstream ISRE binding IRF1 in vitro. However, the HLA-G promoter contains three CRE/TRE elements with binding affinity for CREB/ATF and Fos/Jun proteins both in vitro and in vivo. In transient transfection assays, it was shown that HLA-Gtransactivation is regulated by CREB, CREB-binding protein (CBP), and p300. Moreover, immunohistochemical analysis demonstrated that HLA-G is co-expressed with CREB and CBP in extravillous cytotrophoblasts, revealing the in vivo relevance of this transactivation pathway. This implies a unique regulation of HLA-Gtranscription among the MHC class I genes. major histocompatibility complex antibody activation transcription factor class II transactivator CREB-binding protein CRE, cAMP-response element cAMP-response element-binding protein interferon γ-activated site inducible cAMP early repressor interferon interferon regulatory factor interferon-stimulated response element nuclear factor κB Rous sarcoma virus signal transducer and activator of transactivation 1 12-O-tetradecanoylphorbol-13-acetate TPA-response element The classical HLA class I molecules (HLA-A, HLA-B, and HLA-C) are highly polymorphic and are ubiquitously expressed on most somatic cells (reviewed in Ref. 1Le Bouteiller P. Crit. Rev. Immunol. 1994; 14: 89-129Crossref PubMed Google Scholar). They are essential in the immune response as they present antigen-derived peptides to cytotoxic T-lymphocytes and are important in protection against natural killer cell-mediated cytotoxicity. The nonclassical HLA class I molecules HLA-E, HLA-F, and HLA-G have a limited polymorphism and display a restricted expression pattern (reviewed in Refs. 1Le Bouteiller P. Crit. Rev. Immunol. 1994; 14: 89-129Crossref PubMed Google Scholar, 2Le Bouteiller P. Blaschitz A. Immunol. Rev. 1999; 167: 233-244Crossref PubMed Scopus (168) Google Scholar, 3Le Bouteiller P. Solier C. Microbes Infect. 2001; 3: 323-332Crossref PubMed Scopus (32) Google Scholar). Among the nonclassical HLA class I molecules, HLA-E and HLA-G have been shown to be able to present peptide (3Le Bouteiller P. Solier C. Microbes Infect. 2001; 3: 323-332Crossref PubMed Scopus (32) Google Scholar, 4O'Callaghan C.A. Bell J.I. Immunol. Rev. 1998; 163: 129-138Crossref PubMed Scopus (157) Google Scholar), whereas HLA-F is predominantly expressed as an empty intracellular molecule (5Wainwright S.D. Biro P.A. Holmes C.H. J. Immunol. 2000; 164: 319-328Crossref PubMed Scopus (93) Google Scholar). Although there is increasing evidence that nonclassical class I molecules could be important in protection against natural killer cell-mediated responses (2Le Bouteiller P. Blaschitz A. Immunol. Rev. 1999; 167: 233-244Crossref PubMed Scopus (168) Google Scholar, 3Le Bouteiller P. Solier C. Microbes Infect. 2001; 3: 323-332Crossref PubMed Scopus (32) Google Scholar), their exact role in T cell-mediated immune responses is still under debate. Additionally, HLA-G has been attributed alternative functional properties, such as interaction with and down-regulation of the function of CD4- and CD8-positive T cells (6Riteau B. Menier C. Khalil-Daher I. Sedlik C. Dausset J. Rouas-Freiss N. Carosella E.D. J. Reprod. Immunol. 1999; 43: 203-211Crossref PubMed Scopus (163) Google Scholar, 7Bainbridge D.R. Ellis S.A. Sargent I.L. J. Reprod. Immunol. 2000; 48: 17-26Crossref PubMed Scopus (232) Google Scholar, 8Fournel S. Aguerre-Girr M. Huc X. Lenfant F. Alam A. Toubert A. Bensussan A. Le Bouteiller P. J. Immunol. 2000; 15: 6100-6104Crossref Scopus (429) Google Scholar, 9Le Gal F.A. Riteau B. Sedlik C. Khalil-Daher I. Menier C. Dausset J. Guillet J.G. Carosella E.D. Rouas-Freiss N. Int. Immunol. 1999; 11: 1351-1356Crossref PubMed Scopus (279) Google Scholar, 10Lila N. Rouas-Freiss N. Dausset J. Carpentier A. Carosella E.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12150-12155Crossref PubMed Scopus (291) Google Scholar). Among the MHC1 class I molecules, the expression of HLA-G is particularly restricted and is found in few tissues, including extravillous cytotrophoblast cells (3Le Bouteiller P. Solier C. Microbes Infect. 2001; 3: 323-332Crossref PubMed Scopus (32) Google Scholar, 11Carosella E.D. Dausset J. Kirszenbaum M. Immunol. Today. 1996; 17: 407-409Abstract Full Text PDF PubMed Scopus (113) Google Scholar). Coinciding with the expression of HLA-G in this tissue is a general lack of classical MHC class I expression (3Le Bouteiller P. Solier C. Microbes Infect. 2001; 3: 323-332Crossref PubMed Scopus (32) Google Scholar, 5Wainwright S.D. Biro P.A. Holmes C.H. J. Immunol. 2000; 164: 319-328Crossref PubMed Scopus (93) Google Scholar). Together, this is proposed to be of importance for materno-fetal tolerance (2Le Bouteiller P. Blaschitz A. Immunol. Rev. 1999; 167: 233-244Crossref PubMed Scopus (168) Google Scholar, 3Le Bouteiller P. Solier C. Microbes Infect. 2001; 3: 323-332Crossref PubMed Scopus (32) Google Scholar,11Carosella E.D. Dausset J. Kirszenbaum M. Immunol. Today. 1996; 17: 407-409Abstract Full Text PDF PubMed Scopus (113) Google Scholar, 12Loke Y.W. King A. Curr. Opin. Immunol. 1991; 3: 762-766Crossref PubMed Scopus (53) Google Scholar, 13Ober C. Am. J. Hum. Genet. 1998; 62: 1-5Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The differential expression of HLA-G and classical HLA class I molecules in trophoblasts suggests a tight transcriptional control, which favors the expression of HLA-G and represses the expression of the classical class I genes HLA-A andHLA-B. Transcriptional control of classical MHC class I genes is mediated by conserved cis-acting regulatory elements in the proximal promoter region. These regulatory elements include enhancer A, the ISRE, and the SXY module (14Van den Elsen P.J. Gobin S.J. van Eggermond M.C. Peijnenburg A. Immunogenetics. 1998; 48: 208-221Crossref PubMed Scopus (123) Google Scholar). Enhancer A and the ISRE mediate the constitutive and cytokine-induced expression of MHC class I (15Gobin S.J.P. Keijsers V. Van Zutphen M. Van den Elsen P.J. J. Immunol. 1998; 161: 2276-2283PubMed Google Scholar, 16Gobin S.J.P. Van Zutphen M. Woltman A.M. Van den Elsen P.J. J. Immunol. 1999; 163: 1428-1434PubMed Google Scholar). The recently identified SXY module is important in the constitutive and CIITA-mediated transactivation (17Gobin S.J.P. Peijnenburg A. Van Eggermond M. Van Zutphen M. Van den Berg R. Van den Elsen P.J. Immunity. 1998; 9: 531-541Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). These classical regulatory elements are divergent in HLA-G, rendering this gene unresponsive to NF-κB-, IRF-1-, and CIITA-mediated induction pathways (18Gobin S.J.P. Van den Elsen P.J. Hum. Immunol. 2000; 61: 1102-1107Crossref PubMed Scopus (112) Google Scholar). In this study, we investigated the regulation of HLA-Gtransactivation and searched for alternative regulatory pathways. We assessed the capacity of putative regulatory elements in the promoter of HLA-G to bind transcription factors and to mediate transcriptional regulation of HLA-G. This search lead to the identification of three CRE/TRE elements, which bind proteins of the CREB/ATF and Fos/Jun families of transcription factors. CREB1 was able to stimulate HLA-G promoter activity in vitro, and the coactivators CBP and p300 enhanced this effect, revealing thatHLA-G is under the transcriptional control of the CREB/ATF family of proteins. The trophoblast-derived cell lines JEG-3 and JAR, the teratocarcinoma cell line Tera-2, the cervical carcinoma HeLa (American Type Culture Collection, Manassas, VA), the Burkitt lymphoma cell line Raji, and the T leukemia cell line Jurkat were grown in Iscove's modified Dulbecco's medium supplemented with 10% (v/v) heat-inactivated fetal calf serum (Invitrogen), penicillin (100 international units/ml), and streptomycin (100 μg/ml). Luciferase reporter plasmids used were generated by cloning genomic promoter fragments into pGL3-Basic (Promega, Madison, WI). These constructs contain, respectively, a 1438-bp promoter fragment ofHLA-G (pGL3-G1500), a 302-bpβ 2 m PCR-generated promoter fragment (pGL3-β2m), and a 269-bp AspI-AhaIIHLA-B7 promoter fragment (pGL3-HLA-B). The mutant promoter constructs of HLA-G were generated by overlap extension PCR and are based on pGL3-G1500. In the mutant promoter constructs, the putative CRE/TRE site(s) (underlined) were disrupted by introducing aSmaI site or related sequence (bold). The most upstream putative CRE/TRE site (CRE−1380) was flanked by an additional putative CRE/TRE site (both underlined). Therefore, in pGL3-Gm1, both CRE/TRE sites were mutated, changing the CRE−1380 from 5′-GAGAAATGACACACTCTGACTCATAGTAGCAGG-3′ into 5′-GAGAAACCGCCCACTCCCGGGCATAGTAGCAGG-3′. In pGL3-Gm2, the CRE−930 was changed from 5′-GGGGCATTGTGACTGCACTGAACAC-3′ into 5′-GGGGCATTGTCCCGGGGCACTGAACAC-3′, and in pGL3-Gm3, the CRE−770 was changed from 5′-AAACAAGCGGGAGTCACAGATACACT-3′ into 5′-AAACAAGCGCCCGGGACAGATACACT-3′. The mutant pGL3-Gm1–2-3 contained all three mutations in the CRE/TRE sites. All inserts were verified by sequence analysis. The expression vectors Rc/RSV-CREB1 and pECE/RSV-ATF1, Rc/RSV-CBP, and Rc/RSV-p300 and the Renilla luciferase control plasmid pRL-actin were described previously (19Gobin S.J.P. Van Zutphen M. Westerheide S.D. Boss J.M. Van den Elsen P.J. J. Immunol. 2001; 167: 5175-5184Crossref PubMed Scopus (75) Google Scholar). The expression vector pSG5-ICER was a kind gift of Dr. P. Quinn. Adherent cells were transfected by the calcium phosphate co-precipitation method with a DNA precipitate of 1 μg of pGL3 reporter plasmid, 1 or 0.5 μg of expression vector, and 0.1 μg of Renilla luciferase control plasmid (pRL-actin) per well. The cells were harvested 3 days after transfection. Luciferase activity was determined using a luminometer (Tropix, Badford, MA) and corrected for transfection efficiency with the Renilla luciferase activity values. Nuclear extracts were prepared as described before (16Gobin S.J.P. Van Zutphen M. Woltman A.M. Van den Elsen P.J. J. Immunol. 1999; 163: 1428-1434PubMed Google Scholar). Nuclear extracts (about 5 μg of protein) were incubated in DNA/protein binding buffer (20 mm Hepes, pH 7.9, 50 mm KCl, 10% v/v glycerol, 0.5 mmdithiothreitol, 0.1 mm EDTA) with 200 ng of poly(dI·dC), 200 ng of sonicated single stranded herring sperm DNA, and 1 ng of32P-radiolabeled probe for 30 min at 4 °C. The samples were run on a 6% non-denaturing polyacrylamide gel in 0.25× TBE buffer at 200 V for 2 h. The gels were fixed with a 10% methanol and 10% acetic acid solution, dried onto Whatman 3M paper, and exposed to an x-ray film. The following double-stranded oligonucleotides were used as probes and contained the putative CRE/TRE sites of HLA-G: CRE−1380, 5′-GAGAAATGACACACTCTGACTCATAGTAGCAGG-3′; CRE−930, 5′-GGGGCATTGTGACTGCACTGAACAC-3′; and CRE−770, 5′-AAACAAGCGGGAGTCACAGATACACT-3′. In addition, the following double-stranded oligonucleotides were used as probes for the putative ISRE and GAS sites: ISRE−754, 5′-CACTGTCTGGGAAAGTGAAACTTAAGAGCTTTGTGAGTC-3′; GAS−1020, 5′-ACTAAGTATTCCTAAAAAATATACAC-3′ and GAS−985, 5′-GTGGATACTTCCTAAAAACAGGCAGTG-3′. The double-stranded oligonucleotides used as consensus probes were: GASc, 5′-CCTGATTTCCCCGAAATGATG-3′ and CREc, 5′-AGAGATTGCCTGACGTCAGAGAGCTAG-3′. For the supershift assays, 1 μg of each Ab specifically directed against a member of the ATF/CREB or IRF families of transcription factors was added 30 min after the nuclear extract had been incubated with the probe, and this mixture was incubated for an extra hour at 4 °C. The antibodies used were directed against CREB1 (a kind gift of Dr. J. M. Boss; 20), CREB/ATF (sc-270), ATF1 (sc-243), CREB1 (sc-271), c-Fos (sc-52), c-Jun (sc-822), CBP (sc-7300), and mouse IRF-1 (sc-640) (all from Santa Cruz Biotechnology Inc., Santa Cruz, CA, except the first CREB1 antibody). Formaldehyde-crosslinked chromatin was prepared from 2 × 107 cells as described (20Moreno C.S. Beresford G.W. Louis-Plence P. Morris A.C. Boss J.M. Immunity. 1999; 10: 143-151Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). The chromatin suspension was precipitated with 5 μg of Ab and pulled-down with anti-rabbit and anti-mouse Ig-coupled magnetic beads (Dynal, Olso, Norway) or with protein A-Sepharose beads (Amersham Biosciences). After several wash steps, the samples were analyzed by PCR using the primers specific for the promoter region of HLA-G around CRE−1380 (GF−1461, 5′-TGCTCAAGTGCCTGACATTCTAGAAGCTTCACAAGAATGAGGTGGAGCC-3′; GR−1202, 5′-GCTCCTTTTCCTCACCTCCTGCTCCTCCAGCCCCTTCC-3′). The amplified PCR product is 259 bp. Human placenta tissue sections were prepared short after caesarian section delivery and snap-frozen in liquid nitrogen (tissue material kindly provided by Dr. C. A. Van Meir, Department of Obstetrics and Gynaecology, Leiden University Medical Center, Leiden, The Netherlands). Tissue sections were stored at −80 °C. Serial 5-μm-thick cryostat sections of placenta tissue were cut at −25 °C and collected on gelatin-coated glass slides. Tissue sections were dried overnight at 50 °C, fixed in acetone for 10 min, and blocked with 40% human serum and 3% bovine serum albumin in phosphate-buffered saline for 30 min. Next, the sections were incubated with primary Abs against keratin-7 (OVTL/12/30; IgG1, 1:400; DAKO, Carpintera, CA), HLA-G (G233, IgG2a, 1:200, kindly provided by Dr. J. W. Loke and Dr. A. King), CREB/ATF (sc-270, 4 μg/ml, Santa Cruz Biotechnology), and CBP (sc-7300, 4 μg/ml, Santa Cruz Biotechnology) for 30 min. The Abs DAK-G01 (IgG1; 1:50) and DAK-G05 (IgG2a, 1:100; DAKO) were used as isotype controls. After three washes with phosphate-buffered saline, the tissue sections were further incubated with labeled polymer conjugated to goat anti-mouse IgG (Envision+ System, DAKO) for 30 min. Tissue sections were washed three times and incubated with 0.5 mg/ml 3,3′-diaminobenzidine tetrahydrochloride (DAB; Sigma) in 50 mm Tris HCl, pH 7.6, containing 0.03% H2O2 for 5 min to give a brown-colored reaction product. The sections were counter-stained with hematoxylin. Our previous studies have shown that the classical regulatory elements found in other MHC class I proximal promoter regions, such as enhancer A, the ISRE, and SXY module, were not intact in the HLA-G promoter. Since HLA-G lacked these conserved regulatory regions in its proximal promoter region, we searched for putative regulatory elements in the extended promoter region of HLA-G that could provide for alternative transcriptional control mechanisms. A computer-aided search in the promoter region first led to the identification of one putative ISRE and two putative GAS sites. The two putative GAS sites (designated GAS−1020 and GAS−985) did not show any detectable STAT1 binding after IFNγ stimulation in JEG-3 nuclear extracts, whereas STAT1 binding to a consensus STAT1 binding site was readily detectable (data not shown). In contrast, the putative ISRE site (designated ISRE−754) showed binding of IRF1 using nuclear extracts from IFNγ-stimulated JEG-3 cells, but no binding of IRF2, IRF3, and STAT1 was detected (Fig.1 A). This IRF1 binding to ISRE−754 raised the possibility of this site to mediate IFNγ-induced transactivation of HLA-G. However, in transient transfection assays, no effect has been detected of IFNγ onHLA-G promoter activity in JEG-3 cells (Fig. 1 B). Similarly, induction of HLA-G promoter activity by IFNα and IFNβ was negligible in JEG-3 cells (Fig. 1 B). Previously, a repression of ISRE-mediated induction by IFNγ was detected in trophoblast-derived cell lines (18Gobin S.J.P. Van den Elsen P.J. Hum. Immunol. 2000; 61: 1102-1107Crossref PubMed Scopus (112) Google Scholar). Since this trophoblast-specific repression could be the cause of the repressed IFN induction of HLA-G in JEG-3 cells, we also tested IFN responses in Tera-2 cells, in which classical MHC class I genes are readily induced by IFNγ (16Gobin S.J.P. Van Zutphen M. Woltman A.M. Van den Elsen P.J. J. Immunol. 1999; 163: 1428-1434PubMed Google Scholar). However, also in this cell type, the promoter activity of HLA-G by IFNβ or IFNγ was not induced at greater than 2-fold (Fig. 1 C) despite a strong induction of HLA-B and β 2 mpromoter activity by these IFNs. This shows that these ISRE and GAS boxes do not play a significant role in HLA-Gtransactivation. A further search revealed the presence of three potential CRE/TRE sites (designated CRE−1380, CRE−930, and CRE−770) in the promoter of HLA-G. The most upstream of the three sequences (CRE−1380) was flanked by a second potential CRE/TRE site. Interestingly, this double CRE/TRE site (CRE−1380) was positioned in the region shown previously to be important for the tissue-specific expression of HLA-Gin transgenic mice (21Schmidt C.M. Ehlenfeldt R.G. Athanasiou M.C. Duvick L.A. Heinrichs H. David C.S. Orr H.T. J. Immunol. 1993; 151: 2633-2645PubMed Google Scholar). We first tested the three CRE/TRE sites (CRE−1380, CRE−930, and CRE−770) for binding activity of proteins of the CREB/ATF and Fos/Jun families of transcription factors in vitro. The most upstream CRE/TRE site (CRE−1380) showed binding of complexes containing CREB1, ATF1, and c-Jun (Fig. 2). The second CRE/TRE site (CRE−930) predominantly showed binding of a complex that contained predominantly CREB1, whereas the third CRE/TRE site (CRE−770) showed binding of CREB1 and ATF1 (Fig. 2). This implies that the CRE/TRE sites bind different heterodimers, which could be formed between CREB1, ATF1, and c-Jun, and that CREB1 was the predominant factor in the complexes binding to these sites. To establish the in vivo binding of CREB/ATF and Fos/Jun proteins, we performed a ChIP assay. Using this assay, immunoprecipitation of cross-linked JEG-3 DNA with Abs recognizing CREB/ATF, CREB, c-Fos, or c-Jun showed the in vivo binding of CREB-1 and c-Jun to the upstream of the HLA-G promoter region around CRE−1380 in JEG-3 cells (Fig.3). However, this binding appeared not to be tissue-specific because in vivo CREB binding to theHLA-G promoter was also detected in Tera-2 and Raji cells (data not shown). The contribution of these CRE/TRE sites to HLA-G transactivation was tested in a reporter gene assay using a construct encompassing the 1438-bp promoter region of HLA-G. Both CREB1 and ATF1 could enhanceHLA-G promoter activity (Fig.4 A). However, co-transfection of CREB1 with ATF1 could not further enhance transactivation (Fig. 4 A). Interestingly,HLA-B promoter activity was not significantly induced by CREB1 in JEG-3 cells (data not shown). Co-transfection of CREB1 with the coactivators CBP and p300 also enhanced CREB1-induced transactivation of HLA-G (Fig. 4 B). Neither c-Fos nor c-Jun could significantly stimulate promoter activity ofHLA-G (data not shown). CREB1 could also enhanceHLA-G promoter activity in Tera-2 cells, indicating that for this CREB-mediated transactivation, no trophoblast-specific transcription factors are required (data not shown). The role of CREB in the transactivation of HLA-G was further investigated by testing the effect of ICER, which can repress CREB-mediated transcription (22Molina C.A. Foulkes N.S. Lalli E. Sassone-Corsi P. Cell. 1993; 75: 875-886Abstract Full Text PDF PubMed Scopus (526) Google Scholar). In transient co-transfection experiments, ICER strongly inhibited the CREB-induced transactivation of HLA-G(Fig. 4 C). At higher concentrations (1–8 μg/well), ICER was also able to reduce the basal promoter activity of HLA-G(88–28% of normal; data not shown). To investigate the importance of the individual CRE/TRE sites in the transactivation of HLA-G, we introduced mutations in the CRE/TRE sites CRE−1380, CRE−930, and CRE−770. Transient transfection assays of these modifiedHLA-G constructs, bearing a mutation in the individual CRE/TRE sites (pGL3-Gm1, pGL3-Gm2, and pGL3-Gm3) or in all three CRE/TRE sites (pGL3-Gm1–2-3), revealed that mutation in only one of the three CRE sites resulted only in a strong reduction of transactivation, whereas mutation of all three CRE sites abrogated the ability of CREB to enhance the transactivation of HLA-G(Fig. 4 D). Furthermore, the basal levels of HLA-Gtransactivation of the mutation constructs were reduced to a various extent, being the lowest for the HLA-G construct with CRE−1380 mutated (pGL3-Gm1; Fig. 4 D). This indicates that the contribution of CRE−1380 was most important followed by CRE−930 and CRE−770. Similar results were observed in other cell lines, indicating that the importance of the three CRE/TRE sites for the basal level of promoter activity was ubiquitous (data not shown). Together, this demonstrates that the three CRE/TRE sites in the promoter region of HLA-Gare important for the CREB-mediated transactivation ofHLA-G. The promoter binding and regulation ofHLA-G by CREB in the trophoblast cell line JEG-3 inferred co-expression of HLA-G with its regulating transcription factors in trophoblast tissue. To investigate the physiological relevance of this transactivation pathway, we tested for the in situexpression of CREB, CBP, and HLA-G by immunohistochemical analysis. In caesarian section-derived placenta tissue, the extravillous cytotrophoblast cells were identified on the basis of their morphology and their strong staining for keratin-7 (data not shown). These extravillous cytotrophoblasts also stained strongly for HLA-G, particularly on the cell membrane (Fig.5 B). This expression of HLA-G in the cells coincided with the predominantly nuclear staining of CREB/ATF and CBP (Fig. 5, C and D). The in situnuclear localization of CREB/ATF and CBP indicates that these transcription factors could fulfil their role in HLA-Gtransactivation in trophoblast cells. Detailed studies have shown that HLA-G is found in extravillous cytotrophoblasts and few other tissues. The limited tissue distribution suggests that HLA-G has specialized immunological functions during pregnancy (3Le Bouteiller P. Solier C. Microbes Infect. 2001; 3: 323-332Crossref PubMed Scopus (32) Google Scholar, 11Carosella E.D. Dausset J. Kirszenbaum M. Immunol. Today. 1996; 17: 407-409Abstract Full Text PDF PubMed Scopus (113) Google Scholar, 23Le Bouteiller P. Solier C. Pröll J. Aguerre-Girr M. Fournel S. Lenfant F. Hum. Reprod. Update. 1999; 5: 223-233Crossref PubMed Scopus (102) Google Scholar). Among the MHC class I molecules, the regulation HLA-G is not well understood. HLA-G is not regulated by the transactivation pathways that govern the expression of the classical MHC class I molecules (Fig. 6). Conservedcis-acting regulatory elements that are known to govern the transcription of the classical class I molecules, such as enhancer A, the ISRE, and the SXY module, diverge significantly in HLA-G(18Gobin S.J.P. Van den Elsen P.J. Hum. Immunol. 2000; 61: 1102-1107Crossref PubMed Scopus (112) Google Scholar). The two putative κB sites of enhancer A of HLA-Ghave been shown to bind predominantly the p50 homodimer of NF-κB using nuclear extract of B cells (15Gobin S.J.P. Keijsers V. Van Zutphen M. Van den Elsen P.J. J. Immunol. 1998; 161: 2276-2283PubMed Google Scholar). However, in trophoblast cells, there is no (basal) NF-κB expression, and it is barely induced by tumor necrosis factor-α. 2S. J. P. Gobin, J. E. M. de Steenwinkel, P. Biesta, and P. J. Van den Elsen, unpublished results. Furthermore, p50 homodimers do not posses transactivation properties, which explains the lack of response to tumor necrosis factor-α and the lack of activation by cotransfected NF-κB in reporter gene assays.2 In addition, the most upstream of the two κB sites (κB2) has been found to bind also Sp1 (15Gobin S.J.P. Keijsers V. Van Zutphen M. Van den Elsen P.J. J. Immunol. 1998; 161: 2276-2283PubMed Google Scholar). The directly flanking and downstream positioned ISRE region is partly deleted and has no binding affinity for IRF1, IRF2, p48, or STAT1 (16Gobin S.J.P. Van Zutphen M. Woltman A.M. Van den Elsen P.J. J. Immunol. 1999; 163: 1428-1434PubMed Google Scholar). However, a probe encompassing this ISRE region was able to bind Sp1. It is possible that Sp1 acting through these sites could contribute to the basal level of HLA-G transactivation. The SXY module ofHLA-G is also different from the consensus sequences of the X1, X2, and Y boxes. Although others have detected a weak binding of RFX to the X1 box of HLA-G (24Rousseau P. Paul P. O'Brien M. Dausset J. Carosella E.D. Moreau P. Hum. Immunol. 2000; 61: 1132-1137Crossref PubMed Scopus (13) Google Scholar), our efforts to determine binding of protein complexes to the SXY module with electrophoretic mobility shift assay favoring multiprotein complex formation did not reveal any significant binding of the RFX·CREB/ATF·NFY complex, such as that observed for classical MHC class I genes (19Gobin S.J.P. Van Zutphen M. Westerheide S.D. Boss J.M. Van den Elsen P.J. J. Immunol. 2001; 167: 5175-5184Crossref PubMed Scopus (75) Google Scholar).2 Also, individual binding of CREB/ATF to the X2 box or NFY binding to the Y box of HLA-G was not observed in contrast to the binding of CREB/ATF and NFY to the X and Y boxes of HLA-B (19Gobin S.J.P. Van Zutphen M. Westerheide S.D. Boss J.M. Van den Elsen P.J. J. Immunol. 2001; 167: 5175-5184Crossref PubMed Scopus (75) Google Scholar).2 This explains the lack of induction of HLA-G by CIITA (14Van den Elsen P.J. Gobin S.J. van Eggermond M.C. Peijnenburg A. Immunogenetics. 1998; 48: 208-221Crossref PubMed Scopus (123) Google Scholar, 18Gobin S.J.P. Van den Elsen P.J. Hum. Immunol. 2000; 61: 1102-1107Crossref PubMed Scopus (112) Google Scholar). There is still much debate as to whether HLA-G is responsive to IFN. Previous reports have indicated that HLA-G is weakly induced by IFNγ and IFNβ (25Lefebvre S. Berrih-Aknin S. Adrian F. Moreau P. Poea S. Gourand L. Dausset J. Carosella E.D. Paul P. J. Biol. Chem. 2001; 276: 6133-61399Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). In this study, we identified one putative ISRE and two putative GAS sites. Indeed, the putative ISRE site (ISRE−754) showed binding of IRF1 using nuclear extracts from IFNγ-stimulated JEG-3 cells. However, no STAT1 binding was detected to the two putative GAS sites (GAS−1020 and GAS−985), which is in line with findings by others (26Chu W. Gao J. Murphy W.J. Hunt J.S. Hum Immunol. 1999; 60: 1113-1118Crossref PubMed Scopus (24) Google Scholar). The binding of IRF1 to the ISRE−754 raised the possibility that IFNγ could induce transactivation of HLA-G. However, in transient transfection assays, no effect was detected of IFNγ onHLA-G promoter activity in JEG-3 cells. Similarly, induction of HLA-G promoter activity by IFNα and IFNβ was negligible in JEG-3 cells. Also in Tera-2 cells, the promoter activity of HLA-G was never induced more strongly than 2-fold by IFNβ or IFNγ, which was in contrast to the strong induction of the promoter activity of HLA-B andβ 2 m by these IFNs. We therefore concluded that the extended promoter of HLA-G is not significantly responsive to stimulation by IFN. In our search for alternative regulatory pathways that control the transcription of HLA-G, we identified three CRE/TRE elements in the promoter region of HLA-G (Fig. 6). Using the trophoblast-derived cell line JEG-3, the sites bound CREB1, ATF1, and c-Jun in vitro. The binding of different proteins of the CREB/ATF and Fos/Jun families of proteins allows the formation of various dimers. However, the precise composition of the protein complexes binding to the promoter is not certain, and the participation of other transcription factors cannot be excluded. Furthermore, in ChIP assays, CREB1 and c-Jun were found associated to the upstream promoter region (containing CRE−1380) in vivo. Previously, this region has been associated with the tissue-specific expression of HLA-G in transgenic mice and was shown to display protein binding activity (21Schmidt C.M. Ehlenfeldt R.G. Athanasiou M.C. Duvick L.A. Heinrichs H. David C.S. Orr H.T. J. Immunol. 1993; 151: 2633-2645PubMed Google Scholar, 27Moreau P. Paul P. Gourand L. Prost S. Dausset J. Carosella E. Kirszenbaum M. Hum. Immunol. 1997; 52: 41-46Crossref PubMed Scopus (29) Google Scholar). Our data show that HLA-G transactivation is regulated by CREB since CREB augmented promoter activity and ICER reduced CREB-mediated promoter activity in reporter assays. In addition, the activation by CREB was further enhanced by overexpression of the coactivators CBP/p300. Furthermore, mutational analysis revealed the importance of all three CRE/TRE sites in the regulation ofHLA-G transcription by CREB and the basal level of promoter activity of HLA-G. CREB1 could also enhance HLA-Gpromoter activity in non-trophoblast cell lines, indicating that for this CREB-mediated transactivation, no trophoblast-specific transcription factors are required. However, HLA-B promoter activity was not significantly induced by CREB1 in JEG-3 cells, suggesting a differential regulation of HLA-G versus classical MHC class I promoters in trophoblasts. CREB is thought to be an important factor for the expression of genes in trophoblasts and during differentiation. Similar toHLA-G, the α subunit of glycoprotein hormone is regulated by multiple sites including CRE (28Heckert L.L. Schultz K. Nilson J.H. J. Biol. Chem. 1996; 271: 31650-31656Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 29Knöpfler M. Saleh L. Bauer S Vasicek R. Griesinger G. Strohmer H. Helmer H. Husslein P. Endocrinology. 2000; 141: 3737-3748Crossref PubMed Scopus (45) Google Scholar, 30Budworth P.R. Quinn P.G. Nilson J.H. Mol. Endocrinol. 1997; 11: 1669-1680PubMed Google Scholar). However, the tissue-specific expression of the α subunit of glycoprotein hormone was controlled by an additional regulatory element mediating the trophoblast-specific expression that is not found in the HLA-G promoter. Moreover, CREB/ATF proteins are ubiquitously expressed transcription factors, and ChIP analysis revealed the binding of CREB to theHLA-G promoter in several cell lines not expressing HLA-G. Therefore, they are not likely to account for tissue-specific expression, although it cannot be excluded that a difference between trophoblasts and other cells in the composition of the CREB/ATF dimer complex binding the CRE sites contributes to the trophoblast-specific expression (31Benbrook D.M. Jones N.C. Nucleic Acids Res. 1994; 22: 1463-1469Crossref PubMed Scopus (135) Google Scholar, 32Servillo G. Della Fazia M.A. Sassone-Corsi P. Exp. Cell Res. 2002; 275: 143-154Crossref PubMed Scopus (149) Google Scholar). Therefore, it remains to be determined whether a specific regulatory site or transcription factor(s) is responsible for the trophoblast-specific expression of HLA-G. It could also be envisaged that more general mechanisms play a role in the tissue-specific expression or silencing of HLA-G, such as promoter methylation. Indeed, treatment with 5-azacytidine brought about expression of HLA-G in several cell lines lacking HLA-G expression (JAR, HeLa) as detected by reverse transcriptase-PCR, whereas in other cell lines, no induction of HLA-G expression was observed. 3S. J. P. Gobin, P. Biesta, and P. J. Van den Elsen, unpublished results. However, in vitro promoter methylation of the pGL3-G1438 reporter construct only resulted in a weak reduction of promoter activity and of CREB-induced transactivation.3 Moreover, in several cell lines lacking HLA-G expression (Tera-2, Jurkat), no methylation of the HLA-G promoter was observed.3This suggests that, although it may contribute, promoter methylation is certainly not the sole mechanism securing a tissue-specific expression of HLA-G (33Guillaudeux T. D'Almeide M. Girr M. Rodriguez A.M. Pontarotti P. Fauchet R. Le Bouteiller P. Am. J. Reprod. Immunol. 1993; 30: 228-238Crossref PubMed Scopus (10) Google Scholar). Another general epigenetic mechanism that could play a role in the trophoblast-specific expression of HLA-G is the recruitment of histone deacetylase activity. As stated before, the HLA-G promoter contains two κB sites in enhancer A, which display binding affinity specifically for p50 homodimers (15Gobin S.J.P. Keijsers V. Van Zutphen M. Van den Elsen P.J. J. Immunol. 1998; 161: 2276-2283PubMed Google Scholar). Since the p50 subunit of NF-κB does not possess transactivating activity and NF-κB factors are present in most cell types but not in trophoblast cells, the functional relevance of this binding in non-trophoblast cells has remained unclear. However, a recent study has attributed a role for p50 to establish transcriptional silencing through its recruitment of HDAC1 (34Zhong H. May M, J. Jimi E. Ghosh S. Mol. Cell. 2002; 9: 625-636Abstract Full Text Full Text PDF PubMed Scopus (817) Google Scholar). It could be speculated that in non-trophoblast cells, p50 homodimer binding to the κB sites of HLA-G recruits HDAC1, which represses HLA-G transcription and as such contributes to the tissue-specific expression of HLA-G. Taken together, multiple CRE/TRE sites in the promoter region of HLA-G mediate its regulation by CREB/ATF factors. This is the first report of a regulatory pathway acting through the 1438-bp promoter region of HLA-G. We thank Dr. J. W. Loke, Dr. A. King, Dr. C. A. Van Meir, and Dr. P. Quinn for providing reagents and tissue material. We also thank Dr. N. Van der Stoep for critically reading the manuscript.