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Regular Article |
B and RBPJ in the Rat Pregnancy-Specific Glycoprotein Gene1
a Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei 115, Taiwan
b Graduate Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan
ABSTRACT
The rat pregnancy-specific glycoprotein gene rnCGM3 is primarily expressed in the placenta. Previously, three DNase I footprinting sites (FPI, FPII, and FPIII) were identified in the rnCGM3 promoter region, a yeast one-hybrid screen was performed to identify the nuclear factors binding to the FPIII (5'-GCCTGGGAAAAAACTC-3') element, and RBPJ
, a downstream effector of the Notch signaling pathway, was identified as one of the FPIII-binding factors. In the present study, the NFB member p65 was identified as another FPIII-binding factor. Electrophoretic mobility shift assays showed that NFB members, including p50 and p65, bound to the FPIII site. The core binding sequence in the FPIII element for p50 and p65 is GGGAAA, which overlaps with that for RBPJ. Competition exists between p50 and RBPJ for binding to the FPIII element. Transient expression analyses revealed that p65 significantly stimulated the expression of a reporter gene directed by the NFB core sequence in the FPIII element. However, RBPJ could block this stimulation. These results suggest that the regulation of rnCGM3 expression involves both NFB and RBPJ, and they are mutually exclusive in the FPIII element.
gene regulation, placenta, pregnancy, syncytiotrophoblast, trophoblast
INTRODUCTION
Pregnancy-specific glycoproteins (PSGs) are selectively expressed in the placenta [1]. In situ hybridization has further revealed that PSG is synthesized by the syncytiotrophoblast and spongiotrophoblast in the human and rat placenta, respectively [2, 3]. The concentration of human PSGs in maternal circulation reaches as high as 200–400 µg/ml at term [4]. Based on the sequence identity at nucleotide and amino acid levels, PSG family members and the carcinoembryonic antigen (CEA) family members were categorized into one subfamily within the immunoglobulin (Ig) superfamily [1, 5]. The protein structure of PSG/CEA family members consists of a leader peptide, an Ig variable (V)-like N-terminal domain, various numbers of Ig constant (C)-like domains, and a carboxyl domain of varying length [1, 5]. The carboxyl domain determines whether the PSG/CEA family members are cell-surface bound or secreted molecules. The CEA subfamily members are cell-surface bound molecules, whereas the PSG subfamily members are secreted molecules [1, 5]. Multiple functions have been attributed to the CEA subfamily members, including cell adhesion, receptors for bacteria and mouse hepatitis virus, and inhibition of tumor growth [6]. Although the physiologic roles of PSGs are currently unknown, significant physiologic functions of PSGs are suggested based on the fact that low concentrations of PSGs are associated with poor pregnancy outcomes [7–10].
Because the expression levels of PSG are specifically high in placenta, additional transcription factors might be involved in the upregulation of PSG expression. Two rodent PSG genes, rnCGM6 and rnCGM3, have been characterized, and both are primarily expressed in rat placenta [11, 12]. The protein structures of rnCGM6 and rnCGM3 contain three IgV-like domains, each preceded by a leader peptide, and an IgC-like domain [11, 12]. The 5' flanking region of rnCGM3 has also been sequenced, revealing a TATA box, CATAAA, located at nucleotides -226 to -221 upstream from the translation start site [11]. Moreover, DNase I-footprinting analyses three nuclear protein-binding sites at nucleotides -311 to -290 (FPI), -257 to -239 (FPII), and -108 to -93 (FPIII) [11]. The factor binding to the FPII site had been identified as C/EBPß by expression cloning [13].
To further understand the molecular mechanism of rnCGM3 expression, we recently performed a yeast one-hybrid screen to isolate the nuclear factors binding to the FPIII site. Because the expression patterns of the human and rat PSG genes are very similar, a commercial human placental cDNA library was used in this screen. One of the factors was identified as RBPJ, the downstream effector of the Notch signaling pathway [14]. In the present study, we identified another FPIII-binding factor as the NFB member p65. We also demonstrated that the other NFB member, p50, could bind to the FPIII site and that p65 transactivated FPIII-driven promoter constructs. In addition, RBPJ competed with p65 and p50 for binding to FPIII. Consequently, transactivation of the FPIII-driven promoter by p65 was suppressed by RBPJ. Our data suggest that the FPIII site is a composite cis-element for RBPJ and NFB in the promoter region of rnCGM3.
MATERIALS AND METHODS
Library Screening and Characterization of cDNA Clones
For the yeast one-hybrid screen, six tandem copies of the FPIII element (5'-GCCTGGGAAAAAACTC-3') were placed upstream of the minimal promoter of the yeast HIS3 gene in vector pHISi-1 (Clontech, Palo Alto, CA) to generate the target reporter plasmid, p(FPIII)6HIS3. The YM4271 yeast cells (MATa, ura3-52, his3-200, ade2-101, lys2-801, leu2-3, 112, trp1-903, and tyr1-501) were transformed with 2 µg of linearized p(FPIII)6HIS3 to select for positive reporter strains in a synthetic medium without histidine (SD/-His). One of the strains, YM4271FPIII, has a low level of HIS3 activity and did not replicate on SD/-His plates supplemented with 30 mM 3-aminotriazole (3-AT), a competitive inhibitor of the yeast HIS3 protein. For screening of FPIII-binding factors, the YM4271FPIII cells were transformed with 20 µg of a pACT2 human placental cDNA library (Clontech), in which the cDNAs are fused to the GAL4 activation domain. Approximately 5 x 106 yeast colonies were screened in a synthetic medium minus histidine and leucine and containing 30 mM of 3-AT. Positive colonies were purified after two more rounds of streaking on selection plates. The pACT2 plasmids were rescued from the positive clones, retransformed into Escherichia coli strain HB101, and purified for sequencing.
Plasmid Constructs
The NFB expression plasmids pCMVp65 and pCMVp50 were kindly provided by Dr. T.-H. Tan (Baylor College of Medicine, Houston, TX). The pCMVp5065 chimera construct was built by ligating the DNA-binding domain of p50 with the transactivation domain of p65 as previously described [15]. The coding region of rRBPJ-2N was amplified by polymerase chain reaction (PCR) with the Pfu DNA polymerase (Stratagene, La Jolla, CA) and cloned into pRcCMV to make the pCMVRBP plasmid. The plasmid p(FPIII)6E1bCAT contains six FPIII sites fused to a minimal promoter element from the adenovirus E1B gene and the bacterial CAT (chloramphenicol acetyltransferase) coding sequence. Five copies of M34 (5'-GCTAGGGAAAAAACTC-3') and four copies of M56 (5'-GCCTCCGAAAAAACTC-3') oligonucleotides were inserted in front of the minimal promoter of E1B gene to generate the p(FPIIIM34)5E1bCAT and p(FPIIIM56)4E1bCAT reporter plasmids, respectively. All constructs were verified by DNA sequencing using the Sanger dideoxy chain-termination method [16].
Electrophoretic Mobility Shift Assay
To prepare NFB-enriched nuclear extracts, cells were transfected with pCMVp65 or pCMVp50 or with both plasmids. Cells in a 100-mm culture plate were incubated with 10 ml of a calcium phosphate-DNA coprecipitate containing 25 µg of expression plasmid. Twenty plates of transfected cells were collected for nuclear extracts preparation 48 h after transfection. Nuclear extracts were prepared as described by Ohlsson and Edlund [17]. The p50 and p65 proteins in the nuclear extracts were detected by Western analysis with a p50-specific antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and a p65-specific antibody (Santa Cruz Biotechnology), respectively. In vitro transcription/translation of p65 and p50 cDNAs was performed using the TNT (transcription and translation)-coupled wheat germ extract system (Promega, Madison, WI). For the electrophoretic mobility shift assay (EMSA), end-labeled oligonucleotide FPIII probes (1 ng; 0.5–1 x 105 cpm) were incubated for 20 min at room temperature in binding reaction buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.05% NP-40, 0.5 mM dithiothreitol, and 10% glycerol) containing 1 µg of poly(dI-dC) and 2 µg of nuclear extracts or 1 µl of in vitro-translated proteins. After incubation at room temperature for 20 min, the reaction mixtures were analyzed by electrophoresis on 5% nondenaturing polyacrylamide gels. For competition experiments, the unlabeled oligonucleotides were used at 50- to 200-fold molar excess and incubated with the cell extracts for 20 min on ice before adding radiolabeled probe. For comparison, the signals of binding complexes were quantitated by the image densimeter (Bio-Rad, Hercules, CA). Oligonucleotides used in EMSA were FPIII (5'-GCCTGGGAAAAAACTC), m8 (5'-GGGCACTGTGGGAACGGAAA), and light chain (5'-CAGAGGGGACTTTCCGAGAG).
Cell Culture, Transfection, and Reporter Gene Assays
The human embryonic kidney cell line 293 was obtained from the American Type Culture Collection (Manassas, VA) and maintained at 37°C in Hepes-buffered Dulbecco modified minimal essential medium supplemented with 4% fetal bovine serum, streptomycin (100 µg/ml), and penicillin (100 U/ml). The 293 cells in the 30-mm culture plate were incubated with 2 ml of a calcium phosphate-DNA coprecipitate containing 3 µg of reporter plasmid and various amounts of expression plasmid (see figure captions). The pRcCMV empty vector was included, if necessary, to maintain a constant amount of DNA in the transfection assay. Cells were harvested in the reporter lysis buffer (Promega) 48 h after transfection. CAT activity was assayed by incubating total cellular proteins in the reporter lysis buffer containing 4 mM acetyl coenzyme A and 0.1 µCi of [14C]chloramphenicol. The assay was run for 1 h at 37°C with the amount of extracts required to convert 0.5–50% of the substrate to the acetylated forms. The acetylated compounds were separated from the chloramphenicol by thin-layer chromatography and quantitated by a bio-imaging analyzer (BAS-1500; Fujifilm, Minami-Ashigara-Shi, Kanagawa, Japan). Protein concentrations were measured using the BCA protein assay kit (Pierce, Rockford, IL). The Student t-test was used to determine statistical significance for differences between means. A P value of less than 0.05 was considered significant.
Binding Competition Assay for RBPJ and p50
NH2-terminally His-tagged rRBPJ-2N (HisRBP) proteins were prepared using the baculovirus expression system. The insect cell line Sf9 (Gibco BRL, Grand Island, NY) was maintained in the serum-free medium, Sf-900II SFM medium (Gibco BRL) supplemented with 0.125 µg/ml of amphotericin B, 50 µg/ml of streptomycin, and 50 U/ml of penicillin, as suspension cultures at 28°C. The rRBPJ-2N cDNA was subcloned into the transfer plasmid, pAcSGHisNT-B (Pharmingen, San Diego, CA). Recombinant baculoviruses were established using a commercial kit (Novagen, Madison, WI). Sf-9 cells were infected with the recombinant baculoviruses at a multiplicity of infection of 5 for 72 h. HisRBP proteins were purified using a cobalt affinity column (Clontech). The HisRBP proteins obtained were more than 95% pure and were used in this study. The recombinant p50 proteins were purchased from Promega.
A biotinylated oligonucleotide containing two copies of the FPIII sense-strand sequence was annealed with a complementary oligonucleotide. The annealed oligonucleotide, Biotin-(FPIII)2, was attached to streptavidin-conjugated magnetic beads (Dynal Biotech, Oslo, Norway). For binding competition assay, the beads were incubated with 4 µg of HisRBP proteins and increasing amounts of p50 proteins in the binding solution (50 mM Hepes, pH 7.5, 10% glycerol, 0.05% NP-40, 50 mM NaCl, 5 mM EDTA, 10 mM MgCl2) at 4°C overnight. After extensive washing with the binding solution, the reactions were analyzed by SDS-PAGE and Western assay with a monoclonal antibody specific to His tag (Clontech) and a polyclonal antibody specific to p50 (Santa Cruz Biotechnology).
RESULTS
Isolation and Characterization of the FPIII-Binding Factors
We performed a yeast one-hybrid screen of a human placental cDNA library to identify nuclear factors that bind to the FPIII site in the promoter region of the rnCGM3 gene (Fig. 1A). Ten positive clones out of 5 x 106 yeast colonies screened were further characterized. Six of these clones encoded the NFB family member p65. The other four clones encoded the RBPJ, initiating at different nucleotides in the exon 2 region of the human RBPJ gene [14]. The p65 clones are either the same clone or clones initiating at different nucleotides in the exon 1 region of the p65 gene. Sequence analyses also indicated that these p65 clones encode N-terminally truncated polypeptides containing intact DNA-binding domains. To verify the specificity of the DNA-protein interaction in vivo, we reintroduced into the reporter yeast cells the two positive p65 clones (4B2 and 4B4). In addition, a plasmid containing the mouse p53 gene, pGAD53m, and the parental vector pACT2 (containing the GAL4 activation domain) were also introduced into the yeast cells as negative controls. Colonies appeared only in the sectors of pACT2p65 (4B2 and 4B4) (Fig. 1B). Neither mouse p53 nor the GAL4 activation domain interacted with the FPIII element. These results demonstrate that p65 specifically binds to the FPIII element in vivo.
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Analysis of NFB Family Members Interacting with the FPIII Element
To investigate whether NFB family members directly bind to the FPIII element, we performed an EMSA with a 32P-labeled FPIII probe and in vitro-translated proteins of p65 and p50. The translated protein products all appeared to match the predicted molecular mass (Fig. 2A). Both p65 and p50 specifically bound to FPIII (Fig. 2B, lanes 1 and 4) because the unlabeled FPIII at a 50-fold excess could compete complex formation (Fig. 2B, lanes 2 and 5) but the unrelated oligonucleotide m8 could not (Fig. 2B, lanes 3 and 6). We also performed EMSA with rat placental nuclear extracts and the FPIII probe. However, the NFB-FPIII complex was barely detected (data not shown). It is possible that the level of NFB in the rat placenta extracts was too low for detection.
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To prepare nuclear extracts enriched with NFB, we transfected the human 293 cells with an expression plasmid encoding p65 or p50. The nuclear levels of exogenous p65 and p50 proteins were monitored by Western analysis. The p65 or p50 proteins were only detected in the nuclear extracts of transfected cells (Fig. 2C). EMSA was also performed using nuclear extracts prepared from the p65-, p50-, or p50/65-transfected cells and the 32P-labeled FPIII probe. The p50/50 and p65/65 homodimers (lanes 2 and 8) and the p50/65 heterodimer (lane 14) specifically bound to FPIII (Fig. 2D). Both unlabeled FPIII and NFB oligonucleotides block the corresponding complex (lanes 3, 9, and 15 for FPIII; lanes 5, 11, and 17 for NFB). Moreover, antibodies against p50 or p65 supershifted the corresponding complex (Fig. 2D, lanes 6, 13, 18, and 19). In contrast, the NFB-FPIII complex was not observed in the mock nuclear extracts (Fig. 2D, lane 20). A complex with a faster mobility was also observed in nuclear extracts of transfected and mock cells (Fig. 2D, asterisk), indicating the existence of a constitutive nuclear factor binding to FPIII. This constitutive nuclear factor is likely to be RBPJ because an RBPJ-binding oligonucleotide, m8 [18], blocked the formation of the more quickly migrating complex but not the NFB-FPIII complex (Fig. 2D, lanes 4, 10, and 16). The levels of NFB-FPIII complex were increased in the presence of the m8 oligonucleotide. The signals for p50, p65, and p50/65 were increased by 5.6%, 36.6%, and 19.4%, respectively (Fig. 2B, compare lanes 2 and 4 for p50, lanes 8 and 10 for p65, and lanes 14 and 16 for p50/65). These data indicated that NFB directly bound to FPIII and the constitutive factor, RBPJ, competed with NFB for FPIII.
To identify more specifically the nucleotides in the FPIII region important for NFB binding, we performed competition experiments using p50- or p65-enriched nuclear extracts and a series of mutant FPIII oligonucleotides (Fig. 3A). Mutant oligonucleotides M78 and M910 could not compete efficiently with wild-type FPIII for p50 and p65 (Fig. 3B, lanes 6, 7, 15, and 16). The mutant oligonucleotide M56 could not compete efficiently with wild-type FPIII for p50 but competed partially with wild-type FPIII for p65 (Fig. 3B, lanes 5 and 14). This discrepancy was probably due to the fact that p50 prefers binding to the first half-site of the consensus sequence. These results suggest that a core sequence, GGGAAA, within the FPIII region is important for binding of p50 and p65. However, mutant oligonucleotides M910, M1112, and M1314 competed with wild-type FPIII for RBPJ (Fig. 3B, lanes 7–9 and 16–18), suggesting that overlapping but distinct sequences define the binding sites for NFB and RBPJ.
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Transactivation of the FPIII-Reporter Constructs by NFB
To investigate whether binding of NFB family members to their cognate sites in the FPIII region transactivates gene expression in vivo, transient expression experiments were performed. A reporter plasmid, p(FPIII)6E1bCAT, was constructed consisting of six tandem copies of FPIII placed upstream of the minimal promoter of the adenoviral E1B gene. We examined the CAT activity after cotransfection of p(FPIII)6E1bCAT with an expression plasmid containing either p65 or p50 into 293 cells. CAT expression directed by p(FPIII)6E1bCAT was stimulated by p65 in a dose-dependent manner (Fig. 4A). Induction was 12.4- and 22.7-fold with 0.5 and 1 µg of pCMVp65 DNA over the empty vector, respectively. In contrast, no significant stimulation was observed with p50 (Fig. 4A). Because p65 and p50 can associate as a heterodimer, we also cotransfected pCMVp65 with increasing amounts of pCMVp50. Transactivation by p65 decreased with increasing amounts of p50 (data not shown). This phenomenon could be the result of transcriptionally inactive p50 dimers competing for binding with p65 dimers to limited FPIII sites on the CAT reporter plasmid. Therefore, the pCMVp5065 chimera construct, built by ligating the DNA-binding domain of p50 with the transactivation domain of p65, was tested. A dose-dependent transactivation by the p5065 chimera was observed (Fig. 4A). However, the p5065 chimera is less active than the p65 homodimer in transactivation (Fig. 4A).
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To demonstrate that the transactivation of p65 depends on the core sequence (GGGAAA) in the FPIII region, 293 cells were cotransfected with pCMVp65 and two mutant reporter constructs, p(FPIIIM34)5E1bCAT and p(FPIIIM56)4E1bCAT. The construct p(FPIIIM34)5E1bCAT contains five copies of the M34 mutant oligonucleotide, and the construct p(FPIIIM56)4E1bCAT contains four copies of the M56 mutant oligonucleotide. The CAT activity from p(FPIIIM34)5E1bCAT induced by 1 µg of pCMVp65 was 65.7-fold greater than that of the control (Fig. 4B). No induction was detected with the p(FPIIIM56)4E1bCAT (Fig. 4B). These results suggest that p65 specifically transactivated reporter gene via the NF core sequence in the FPIII region.
Competition of p50 and RBPJ for FPIII Element
Previously, we identified RBPJ as an FPIII-binding factor and demonstrated that the core sequence in FPIII for RBPJ is CTGGGA [14], which overlaps with the core sequence (GGGAAA) for p50 and p65. In the present study, we tested whether NFB and RBPJ compete with each other for FPIII. Because p50 and p65 have similar binding characteristics and because pure p50 protein is commercially available, this factor was used in competition binding experiments. Four micrograms of HisRBP protein was incubated with a biotinylated FPIII fragment and increasing amounts of p50 protein. The HisRBP-FPIII complex was replaced by the p50-FPIII complex as the amount of p50 protein increased (Fig. 5). This result suggests that RBPJ and p50 are mutually exclusive on the FPIII site.
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Effect of RBPJ on FPIII-Reporter Constructs
To determine whether RBPJ competed with NFB for binding to FPIII in vivo, we examined CAT expression of p(FPIII)6E1bCAT stimulated by p65 in the presence of rRBPJ-2N. The CAT activity stimulated by p65 was suppressed when increasing amounts of rRBPJ-2N were present (Fig. 6). These results indicate that the FPIII site in the rnCGM3 promoter is a functional cis-composite element for NFB and RBPJ.
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DISCUSSION
In this study, we identified NFB as a potential FPIII-binding factor for rnCGM3 expression. Several lines of evidence support this conclusion. First, the NFB member p65 isolated from the yeast one-hybrid screen could interact with the FPIII site in vivo when retransformed into the yeast reporter strain. Second, both of the NFB members p65 and p50 specifically bound to the FPIII fragment in vitro. Third, p65 could transactivate reporter-gene expression via the FPIII site. NFB was originally described as the nuclear factor bound to an intronic enhancer element of the Ig light chain gene [19]. The mammalian NFB is composed of a family of structurally related proteins, including c-Rel, Rel B, p65, p50/105, and p52/p100 [20]. NFB exists as homo- or heterodimers that are sequestered in the cytosol with the inhibitory proteins IBs through noncovalent interactions [20]. The latent NFB can be released in the presence of extracellular stimulants, which leads to the degradation of IBs [21]. Therefore, the sequestration of NFB by IB in the cytosol may be attributed to the low level of NFB in the rat placental nuclear extracts used in this study.
We defined the core sequence for p65 and p50 as the GGGAAA motif within the FPIII site. The sequence of the FPIII site (GGGAAAAAAC) matches 7 of the 10 nucleotides in the consensus NFB sequence, 5'-GGGPuNNPyPyCC-3' [22]. Studies have shown that p50 prefers binding to the first half-site of the consensus sequence [23]. Although p50 binds to FPIII, it was not isolated in the yeast one-hybrid screen. There are several possible reasons for this finding: placenta may have a higher level of p65 than p50, the conditions in yeast nuclei may favor p65 binding, or the p65 activation domain, together with the GAL4 activation domain, may synergistically activate HIS3 gene expression.
In our previous study, we identified RBPJ, the downstream effector of the Notch signaling pathway, as a constitutive nuclear factor binding to the FPIII site in the rnCGM3 promoter [14]. Notch functions as a transmembrane receptor, which can be activated by ligand stimulation [24]. The interaction between Notch and its cognate ligand results in proteolytic cleavage, with release and nuclear translocation of the intracellular domain of Notch protein (NotchIC) [25]. RBPJ is identical to CBF1 and KBF2 and can associate with a variety of proteins, including the Drosophila hairless protein, the EBV nuclear antigen 2 (EBNA2), and NotchIC [26–29]. EBNA2 and NotchIC do not bind directly to DNA. However, the associated RBPJ/EBNA2 and RBPJ/NotchIC complexes can activate target genes through the RBPJ site. Recently, potential RBPJ binding sites have been identified within the NFB sites in the promoter region of genes, including the mouse major histocompatibility complex, ß2-microglobulin, interleukin (IL) 6, NFB2, and CYP2B1 [26, 30–34]. Moreover, Oswald et al. [33] demonstrated that RBPJ binds to the NFB binding sites in Bcl-3, IB
, IFNß, and mRBPJ. Therefore, a subset of NFB binding sites may also contain an overlapping RBPJ site. In the present study, we collected evidence that the FPIII site of the rnCGM3 promoter is also a composite element containing overlapping sequences for NFB (GGGAAA) and RBPJ (CTGGGA). In EMSA, when the endogenous RBPJ proteins in the NFB-enriched nuclear extracts were titrated out with an RBPJ-specific oligonucleotide, formation of the NFB-FPIII complex increased dramatically. In transient expressions, p65 activated CAT expression directed by p(FPIII)6E1bCAT or p(FPIIIM34)5E1bCAT by 22.7- or 65.7-fold over the control, respectively. This result suggests that p65 activates reporter gene expression via the NFB core sequence in the FPIII element more efficiently when the RBPJ core sequence is mutated. These results and those of the in vitro-binding competition assay using pure p50 and RBPJ proteins (Fig. 5) indicate that NFB and RBPJ are mutually exclusive for binding to FPIII. We also observed a 1.5- to 2-fold stimulatory effect by p65 in JEG3 and BeWo cells on the reporter construct directed by a native rnCGM3 promoter fragment (nucleotides -326 to -33 relative to the translation initiation site), which contains the FPIII element (data not shown). Thus, endogenous RBPJ may obscure the positive effect of p65 using this native promoter construct.
The C/EBPß transactivator is a positive regulator that binds to the FPII element of the rnCGM3 promoter. Physical and functional interactions between NFB and C/EBP families have been demonstrated in the control of cytokine gene expression such as that of IL-6 and IL-8 [35–38]. The regulatory region of IL-6 contains one NFB and two C/EBPß binding sites, and the two factors activate IL-6 synergistically [39]. The FPII and FPIII sites in the promoter region of rnCGM3 contain recognition sites for C/EBPß and NFB, respectively, suggesting that the two factors play important roles in mediating rnCGM3 expression. Both NFB and C/EBPß are responsible for regulating a variety of genes involved in inflammatory and immunologic responses, e.g., cytokines and acute phase protein genes. Although the physiologic functions of rnCGM3 are not known, rnCGM3 may belong to this category of genes based on the mechanism of transcriptional regulations.
In our previous studies, we also demonstrated that RBPJ represses reporter gene expression through the FPIII site and that NotchIC can counteract this repression [14]. Moreover, NotchIC enhances the transcriptional activation of C/EBPß on rnCGM3 [14]. The physiologic role of NFB- and RBPJ-mediated gene expression via the FPIII composite element in the rnCGM3 promoter is not clear. Recently, a dynamic expression of Notch genes has been detected by in situ hybridization during trophoblast differentiation [40]. The Notch signaling pathway is involved in determination of the fate of many cell types, including the inhibition of myoblast differentiation, the maturation of T-cell development, and the singling out of neural precursor cells in Drosophila [41]. RBPJ might repress rnCGM3 expression until ligands for the Notch receptor induce the cleavage of the intracellular domain of the Notch receptor, which subsequently can associate with RBPJ. The association of NotchIC and RBPJ activates rnCGM3 expression, which might be considered an outcome from trophoblast differentiation controlled by the Notch signaling pathway. As pregnancy proceeds, a variety of cytokines, such as IL-1, IL-2, IL-6, tumor necrosis factor , transforming growth factor ß, and interferon 
, are released from uterine epithelial cells, trophoblasts, and lymphocytes to modulate trophoblast proliferation and invasion and maternal immune responses. Therefore, these extracellular stimulants might activate the latent NFB and consequently activate rnCGM3 expression to maintain a high level of PSG proteins during pregnancy. The effects of C/EBPß, NFB, Notch, and RBPJ on rnCGM3 expression in trophoblasts at different gestation stages are currently under investigation.
ACKNOWLEDGMENTS
We thank Dr. Hsou-min Li for critical reading of the manuscript.
FOOTNOTES
First decision: 26 January 2001.
1 This work was supported by grants (to H.C.) from the National Science Council (89-2311-B-001-229) and Academia Sinica of Taiwan. 
2 Correspondence. FAX: 011 886 2 27889759; hwchen{at}gate.sinica.edu.tw
Accepted: June 18, 2001.
Received: December 8, 2000.
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