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a Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina
b Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS/INSERM U184/ULP, BP 163 67404 Illkirch Cedex, C.U. de Strasbourg, France
c Unite INSERM U.384, Faculté de Médecine, U.F.R. de Medecine et de Pharmacie, B.P 38 F-63001 Clermont-Ferrand Cedex, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, the molecular cloning of the first human transcription factor that interacts with the promoter of PSG genes has been accomplished [6]. This factor, whose corresponding gene has been renamed COPEB, is localized to human chromosome 10p15 [7]. Human core promoter binding protein (hCPBP) mRNA is highly enriched in placenta, which constitutes the major PSG expression tissue, suggesting a relevant role of this factor for the transcriptional regulation of PSG genes in vivo [6]. Human CPBP is a member of the Krüppel-like class of transcription factors, which share a signature motif consisting of three zinc fingers at their C-terminal domains. Additionally, this motif constitutes the DNA-binding domain that recognizes GC-rich regions present in a number of promoters [8]. Most significantly, it has been demonstrated that several proteins of the Krüppel-like family have functional relevance as regulators of cell differentiation and tissue morphogenesis [9–12].
Recently, cDNAs encoding polypeptides sharing strong sequence homology with CPBP have been isolated from human (Bcd and UKLF) [13, 14] and rat tissues (Zf9) [15]. Additionally, a molecule identical to hCPBP—GBF—was isolated by its ability to interact in vitro with the GC-rich region of HIV promoter sequences [16]. Interestingly, it was shown that Bcd encodes a highly mutated version of hCPBP, which was obtained from a patient with B-cell chronic lymphocytic leukemia and behaves as an oncogene due to its capacity to transform NIH3T3 cells and induce tumors in nude mice [13]. In addition, the Zf9 cDNA encodes a polypeptide that shares 98.5% of identity with hCPBP at the amino acid level and could represent a variant of the same gene. The Zf9 expression and biosynthesis was induced in vivo in hepatic stellate cells after liver injury in rats [15]. The Zf9 was also shown to regulate the promoters of transforming growth factor ß1 (TGFß1) and its receptors [17]. The UKLF cDNA was isolated from human vascular endothelial cells, and its encoded polypeptide shows a ubiquitous expression pattern in contrast to the preferential mRNA enrichment of hCPBP in placenta. While the structural resemblance between hCPBP and its related proteins might predict some similarity of their functional activities, their mRNA tissue distribution is quite different, suggesting that they play specific biological roles in particular physiological and/or pathological conditions.
As an essential step in establishing an animal model to better define the role of CPBP in vivo, we undertook the molecular cloning of the mouse CPBP (mCPBP) cDNA and studied its spatiotemporal expression during mouse development and in adult tissues.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A 543-base pair (bp) cDNA fragment comprising nucleotides 377–920 of mCPBP was initially isolated by RT-PCR from mouse placenta total RNA using the following hCPBP-derived primers: 5'-GCCTGAACTCAGATGTCAGC-3' and 5'-CAGAGGTGCCTCTTC-3'. This fragment was cloned into the pBluescript plasmid (Stratagene, La Jolla, CA), and its complete nucleotide sequence was obtained for both strands by automatic sequencing using T7 and T3 RNA polymerase primers.
To isolate the mCPBP cDNA, a 383-bp EcoRV-PstI restriction fragment that does not contain the conserved zinc-finger coding region was purified, labeled with [32P]ATP [18], and used as a probe to screen a lambdaZAP II (Stratagene) mouse placenta cDNA library according to standard procedures. Briefly, roughly 250 000 plaque-forming units (pfu) were screened by hybridization at 42°C in 6-strength SSC (single-strength SSC is 0.15 M sodium chloride, 0.015 M sodium citrate)/50% formamide. Filters were washed at 65°C in double-strength SSC/0.1% SDS for 60 min. Positive clones were picked, purified, and obtained as pBluescript derivatives [19].
A 1007-bp cDNA fragment was isolated and sequenced as described above. DNA and protein sequence analyses were performed using the DNASIS software package (Hitachi, Tokyo, Japan).
RNA Analysis
Total RNA was purified from both mouse placenta and total embryos essentially as described [20] and then analyzed by Northern blot [21]. Poly(A)+ RNA samples derived from adult mouse tissues (Clontech, Palo Alto, CA) were also analyzed. Blots were hybridized under the same conditions as those indicated for the library screening procedure, using as a probe the 32P-labeled 383-bp EcoRV-PstI cDNA fragment obtained as described above. As an internal control, the blots were probed with the mouse GAPDH cDNA. The intensity of the bands was quantified by the public-domain NIH Image program (http://rsb.info.nih.gov/nih-image/).
Mouse Embryo and Placenta Collection
CD1 mice were mated naturally overnight. Pregnant females (morning of vaginal plug was considered as 0.5 days postcoitum [dpc]) were killed by cervical dislocation, and the fetuses were collected in PBS after cesarean section. The specimens were placed in molds containing tissue-freezing medium (Tissue-Tek; Sakura Finetek, Torrance, CA) and frozen on dry ice, as described [22]. Serial cryostat sections (10 µm thick) were collected on gelatin/chrome alum-coated slides and stored at -80°C until use. Mouse conceptuses were sectioned in utero (from 6.5 to 15.5 dpc) or explanted from the uterus (from 15.5 to 18.5 dpc) and sectioned together with the placenta and extraembryonic membranes.
In Situ Hybridization
The spatiotemporal tissue distribution of CPBP transcripts was analyzed by in situ hybridization at different stages of mouse embryogenesis (6.5 and 18.5 dpc) according to the procedure described by Niederreither and Dollé [22]. Briefly, the antisense and sense RNA probes were in vitro-transcribed by standard T7 and T3 RNA polymerase reactions, respectively, using as template the mCPBP cDNA cloned into the pBluescript plasmid (Stratagene). During RNA synthesis, [
-35S]CTP (Amersham, Buckinghamshire, England) was included in the reaction mix to obtain labeled probes at roughly 5 x 108 cpm/mg of specific activity. The average probe length was limited to about 150 nucleotides by partial alkaline hydrolysis in 0.1 M triethanolamine (pH 8.0)/0.25% (v:v) acetic anhydride. Cryostat sections were also immersed in the same solution for 10 min, dehydrated in ethanol, and air-dried. Labeled probes diluted up to 25 000 cpm/ml were applied to each section in 50 µl of the hybridization buffer (50% formamide, single-strength Denhardt's solution, 500 mg/ml tRNA, 10% dextran sulfate, and 10 mM dithiothreitol). Sections were covered and incubated in humid chambers at 50°C overnight. After hybridization, the probes were immersed at 55°C in washing buffer (50% formamide, single-strength SSC) for 2.5 h. They were rinsed twice for 5 min in double-strength SSC at room temperature, treated for 30 min with 100 mg/ml ribonuclease A (Sigma Chemical Co., St. Louis, MO) at 37°C, and washed for 2 h at 55°C in 50% formamide, double-strength SSC. The slides were then placed for 15 min at 55°C in 0.1-strength SSC, dehydrated, coated with Kodak NTB2 emulsion (Eastman Kodak, Rochester, NY), and stored at 4°C for 2–3 wk. They were developed in Kodak D19 and stained with toluidine blue. The sections were examined at various magnifications with a Nikon microscope (Nikon Inc., Melville, NY) under brightfield illumination to observe the histology, and under darkfield illumination, which allows the autoradiography signal grains to appear as white dots.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A cDNA fragment of 543 bp was initially isolated by RT-PCR using primers deduced from the hCPBP cDNA encompassing a region with a relatively high degree of sequence conservation in other cDNA clones encoding CPBP-related proteins. Sequencing analysis confirmed that this cDNA encodes the mouse counterpart of hCPBP polypeptide. To further extend these results, a cDNA of 1007 nucleotides was cloned from a mouse placenta cDNA library using as a probe a 383-bp EcoRV-PstI restriction fragment that does not have significant sequence homology with any known gene. This region encodes the serine/threonine-rich domain, which is not conserved in other Krüppel-like proteins. According to sequence data base analysis, the isolated cDNA encodes a complete open reading frame of 283 residues previously unknown in the mouse. The sequence conservation of this molecule between human and mouse species reaches 87.1% and 94.4% identity at the nucleotide level and the amino acid level, respectively. Importantly, the amino acids involved in the Cys2-His2 zinc-finger structure at the C-terminal region of both molecules are identical (Fig. 1). Moreover, the acidic N-terminal region that is able to mediate transcriptional activation in hCPBP (unpublished observations) shares 95.4% identity, suggesting a conserved evolutionary role of these domains in the CPBP function (Fig. 1). Sequence alignment indicated that the mCPBP polypeptide is highly homologous to, in addition to its human counterpart, other Krüppel-like family members whose cDNAs have been recently isolated (i.e., hUKLF and rZf9) [14, 15]. The rZf9 and mCPBP sequences are nearly identical, showing 98% homology probably representing isoforms encoded by orthologous genes. In contrast, the overall sequence homology between mCPBP and hUKLF is more divergent, reaching 50% identity. However, the zinc-finger DNA-binding domain and the first 47 amino acids of the acidic N-terminal region are highly conserved between both molecules, showing 95% and 91% homology, respectively (Fig. 1).
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In sum, a cDNA encoding a novel mouse protein, mCPBP, was isolated and found to be almost identical to its human counterpart, indicating that this polypeptide may play a conserved biological role in both species.
Specific Expression of mCPBP in Developing Placenta During Mouse Gestation
As an essential step toward understanding CPBP function in vivo, we first analyzed its expression pattern in adult mouse tissues by Northern blot and additionally during different stages of mouse development using in situ hybridization techniques. Northern blot analysis showed a unique 4.5-kilobase (kb) transcript in all RNA samples, suggesting that the CPBP gene is ubiquitously expressed (Fig. 2). Interestingly, one can also conclude from this result that CPBP gene transcription is differentially regulated in distinct tissues and that specific mechanisms must exist to express particularly high mRNA levels exclusively in placental cells. In contrast, the transcript level was nearly 10-fold reduced in embryos (17 dpc; Fig. 2). This low level of expression was also observed in the other tissues from adult mice except for heart, in which the mRNA abundance represents roughly the 25% of the maximal level detected in placenta (Fig. 2).
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To further examine mCPBP placental expression, in situ hybridization experiments were performed at different developmental stages between implantation (6.5 dpc) and just before delivery (18.5 dpc). From 6.5 to 7.5 dpc, the mCPBP gene was expressed in the ectoplacental cone, and no specific labeling was detected in the yolk sac (data not shown). At this and later times (10.5 dpc), an additional signal was observed in a reduced and specific region of the endometrium, around all of the conceptus (zone of implantation; Fig. 3, A and C). At 8.5 dpc, specific expression began in the trophoblastic giant cells (Fig. 3A). Expression of mCPBP persisted in the primitive placenta during all early placentation, except for the region in which the fusion between the allantoic bud and the chorion occurred (Fig. 3, C and G). At 12.5 dpc, the placenta, including its distinguishable trophoblastic giant cells, was still clearly labeled (Fig. 3G). No specific labeling of these structures was observed using the mCPBP sense riboprobe at 10.5 dpc (Fig. 3E) or at later stages (not shown). Most of the expression domains that were established at 12.5 dpc can still be described at later stages. Thus, from 12.5 to 18.5 dpc, mCPBP was strongly expressed in the spongiotrophoblastic and labyrinthine zone of the definitive placenta (Fig. 3K), whereas the yolk sac membranes and the uterus remained unlabeled (Fig. 3, I and K). It is important to note that all the intermediate developmental stages showed essentially the same distribution of mCPBP transcript as those described above (data not shown).
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At the embryo proper, a diffuse and low signal was observed without tissue specificity (Fig. 3C), consistent with the scarce amount of CPBP transcript detected in RNA samples purified from total embryos (Fig. 1, lane 2). This result may reflect a low level of ubiquitous mCPBP expression at the embryo during development that is not detected in a particular structure by in situ hybridization. Although no specific organ of the embryo appeared to show substantial CPBP expression, even at the end of gestation, the CPBP transcript was clearly detected in all the tissues analyzed from adult mice (Fig. 2, lanes 3–9).
Our results demonstrate that under normal conditions the CPBP gene displays a differential expression pattern, being primarily enriched in placental cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sequence comparison analysis indicated that the isolated CPBP cDNA encodes a previously unidentified mouse protein that is the counterpart of the human CPBP polypeptide. The overall sequence homology between both molecules reaches 94.4% at the amino acid level and shows the same modular structure previously reported for hCPBP. Moreover, the mCPBP expression level, like its human counterpart, was maximal in placenta [6]. Accordingly, no specific labeling was detected in any structure of the embryo by in situ hybridization from the beginning to the end of gestation. Its transcript level was markedly reduced in total embryos as well as in all tissues from adult mice, as shown by Northern blot assay. This observation indicates that similar gene control mechanisms operate in mammalian species to produce a high level of CPBP mRNA in placenta, and further emphasizes the evolutionary significance of this transcription factor for placental function.
Recently, several cDNAs encoding polypeptides highly homologous to CPBP, such as Bcd, Zf9, and UKLF, have been isolated because of their differential expression patterns in particular pathological and physiological conditions unrelated to pregnancy [13–15]. According to their structural features, all these molecules, as well as CPBP, belong to the Krüppel-like family of transcription factors that have functional relevance as regulators of cell differentiation and tissue morphogenesis [9–12]. Our data strongly suggest that under physiological conditions the main biological role of CPBP might be in the transcriptional control of specific genes that are important for growth and differentiation of trophoblast cells. This hypothesis is further reinforced by the recent finding that Zf9, an isoform nearly identical to the CPBP protein, is able to activate the TGFß1 gene [17], whose product has a remarkable spectrum of effects on growth and development of several tissues including placenta [25–27].
Finally, the striking homology in amino acid sequence and expression pattern between human and mouse CPBP as it is described here should lay the foundation for future functional studies by transgenic and gene targeting strategies in mice.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: J.L. Bocco, Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Pabellón Argentina—Ciudad Universitaria (5000), Cordoba, Argentina. FAX: 54 351 4334174; jbocco{at}fcq.unc.edu.ar
3 Current address: Hormone and Metabolic Research Unit, International Institute of Cellular and Molecular Pathology and University of Louvain Medical School, 75, Avenue Hippocrate, B-1200 Brussels, Belgium.
Accepted: August 10, 1999.
Received: April 26, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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