Structure of the Murine CD156 Gene, Characterization of Its Promoter, and Chromosomal Location*

(Received for publication, February 25, 1997, and in revised form, April 28, 1997)

Masashi Kataoka , Kazuhiro Yoshiyama , Keiko Matsuura , Naoki Hijiya , Yasunori Higuchi and Shunsuke Yamamoto Dagger

From the Department of Pathology, Oita Medical University, Hasama-machi Oita 879-55 Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The murine cell surface antigen mCD156 is a glycoprotein that is expressed in monocytic cell lines and consists of a metalloprotease domain, a disintegrin domain, a cysteine-rich domain, and an epidermal growth factor-like domain in the extracellular region. The mCD156 gene is composed of 24 exons and 23 introns and spans approximately 14 kilobases. The first exon encodes most of the signal peptide sequence, and the transmembrane region is encoded by a single exon (19). In contrast, the other regions are composed of multiple exons. Of these, exons 7-12 and 12-15 encode a metalloprotease domain and a disintegrin domain, respectively. Sequence analysis of the 5'-flanking DNA revealed many potential regulatory motifs. Chloramphenicol acetyltransferase analysis demonstrated that nucleotides at positions -183, -334, and -623 contained cis-acting enhancing elements in a mouse monocytic cell line, aHINS-B3. Nucleotides at positions -183 and -390 contained elements responsible for lipopolysaccharide (LPS) inducibility, although several other 5'-flanking regions were also involved in LPS responsiveness. Regions -202, -507, and -659 play a role in interferon-gamma inducibility. Some of the potential regulatory motifs and other unknown cis elements may be involved in the constitutive expression, and LPS and interferon-gamma inducibilities. The mCD156 gene was mapped to chromosome 7, region F3-F4.


INTRODUCTION

The mouse CD156 (mCD156)1 (MS2) and human CD156 are type I transmembrane glycoproteins found on myelomonocytic cell lineage (1, 2). The mCD156 cDNA is 3.2 kb long with an open reading frame encoding 826 amino acids. The extracellular region consisting of 644 amino acids has a metalloprotease (MTP) domain containing the zinc-binding consensus histidine and protease catalytic glutamic residues and a disintegrin domain containing a platelet aggregation inhibitor-like structure, whereas the cytoplasmic region consisting of 143 amino acids contains a proline-rich amino acid sequence containing consensus SH3 (Src homology 3) binding sequence. mCD156 plays a role in infiltration of leukocytes by leukocyte adhesion to endothelial cells and degradation of vascular basement membrane (3).

CD156 is a member of a family of proteins (ADAM) which are characterized by a conserved structure in hemorrhagic snake venom proteins. Following our report of mCD156 (ADAM 8), a number of ADAMs have been reported (1, 4-14). ADAMs 1-7 are expressed in reproductive organs, mainly the testes, and are implicated in sperm-egg fusion and spermatogenesis. ADAMs 8-12 (meltrin-alpha ) and 15 (metargidin) are of non-reproductive cell origin, including myeloid, muscle, brain, and breast cells. ADAM 12 has been shown to mediate myocyte cell fusion. Recently, a new family member designated TACE has been reported to function as a tumor necrosis factor-alpha -converting enzyme that liberates 17-kDa tumor necrosis factor-alpha from 26-kDa membrane-bound pro-tumor necrosis factor-alpha (15, 16). TACE activity is also found in ADAM 10, which is originally separated as an enzyme for degradation of myelin basic protein (17). Another new family protein designated ADAMST-1 appears to contain thrombospondin-like structures and play a role in tumor cell migration (18).

We report here the isolation and characterization of the mCD156 gene and the presence of the cell type-specific, lipopolysaccharide (LPS) and interferon (IFN)-gamma -inducible promoter activities in its 5' upstream sequence. We have also determined the mCD156 gene location.


EXPERIMENTAL PROCEDURES

Materials

Restriction enzymes were purchased from Takara (Kyoto, Japan), Toyobo (Osaka, Japan), Life Technologies, Inc. (Gaithersburg, MD), and Wako Pure Chemicals (Osaka, Japan). Agarose ultrapure DNA grade was from Takara. The DNA ligation kit, reverse transcriptase, S1 nuclease, and mung bean nuclease were obtained from Takara, and bovine alkaline phosphatase was obtained from Boehringer Mannheim (Tokyo, Japan). LPS (Escherichia coli serotype 055:B5) was obtained from Sigma Chemical Co. (St. Louis, MO). Mouse IFN-gamma was purchased from Genzyme (Cambridge, MA). Radioactive nucleotides [alpha -32P]dCTP (3,000 Ci/mmol) and [gamma -32P]ATP (6,000 Ci/mmol) were obtained from NEN Life Science Products. Chemicals used for DNA sequencing were obtained from Toyobo. X-ray film (XAR-351) was obtained from Kodak (Rochester, NY).

Cell Lines

Murine macrophage cell line aHINS-B3 and murine glioblastoma cell line G203 were grown in Dulbecco's modified Eagle's medium containing fetal calf serum.

Production of Genomic Libraries and Isolation of mCD156-associated Clones

The bacteriophage EMBL-3 murine liver library was produced as described previously (19, 20). Briefly, genomic DNA prepared from BALB/c murine liver was partially digested with MboI, followed by size fractionation via sucrose gradient ultracentrifugation. The DNA fragments in the size range of 12-20 kb were inserted into the BamHI site of EMBL-3 arms. The annealed DNA was packaged using a commercial packaging extract (Stratagene Cloning Systems, La Jolla, CA), and then the phage was grown in E. coli Q359 and screened as described. Radioactive probes were prepared by 32P labeling cDNA inserts from an mCD156 clone (1) using nick translation (21), with a specific activity of 108 cpm/µg of DNA. Filters containing recombinant plaques were screened according to the procedure of Benton and Davis (22), prehybridized at 42 °C in 6 × SSC, 5 × Denhardt's solution for 4 h, and hybridized with 106 cpm/ml 32P-labeled insert at 42 °C for 20 h. The filters were exposed to Kodak XAR-5 film with a Fuji intensifying screen at -80 °C for 2 days. Phage clones were subsequently purified by repeated screening cycles. Large scale phage DNA and minipreparations were obtained as described by Maniatis et al. (23).

Plasmid Subcloning and Sequencing

DNA fragments originating from the genomic DNA inserts of phage clones were subcloned into pUC118/119. The pUC118/119 vectors were linearized and ligated for 12 h at 16 °C using a ligation kit purchased from Takara. The ligated DNA was used to transform MV1184 cells. White bacterial colonies were selected. Nucleotide sequences were determined by dideoxy chain termination (24, 25) using single-stranded plasmid DNA with modifications as described in the Sequenase technical manual (Toyobo). Nucleotide sequences were determined on DNA subcloned in pUC118/119 vectors using pUC primers. Other oligonucleotides used as primers for sequencing were synthesized using an automated DNA synthesizer (model 8700, Biosearch). Sequences were determined on both strands.

S1 Nuclease Mapping

S1 nuclease mapping was performed according to a modified method of Berg and Sharp (26). A single-stranded DNA corresponding to the SalI-BamHI fragment containing the region from the 5' upstream to intron 1 of the mCD156 gene was prepared using M13KO7 phage as a vector and annealed with an end-labeled oligonucleotide primer, a synthetic 30-base single-stranded oligomer (3'-TGGTGTCCATAAGACGCTGAGCAGCCAGAG-5') corresponding to bp +143 to +172 of the first exon of the mCD156 gene. The annealed DNA was incubated with 400 µM dNTPs and 10 units of the Klenow fragment of E. coli DNA polymerase for 30 min at 37 °C. The mixture was heated to 65 °C for 5 min to inactivate the Klenow fragment and then chilled on ice. After digestion with PstI and alkali denaturation, the extended primer was separated on alkaline gel electrophoresis. Total RNA, 50 µg from aHINS-B3 cells, was then hybridized for 16 h at 50 °C with the extended primer in 40 mM Pipes, pH 6.4, 0.4 M NaCl, 1 mM EDTA, and 80% formamide. Following hybridization, the reaction was diluted 10-fold with S1 nuclease buffer (300 mM NaCl, 30 mM sodium acetate, pH 4.5, and 3 mM ZnCl2), and 10 µg of salmon sperm DNA. S1 nuclease (130 units) was added. The reaction mixture was then incubated for 1 h at 37 °C. The reaction mixture was terminated by the addition of termination buffer (2.5 M ammonium acetate and 50 mM EDTA), and the DNA·RNA hybrids were extracted with phenol, precipitated with ethanol, resuspended in 80% formamide, heated to 90 °C, and resolved on a 6% acrylamide, 8 M urea gel.

Primer Extension Analysis

The probe for primer extension analysis was the end-labeled synthetic 30-base single stranded oligomer used in the S1 nuclease protection. Total cellular RNA was isolated from aHINS-B3 cells. The primer was annealed to 100 µg of total RNA by heating the reaction mixture for 15 min at 80 °C in 20 µl of 80% formamide, containing 400 mM NaCl, 40 mM Pipes, pH 6.4, and 1 mM EDTA. The resulting DNA·RNA hybrid was ethanol precipitated and dissolved in reverse transcriptase buffer (50 mM Tris-HCl, pH 8.0, 20 mM 2-mercaptoethanol KCl, 10 mM MgCl2) in the presence of 1 mM deoxynucleotides, 100 units of reverse transcriptase, and 60 units of RNase inhibitor. After 90 min at 42 °C, the DNA·RNA hybrids were phenol extracted, ethanol precipitated, dissolved in loading buffer (95% formamide, 20 mM EDTA, 0.05% bromphenol blue, 0.05% xylene cyanol FF), heated to 90 °C, and resolved on a 6% acrylamide, 8 M urea sequencing gel.

Construction of mCD156-CAT Plasmids

mCD156G1 clone containing the 2776-bp nucleotides corresponding to bases -2279 to 498 of the 5' upstream, exon 1 and intron 1 sequence of the mCD156 gene was digested with XbaI and SphI. The insert in the linealized DNA was 3' deleted with exonuclease III. Aliquots (2.5 µl) were removed 1-min intervals and mixed with 50 µl of 2 × mung bean nuclease buffer. After heating for 5 min at 65 °C, the samples were incubated with mung bean nuclease for 60 min at 37 °C. The 3'-deleted samples were then digested with the Klenow fragment followed by T4 DNA ligase treatment. To determine the extent of deletion, each mutant was sequenced as described previously (19). Selected mutants was digested with HindIII and inserted into the HindIII site of the multicloning site containing pSVmCAT. This plasmid containing the 2033-bp nucleotides corresponding to bases -1943 to 90 was digested with KpnI-SmaI and subjected to successive treatment with exonuclease III, mung bean nuclease, and Klenow fragment as above. Each 5'-deleted DNA was ligated with T4 DNA ligase. Plasmid DNA was prepared by alkaline lysis followed by two centrifugation steps through CsCl to isolate supercoiled plasmid DNA.

DNA Transfections and CAT Assay

Plasmid DNA was transfected into the mouse macrophage line aHINS-B3 and the mouse glioblastoma line G203 by calcium phosphate coprecipitation as described previously (19). Cells were seeded at 2 × 106 cells/plate. For transient transfections, a mixture of the test CAT hybrid gene (20 µg equivalent) and the transfection control plasmid (20 µg) pCH110 containing the beta -galactosidase gene was precipitated in Hepes-buffered saline, pH 7.1, and then added to the plates. After 4 h, the cells were given a 1.5-min glycerol shock followed by a wash with ice-cold phosphate-buffered saline. Fresh medium was then added and the incubation continued for 24 h. 24 h after transfection, the cells were stimulated with LPS (100 ng/ml) or IFN-gamma (200 units/ml) for 16 and 24 h, respectively. Cells were washed three times with phosphate-buffered saline, incubated with 40 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA for 5 min on ice, harvested by scraping and centrifugation, and resuspended in 180 µl of 0.25 M Tris-HCl, pH 8.0. Extracts were prepared by freeze-thaw and centrifuged. Supernatants were collected and assayed for protein according to the method of Bradford (27). CAT was assayed as described previously (19). The extract was incubated with [14C]chloramphenicol and 0.2 mg of acetyl-CoA for 1.5 h at 37 °C, and the products were separated by thin layer chromatography. The CAT activity for each construct was normalized to beta -galactosidase activity from the same sample. The relative CAT activity for each deletion mutant was expressed as a percentage of that of wild type -390/+90CAT stimulated with LPS.

In Situ Hybridization and FISH Detection

Mouse chromosomes were prepared according to a published procedure (28). Briefly, lymphocytes were isolated from mouse spleen and cultured at 37 °C in RPMI 1640 medium supplemented with 15% fetal calf serum, 3 µg/ml concanavalin A, 10 µg/ml LPS, and 5 × 10-5 M 2-mercaptoethanol. After 44 h, the cultured lymphocytes were treated with 0.18 mg/ml bromodeoxyuridine (Sigma) for an additional 14 h. The synchronized cells were washed and recultured at 37 °C for 4 h in alpha -minimal essential medium with thymidine (2.5 µg/ml) (Sigma). Chromosome slides were made by the conventional method used for human chromosome preparation (hypotonic treatment, fixation, and air drying).

The genomic DNA of mCD156G2 containing a 15-kb insert spanning nearly the entire coding regions except the most 5' end (see Fig. 1) were biotinylated with dATP using the Life Technologies, Inc. BioNick labeling kit (15 °C, 1 h) (29). The procedure for FISH detection was performed according to the method of Heng et al. (30) and Heng and Tsui (29). Briefly, slides were baked at 55 °C for 1 h. After RNase A treatment, the slides were denatured in 70% formamide in 2 × SSC for 2 min at 70 °C, followed by dehydration with ethanol. The probe was denatured at 75 °C for 5 min in a hybridization mix consisting of 50% formamide, 10% dextran sulfate, and human cot I DNA, and prehybridized at 37 °C for 15 min. The probe was then loaded on the denatured slides. After overnight hybridization, slides were washed and detected as well as amplified using a published method, FISH signals and the DAPI banding pattern were recorded separately by taking photographs, and the assignment of the FISH mapping data with chromosomal bands was achieved by superimposing FISH signals on DAPI-banded chromosomes (30, 31).


Fig. 1. Exon and intron organization of the mCD156 gene. The top line shows a schematic diagram of the mCD156 gene. The bottom line demonstrates a schematic diagram of mCD156 mRNA. Exons are boxed. Filled boxes in the gene are coding regions. Open boxes in the gene are untranslated regions. Abbreviations for mRNA: UT, untranslated region; SP, signal peptide; PMTP, pro-MTP domain; MTP, MTP domain; DI, disintegrin domain; CR, CR domain; EGF, EGF domain; TM, transmembrane domain; CY, cytoplasmic domain.
[View Larger Version of this Image (11K GIF file)]


RESULTS

The mCD156 Gene Structure and Organization

The mCD156 mRNA was 3.1 kb (Fig. 1), from which mCD156 cDNA was cloned and characterized (1). A genomic library, prepared from an MboI partial digest of BALB/c mouse DNA inserted into EMBL-3, was screened using randomly labeled mCD156 cDNA. Two positive clones, designated mCD156G1 and mCD156G2, were selected for further analysis. Mapping and sequence analysis of these inserts established that clone mCD156G1 contained nearly the entire 5' half of the message for mCD156 but not for the 3' half of the gene, whereas clone mCD156G2 contained a 15-kb insert spanning nearly the entire coding regions except the most 5' end.

The mCD156 gene is composed of about 14 kb, 24 exons and 23 introns. Fig. 1 is a line drawing of the intron-exon organization of the gene. The introns range in size from 71 (intron 11) to 2,687 (intron 22) bp. Large introns (intron 1, 907 bp; intron 2, 1,340 bp; and 22) occur on the 5' and 3' side of the gene, and a cluster of exons is found between them. Analysis of splice junction sequences revealed that all intron-exon junctions followed the normal consensus sequence rules (32), except at intron 11 where the acceptor splice site AG was replaced by TA (Table I). Exons comprise approximately 22.8% of the gene; about 18% of the total gene actually codes for amino acids.

Table I. Exon-intron organization of the mCD156 gene

Codon numbers are above the nucleotide sequence.

Exon no. Exon size 5' Splice site Intron size 3' Splice site

bp bp
    -1         1
1 173 CACCAGgtgagt 907 ctgaagCAGTAG
  P  A         V  A
   24        25
2 104 CACTGGgtaagt 1340 atccagGGCCAG
H  W       G  Q
    60        61
3 77 GAACAGgtaagc 142 ttccagGGACCT
 N  R        D  L
   86       87
4 79 GAGCAGgtatgg 77 ccttagGACCAT
E  Q       D  H
        112        113
5 77 CCTCAGgtatnt 477 cctcagGGGCTT
 L  R        G  F
   173       174
6 184 CCACGGgtgagc 216 ccctagAACTGG
P  R       N  W
   194       195
7 63 CAAGAGgtatgg 82 ttccagTTCCAG
Q  E       F  Q
   217       218
8 69 GACAAGgtgggt 173 ctgcagCTTTAT
D  K       L  Y
    273        274
9 167 TATCACgtgagt 166 ttccagGGGGGT
 I  T        G  V
   300       301
10 82 AATCAGgtaact 245 tcatagGACCAC
N  Q       D  H
     350         351
11 148 GCATCGgtagtc 71 cccataGCTCCA
  I  G         S  K
   409       410
12 179 CCTCAGgtctgt 291 tcccagGACTGT
P  Q       D  C
   439       440
13 90 TGCAAGgtaagt 96 gaccagGTGAAG
C  K       V  K
     503         504
14 190 GGCCAGgtgagg 84 ccacagGTGCTC
  P  G         A  R
    528        529
15 76 TGGCAGgtaagc 264 ctgcagGATCAA
 G  R        I  N
   583       584
16 166 GGAAAGgtgagt 392 ttccagGTTTGC
G  K       V  C
   609       610
17 78 CATGGGgtatgt 397 ctccagGTATGC
H  G       V  C
     641         642
18 94 AAGCAGgtaagg 498 tcccagCGTCTA
  A  A         S  T
    679        680
19 115 GAGGAGgtgagg 693 gcctagGAGTGT
 R  R        S  V
   732       733
20 160 CCTGCAgtaagt 461 ctcaagCCTCCA
P  A       P  P
   754       755
21 66 AATCAGgtaagc 473 caatagTTTAGA
N  Q       F  R
   771       772
22 51 AAGCAGgtaagg 2687 tcccagGTCAAG
K  Q       V  K
   796       797
23 75 ACTCAGgtgagc 424 tggcagGGAGCT
T  Q       G  A
24 587 ATAGATcaatca

Exons are often associated with separate structural or functional domains of a protein (32), such as in the immunoglobulin (33). However, the exon organization of the mCD156 gene is partially predictable based on the predicted protein structure (Figs. 1 and 2). The mCD156 protein is composed of a signal peptide, a pro-MTP, an MTP, a disintegrin, a CR, an EGF, a transmembrane, and a cytoplasmic domain. The first exon encodes most of the signal peptide sequence, and the transmembrane domain is encoded by a single exon (25). In contrast, other regions are comprised of multiple exons. Exons 2-6 encode a pro-MTP domain; exons 7-12, an MTP domain; exons 12-14, a disintegrin domain; exons 14-18, a CR domain, including an EGF domain within exons 17-18. The cytoplasmic region shows a proline-rich amino acid stretch that is encoded by exons 20-23. The carboxyl-terminal residues that have little proline and the total 3'-untranslated region are encoded by exon 24. 


Fig. 2. Amino acid sequence alignment of CD156 with ADAM 11 and boundary of exons. Consensus alignment of CD156 (D26579) and ADAM 11 (human, MDC-769) (D31872). Reversed print reflects identical residues. Amino acid positions are indicated at the right margin of each line. Sequences were aligned using the NBRF-PDB with the GENETYX system (Software Development Co., Ltd., Tokyo). For maximum alignment, gaps were introduced. The transmembrane regions are underlined. Open circles above the sequence indicate the consensus MTP active site sequence of mCD156. The vertical bar above and below amino acid sequences indicates the boundary of each exon. The number above and below amino acid sequences represents the exon number.
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The exon-intron organization of the ADAM 11 gene has been reported (34). The numbers of its exons and introns were 25 and 26, respectively, resembling those of the mCD156 gene. Therefore, we compared amino acids encoded by each exon between mCD156 and ADAM 11 (Fig. 2). It is of note that 12 exons out of 24 of mCD156 correspond to the homologous regions of exons of ADAM 11; for example, the boundary between exons 3 and 4 of the mCD156 gene corresponds to that between exons 1 and 2 of the ADAM 11 gene.

Determination of Transcription Initiation Site

The transcription start sites of the mCD156 gene were analyzed by S1 nuclease protection. An antisense single-stranded DNA probe (231 bp) corresponding to the region spanning from site (+172) to an upstream PstI site (-183) (see "Experimental Procedures") was constructed. The results suggested the presence of a major transcription start site approximately 114 nucleotides upstream of the translation start site (Fig. 3A). The transcriptional initiation site demonstrated by S1 nuclease protection was confirmed by primer extension (see "Experimental Procedures"). An antisense synthetic oligonucleotide was used for primer extension using RNA isolated from aHINS-B3 cells and reverse transcriptase. A single transcriptional initiation site 134 nucleotides upstream of the translation start site was identified (Fig. 3B), which confirmed the S1 nuclease result.


Fig. 3. S1 nuclease mapping and primer extension analysis of mCD156 mRNA. Panel A, an antisense 231-base DNA probe was hybridized to total RNA from aHINS-B3 (see "Experimental Procedures"). After digestion of single-stranded DNA with S1 nuclease, the protected fragments were electrophoresed. Dideoxynucleotide sequencing reactions were electrophoresed in parallel as markers. Lane S, products of S1 nuclease digestion in the presence of total RNA from aHINS-B3. Arrowhead indicates the site corresponding to that of the most predominant band in S1 nuclease mapping. Panel B, total RNA from aHINS-B3 was annealed to a 30-base oligonucleotide specific for a 5' region of mCD156 mRNA. The oligomers were extended and analyzed as described under "Experimental Procedures." Dideoxynucleotide sequencing reactions were electrophoresed in parallel as markers. Lane S, aHINS-B3 RNA with reverse transcriptase. Arrowhead indicates the 5' transcriptional site (nucleotide +1, see Fig. 4).
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Characterization of the 5'-Flanking Region

The 5' upstream region contained a number of potential regulatory sequences, as shown in (Fig. 4). TATA box-related elements (TATAGA) were identified at positions -25 to -20, possibly directing transcription from the indicated position +1 (Fig. 4). Although it differs from the canonical sequence by a G in the 5th position and although G and C are rare in TATA sequences and are associated with down-mutations in some promoters, the human beta -globin promoter TATA box contains a G (35). Other TATA box-related sequences, TATAA and AAATTT, are found further upstream, although these sequences appears to be too distant to direct transcription from the cap site. Another regulatory element, the atypical GC box-related motif GGGAGG (CCTCCC), is located at positions -295 to -290 and -231 to -226. Commonly, binding sites for Sp-1 are located near the TATA box, but in some cases, such as hydroxymethylglutaryl-CoA reductase, they are further upstream (36). The CAAT (ATTG) box and its reverse sequences are found at positions -225 to -222, -217 to -214, -168 to -164, and -112 to -109 (37). The PU box GAGGAA (TTCCTC) that is recognized by the macrophage and B cell-specific transcription factor PU.1, which is related to the ets oncogene (38), is located at positions -484 to -479 and -279 to -274. An NF-1 binding site exists at positions -49 to -37 (39). The interleukin (IL)-6 response element motif, CTGGGA (TCCCAG), is found at positions -621 to -616 and -200 to -195. The IL-6 response element is present in many acute phase response genes such as mouse serum amyloid A, rat alpha 2-macroglobulin, and C-reactive protein (40), and in the complement component 5 gene. The NF-IL-6 binding site and its reverse sequence are located at positions -591 to -588, -422 to -415, and -324 to -318. A minimal motif of IFN-stimulated response element, GAAANN, and its reverse sequence exists in the region between -358 and -338 (41). An interferon regulatory factor-1 binding sequence is located at positions -38 to -33 (42-44). A heat-shock element, GAANNTTC, shares elements with AAATTT at positions -320 to -315 (45, 46). The further upstream sequence (-531 to -691) has no consensus regulatory elements.


Fig. 4. Nucleotide sequence of the 5' region of the mCD156 gene. Nucleotides are numbered relative to the ultimate 5' transcriptional site, which is designated +1. Shaded area represents exon 1.
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CAT Analysis of Transcriptional Regulatory Regions Upstream of the mADAM 8 Gene

To examine the regions essential for transcription of the mADAM 8 gene, we fused various portions of the 5'-flanking region to the bacterial CAT gene as a heterologous reporter gene (see "Experimental Procedures" and Fig. 5). The constructs were transfected into aHINS-B3 cells, which express mADAM 8, and G203 cells, which express no mADAM 8. The region including 44 bp immediately upstream of the transcription start site (-44CAT) was incapable of directing CAT synthesis in aHINS-B3 cells, whereas -183CAT clone containing further 139 bases upstream of the -44CAT clone had a significant CAT activity. Stretch of the upstream sequence greatly diminished the CAT gene expression. However, the CAT gene expression increased by including position up to -334. Further elongation of the 5' upstream sequence reduced the reporter gene expression, but the -623CAT clone showed a significant CAT synthesis. Very little or no CAT synthesis was observed in G203 cells.


Fig. 5. CAT activities of 5' deletion mutants of the mCD156-CAT fusion gene in aHINS-B3 and G203 cells before and after stimulation with LPS or IFN-gamma . A series of chimeric plasmids was constructed and transfected into aHINS-B3 and G203 cells. Panel A, before and after stimulation with LPS (100 ng/ml) for 16 h. Panel B, before and after stimulation with IFN-gamma (200 units/ml) for 24 h. The relative CAT activity for each deletion mutant was expressed as a percentage of that of wild type -390/+90CAT stimulated with LPS. Values at right indicate the fold induction of CAT activity stimulated with LPS or IFN-gamma versus untreated but transfected cultures. The values presented are the mean from three independent experiments.
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We showed previously that expression of mMS2 mRNA in mouse macrophage cell line is enhanced by LPS and mouse IFN-gamma (1). We examined the effect of LPS and IFN-gamma on CAT expression. The CAT gene-transfected aHINS-B3 and G203 cells were stimulated with LPS (100 ng/ml) for 16 h (Fig. 5A). CAT synthesis in aHINS-B3 cells transfected with -183CAT and -390CAT clones after stimulation with LPS was high. -202CAT, -270CAT, -350ACT, -419CAT, -432CAT, -507CAT, and -559CAT clones were also capable of mediating the LPS-induced CAT synthesis. Fold induction of -183CAT and -334CAT was relatively low because of high basal activity. The CAT gene-transfected aHINS-B3 cells were stimulated with IFN-gamma (200 units/ml) for 24 h (Fig. 5B). CAT activities in aHINS-B3 cells transfected with -202CAT, -507CAT, and -659CAT clones were significantly enhanced after treatment with IFN-gamma . CAT synthesis in G203 cells was not enhanced by LPS and IFN-gamma treatment.

Chromosomal Localization of the mCD156 Gene

Under the conditions used, the hybridization efficiency was 90% for the probe (among 100 checked mitotic figures, 89 showed signals on one pair of the chromosomes). Since the DAPI banding was used to identify the specific chromosome, the assignment between the signal from the probe and the mouse chromosome 7 was obtained. The detailed position was determined further based on the summary from 10 photos (Fig. 6A). According to the summary, this gene is located at chromosome 7, region F3-F4. An example of the mapping results is presented in Fig. 6B. The mouse chromosome 7 broadly distributes in human chromosomes including chromosomes 10, 11, 15, 16, and 19. The human and mouse ornithine aminotransferase genes have been mapped to human chromosome 10q26 and mouse chromosome 7, respectively, and the human and mouse cytochrome P450, subfamily IIE genes have been mapped to human chromosome 10 and mouse chromosome 7, respectively (47). Thus, compared with human CD156 locus, there is a strong site relationship between human (chromosome 10q26.3) (2) and mouse genome.


Fig. 6. FISH mapping results. Panel A, diagram of FISH mapping results. Each dot represents the double FISH signals detected on mouse chromosome 7. Panel B, an example of FISH mapping. Panel a shows the FISH signals on chromosome; panel b shows the same mitotic figure stained with DAPI to identify chromosome 7.
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DISCUSSION

We have characterized the structure and determined the chromosomal location of the mCD156 gene. The gene spans approximately 14 kb and consists of 24 exons and 23 introns. The exon of this gene encode the mCD156 (MS2) cDNA that had been cloned from mouse myelomonocytic cells (1). The site of transcription initiation site, as determined by S1 nuclease protection and primer extention, agreed well with the location of a TATA box-like element TATAGA at nucleotide position -25 to -20 within the 5'-flanking sequence of the mCD156 gene. Other sequences that might interact with factors that regulate transcription were also present within the 5'-flanking region of the gene. Indeed, CAT analysis suggested the involvement of several regions that regulated the expression of the mCD156 gene in aHINS-B3 cells in the presence or absence of LPS or IFN-gamma .

We also localized the mCD156 gene to mouse chromosome 7, region F3-F4. We localized the human CD156 gene to the distal region of chromosome 10q26.3 (2), an area syntenic to mouse chromosome 7, region F3-F4, the region of chromosome 7 on which the mCD156 gene is found. ADAM 11 has been mapped to human chromosome 17q21(12), where many genes including the myeloperoxydase and homeo box region 2 genes are mapped (47, 48). However, the mouse homologous loci of these genes reside in chromosome 11, which also accommodates homologous loci of human chromosomes 2, 5, 7, 16, and 22. Two forms of transcripts generated by alternative splicing MDC-542 and MDC-769 are identified for ADAM 11: transcripts for MDC-542 encode 542 amino acids, which lack a transmembrane and cytoplasmic region, whereas that for MDC-769 encodes a protein of 769 amino acids which contain full regions from pro-MTP to the cytoplasmic region (34). The size and number of exons in the MDC-769 gene are strikingly similar to those of the mCD156 gene, although exon 1 of MDC-769 has splice variants. They consist of 25 exons and 24 introns: for MDC-769, a pro-MTP domain is encoded by multiple exons including 1b, 1c, 1d, and 2-7; an MTP domain by exons 7-13; a disintegrin domain by exons 14-17; a CR domain including an EGF-like repeat by exons 18-22; a transmembrane domain by exon 23; and a cytoplasmic domain by exons 24 and 25. Alignment of amino acids between mCD156 and ADAM 11 revealed that 12 exons out of 24, in particular, in central region of mCD156 completely corresponded to those of ADAM 11. Thus, the ADAM family genes are probably derived from the same ancestral gene. Since the mouse ADAMs 1, 2, 4, and 5 genes have been mapped to chromosomes 5, 14, 9, and 8, respectively (49), ADAM genes appear to be distributed to different chromosomes after duplication.

ADAM family proteins are a group of proteins exhibiting a hemorrhagic snake venom-like structure, with a widespread cell distribution including sperm, epididymal epithelium, placenta, ovary, breast, skeletal muscle, lung, heart, liver, kidney, small intestine, colon, brain, thymus, spleen, and leukocytes, and a high degree conservation throughout evolution (8, 50). Transmembrane and cytoplasmic domains have been added on the COOH-terminal side of mammalian ADAMs with the process of evolution, and new functions have been acquired. It might be possible to classify ADAMs into five subgroups on the basis of structure and function. The group 1 family of ADAMs (ADAM 10, TACE, KUZ, and possibly CD156) plays a role in the protein degradation by conservation of MTP activity (11, 15, 16, 51). The group 2 family (ADAMs 1 and 12) has a short hydrophobic region similar to viral fusion peptide and is mostly involved in membrane fusion (4, 13). The group 3 family (ADAM 2) induces cell adhesion. The group 4 family (ADAMs 9, 10, 12, and 15) mediates intracellular signaling or cytoskeletal attachment via proteins containing SH3 domains (10, 11, 13, 14). mCD156 could also belong to this class because the cytoplasmic tail of mCD156 has short proline-rich sequences resembling to SH3 ligand motifs. The group 5 (KUZ, ADAMs 7 and 12) facilitates differentiation and maturation of cells including nerve, sperm, and myogenic cells (7, 13, 51). Other ADAM family proteins will be subdivided into these groups in the future.

Some regions -183, -334, and -623 contained CAT active elements. Since the position up to -334 contains a PU box, the box would contribute to enhanced CAT expression. PU.1 has been shown to play a critical role in myeloid cell-specific expression of CD11b (Mac-1alpha chain) (52), macrophage colony-stimulating factor receptor (53), IL-1beta (54), Fcgamma .RIIIA (55) and Fcgamma .RIb (56). These results, therefore, suggest that the regions -183, -334, and -623 are involved in the cell type-specific expression of the mCD156 gene. The TATA box-like sequence, IL-6 response element, and NF-IL-6 binding motif are present in the region -623 to -507, whereas CAAT and inverted CAAT boxes are found in the region -183 to -94.

The regions -183 and -390 efficiently regulate the response to LPS, although many other regions also appear to contain elements responsible for LPS inducibility. Several elements including NF-kappa B (57-59), Oct-2 (60), AP-1 (61), and NF-IL-6 (59) binding sites have been shown to mediate response to LPS in several systems, although the role of NF-kappa B and NF-IL-6 binding site is controversial (62, 63). Of these, the 5'-flanking region contains three NF-IL-6 binding sites. NF-IL-6 binding sites may be involved in LPS inducibility, although the regions -183 and -390 have no such elements. Therefore, other cis regions and/or novel elements may confer LPS responsiveness.

A few restricted regions play a role in IFN-gamma -mediated CAT synthesis, in particular, the distal two regions -507 and -659 being important. The 5' upstream region of the mCD156 gene contains elements responsible for IFN-gamma inducibility such as NF-IL-6 binding sites (58, 63) and IFN-stimulated response element (64). However, none of these elements is found in the distal regions. Further analysis is needed to localize cis regions for IFN-gamma -stimulated responsiveness.


FOOTNOTES

*   This work was supported in part by Grant 02454169 from the Ministry of Education, Science, and Culture.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s)  D10911.


Dagger    To whom correspondence should be addressed. Tel.: 0975-86-5690; Fax: 0975-86-5699; E-mail: yamamoto{at}oita-med.ac.jp.
1   The abbreviations used are: mCD156, murine CD156; kb, kilobase(s); MTP, metalloprotease; LPS, lipopolysaccharide; IFN, interferon; bp, base pair(s); Pipes, 1,4-piperazinediethanesulfonic acid; CAT, chloramphenicol acetyltransferase; CR, cysteine-rich; EGF, epidermal growth factor; NF, nuclear factor; IL, interleukin; FISH, fluorescent in situ hybridization; DAPI, 4',6-diamidino-2-phenylindole.

ACKNOWLEDGEMENTS

We thank Dr. M. Setoguchi for useful discussions and T. Iwao for the photography.


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