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From www.bloodjournal.org by guest on June 18, 2017. For personal use only. The Murine Interleukin-3 Receptor a Subunit Gene: Chromosomal Localization, Genomic Structure, and Promoter Function By lkuko Miyajima, Lee Levitt, Takahiko Hara, Mary A. Bedell, Neal G. Copeland, Nancy A. Jenkins, and Atsushi Miyajima The interleukin-3 receptor (IL-3R) is composed of (Y and p subunits, members of the class I cytokine receptor family. Here we describe isolation andcharacterization of the chromosomal gene for the mouse IL-3R (Y subunit (mlL-3Ra). Whereas the human IL-3Ra gene is tightly linked with the granulocyte-macrophage colony-stimulating factor receptor (Y subunit (GM-CSFRa) gene in the pseudoautosomal region of the X and Y chromosomes, the mlL-3Ra gene (113ra) is located in the proximal region of mouse chromosome 14, separated from the mouse GM-CSFRa gene, which is on chromosome 19. The mlL-3Ra gene spans about 10 kb and is divided into 12 exons. All the exon-intron boundaries possess the splicing junction consensus sequences (5’GTAG3 ’ 1, and the wholegenomic structure is similar t o those of the previously reported class I cytokine receptor genes. There are t w o major transcription initiation sites that are located at 215 and 188 nucleotides upstream ofthe initiator codon. The promoter region is GC-rich and contains potential binding sites for GATA, Ets, c-myb,, Spl, Ap-2, and G-C boxes, but not a typical TATA or CAAT sequence. A fusion gene containing 0.8 kb of the 5 ’ noncoding sequence linked to the firefly luciferase gene directed the transcription in mouse mast cells but not in fibroblasts or T cells, suggesting that this promoterfunctions in a cell type-specific manner. Further sequential deletion of the 5’ region suggests t w o potential regulatory regionsfor transcriptionof the mlL-3Ra gene. 0 7995 b y The American Society of Hematology. H shared by the three receptors.* The high affinity receptors for IL-3, IL-5, and GM-CSF are composed of two subunits, a and p, both of which are members of the class I cytokine receptor family. The a subunits (IL-3Ra, IL-SRa, and GMCSFa) are cytokine-specific and bindtheir specific ligand p withlowaffinity by themselves, whereasthecommon subunit (pc,originally termed KH97) isrequired for high affinity binding as well as signaling by these three receptors in the human. Thus, pc is responsible for common functions of these cytokines, and expression of the a subunits is responsible for specificity to cytokines.’ In contrast to the human receptors, there are two distinct p subunits in the mouse. Likethe humanreceptors, themousehas three cytokinep subunit (pc,originally specific a subunits and the common termedAIC2B). In addition, mouseIL-3(mIL-3) specific p subunit originally termed AIC2A), which is 91% identical to p, at theaminoacid level, is presentin the binds mIL-3 with low affinity andinteracts mouse.’..’ pIL3 mGMwithonly themIL-3Ra5 but not witheitherthe CSFRa6 or the mIL-5Ra7 subunit. However, no clear functional difference hasbeen found betweenthe two distinct high affinity mIL-3Rs formedwith either pcor plLl.’ The two p subunit genes are closely linked on mouse chromosome 15, and their genomic organization as well as surrounding sequences arewell conserved,’ suggesting that the twogenes arose by duplication. As receptor expression is a prerequisite for cytokine response and IL-3 interacts with an early multipotential hematopoietic progenitor, the IL-3R expression may be an excelfor hematopoietic development. In fact,the lentmarker IL-3R expression seems to be restricted mainly to myeloid lineages. By using antibodies against receptor subunits, we have previously shown that IL-3a and pc are widely distributed in myeloid cell lineages, as well as early progenitors in human bone marrow and cordblood cells. In the lymphoid compartment, IL-3R expression is found in a minor fraction of cells with B-cell marker CD19, but not in cells with Tcell marker CD3.’ To study the developmental and cell-type specific expression of the IL-3R, we have characterized the chromosomal gene (113ra)for the mIL-3Ra subunit and analyzed the transcriptional activity of its promoter. EMATOPOIESIS isan excellent model system tostudy the mechanism of cell proliferation and differentiation, because a pluripotent stem cell differentiates through a series of committed progenitors towardterminally differentiated cells of each blood cell type. Each process is regulated by cytokines that are produced by a wide variety of cell types, either constitutively or inducibly in response to hematologic or immunologic stimulation, and exhibit pleiotropic functions.’ Among those cytokines, interleukin-3 (IL-3), IL5, andgranulocyte/macrophage colony-stimulating factor (GM-CSF) are major hematopoietic cytokines that are secreted by activated T cells and mast cells and play an important role in hematopoiesis associated with the inflammatory reactions. Whereasthesethreecytokinesshow no significant aminoacidsequencehomology, the genesare tightlylinked onthesamechromosome, theexon-intron structures are similar, and they exhibit similar functions in common target cells.’ Common biologic function exhibited by these three cytop subunitthatis kinesisnow explained by the common From the DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA; and the Mammalian Genetics Laboratory, ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD. Submitted January 18, 1994; accepted October 28, 1994. Supported in part by the National Cancer Institute, Department of Health and Human Services, under Contract No. Nol-CO-74101 with ABL. M.A.B. was supported by a fellowship fromthe Foundation for Advanced Cancer Studies, Inc. DNAX Research Institute i s supported by Schering-Plough. L.L. is presently at the Department of Hematology, Stanford University School of Medicine, Stanford, CA. Address reprint requests to Atsushi Miyajima, PhD. DNAX Research Institute of Molecular and Cellular Biology, 901 California Ave, Palo Alto, CA 94304. The publicationcosts of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1995 by The American Society of Hematology. 0006-4971/95/8505-0026$3.00/0 1246 Blood, Vol 85, No 5 (March l), 1995: pp 1246-1253 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 1247 MURINE IL-3 RECEPTOR a SUBUNIT GENE MATERIALSANDMETHODS Cell culture. A mouse mast cell line, PT18" was cultured in RPM1 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 50 pmoUL 2-mercaptoethanol (2-ME), and 50 U/ mL mGM-CSF. A mouse fibroblastic cell line, NIH3T3, was cultured in Dulbecco's modified Eagle's medium (DMEM) with 5% FCS. An IL-2-dependent T cell line, CTLL2," was maintained in RPM1 1640 medium containing 10% FCS, 50 pmol/L 2-ME, and 1 0 0 U/ mL mIL-2. Isolation and subcloning of genomic clones. A Balb/c sperm genomic library in Charon 4A (provided by Dr Mark Davis, Stanford University, Stanford, CA) and an adult Balbk liver genomic library in A SP6n7 (Clonetech, Palo Alto, CA) were screened with the 1.3kb fragment encoding the entire IL-3Ra. Furthermore, a C129 mouse genomic library in AEMBL was screened with a 0.6-kb NCOI-Sma I fragment of cDNA. The probes were labeled with [a-'*P]dCTP by the T7 Quick Prime kit (Pharmacia, Alameda, CA) to a specific activity of greater than 4 X 10' c p d p g . Plaques were transferred to a nitrocellulose filter and hybridized for 18 hours at 42°C in hybridization solution (6X SSPE, 50% formamide, 5X Denhardt solution, 100 pg/mL yeast tRNA, 0.2% sodium dodecyl sulfate [SDS]). Positive plaques were purified, and phage DNA was prepared by a standard plate lysate procedure. The mIL-3Ra gene was subcloned into pUCl8 for further characterization and sequencing. Characterization of cloned DNA fragment and DNA sequencing. The subcloned DNA fragments were mapped by restriction enzyme analyses, andthe exons were localized by Southern hybridization using mIL-3Ra cDNA as a probe. Supercoiled plasmid DNA was alkaline-denatured and sequenced by the dideoxy chain termination method using Sequenase (United States Biochemical Corp, Cleveland, OH) for all the intron-exon junctions and short introns. The sizes of introns were determined by polymerase chain reactions (GeneAmp PCR reagent kit: Perkin Elmer Cetus, Norwalk, CT) using several oligonucleotide primers corresponding to mIL-3a cDNA. The reaction mixture including 1 ng template DNA and 100 pmol/L each primer were incubated in a thermal cycler (Perkin Elmer Cetus) for 30 cycles (denatured for 1 minute at 9 4 T , annealed for 2 minutes at 5 0 T , elongated for 3 minutes at 72°C). Products were analyzed by electrophoresis on a 1.6% agarose gel in the presence of ethidmm bromide. Southern hybridization analysis of mouse genomic DNA. Genomic DNA (10 p g ) from liver of adult Balbk (Clonetech) and C129 SV mice were digested to completion by the restriction endonucleases (EcoRI, BarnHI, and HindIII). After electrophoresis on a 1% agarose gel, the DNA was treated with 0.25 mom HC1, alkalinedenatured (0.5 m o m NaOH, 1.5 mol/L NaCI), neutralized (0.5 mol/ L Tris HC1, pH 7.5; 1.5 m o m NaCl), transferred to a Hybond-N nylon membrane (Amersham, Arlington Heights, IL) using 20x SSPE, crosslinked by UV transilluminator (Stratacross-linker; Stratagene, La Jolla, CA), and then hybridized with the 32P-labeled1.3kb mIL-3Ra cDNA. 5'-rapid amplification cDNA ends (5'-RACE) assay. PolyA+ RNA was prepared from PT18 cells by the Fast Track mRNA Isolation kit (Invitrogen Corp, San Diego, CA). The d L - 3 R a cDNA was reverse transcribed from 10 pg of polyA+ RNA together with an 18-mer oligonucleotide primer complementary to a sequence of the third exon (ST104; 5 'GCTCAGTGTGTAGTGGGCGGG3'). The cDNA purified by agarose gel electrophoresis was then ligated with AmpliFINDER anchor according to the manufacturer's protocol (Clonetech). The cDNA including 5 '-end was amplified by polymerase chain reaction (PCR) as follows. One tenth of the ligation product was amplified using a set of a 38-mer anchor primer and an antisense 18-mer primer (ST-125; S'GAGTGGGATGCCAGGAGCCCC3') within the second exon, under the following condition: 35 cycles of 94°C for 1 minute, 57°C for 1 minute, 74°C for 3 minutes, and eight more cycles with additional enzyme. The first amplified product was then diluted one tenth and further amplified using another nested antisense primer (ST-143; 5 'GCCTCGGAGGCCTGGGGGCGG3') under the same condition except for annealing temperature at 55°C. The final amplified product was treated with T4 DNA polymerase to create blunt ends. After heat inactivation of polymerase, the DNA was cleaved with EcoRI, as the anchor sequence includes an EcoRI site. The resultant DNA fragments were purified by agarose gel electrophoresis and spin column (Microspin S-200: Pharmacia) and then subcloned into the EcoRI-Sma I sites of pUCl8. The cloned DNAs were sequenced to identify the site(s) of 5 '-cDNA ends. Interspec$c backcross mapping. Interspecific backcross progeny were generated by mating (C57BL/6J X Mus spretus)Fl females and C57BL/6J males as described.'* A total of 205 Nzmice were used to map the 113ra locus (see Results for details). DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer, and hybridization were performed as de~cribed.'~ All blots were prepared with Zetabind nylon membrane (AMF-Cuno, Meriden, CT). The probe, a 1.3-kb Xho I fragment of mIL-3Ra cDNA (SUT-l), was labeled with [a"P]dCTP using a random primed labeling kit (Boehringer Mannheim, Indianapolis, IN); washing was performed to a final stringency of 1 .OX SSCP, 0.1% SDS, 65°C. A major fragment of 7.6 kb was detected in Bgl 11-digested C57BW6J DNA, and a major fragment of 11 .0 kb was detected in Bgl 11-digested M sprerus DNA. The presence or absence of the 11.0-kb M spretus-specific Bgl I1 fragment was followed in backcross mice. A description of the probes and restriction fragment length polymorphisms (RFLPs) for the loci linked to 113ra, including retinoic acid receptor beta (Rarb, formally referred to as Hap), plasminogen activator urokinase (Plau), and SP-A pulmonary surfactant protein ( S f t p l ) , has been reported previo~sly.'~.'~ Recombination distances were calculated as described16 using the computer program Spretus Madness. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns. Transient expression assays. For the analysis of promoter activity, the fragment containing the 5' noncoding region of the mIL3Ra gene was fused to the firefly luciferase gene. The luciferaseexpression vector pGL2-Basic Vector (Promega Corp, Madison, WI) was used. Cells (5 X 10' PT18 cells, 5 X IO6 CTLL-2 cells, lo6 NIH3T3 cells) were transfected with supercoiled plasmid DNA (40 pg for FT18,40 pg for CTLL-2,30 p g for N1H3T3) by electroporation. At 18 hours after transfection, the luciferase activity was assayed using the luciferase assay kit (Promega Corp, Madison, WI). The SRa promoter (hybrid promoter containing SV40 and HTLVLTR)-luciferase fusion gene was used as a positive control. RESULTS Chromosome mapping. The mouse chromosomal location of the IL-3Ra gene (I13ra) was determined by interspecific backcross analysis using progeny derived from matings of [(C57BL/6J X M spretus)Fl X C57BL/6J] mice. This interspecific backcross meiotic mapping panel has been typed for over 1,500 loci that are well distributed among all the autosomes as well as the X chromosome.'* This indicates that, on average, markers are spaced about 1 centiMorgan (CM) apart over the genome. To place a new marker, an RFLP is established by Southern blot analysis of the two parental strains in the cross, and the backcross mapping panel is then typed for the informative RFLP. The segregation patterns of the new marker are then compared with known From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 1248 MlYAJlMA ET AL segregation patterns of all other markers. Gene order is determinedby minimizing the number of double or multiple crossovers required to explain the new probe distribution. C57BU6J and M spretus DNAs were digested with several enzymes and analyzed by Southern blot hybridization for informative RFLPs using a mouse IL-3Ra cDNA probe. An 11.O-kb M spretus Bgl I1 RFLP (see Materials and Methods) was used to follow the segregation of the I13ra locus in backcross mice. The mapping results indicated that Il3ra is located in the proximal region of mouse chromosome 14 linked to Rarb, Plau, and Sftpl.Although 161 mice were analyzed for every marker and are shown in the segregation analysis (Fig l), up to 185 mice were typed for some pairs 0 0 11 7 0 2 of markers. Each locus was analyzed in pairwise combinations for recombination frequencies using the additional data. The ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene order are as follows: centromere-Rarb-0/174-I13ra-2/185-Plau-19/18314 S'pl. The recombination frequencies (expressed as genetic distances in centiMorgans [CM] 2 the standard error) are (Rarb, IZ3ra)-l.l 2 0.8-Pluu-10.4 +- 2.3-Sftpl.No recombinants were detected between Rurb and I13ra in l74 animals typed in common, suggesting that the two loci are within 1.7 CM of each other (upper 95% confidence limit). Rarb 3p24 We have compared our interspecific map of chromosome I13ra lOq24qter Plau 14 with a composite mouse linkage map that reports the map location of many unclonedmouse mutations.'haI13ru mapped in a region of the composite map that lacks mouse mutations with a phenotype that might be expected for an alteration in 10.4 l - 1 this locus (data not shown). Isolation of chromosomal gene for the mIL-3Ra gene. Of 5 X IO5 independent phage plaques of the Balb/c sperm library screened with the 1.3-kb cDNA (SUT-l) fragment 1Oq21-q24 of mIL-3Ra as a probe, one positive clone (Xsut-l) was obtained. Using the same probe, three positive clones (Xsut3, -4, and -6) were obtained from 2 X 10' plaques of the Balb/c liver library. These four clones were plaque-purified, and the X DNA were analyzed by restriction enzyme mapping and Southern hybridization. Three clones (Xsut- I , -3, and -4) were overlapped, covering the 3 ' region of the gene, Fig 1. 113ramaps in the proximal region of mouse chromosome 14. 113rawas placed on mouse chromosome 14 by interspecific backcross while one clone (Xsut-6) contained the 5 ' portion (Fig 2). analysis. The segregationpatterns of N3ra and flanking genes in 161 These four clones covered most of the cDNA sequence of backcross animalsthat were typed for all loci are shown at the top mIL-3Ra, except for the short fragment located in the middle of the figure. Forindividual pairs of loci, more than 161 animals were of cDNA including the exon V. To isolate the DNA for this the chromosome identityped (see Results). Each column represents missing portion, 10' plaques of the C129 mouse genomic fied in the backcross progeny that was inherited from the (C57BLI 6J x M spretus)Fl parent. Thesolid boxes representthe presence of a library were screened with the 0.6-kb Sma I-NCOl fragment C57BL/6J allele, and open boxes represent the presence ofMspretus of mIL-3Ra. Only one positive clone (Xsut-7) was isolated, allele. The number ofoffspring inheriting each type of chromosome and it contained the sequence that was missing in the other is listed at the bottom of each column. A partial chromosome 14 clones (Fig 2 ) . linkage mapshowing the location of 113ra in relation to linked genes is shown at the bottom of the figure. Recombination distances beTo confirm that the cloned DNA fragments represented tween loci in CMare shown to the left of the chromosome, and the the mIL-3Ra gene locus, mouse genomic DNA from the positions of loci in human chromosomes, where known, areshown liver of Balb/c and C129 mice was digested with EcoRI, to the right. References for the human mappositions of loci mapped BamHI, or HindIII and analyzed by Southern blotting using in this study canbe obtained from Genome DataBase (QDB), a computerizeddatabase of human linkage information maintainedby The the mIL-3Ra cDNA as aprobe. The EcoRI or HindIII cleavWilliam H. Welch Medical Library of The Johns Hopkins University age generated a single fragment of approximately 18 kb and (Baltimore, MDLU It should be noted that, while the region of the approximately 13 kb, respectively, whileBamHI cleavage mouse chromosome 14 where113ra is placed is homologous to huproduced two fragments of 7.5 kb and4 kb (data not shown). man chromosome 3p24, the human 113ra resides in the pseudoautosomal region of X and Y chromosomes, as described in Results. The 10-kb cloned chromosomal gene of mIL-3Ra, which om o m m 0 U. 0. Om B O BO O B B O BO BO From www.bloodjournal.org by guest on June 18, 2017. For personal use only. MURINE IL-3 RECEPTOR l 2 3 4 5 (Y 1249 SUBUNIT GENE 6 70910 U 1112 nn \ B P S I B P P AsUnr P R HB 1 kb U " " Fig 2. Genomic structure of the mouse IL-3Ra gene. Exons are shown byboxes. Solid boxes indicate the noncodingregion, and open boxes are coding exons. The restriction sites for & d l , Pot 1, Stu 1, Hindlll, and € d l are shown as B, P, St, H, and R, respectively. The clones used for the analysis are shown by bars.There is a single BamHl site present in exon 4, but no Hindlll or € d lsite within about 10 kb of mlL3Ra gene from exons 1 to 12. Clones h u t 3 and h u t 6 are derived from the Balb/c, and Asut7 is derived from C129. The dashed lines represent A arms. spans 12 exons, has no recognition site for EcoRI or HindIII and has a single site for BamHI (Fig 2), whereas an EcoRI site, a HindIII site, and two BamHI sites are located nearby in the surrounding regions, which indicates that one copy of the mIL-3Ra gene is present per genome and is covered by the cloned DNA fragments. Structure of the mIL-3Ra gene. The overall structure of the mIL-3Ra gene was determined by Southern hybridization, PCRs, and DNA sequencing: PCR and restriction enzyme analyses were performed to determine intron length using the cloned A DNAs and the subcloned plasmid DNAs. All exon-intron borders and several smaller introns were sequenced entirely. As shown in the schematic diagram of the gene and restriction map (Fig 2), the estimated length of the gene from the transcription start site to the polyadenylation site is approximately 10 kb. The gene contains 11 introns, and all the sequences of intron-exon junctions conform with the consensus sequences of eukaryotic splice junctions (Table 1). Exon I encodes 5 "untransiated sequence, and exon I1 encodes more 5 "untranslated sequence and the signal peptide. The extracellular domain is encoded by seven exons (exons I11 to IX), the transmembrane domain by a single exon (exon X), and the cytoplasmic domain by two exons (exons XI and XII). Exon XI1 also contains the 3 "untranslated sequences. Identijcation of the transcriptional srart site(s). To identify the functional promoter region that regulates mIL-3Ra gene expression, we attempted to map the 5'-end of the transcript by S 1 nuclease protection assay and primer extension. However, the results were rather ambigous, probably due to the presence of the G/C rich sequences in the 5' region. Therefore, we mapped the transcriptional start site(s) using the 5 ' RACE assay. First, the 5 '-ends of the mRNA were reverse transcribed from polyA+ RNA of PT18 cells. The cDNA was amplified and subcloned into pUC18. Plasmid DNA from 13 individual clones was isoIated, and the cloned DNA fragments were sequenced. Six clones had the same 5 '-end at 215 nucleotides, and seven clones had the 5 '-end at 188 nucleotides upstream from the initiation codon (Fig 3), suggesting that the transcription starts at these two positions. Sequences of the S'janking regions. Although there are no conventional TATAA and CCAAT sequences in the 5 'flanking region of the mIL-3Ra gene (Fig 3), the potential promoter region carries several noteworthy features. First, the overall G + C content (65%) is unusually high. There are several GC boxes,I7 Spl binding sites (G/TG/AGGCG/ TG/AG/AG/T)," AP-2 binding sites (CCCA/CNG/CG/CG/ C),'8 and a G-stretch of a consensus sequence (CS) for erythrocyte-specific binding protein BGP1.I9 Second, an A T stretch of 21-mer is present and includes a CS (C/TA/GGTTTCA/GC/"TC/TC/TN) for interferon CS binding protein ICSBP.20Third, two known recognition sequences are present tandemly: GATA (A/TGATAA/G)2' andEts (G/CA/ CGGAA/TGT/C)," two Myb sequences (T/CAACG/TG)'* that compose a short palindrome, CaGTTGCAACgG. Moreover, there is a 21-bp potential palindromic sequence (GaGGCCCCGgcgCGGGGCCaC) just upstream of the GATAEts sequences. An NFKB site (GGA/GA/GAT/GT/CCCCC/ T)" is also found at about -350 bp. The promoter region contains several repetitive sequences, including some CS for known transcription factors: three GC boxes, three y-interferon responsive elements (CA/TGG/ + Table 1. Exonllntron Boundaries No. Exon Size (bp) Donor 1 2 3 4 5 6 7 8 9 10 11 12 135 178 156 123 130 233 119 30 115 103 67 ACTCAG GAT CTG G GTC TGG GAT GAT G GAT GTG CG CGC GCA G AAC CAG TAC AAT GGC CTC G TGG TGG AG GAG ATG Intron Size Ikb) gtccggtccg-0.5-accatgacag gtaggtgtct-0.6-ttgacaacag gtgaggggca-0.8-gaccccgcag gtgggtgagg-0.25-aggcccacag gtgggtttta-0.7-ctcgcaccag gtgagggcct-0.4-ccgcccccag CTG gtgcggggtg-0.12-ctgcccccag gtgagttggg-0.09-gcccaccccag gtgagtgggt-0.8-cttcccgcag gtgagggcct-3.0-gtccccacag gtgagggggc-0.27-ctctctgcag Acceptor GTC GAACCA CT GCG GTC GCT GAC GT GGC GAC G CTC GGT AG AGC TCC GTC TCC TC TGC CCC G AAG TCG GTC GTG All exon/intron boundaries were sequenced. Spaces in the splicing donor and acceptor sequences indicate reading frame of the protein. From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 1250 MlYAJlMA ET AL C.T...G(;AEiCACAGG ACAAATTCTG AAAAACGGAA ACGGAAATAA GGCCGCCTGT GAGGCAT$W$$GC$TATCC ,TGGGCGGGGT,GGGGCCGGCC TCCTAAGCACJ, GC-BOX CIFTGTGACG TCCGCTGTGA PVUII GCTCTGCTC CAGAGTGGGG GGGGGGGGGG GGGATGAAGA CAGGGCAGGG -A0 BOP 1 TCCGCTCACA AGTTAAGTTC CGTGGGTCTC AATGGGATGC CTTCAGGCAT *..***.* GGATGGCAGG TAGTGAACAA CCGCTACAAA e.*.* GCAGGGGTAT GATGGC&G.Cx. ....FTCAGTGACT GCGGGGTAAA GAGCGTGAGG - C G A G T ~ A G TT&AACG&T GACAGGAGAG CGACTTCCTG TCTGCGGCAG GACGTCACGF CCGCTCGCCC GTGACTGACGGGAACGACTT CCAGTTTCAT ICSBP -250 - 4 TCCCTCCCGC GTGACTCCCG A G G X C ~ ~ ~ C " ~ C G G G G C $ C TATC%AGGA -50 BGCTCAA TCTCGGGACC ATGGGCGCGG T ~ G G G C G G GGF GGGGCTCT ' GTTCCGACCC ' GGGGTCACG GCAGGGTCFCAGECCTCTGCCCTGTAAGCA +l E GC-Box t10 CCAGACCCFC GAGGACAGGA E% +50 * AE~ACGCCG .&f" transfected cell lysates was measured 18 hours after transfection. As shown in Fig 4, luciferase activity was clearly detected in PT18 but not in CTLL-2 or NIH3T3 cells transfected with the Pst-luc plasmid. The plasmid with the Pst I-Stu I fragment inan opposite direction (shownas a Pst-luc-) generated a significantlylow level of luciferase activity, suggesting an orientation dependency of thepromoter. The results also suggest that the 0.8-kb fragment is able to direct transcription in a cell type-specific manner. To locate the promoter function within the 0.8-kb Pst IStu I fragment, the 0.4-kb Pvu 11-Stu I and the 0. 17-kb Eag I-Stu I fragments were also fused to the reporter gene, and the resultant plasmids, Pv-luc and E-luc (Fig 4), were used to evaluate the promoter function. Pv-luc directed production of luciferase in PT18 cells, but not in CTLL or NIH3T3 cells. The luciferase expressed in PT18 by Pv-luc was about half of that produced by Pst-luc. In contrast, luciferase produced by E-luc in PT18 was about the same level as that produced by Pst-Iuc- (Fig 4). These results suggest that multiple DNA elements may be required for the transcription of the IL-3Ra gene in hematopoietic cells. CCAGCGCCGT GGGCGCCACT ,.."""2 ....At+++ ++++++++++ Fig 3. Nucleotidesequence ofthe 5"flanking region of the mouse IL-3Ra gene. The transcriptional start sites determined by 5' RACE are indicated by arrowheads. The numbering (bp) in sequence is based onthe longer 5'-cDNA end as +l. A sequence of the previously isolated cDNAis marked with pluses. The potential binding sites for various transcription factors are shown. An arrow indicates the apparent palindromicstructure. NF-IL6-like sequences (see Results) are marked by asterisks. The Pst 1, Pvu II, Eag I, and Stu I sites used for the luciferase constructs in Fig4 are also indicated. TAAGT/C),22five AP-2 binding sites, and eight GCF CS (C/GCGC/GCIGC/GC)." An imperfect direct repeat ofan 1 l-bp motif, GCTGTGACGTCCGCTGTGAGCTCexists at around -570, and a sequence of 5 'GCGCCGTGGGCG3 ' is present at around +80 and -610. In addition, the sequence (T/G)(G/A)(A/T)GGCAG, which is similar to the NF-IL6 CS (TT/GNNGNAAT/G)," is repeated six times between -320 and -470 (Fig 3). Cell type-specijic function of the promoter. IL-3Ra mRNA is expressed in various hematopoietic cell lines, including mast cells, myeloid progenitors, pro-B cells, and early progenitors, and in nonhematopoietic cells such as ES Cells5.23 and endothelial but not in mature T cells, mature B cells, and fibroblasts.' To study whether the 5'flanking region of the mIL-3Ra gene has transcriptional activity, the 0.8-kb Pst I-Stu I DNA fragment was fused to the reporter gene, the firefly luciferase gene. Pst I and Stu I are located in the 5 '-flanking region (Figs 2, 3, and 4). This fragment contains one of the transcription start sites mapped by the S'RACE assay. The resultant plasmid, Pst-luc, was transfected into the mouse mast cell line PT18, the T-cell line CTLL-2, and the fibroblastic cell line NIH3T3. As a control, pME-luc, which has the SRa promoter (ie, a hybrid promoter between SV40 and HTLV-LTR25), was also transfected into these cell lines. Luciferase activity in the DISCUSSION The genes for IL-3, IL-5, and GM-CSF are tightly linked on human chromosome 5 and mouse chromosome 1 l.' In contrast, while the human IL-3Ra (fL3RA)and GM-CSFRa (CSF2RA) genes are also tightly linked on the pseudoautosothe ILmal region of the human X and Y chromosomes,2h-28 5Ra gene (fL5R)is on 3p24-3~26.~' All the mouse a subunit genes are located on different chromosomes: mIL-3Ra (IZ3ru), mGM-CSFRa (Csfzra),'" and mIL-5Ra (fL5r)'' are on mouse chromosomes 14, 19, and 6, respectively. Interestingly, however, the proximal region of mouse chromosome 14, where 113ru and Rarb genes reside, shares regions of homology with human chromosome 31324, and humanRARB has been placed on 3p24. Although the human IL3RA gene is located on psuedoautosomal regions of X and Y chromosome~,~'.~* the human fL5-R gene resides on 3 ~ 2 4 . ~While ' this is an interesting finding, it is unknown whether this is due to a result of recombination between these loci or is a simple coincidence. The mIL-3Ra gene spans about 10 kb and is divided into 12 exons. As shown in Fig 5, the overall structure of the gene is similar to that of previously reported cytokine receptors, including human and mouse erythropoietin receptors (h/m E ~ o R ) , ' human ~ . ~ ~ IL-2 receptor P subunit (hIL-2RP)," mIL4R," h/m IL-7R," human G-CSF receptor (hG-CSFR)," and two mouse P subunits, AIC2A and AIC2B.s In general, the gene encoding the class I cytokine receptor motif is divided into four exons, except for hIL-2RP and mIL-3Ra (Fig S ) . In IL-2RP, one of the two exons encoding the conserved cysteine residues is split into two exons." In contrast, there is a small exon of 30 nucleotides in front of the exon for the WSXWS motif in the mIL-3Ra gene. It would be interesting to see whether a unique small exon is also present in the GM-CSFRa and IL-5Ra genes. When linked to the promoterless firefly luciferase gene, the 0.8-kb Pst I-Stu I fragment, including a transcription initiation site and a 14-bp 5"untranslated region, directed From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 1251 MURINE IL-3 RECEPTOR n SUBUNIT GENE Xhol I Pstl Pvull I I Eagl Stul I -1640 murine IL-3Ra I 0 +l4 start - Luciferase -776 -394 I Pst-luc P Pv-luc -160 - 1 E-IUC Relative Luciferase Activity (SRa=l) 0 Pst-luc+ 0.1 0.2 0.3 0.4 a i NIH3T3 Pst-lucPv-luc Fig 4. Promoter activity of the 5"flanking region of themlL3Ra gene. The 0.8-kb Pst I-StuI, 0.4-kb Pvu Il-Stu 1, or 0.15-kb Eag I-Stu I fragment containing IL-BRa gene was fused the 5"flanking region of the t o the luciferase gene as described in Results. Pstluc' and Pst-luc- indicate the correct and reverse orientationofthe fragments, respectively. Plasmid DNA was transfected intoNIH3T3 (solid bars), CTLL2 (shaded bars), or PT18 cells (open bars) and the luciferase activity in cell extracts was determined at 18 hours after transfection. The results are shown as relative luciferase activity; ie, luciferase activity obtained with SRa-luc (raw value of luciferase activity/100 p g cell extract protein) is regarded as1.0. Values represent the average of three t o four independent experimentswith standard deviation. E-Iuc Pst-luc+ 0CTLL-2 Pst-lucPv-luc E-Iuc Pst-luc+ 0PT18 Pst-lucPv-luc E-IUC the transcription in mouse mast cells but not in either mouse fibroblasts nor T cells in an orientation-dependent manner (Fig 4). In addition, another IL-3-dependent hematopoietic cell line, OTT-l , also expressed luciferase after transfection of Pst-luc at a level comparable to PT18 (data not shown). These results indicate that the Psr I-Sru I fragment contains the promoter that functions in a hematopoietic cell-specific manner. In addition, there are at least two regions of positive regulatory elements within 0.8 kb, as the 0.4-kb f v u IISru I fragment produced less luciferase than the f s r I-Stu I fragment (Fig 4). Although there is no typical TATAA and CCAAT sequence, TAATAA and GCAAT are present at -200 and - 140, respectively. Instead, like many housekeeping genes, the most proximal region to the start site is GCrich (GC content is 70%). Although there are many potential recognition sites for transcription factors as described above (Fig 3 ) , the role of these elements for cell type-specific expression is unknown. Identification of the cis elements for cell type-specific transcription is important to understand the regulation of the IL-3Ra. Because IL-3 interacts with very early multipotential hematopoietic progenitors, the IL-3 receptor must be present on these cells. In fact, the early human hematopoietic progenitors with a hematopoietic stem cell marker CD34' express IL-3Ra andIL-3RDsubunits.') The 0 subunit RNA is not present in mouse embryonic stem (ES) cells. and it appears on day 7 of in vitro development after initiation of embryoid body culture. Likewise, 0 RNA is not detectable in blastocysts but is induced byin vitro culture of blastocysts. Timing of the appearance of the 0 subunit RNA is consistent with the onset ofhematopoiesis.".3"J" In contrast, the IL-3Ra transcript is detectable in ES cells as well as blastocysts.'3 However, the IL-3Ra expression is clearly tissue-specific andis restricted mainly to myeloid lineages.'.' Thus, transcription of the IL-3Ra and IL-3RP subunit genes appears to be differentially regulated during development. Consistent with the differential regulation, there is no significant similarity of the 5' upstream sequences between the mIL-3Ra andthe mlL-3RP genes (AIC2A and AIC2B),* although the GATA motif, frequently found in hematopoietic specific genes, is present in both genes. Another interesting feature of the 5 ' upstream sequence of the mIL-3Ra gene is a high frequency of CpG dinucleotide. Whereas CpG dinucleotides are usually found one fifth as From www.bloodjournal.org by guest on June 18, 2017. For personal use only. MlYAJlMA ET AL 1252 26 42 63 45 53 52 45 57 52 30 19 35 353 oo~moo81oo 19 52 41 43 77 39 10 38 33 22 18 frequently as GpC in mammalian DNA (CpG:GpC ratio is 0.2);' the CpG:GpC ratio in the region between - I to -200 of the IL-3Ra gene is as high as 1.0. Interestingly, like IL3Ra. the mouse pim-l gene, one of the hematopoietic specific genes, also contains a high G C content and has a high CpG:GpC ratio (1.0) in the corresponding region." In contrast, the CpG:GpC ratio of the S ' flanking regions of the 0 subunit genes between -1 and -200 is 0.27.' AsDNA methylationofteninhibits gene expression andCpG is known to be a common target of DNA methylation,"3 high frequency of CpG in the S'-flanking regionmayindicate gene inactivation by methylation. If methylation occurs in a cell type-specific manner during differentiation, it may explain why ES cells express IL-3Ra RNA but the differentiatednonmyeloid cellsdo not.5"." However,exogenously introduced unmethylated plasmid DNAs containing themIL3Ra-luciferase fusion genedid not expressluciferase in NIH3T3 cells. Thus. cell type-specific methylation, if it is present. does not solely explain the cell type-specific function of the mlL-3Ra promoter. Presumably, cell type-specific factors are required for regulated expression of mIL3Ra. either positively or negatively. Identification of such transcription factors is critical to understanding the mechanism of IL-3 receptor expression. ACKNOWLEDGMENT Wethank DJ. Gilbert and D.Thompson for excellent technical assistance, D. Robison for oligonucleotide synthesis,and G. Budget for preparation of the manuscript. We also thank Drs T. Iwamoto, D. Gorman. E. Masuda,and N. Araiforhelpfuldiscussionand critical reading of the manuscript. Fig 5. Structures of the cytokine receptors. Schematic representationsof the members of the class I cytokine receptor family, whose genomic structure has been published. Boxes represent exons. Hatched boxes indicate signal sequences. Exons encoding the transmembrane domains are shown as solid boxes. CC and WS indicate the exonsencoding the conWS motif, respectively. served cysteine residues and Stippled boxes in the most 5'-side represent exons that carry a signal peptide. The G-CSF receptor contains additional motifs in the extracellular domain, and only the exons encodingthe class I cytokine receptor motif are shown. Although the exons encoding the N-terminal and C-terminal domains also contain noncoding regions, the figure shows only protein-coding regions. 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For personal use only. 1995 85: 1246-1253 The murine interleukin-3 receptor alpha subunit gene: chromosomal localization, genomic structure, and promoter function I Miyajima, L Levitt, T Hara, MA Bedell, NG Copeland, NA Jenkins and A Miyajima Updated information and services can be found at: http://www.bloodjournal.org/content/85/5/1246.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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