T Cells and Hematopoietic Stem Cells - Molecular and Cellular Biology

Vol. 10, No. 10

MOLECULAR AND CELLULAR BIOLOGY, OCt. 1990, p. 5150-5159

0270-7306/90/105150-10$02.00/0 Copyright X 1990, American Society for Microbiology

Characterization of Promoter Elements of an Interferon-Inducible Ly-6E/A Differentiation Antigen, Which Is Expressed on Activated T Cells and Hematopoietic Stem Cells KHUDA DAD KHAN, GLEN LINDWALL, STEPHEN E. MAHER, AND ALFRED L. M. BOTHWELL*

Section of Immunobiology, Departments of Biology and Pathology and Howard Hughes Medical Institute, Yale University Medical School, 310 Cedar Street, New Haven, Connecticut 06510 Received 22 February 1990/Accepted 11 July 1990

The Ly-6E/A antigen is expressed on activated murine T cells. Using probes made from the previously characterized cDNA, we have isolated a genomic DNA clone encoding the Ly-6A antigen. We determined the DNA sequence of the genomic clone and conducted a functional analysis of the promoter region. Mouse fibroblast BALB/3T3 cells transfected with this genomic clone constitutively expressed Ly-6A antigen on their cell surface. This expression was inducible by alpha/beta and gamma interferons. The Ly-6E 5'-flanking region was analyzed by chloramphenicol acetyltransferase assays in fibroblast cells for cis-acting elements. At least two positive elements were found to be needed for maximum constitutive promoter activity in L cells. One of the positive elements was specifically bound by a CCAAT box-binding protein from crude nuclear extract, as shown by electrophoretic mobility shift assays and footprinting. The other element, which contains a GGAAA motif and has homology to various known enhancers, also showed a specific binding activity. This second positive element when multimerized became a very powerful enhancing element. Interferon treatment could enhance expression of the chloramphenicol acetyltransferase gene fused to the Ly-6E 5'-flanking region in stably transfected BALB/3T3 cells. The elements responsible for this enhancement lie, at least in part, between positions -1760 and -900 of the gene. Surprisingly, there is no sequence homology between this region of Ly-6E and the established consensus for the interferon-stimulated response element, which has been shown functionally important to all previously characterized alpha/beta interferon-inducible promoters. The Ly-6E gene may prove to be a novel system for the study of interferon induction. The Ly-6-encoded proteins are members of a multigene family which are anchored on the cell surface via a carboxyterminal phosphatidylinositol moiety (16, 25). These molecules are expressed at critical times in the differentiation of hematopoietic cells, and some of them are restricted to the cells of lymphoid lineage (23). Antibodies directed against both Ly-6E/A and Ly-6C antigens stimulate normal heterogeneous peripheral T lymphocytes to proliferate in the presence of appropriate second signals (18). As a consequence, one of these antigens, Ly-6A.2, has been called TAP (T-cell activation protein). The role of Ly-6 molecules in antigen-stimulated T-cell receptor-mediated responses was studied by selecting T-T-cell hybrid clones for the specific loss of surface Ly-6A.2. These mutants had markedly impaired antigen- and T-cell receptor-stimulated responses (28). Ly-6A.2 is expressed primarily on a subset of peripheral T cells and on most activated lymphocytes. In the thymus, this antigen is restricted to a relatively mature population of thymocytes (23). There is a similar pattern of expression for Ly-6E.1. The cDNAs for the genes encoding Ly-6A.2 and Ly-6E.1 are highly homologous and differ at only three nucleotides in the coding region, resulting in two amino acid differences (16, 19, 21). One of the amino acid differences is located in the carboxy-terminal end of the protein and is thought to be lost during the addition of phosphatidylinositol anchor. The mature proteins differ in only one amino acid, which produces the serologic polymorphism in the gene products. Recently, monoclonal antibody (MAb) Sca-1

*

Corresponding author.

(stem cell antigen) was used to isolate a pure population of pluripotent hematopoietic stem cells from murine bone marrow. These Sca-1+ cells were the most primitive precursors with the capacity to reconstitute the complete hematolymphoid system in the irradiated recipient mice (24). Sca-1 actually recognizes both allelic forms, Ly-6A.2 and Ly-6E.1, on cells from appropriate strains of mice (22). The availability of Ly-6 DNA probes allowed analysis by Northern (RNA) blot of a variety of tissues and cell lines (15). Ly-6E/A antigens are expressed constitutively in T cells, fibroblasts, and kidney cell lines. In addition to constitutive expression, Ly-6 gene expression can be further induced in T cells, B cells, and fibroblasts by alpha/beta and gamma interferons (IFN-a/,B and --y). Activated T cells and B cells express much higher levels of Ly-6E/A antigens than do resting populations. Among the various nonlymphoid organs examined by immunohistochemistry, only kidney parenchymal cells express surface Ly-6E/A antigens (22). Ly-6 genes are regulated in a complex fashion, as demonstrated by the diversity of both the tissues and the various developmental stages in which these antigens are expressed. The molecular mechanisms involved in the tissue-specific, differentiation-dependent, and inducible expression of Ly-6 antigens provide one of the most interesting systems for studying gene regulation. To elucidate the mechanism(s) that regulates the expression of the Ly-6E/A gene, we have isolated and characterized a genomic clone that encodes Ly-6E/A antigen. Analysis of the 5'-flanking sequence was performed by using the chloramphenicol acetyltransferase (CAT) reporter gene. The results demonstrated that at least two distinct regions of the Ly-6E promoter are involved in constitutive expression. One of these regions contains a 5150

PROMOTER ELEMENTS OF A Ly-6E/A GENE

VOL. 10, 1990

12 3 4

I

EcoR I

1I1

Bgl 11

I

I

I 11

1 I I

Kpn I

I

I

Hind III

I

BamH I

I

I

1 kb

FIG. 1. Restriction

map

of the murine Ly-6EA

gene.

The

map

represents 27 kb of DNA derived from two recombinant phages fused at the BgllI site between exons 3 and 4. The four exons are numbered. The bold line at the top indicates the region of the cloned

DNA that

was

used for stable transfections.

CCAAT box which is present in many different promoters, both regulated and constitutive. The second element is a purine-rich region and contains a GGAAA motif which exists in several well-characterized cellular and viral enhancers. We have also detected DNA-binding proteins that specifically recognize these transcriptional elements in electrophoretic mobility shift assays. Analysis of the stable clones derived by transfecting BALB/3T3 cells with Ly-6ECAT hybrid constructs has allowed the localization of IFNstimulated response element(s) (ISRE) for both IFN-a/,B and IFN--y in the -1760 to -900 region of the Ly-6E gene. This region lacks any apparent sequence homology to the consensus ISRE; a novel sequence may be responsible for IFN-a/, of this gene. MATERILS AND METHODS Isolation of genomic DNA clones and DNA sequencing. The genomic clones were isolated by standard procedures used previously to identify the Ly-6C.J gene (15). The nucleotide sequence was determined in part by the Maxam-Gilbert method and in part on the Applied Biosystem model 370A DNA sequencer, using fluorescent primers obtained from the manufacturer and a Taq polymerase-dideoxy sequencing kit (Promega Corp.). Plasmid constructions. The 5'-flanking sequences were subcloned into reporter plasmid pUC-CAT (2). This plasmid contains a promoterless bacterial CAT gene. The sequence of the polylinker has restriction sites in the order 5'-HindIIIPstI-SalI-XbaI-BamHI-SmaI-3' relative to the transcription of the CAT gene. The first derivative contains the sequence -81 to +7 (Fig. 1) and was constructed by subcloning a 99-base-pair (bp) oligonucleotide into XbaI and XmaI sites of pUC-CAT. A single nucleotide substitution was made at the 5' end of this sequence to create a unique Bsu36I site for further constructions. This oligomer and the other oligonucleotides used in construction of the recombinant plasmids were synthesized by the phosphoramidite method on an Applied Biosystems model 381A DNA synthesizer and were then purified on a preparative gel (10% acrylamide-7 M urea). This plasmid, referred to as pCAT-1, was used to generate two additional recombinants. In the first, the further upstream sequence -167 to -81 was subcloned between the Hindlll and Bsu36I sites. This sequence was also synthesized as an oligomer, and a single nucleotide substi-

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tution in this sequence was made to create a unique NheI site to facilitate analysis. Hence, this plasmid, pCAT-2, contains the sequence -167 to +7. Plasmid pCAT-2 was used to make a shorter construct by digesting the plasmid with HindIII and NheI, filling the overhangs with Klenow polymerase, and ligating the blunt ends. The new derivative, pCAT-3, contains the sequence -138 to +7. An oligomer containing the sequence -113 to -88 repeated three times was synthesized. To make the trimer plasmid (pCAT-7), this oligomer was subcloned between the HindlIl and XhoI sites of the original derivative, pCAT-1. An XmaI fragment containing the sequence -113 to +7 was excised from the trimer plasmid and subcloned into the XmaI site of pUC-CAT; this plasmid is called pCAT-8. A larger construct was made by subcloning a 260-bp Bcll fragment into the BcI site of pCAT-2. The resulting plasmid, pCAT-4, contains the sequence -420 to +7. A 490-bp HindIII-AvrII fragment derived from a genomic clone was subcloned into pCAT-4 to give pCAT-5, with the sequence -900 to +7. An additional upstream sequence, the 800-bp Asp7l8-AvaI fragment from the genomic clone, was bluntend ligated into the HinclI site of pUC19 and then transferred into pCAT-5 between the HindIll and XbaI sites. This recombinant, pCAT-6, is the largest of our constructs, containing 1,760 bp of the 5' flanking sequence of the Ly-6E gene subcloned upstream of the coding region of the reporter CAT gene. The DNA sequences of the recombinant constructs pCAT-1, -2, -3, -7, and -8 were confirmed by MaxamGilbert sequencing. DNA transfection for transient expression. Mouse Ltkcells (referred to as L cells) were grown in Dulbecco modified Eagle medium with 10% fetal bovine serum. One day before transfection, the L cells were seeded at a density of 2 x 106 cells per 60-mm culture dish. Cells in each dish were transfected with a mixture of 10 ,ug of appropriate test plasmid DNA, 1.5 ,ug of internal control plasmid pXGH, and 40 mg of liposomes. DNA and liposomes were each separately diluted in 1.5 ml of serum-reduced medium and then mixed (liposomes and serum-reduced medium are commercially available as lipofectin and Opti-MEM from Bethesda Research Laboratories, Inc.). Cells that had just reached confluency were washed with phosphate-buffered saline, and then 3 ml of the lipid-DNA mixture was added to the cells. The cells were incubated for 18 to 24 h at 37°C, after which 2 ml of Dulbecco modified Eagle medium with 10% fetal bovine serum was added. Cells treated with IFN received 1,000 U of IFN-aI1 (Lee Biomolecular) per ml or 500 U of recombinant murine IFN--y (Amgen) per ml. Lipofection was performed as described previously (10). Plasmid pXGH, used as internal control, contains the human growth hormone (HGH)-coding region under the control of the metallothionein gene promoter. After 48 h of transfection, CAT assays and HGH assays were performed. Assay of CAT activity. Forty-eight hours after transfection, cells were harvested with trypsin, washed with phosphatebuffered saline, and suspended in 100 ,ul of NP-40 lysis buffer. The samples were spun for 10 min in a microcentrifuge, and the supernatants were assayed for CAT activity as described by Gorman et al. (14), with some modifications. A 100-,l sample of cell lysate was assayed in 250 pl with the addition of 100 ,ul of 1 M Tris hydrochloride (pH 7.8), 45 ,ul of 4 mM acetyl coenzyme A (Sigma Chemical Co.), and 5 ,u1 of [14C]chloramphenicol (60 mCi/mmol). After 2 h at 37°C, the chloramphenicol was extracted with 1 ml of ethyl acetate. The organic layer was dried under vacuum, dissolved in 30 RI of ethyl acetate, and spotted onto silica gel thin-layer

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plates. Ascending chromatography was performed by using chloroform-methanol (95:5) for 30 min. CAT activity was quantified by densitometric scanning of the autoradiographs. HGH levels were detected in the supernatants of the transfected cells by immunoassay, using a commercial kit (Nichols Institute). Gel mobility shift assay. Crude nuclear extracts were prepared from the T-cell lymphoma cell line YAC-1 as described by Dignam et al. (18). Gel mobility shift assays were done by the method of Fried and Crothers (11) as modified by Ballard et al. (13) except that the binding reaction contained 50 mM NaCl and dithiothreitol was omitted. Restriction fragments used as probes were prepared from Ly-6E promoter-CAT constructs and labeled with 32P, using the Klenow DNA polymerase; these fragments were used at 1.7 x 1010 to 4.5 x 10-10 M. Nuclear extract (protein concentration, 2 ,ug/,ul) was incubated with the probe in the presence of nonspecific competitor, either dextran sulfate (0.02 to 0.1 mg/ml) or poly(dI-dC) poly(dIdC) duplex (0.02 to 0.15 mg/ml; Pharmacia). For specific competition, restriction fragments derived from Ly-6E promoter-CAT constructs were included in some reactions. Stable transfections and immunofluorescence. Transfections were performed by using the CaPO4 coprecipitation procedure (2). Actively growing BALB/3T3 cells were plated at 40% confluency (4 x 106 cells per T-75 flask) one day before transfection. A 50-,Ig sample of plasmid DNA was cotransfected with 5 ,ug of pSV2neo DNA, which confers resistance on eucaryotic cells to the antibiotic G418. Cells were washed once with phosphate-buffered saline 8 h after the addition of DNA-CaPO4 precipitates, and then Dulbecco modified Eagle medium containing 10% fetal calf serum was added. Cells were selected for drug resistance after 48 h by addition of G418 (1 mg/ml; GIBCO Laboratories). After 2 weeks, drug-resistant colonies were picked and expanded. Cells were analyzed for surface expression of Ly-6 antigens by immunofluorescence as described previously (26), using fluorescence-activated cell sorting (FACS) either with or without treatment with IFN. MAbs used were 34.11.3 (anti-Ly-6A.2) and SK70.94 (anti-Ly-6E.1), of mouse origin. Their binding is detected with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse secondary antibody. RESULTS Isolation of the Ly-6E/A gene. A mouse genomic DNA library, when screened with a probe which contained the 5' 420 bp of the Ly-6E.J cDNA, yielded several independent bacteriophages. These recombinant phages also showed strong hybridization to a 35-mer oligonucleotide corresponding to the amino terminus of the Ly-6E protein (16). Restriction mapping of these phage DNAs indicated that most of the phages contained the same sequences as previously characterized in the Ly-6C.J gene (5). However, one phage, clone 50, had DNA sequences located in three exons that corresponded exactly to those found in the first 274 bp of the Ly-6E.J cDNA (16) and Ly-6A.2 (19, 21). A different library was screened with the entire 750-bp EcoRI cDNA probe, and clone 51, containing the exon 4 of the Ly-6A.2 gene plus an additional 15 kilobase pairs (kb) 3' to that exon, was isolated. A restriction map of these two phages is shown in Fig. 1. A 6-kb BglII fragment, containing the 3.2 kb of 5'-flanking sequence and the first three exons with intervening sequences derived from clone 50, was subcloned into pUC19. A 2.3-kb BglII fragment, containing the exon 4 and 3'-flanking sequence from clone 51, was also subcloned into

MOL. CELL. BIOL.

pUC19 to assemble the entire gene that encodes Ly-6A.2 specificity. The entire DNA sequence present in this plasmid is overlined in Fig. 1. This plasmid was used to make stable transformants for use in studies of gene expression. Constitutive expression and IFN induction of the transfected genomic clone in stable transformants. To determine whether the cloned gene was functional, transfection studies were performed on BALB/3T3 cells. This fibroblast cell line expresses the endogenous Ly-6E.1 antigen, which is recognized by MAb SK70.94. The surface expression of Ly-6E.1 can be markedly augumented by treatment of cells with IFN-oa/, and --y. The transfected clone, which encodes the Ly-6A.2 antigen, can be specifically detected by MAb 34.11.3, since the antibody does not stain the parental BALB/3T3 cell line. The genomic clone containing the entire Ly-6A-coding region plus 3.2 kb of 5'- and 1.5 kb of 3'-flanking sequences subcloned in pUC19 was cotransfected with plasmid pSV2neo. Individual clones resistant to the antibiotic G418 were isolated and analyzed by FACS. A representation of positive clones is shown in Fig. 2. The stably transfected clones expressed significant basal levels of Ly-6A.2 antigen, which could be increased by IFN treatment. This result confirmed that the transfected genomic clone contained the cis-acting elements essential for the full promoter-enhancer activity, including responsiveness to IFNs. Analysis of the nucleotide sequence. To analyze the DNA elements involved in control of the tissue-specific expression and IFN responsiveness of this gene, the DNA sequence of the expressed clone was determined (Fig. 3). Exons and introns were determined by comparison with the Ly-6E.J cDNA sequence. The organization of the Ly-6E/A gene is very similar to that of Ly-6C.J (5). Sequences upstream of the initiation site were analyzed for homologies to known promoter and enhancer elements. No previously defined elements were found except CCAAT boxes. They occur in both orientations, dispersed throughout the 5' region, with the most proximal one located at -127 to -123 (Fig. 3). An interesting aspect of Ly-6E/A gene regulation is its tremendous inducibility by both IFN-a/, and -y. A 30-bp Friedman-Stark consensus sequence has been found 5' of some IFN-inducible genes, such as class I and class II major histocompatibility complex and metallothionein genes (12). The DNA sequence reveals that the 5'-flanking sequence of the Ly-6E gene does not contain this consensus sequence. The IFN-y-inducible class II major histocompatibility complex genes contain two well-conserved 5'-flanking sequence elements, termed X and Y. These elements and another cis-acting element (W or Z box), located just upstream of the X box, are considered necessary for the induction by IFN--y (27). A sequence with considerable homology (11 of 14 bp) to the Y box is present between -261 and -247 of the Ly-6E gene (Fig. 3). Recently, another ISRE has been described on the basis of comparative sequence analyses and transfection studies of a number of IFN-a/,-responsive genes (20). Surprisingly, no such sequence conserved in its entirety is present in 3.2 kb of 5'-flanking sequence of the Ly-6E gene (Fig. 3). There is an element with significant homology to this ISRE located between -109 and -95 (Table 1). Although the -113 to -88 region of the Ly-6E gene acted as a

positive element in transient CAT assays (see below), it did not show any IFN inducibility. Perhaps entirely different sequence elements are responsible for IFN induction of the Ly-6E gene. This is demonstrated by the inducibility of


VOL. 10, 1990

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Ly-6A. 2 Basal

Interferon

60

120

180

240

FIG. 2. FACS analysis of cells for expression of the endogenous Ly-6E gene and of the transfected Ly-6A gene. (A) Negative control, Parental BALB/3T3 cells stained with FITC-conjugated rabbit anti-mouse antibody only; Basal, cells stained for endogenous Ly-6E antigen with anti-Ly-6E MAb SK70.94 without IFN treatment; Interferon, cells stained for the Ly-6E antigen after treatment with 500 U/ml of mouse recombinant IFN--y for 24 h, purified mouse IFN-oa/3 (1,000 U/ml) gave similar results. (B) Negative control, Parental BALB/3T3 cells stained with anti-Ly-6A MAb 34.11.3; cells stained with FITC-conjugated rabbit anti-mouse antibody gave a similar pattern. Basal, Stably transformed BALB/3T3 cells stained for the surface expression of the transfected Ly-6A gene before IFN treatment. Interferon, Transfected cells stained for Ly-6A after incubation with IFN as described above.

pCAT-6, which contains 1,760 bp of the Ly-6E 5'-flanking sequence. Functional analysis of the 5'-flanking sequence of the Ly-6E gene. Ly-6 genes are constitutively expressed in mouse fibroblast cell lines, including L cells, as shown by Northern blot analysis (15). We chose the L-cell line to delineate cis-acting elements responsible for basal expression of the Ly-6E gene. Various portions of the 5'-flanking region of the Ly-6E gene were fused with the coding region of the bacterial CAT gene in the plasmid pUC-CAT (2). These constructs were transfected into the L cells. Three controls were used: mock-transfected cells, cells transfected with the promoterless pUC-CAT, and cells transfected with pRSV-CAT, in

which expression of the CAT gene is driven by promoterenhancer sequences derived from 5' long terminal repeat of Rous sarcoma virus. pUC-CAT by itself gave a low level of CAT activity; this was subtracted from the reported CAT activities of the recombinant constructs. As an internal control, all of the recombinants were cotransfected with plasmid pXHGH, which expresses HGH under the control of the metallothionein gene promoter. CAT activities were normalized to HGH levels for transfection variations. Each experiment reported was done multiple times with consistent results. Figure 4 depicts the structure of the 5' region of the Ly-6E gene; Fig. 4A shows the restriction sites used to generate the

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MOL. CELL. BIOL.

Bgl II

AGATCTGCTTTTGTCTGTTTTCTTGGGGGATCACTGACATTTCCCTATTATGAGAGTTGAACAAAATATTAAGGCCCTTCACGAACTCTGCGACAATTTGAGGTCCCAATATGCCTTCAATAATCATGCTTTCTGGTACTCCAMCCCCCT 151 301

451 601 751

Spe I CATTGC CTGGCTCT TGTCCTTGATGGGAAGCCATCGGAGC CCCCCTCTTTCTCACC CTATGTATTATACAGTTTCTCAAAAACAACATACATATAAGTCACAAAAACCACTTACTGGTACAATGACAAGC TATT CCTAGAGACTAG Bgl I TCTAACACTTATGACACAGCCCCATTCTTGTGTGTTCC CAAGACAGATGAGGGACAGTAGAGCATT GACTGTTGTGCTTAGAAAATAAAAAMAAGC CCGGAATGTGTTTGGTGGAGGCAGCTAT GATGAGGGGGAAGGGGGT GC CACT GA Nde I GGCCCATGCTGAGGCATTCCTTTCCC CTGAGGTAACAGACATATGATGTGTAGAGTATAGAATAGCTTTTACTTAGGGCATGGGGAGGGGATTTGTTGTC CTTAGTGGAGATAGAGAAAGGTGGGGGC GGGGGGTAGAGGCCAT GT GGAG Apa I AAGCAAAGAGACAGTGAGGAGGGCCCAAGCAGCC CATTTTACAGTGAGTCGGGCATCCCAGGATGTTGCTAGGTAACTACAGGCAGAGCC TAGAAG AGAGAGGGAMGAGAATGGGGAAAAAAGGAACAGGGAGGAAGAAAGG Pst I AAATGCTAATACTACC CATACTCCTGGACAGCTCTC CTAGAGCACTGCAGGAGTGGGGATGTTCAGGACC CAAATCAGGCTTGGGCCTACTCAACTCCACAGAGACAAAAGC CAAGTCAC GATAAGGCCAGTTAGGTT TCTG TT TCCC CA

901 1051

BstE II TCAATAAAGGAGGTGACCTCCAACACAGTCTTTTGAATCATTTATGCC CACCATCCCTGCAACACCCTGCTCCCAGCTGC TCTGCATCCCAAGACCACAGCACATGGGGGAGGGGCAAGTGGCATTGC GAGGAAGTACAC TTAGTTMAAA

1351

Hpa I TGAAACCTTTGCTT TATGACTCTTGGTAGTGAGGAAMAAATATGTGACCCTGGCTAGTTAACACAGGTCATTCATTCTCTGAATAGGGGCATGGGC TTGACTAAGGTTTGACCTATTGAG TGTGAGAATAGTCAGG CT TCAAGGTGAACT Pst I Kpn I GGAAGGCAGGAGAGAGGGCTGGGTGTGTTTTTGTCTTGCATGAAGACTTCTCTGCAGAGGGCCT GGCTTCTCTAGCACAAGCCTGGCAACATCTGGTACCTC TAACTC TAAGATTACTCAAGTACACTGTAGCC CTCT CCCAGAAGAGT T

1501

AMTGTGAGTGGTCT GTCAGTAGAATGGGCAGGAGTCCACCACTAAGGGAAGCTAGTTCCCCAACAMTGGGGCTGGGTGGAAMCTGGAGGACTCATGAGAATC CCTAGTTTAAGACTTTTAGAGAAACAGT GCACGGCACT GT GGTTATAT

1651

GGCCTTTGCCTC CAAAGAGAAGGTGATGGCCCTGTTGTCATCCTGGAGTAGGGATAATGTTGGCCCAGGAGCCCTGGCMATAAATAGAGTCAGAAGAGCAAAACAGCAACAGGT CTGTGATAAT GGGCAGAACT CT CTATACTCAGAAG G

1801

GAATAC TGTCACCTGGGGTTCCAGCCATGACT CCCTCCTGTTCTCCTCCAAGCACAAGTGGT CTCAGAAGATACTAGAATATAGAGGATACAGAGGATTTAC TGAAAGAGGGAC TCCGTGTACTGC TTTTATGATGGGGT GAGATT TGG T Nai I GGTGACTAAGCTGC TCAGAATTTATGCATATTCCTGTAAGTGACCTCACC CATCCTCT GGGAAAAGCCTCATAAC CTCATTGGGTGGTGACATTGGCAGGGTTTATCACTT GGAT CTTTCC TTCCCGCTTTTTCTTT GT TTGCAG Nai I AGGCTT GCTGTCTCCTGCTCTTTCTTTCACATACACTTCTTCATGCATGAGCCAATGC CT GCACAGCATGTGACAGAATTAAAGAGACAACATTTCTT GCTTGCTCTC TTCCTCCTAC CACTGTGC TGGGTTATGC GG TGTT GGAGGCAA Ava I Nco I ACTGTTTGCT TAAkATGATATATTATAGGGTAAGAAGGAAkGGGTGCTCGGGCAGAGGCCATGGAGTGTAAGCAGC CTTCCCTGCTTAAGTGGGGTGC GCTCAGCTTCAC TGATGCTGTGGTCC GTTT GGGGAC TCAGTT GT CCTGTGAGCA

1201

1951 2101

2251 2401

CAGCTGTTCATTTGCTGTGGTATCATCTCAGGATGATTTCCAGC TCAGGTCTCTAC CTCTGTCCAGCACC CACAGCCCCATACATCCCCAAACACATCAGACAC TG CTTGGTAACT TCCATCCCAGTT GC CAGT TAGT TCTT GC CTCAG G

25 51

ACTGTCACTTTGTGGATGCAAGAGCCTACTGGGGGCTT GC TCACCAGAGCCAGT CTTTAGGGATTT GTAAATTGTTTGCCTGCTTATAGGAAGGAGGCCATT GGCT CT GAGCCACT GCAAAC CATACC TT CTATTTAGAAAC AGAAAAAA Bcl I GC CAGGTAGT GGAGGCAGAAGTATCAGGGGCT GTTGTGCACC CCTTTGATAGCTGT GTGT GTTGGAGCCAGC CTGGTCTACT GATCAAGTCC TAGGACAACCAAAGCTACACAGAGAAACTT TGTCACAAAAG GCAAAAGG CAAA Y box Hinc II AAATAAAAATAAAAAC CAAAATAAACAGACAAAAAACACGAAGCTAAMCAAAACAAAAAC TAGGAAAGGGCTAAGCAACT TGACTTCT TCCCTTGC TT TCTAAT TGGCAAGCACAAGT CAACTG TG GCCT CTGC CCCT CGGC CT CTCAG T

27 01 28 51

3001

TTTCCTGC CTCATG GT TCCT CCCCAACT GCTATAAMTC TGGCTTGATCAGGT CACAAMCAAMTC TTGCTACC TCTTAMCCAATAAMCATG GGTGGCCT GGAAAGGTTAAGTAC TGAAACCC CTCC CT CTTCAGGATG CCAGCT GGGAG G

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T TGCGAACGC AGCTGAAGGAAMTTAAAGTACTTCAGTC CACATCTGACAGAACT TGCCACTGTG

3301

GGCGTTGTGATGACAAMGGGAAGAC CC TCAGGATAGG GC TGGG GT TGGGAG EcoR I Bcl I TG TGGGAT TAGGAAGGAAGAGC TGGGTGGGTGGT GGGT GAGAGAAGTCAGGCAGACAT GAAT TC CTCAGGGAAACG TGTGTAG.AGAAT TGGAGGGAGGGAAGAT TG GATGCT TGAGCT GAGGGAGAGC CCAGGGAT GT GATCAG GG GTC T

34 51

ATTAACTGGTTCCAACTTCCAAGTATC

36 01

GGATACAATGGAGATGGAAMAGGGCCTCAGGGTTGTGGGTACACCTATACTTCCATTGTGTGCCCCGTGCCTGAGGTTTGTAGGGGGTTGGGAATTGGTTTGTGGTGGGTGAAGCCTTCTGTGG TGGGGAGCTTTACTGGGTCT GTAAAC

3751

Sac I Bal I CTGTCAGAACCTGCCTTGCAACCATCACCACATACCAAAGGACACTT GGAGCTCAGTATCTCT GC CACAGAACCTGGCCAC CAGAGCAGCCTG TCCTTAGACTCTACCC TTCT GCACTGGC CTCC CAGT CAGGACACTG GC CTGT GTG C

M

3901

D T S H T T TG ACACTTCTCACAC

K

C

S

L

L

I

L

L

V A L L GTGGCCCTA

C

A

E

R

TGAGGTAGAAAATGAGAGAGGGGCAGAA

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ACACAAAC TGGAAGAGCCTTCTTTTT CTCCCCAGATGCCAAG TCAATGGATTTGGAAT CCTTAT TGCC TGGACTTTGACTTT TC CTTATGTCCCAGCCCATGCCACAT GAGACC CAAATGCTAT GC CT TCTGTACT CTAAGAAATT CT GA Sac I GCTC TAAGATAC GC CTATAC TT GTAGTTACAT GTGTAT TGCAGGAAGC CTCATGGT GAGCCTCAGC TTTAAGCT GACTCT GGGAAG TTCT GTGC TT TGTCAT GC TCCAATAC CCAGGG GC CAGC TC TGCAGG TG TAGGGTAG GACA GGT C

4201

AGAAGAGC CCTC TCTATTTGGTGGCCCCAGCATCTACTTACACACC CACT CACT GC TTCAGC GGACATTT GGTATC TATATC TTCC CAATAT GGTGGCTGTC TC TTTAATAT CT CCTG GG GTGCACAT GT CTCC TGTG GGGC TG TGTA GC

4351

TGAGACAGCAGTACCAGGCATCTTTAAACAACAGTGGT TC TTGGTT CACTCT TGAC TGAACAGTAATGTC CTTGCTCT GACCTAGCAG CCAGCAGCTGTGTG TGCAAGTAAAGACATT GGAGAG GAGGGG TC TG GGCTAGGAAT TG CAT G

4501

A Q G TACTAGCCCTCATGTATTGGCTTGTCTCTCA

L

E

C

G

V

P

4801

S C P S I T C P Y P D G V C V T Q E A A V I V G Nco I TAAGTT CTTTGT GGCC TATATAAMGATGGCTGAGGGTGGCTT TGGAGAAAAT GT CGAGTCTGGGCACT TGTT GT GGCCATGGTT CC CCTCCCCC CAAC TAGAGCTT TC CTTGTGTTGGAGGAGAAC TCTT GCAAAGTGGG GG CTGT TGG G Bcl I GTCCAG GGATGTGC TGGGATGT GATCATCAAGCAGGGC TGCC CATTGGCATT CACAGT GTTTAT CATC CCATTTTGTT GCCAGT GTAG TCAC GTGATGAT CC TGCC TATACT TTAGGT CTAT GGTC TCAC TG GCTCAC TGACAGAC CAG G

4951

AGAGTC GGAAGCAT CTAGTGAGTCTTTGCATATAATGAAGGAATCTGGAGTT TAGGCTGAAGGAAT GACTTAGGAATGTT GATAAAMAGAGATATTAGTACATATCAGATCCTC CAAMGTTTCTTT GGTTTC TAGGCATATC CT CTGATA

5101 5251

TACAAGGCAGGAGCAGCCCCACCCTGCTATCCAGTTTC CCTTCCCT GAGCATATCC TG CCTGTGTCCTGTCCCTAGGAAATAATTGCC TGGT GAACTGAAAGAG TC TGAGATGCTGGTTGCCCTTT CT GCTGTGCCAC TGGG CGAT CT TA Nde I CACTGGGC TGTGTCTTTGTGGT GTGTGCATGTGTGTGTGTCCATAAATGT GGGGTACACATGTATGAACATATGTGAT GTAATGTATACAAMGGTCAGAGGACATC CTTAACTACT GATC CTCAGGCATTGCTTGCTGTT TTAT GCAAAT

5401

Hpa I Bgl1 I I GAGC CT TGGATC TC CAGAAMCTGGAATTAGGAATGGTT GTAAACCAGGATGTGTGT CTCAGAAC CAAMCCCTGGGCTT CTGTAAGAGCAACAAGTACTGT TAAC TG CTGTGT CATGTCTC CAAC TC GTTTACTGAGACAAGATC TT TCAC

4651

5551

5701

Y

Q

C

Y

F

E

T

D S Q T R K CAGGACCTAGGATTTGCCAACTATGCTGGGTGGGCCAGAGTT TGGGAAGC TC TGTTGTCCCTGCATAAGAAGTGAGTCACTCC CTGA rTTCTTGCAAGG Hinc I I Nco I N I E S M E I L G T K V N V K T S C C Q E D L C N V A V P N G G S T W T M A

V

K

N

N

L

C

L

P

I

C

P

P

GAACAATCTTTGCTTACCCATCTCCCTCC G

V

L

L

F

S

Sac I L S S

V

L

L

TAATATTGA ATATGGA ATCCTGGGTACTAAGGTCAACGTGAAGA TCTTTGC AGAACCGCAATGTAG AGTCC AAGGAGGCAGC ACCTGGACC TGAGGGGTG TTCTGTTC GCCTGAGCTCAGTC CTCCT

Pst I Q T

L

La

5851 6001

TTTATA GrrrTGrT CST AGGTA GTSrTTAATrrTCTT

61 51

TGCTATCACCATCCACACATAAGTATCTGGGGTCCTGCAATGTTCCCACATGTATCCTGAATGTCCCCCTGTTGAGTCCAATAAACCCTTTGTTCTCCC

..CTTTr AAr.TrTnTArTTrrTA r^TT

TTTSTT^T=A TTar-TGrTcAS

rrTr.rsAaCr A AaGrcArcAarACSA

CAnA ATTrrA A GCT ATT A

624 9

FIG. 3. Nucleotide sequence of the expressed Ly-6EIA gene. The four exons are defined by comparison with the cDNA sequence and are underlined (16). The translated amino acid sequence in single-letter code in exons 2 to 4 is shown above the DNA sequence. The CAT box (CCAAT) sequences in either orientation, and the regions with homology to the Y box are indicated by lines above the sequence. The first amino acid (D) in exon 4 is responsible for the allelic difference between Ly-6E and Ly-6A antigens distinguished by MAbs.

fragments subcloned into pUC-CAT. Both plasmids pRSVCAT, the positive control, and pCAT-6, the largest of the constructs studied, were able to efficiently direct CAT synthesis in L cells. Plasmid pCAT-6, which contains 1,760 bp of Ly-6E 5'-flanking sequence, expressed 31% as much CAT activity as did pRSV-CAT. Plasmids with endpoints at positions -900, -420, and -167 yielded CAT activities similar to that of pCAT-6, suggesting that no major regula-

tory sequences are located upstream of position -167 for expression in L cells. Interestingly, the construct with sequence deleted to -138 had significantly greater CAT activity. Further studies will be required to unequivocally demonstrate negative regulatory sequences in the -167 to -138 region of the gene. Additional sequence deletion to -113 gave a reduction in CAT activity back to a level comparable to those of the larger constructs. This plasmid

VOL. 10, 1990

I

TABLE 1. Homologies in 5'-flanking regions of transcriptionally regulated genes Sequencea Gene or sequence RGGAAANNGAAACT IRSE consensus ....................... Ly-6E (this report) .....................-109 TGGAAAGGTTAAGT -95 Human c-fos ........................-282 TGGAAACCTGCTGA -295 Human heat shock protein gene .. -55 GGGAAAAGGCGGGT -42 Human interleukin-2 gene -141 AGAAGAGGAAAAATG -127 (proximal) ...................... Human IFN-a gene ................... -80 TGGAAAGTGGCCCA -67 Human interleukin-2 receptor gene ......................-177 AGAAAGGATTCATAA -159 Simian virus 40 enhancer core .... -174 TGGAAAGTCCCCAG -161 Cytomegalovirus enhancer ......... -93 TGGAAAGTCCCGTT -106 Human immunodeficiency virus long terminal repeat ............... -79 TGGAAAGTCCCCAG -92 a Taken from references 13 and 20 unless otherwise indicated.

A Asp 718

I

Ava

Nco I

B


defines the minimal Ly-6E promoter essential for significant constitutive expression in transient CAT assays, since the construct with its endpoint at -81 had no promoter activity. In the past, various anomalies have been suggested to arise from analysis of gene expression in transient transfection assays (1). To address this issue, we made stable transformants from pCAT-5, pCAT-3, and pCAT-8 containing integrated copies of the chimeric CAT gene. Their expression efficiencies were consistent with those from the transiently transfected cells (data not shown). Characterization of the region between -138 and -81 of the Ly-6E gene. Deletion analysis shows that the principal region required for constitutive promoter activity is located between -138 and -81. This region appears to contain two distinct elements when divided at -113. The most proximal element of the Ly-6E gene that has the ability to stimulate transcription lies between -113 and -81. This sequence contains a purine-rich region with considerable homology to many enhancers. It seemed possible that any factor-binding Bsu36

BcI

XmaI

B EdI

I

I

Avr II

Nhe

% CAT activity

Polylinker

(N)

TCAT Gene \C

pUC-CAT

-1760

5155

_

0

12

pCAT-6

31.2

3

pCAT-5

29.4

5

pCAT-4

27.0

8

pCAT-2

23.3

10

pCAT-3

59.9

10

pCAT-8

33.3

10

pCAT-1

0

12

+7

-900

-420

-167

-138

-113

-81

pRSV-CAT

100.0 12

FIG. 4. Deletion analysis of the Ly-6E 5'-flanking region. The restriction fragments were subcloned into the polylinker of pUC-CAT, and their relative CAT activities were determined. Acetylated forms of chloramphenicol were quantitated by densitometric scanning of autoradiographs. Although pUC-CAT does not have any promoter sequence, it still showed a low level of CAT activity. For comparative analysis, this activity was subtracted from the CAT activities of all recombinant constructs. The positive control pRSV-CAT yielded the highest level of CAT activity in L cells; its activity was therefore arbitrarily set to 100%0. The values for all other constructs are shown as a percentage of this CAT activity. The relative CAT activities shown are the values obtained after normalization for transfection efficiency. N, Number of determinations.

5156

KHAN ET AL.

MOL. CELL. BIOL.

site in this region would show further enhanced transcriptional effect if present in multiple copies. We inserted three tandem copies of a 27-bp synthetic DNA fragment containing the -113 to -88 sequence into pCAT-1. The new derivative plasmid, pCAT-7, showed tremendous CAT activity in L cells and all other cell lines tested (BALB/3T3, YAC-1, El-4, BW5147, and HeLa; Fig. 5 and unpublished data). These results suggest that some ubiquitous nuclear factor(s) binds to this region. To screen for a nuclear factor(s) that binds to these transcriptional elements, an electrophoretic mobility shift assay was used. In this assay, DNA-protein complexes migrate slower than the free probe. To reduce nonspecific binding of the proteins to the labeled probe fragment, an excess of the synthetic copolymer poly(dI-dC) poly(dI-dC) duplex or dextran sulfate was included in the binding reaction. Crude nuclear extracts of the T-cell lymphoma cell line YAC-1 were incubated with an end-labeled 52-bp XmaI fragment derived from pCAT-7. The sequence of this probe is equivalent to -113 to -62 of the Ly-6E promoter (Fig. 5A). The specificity of these complexes detected was tested by comparing three reactions: one in which no heterologous competitor sequence was added and two others which contained an approximate 24-fold molar excess of either specific unlabeled competitor DNA sequence derived from trimer plasmid pCAT-7 or nonspecific competitor DNA sequence. The uppermost retarded band was eliminated by specific competition, whereas an unrelated restriction fragment only weakly interfered with that band (Fig. SC). Our analysis of the Ly-6E promoter showed a second positive transcriptional element between -138 and -113. This region contains a CCAAT box and shows a specific binding activity in gel mobility shift assays. Crude nuclear extract was incubated with an end-labeled 66-bp fragment extending from -136 to -71 in the Ly-6E promoter, the NheI-Bsu36I fragment in Fig. SA. Specificity of the complexes was tested by competition with different unlabeled restriction fragments of the promoter region. Only fragments that contained the CCAAT box region were able to compete for the binding that gave the uppermost band (Fig. SD), whereas three other fragments lacking the CCAAT box region had no effect on that binding. Methylation interference footprinting showed the binding protein to make critical contacts within the CCAAT pentanucleotide sequence (unpublished results). IFN inducibility of the Ly-6E-CAT chimeric constructs. Expression of the Ly-6E gene is inducible by both IFN-a/& and --y. We wished to assess the contribution of the 5'flanking region of Ly-6E gene to IFN-induced augmentation of Ly-6E gene expression. To do so, we tested the CAT activities of pCAT-6 and pCAT-5. Plasmid pCAT-6 contains the Ly-6E 5' flanking region extending to position -1760, whereas pCAT-5 is a shorter construct extending to -900. These chimeric constructs were used in transient expression assays in BALB/3T3 cells. Comparison of CAT activities from untreated and IFNtreated cells demonstrated the inducibility of pCAT-6 with both IFN-a/, and -y, whereas pCAT-5 was not inducible by either IFN. Overall signals were, however, very low (data not shown). Conclusions concerning IFN inducibility from such low levels of CAT activities are questionable and might be explained simply by experimental variations in transient assays. Furthermore, transient expression assays do not reflect steady-state events in the cell. To avoid such problems, we tested these constructs in stably transfected cell lines. The gene constructions were cotransfected with -

pSV2neo as a selectable marker for stable transformation into the BALB/3T3 cell line. Stable cell lines were cloned from single colonies and assayed for basal and IFN-inducible CAT expression (Table 2). Of the clones derived from pCAT-6 that expressed basal levels of CAT, all but one were also inducible by both IFN-ax4 and -y. None of the clones derived from pCAT-5 showed any significant inducibility by either IFN, although almost all of them had considerable basal CAT expression. There was noticeable quantitative variation in the extent of induction by IFN-co3/ or --y in different clones containing the pCAT-6 construct (Fig. 6), but the response to IFNs was conserved in all but one clone. These data clearly establish that IFN-ot/, and -y inducibility can be conferred on a marker gene by sequences originating entirely from the 5'-flanking region of the Ly-6E gene. The elements required for this response are located 5' of position -900. Further deletion analysis of this region to define the 5' and 3' boundaries of the inducible element(s) is under way. DISCUSSION We have isolated and characterized a genomic clone that encodes the Ly-6A antigen. Analysis of BALB/3T3 cells stably transfected with the genomic clone confirmed the presence of an intact gene with all functional elements essential for constitutive gene expression and for induction by IFN-o1/, and --y. Transient CAT assays revealed a functional promoter for basal expression within a 1,760-bp fragment of 5'-flanking sequence of the Ly-6E gene. A series of successive sequence deletions from the 5' end of this construct demonstrated that the minimal sequence essential for constitutive promoter activity in L cells lies downstream of -138. The smallest construct that we examined extends to position -81 and is unable to promote expression of the reporter CAT gene. Hence, the functional elements for the Ly-6E promoter exist between -138 and -81. Dissection of this region allowed the identification of two positive transcriptional elements: an element located between -138 and -113 and a second element located between -113 and -81. The positive element located between -138 and -113 contains a CCAAT box. Many eucaryotic promoters possess the pentanucleotide sequence CCAAT as a transcriptional element (17). Several proteins that specifically recognize elements containing a CCAAT box have been described (9). To further characterize the -138 to -113 region of the Ly-6E promoter, gel mobility shift assays were used. A specific DNA-binding activity was detected in crude nuclear extracts of YAC-1 cells. Both competition by different DNA sequences and methylation interference analysis confirmed this protein to be a CCAAT box-binding protein. Recent evidence suggests that different CCAAT box-binding nuclear factors are capable of distinguishing between these elements. Consensus sequences for binding sites of some of these proteins have been described by comparison of DNA footprinting patterns and binding affinities to elements derived from different promoters (6). The CCAAT box-binding site of the Ly-6E promoter is very similar to the consensus CP1-binding site. Competition with known CP1 elements and mutagenesis of the critical residues should allow the identification of this protein. The second positive element of the Ly-6E promoter identified by CAT assays resides between -113 and -81. In this region, the segment between -109 and -95 bears significant homology to enhancers of many transcriptionally regulated genes (Table 1). The GGAAA motif is especially intriguing


VOL. 10, 1990

5157

A

dibjihSSAim C

Sr D

FIG. 5. Characterization of the Ly-6E proximal promoter elements by CAT assays and by DNA-binding proteins in gel mobility shift assays. (A) DNA sequence of the -150 to -70 region of the Ly-6E gene. Restriction sites in this region used to derive DNA fragments are shown. The CCAAT box and the sequence with homology to known enhancers are marked by lines. (B) Comparison of CAT activities of pCAT-1 and pCAT-7 constructs; pCAT-1 contains the region -81 to +7 of the Ly-6E gene, whereas pCAT-7 contains, in addition to this region, the sequence between -113 and -88 repeated three times upstream of -81. Quantitative estimation was done as described for Fig. 4. Chi, Unacetylated ['4C]chloramphenicol; Ac-Chi, acetylated ['4C]chloramphenicol. (C) Gel mobility shift assays for detection of a DNA-binding protein(s) in YAC-1 cell crude nuclear extracts. An Xmal 52-bp fragment was derived from pCAT-7 and end labeled. This is equivalent to the sequence - 113 to - 62 of the Ly-6E promoter. Dextran sulfate (0.025 mg/ml) was included to minimize binding of nonspecific DNA-binding proteins. For the binding reactions, no competitor DNA was included in lane 1. The other two lanes contained approximately 24-fold excess of competitor DNA. Lane 2, A 100-bp nonspecific DNA competitor derived from pUC19; lane 3, a 96-bp fragment derived from pCAT-7 containing three copies of the sequence - 113 to - 88 in tandem repeat. The arrow identifies a specific complex. The lowest band in all lanes is the free probe. (D) Detection of a CCAAT box-binding protein by gel mobility shift assay. A 66-bp NheI-Bsu36I fragment containing the sequence -136 to -71 from the Ly-6E promoter was used as a probe. YAC-1 crude nuclear extracts were used, and dextran sulfate (0.06 mg/mi) was included as a nonspecific competitor in all lanes. Synthetic copolymer poly(dl-dC) poly(dI-dC) (0.06 mg/ml) as a nonspecific competitor gave identical results, though the bands were less sharp (not shown). In addition, competitor DNA fragments derived from the Ly-6E 5'-flanking region, at 33-fold molar excess, were included as follows: lane 1, none; lane 2, 31 bp (-136 to -106) containing the CCAAT box; lane 3, 45 bp (-106 to -67); lane 4, 36 bp (-106 to -71); lane 5, 64 bp (-169 to -106) containing the CCAAT box; lane 6, 66 bp (-136 to -71), exactly the same fragment as used for the probe; lane 7, a 58-bp fragment derived from pUC19. The arrow identifies the specific complex. The lowest band in all lanes is the free probe.

5158

KHAN ET AL.

MOL. CELL. BIOL.

TABLE 2. Analysis of cloned cell lines CAT

Construct

No. resistant to G418 Neo

Bal

pCAT-6 pCAT-5

13 11

10/13 9/11

activity' 9/10 0/9

9/10 0/9

a Basal, Number of clones positive for basal CAT expression; ac/a and Y, number of clones showing basal and IFN-inducible CAT activity.

because it is conserved in enhancer elements that respond to very different inducers, e.g., serum, virus or doublestranded RNA, IFN-a/3, and T-cell mitogens (13, 20). The importance of this sequence (-113 to -88) was further supported by specific binding of a nuclear protein in gel mobility shift assays. This sequence, when repeated three times, is a powerful enhancing element in transient CAT assays in all of the cell lines tested. Though multimerization might lead to the creation of artificial binding sites, the transcriptional enhancement and binding observed to this sequence when present as a single copy in its natural context strongly support the view that some ubiquitous factor does bind to this sequence. We are currently testing this element for other properties common to enhancers. 4

'~2t.

~4 pCAT 1r.I

6

A 9N Ii B

I,

,

s-. 'T' NX Ol

C A

N

t 1 f ,

i 4-;F~.

FIG. 6. IFN inducibility of Ly-6E-CAT hybrid constructs in stably transfected clones. BALB/3T3 cells containing the integrated Ly-6E-CAT hybrid plasmids were established as described in Materials and Methods. Individual clones were treated with IFNal or --y for 36 to 48 h before being harvested for CAT assays. Quantitative estimation of CAT activities was done by densitometric analysis of the autoradiographs. B3 and B9 represent two independent clones derived from the same plasmid, pCAT-6, which contains 1,760 bp of the 5'-flanking region of the Ly-6E gene fused to the reporter bacterial CAT gene. N4 is one of the stable clones derived by transfection of the pCAT-5 construct which contains 900 bp of the 5'-flanking region of Ly-6E. -, Untreated cells; ac, cells treated with 1,000 U of purified IFN-a/,B per ml; -y, cells treated with 500 U of recombinant IFN--y per ml.

Some of the functional elements essential for Ly-6E gene expression have been identified, but regulation of gene expression in vivo may be much more complex, involving still unknown sequence elements. For instance, a possible negative regulatory element exists between -167 and -138. Complex regulation is expected because of the presence of this antigen on hematopoietic stem cells (24) and subsequently on mature peripheral T lymphocytes (23). This tissue distribution also suggests involvement of Ly-6E/A antigens in the development of lymphoid cell lineages. The question of tissue-specific expression of this gene cannot be easily addressed in vitro because many established tissue culture cell lines express the Ly-6E gene, especially with IFN treatment. Hence, tissue specificity can be addressed only in vivo. We have created transgenic mice with a fusion gene containing 6 kb of 5'-flanking sequence of the Ly-6E gene linked to the herpes simplex virus thymidine kinase gene (unpublished results). Cells expressing the latter gene can be eliminated after exposure to certain antiviral drugs (24), enabling us to study ontogenic expression of the Ly-6E gene. The expression of Ly-6E antigen is highly induced by both IFN-a/p and --y. Although accumulation of Ly-6E transcripts increases upon treatment with IFN (15), it is not yet clear whether this enhancement is entirely the result of an increased rate of transcription or combination of increased transcription and stabilization of mRNA. An ISRE has been identified in the 5'-flanking region of the genes that are induced transcriptionally by IFN-ao/,B. Deletion analysis, point mutagenesis, and sequence comparisons have established a consensus for the ISRE (7). We sequenced 3.2 kb of the 5'-flanking region and the entire body of the expressed Ly-6E gene to identify any elements involved in regulation of this gene. Analysis of the sequence did not show any sequence element fully homologous to the consensus ISRE. We analyzed the Ly-6E-CAT chimeric constructs for IFN inducibility in BALB/3T3 cells by transient and stable transfections. Transient expression assays demonstrated the inducibility of pCAT-6 (-1760-CAT) by IFNs. These results were confirmed by screening a panel of stably transfected BALB/3T3 clones derived from pCAT-6. Truncation of the 5'-flanking region from position -1760 to position -900 resulted in the loss of inducibility of the reporter gene by both IFN-ox/p and --y. It can be concluded from these data that induction of Ly-6E gene expression by IFN-a/,B and --y is, at least in part, at the transcriptional level, although additional posttranscriptional effects of IFNs cannot be excluded. The ISRE(s) for the Ly-6E gene resides between -1760 and -900. This location in itself is quite unusual, since all previously characterized IFN-inducible genes have the IRSE within 500 bp of the transcription initiation site. The nucleotide sequence of the -1760 to -900 region of the Ly-6E gene suggests that a novel sequence is responsible for induction by IFN-a/p of Ly-6E. The relationship between sequence elements required for IFN-y inducibility and IFNalp inducibility is unknown. Since the -1760 to -900 region of the Ly-6E gene can mediate induction by both IFN-a/I and -y at least in the context of its homologous promoter, the Ly-6E gene provides a valuable system for understanding the mechanism of interaction between the two types of IFN. ACKNOWLEDGMENTS We thank Margot Bridgett for preparing the anti-Ly-6 MAbs and William Philbrick for helpful suggestions and technical comments throughout the experimental work. This work was supported by a Public Health Service grant GM40924 from the National Institutes of Health. K.D.K. is sup-

VOL. 10, 1990

ported by a fellowship from the Ministry of Science and Technology of the Government of Pakistan. A.B. is an Associate Investigator of the Howard Hughes Medical Institute. LITERATURE CITED 1. Alwin, J. 1985. Transient gene expression control: effects of transfected DNA and trans-activation by viral early proteins. Mol. Cell. Biol. 5:1034-1042. 2. Baldwin, A. S., Jr., and P. A. Sharp. 1987. Binding of a nuclear factor to a regulatory sequence in the promoter of the mouse H-2Kb class I major histocompatability gene. Mol. Cell. Biol. 7:305-313. 3. Ballard, D. W., W. M. Philbrick, and A. L. M. Bothwell. 1988. Identification of a novel 9-kDa polypeptide from nuclear extracts. J. Biol. Chem. 17:8450-8457. 4. Borreli, E., R. Heyman, M. Hsi, and R. M. Evans. 1988. Targeting of an inducible toxic phenotype in animal cells. Proc. Natl. Acad. Sci. USA 85:7572-7576. 5. Bothwell, A., P. E. Pace, and K. P. LeClair. 1988. Isolation and expression of an interferon-responsive Ly-6C chromosomal gene. J. Immunol. 140:2815-2820. 6. Chodosh, L. A., A. S. Baldwin, R. W. Carthew, and P. A. Sharp. 1988. Human CCAAT-binding proteins have heterologous subunits. Cell 53:11-24. 7. Cohen, B., D. Vaimen, and J. Chebath. 1989. Enhancer functions and in vitro protein binding of native and mutated interferon-responsive sequences. Nucleic Acids Res. 17:1679-1695. 8. Dignam, J. D., R. M. Lebovitz, and R. G. Roeder. 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11:1475-1489. 9. Dorn, A., J. Bollekens, A. Staub, C. Benoist, and D. Mathis. 1987. A multiplicity of CCAAT box-binding proteins. Cell 50:863-872. 10. Feigner, P. L., T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P. Northorp, G. M. Ringold, and M. Danielson. 1987. Lipofection: a highly efficient, lipid mediated DNAtransfection procedure. Proc. Natl. Acad. Sci. USA 84:74137417. 11. Fried, M., and D. Crothers. 1981. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 9:6505-6525. 12. Friedman, R., and G. Stark. 1985. a-Interferon induced transcription of HLA and metallothionein genes containing homologous upstream sequences. Nature (London) 314:637-639. 13. Fujita, T., H. Shibuya, T. Ohashi, K. Yamanishi, and T. Taniguchi. 1986. Regulation of human interleukin-2 gene: functional DNA sequences in the 5' flanking region for the gene expression in activated T lymphocytes. Cell 46:401-407. 14. Gorman, C. M., L. F. Moffat, and B. H. Howard. 1982. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2:1044 1051.


5159

15. LeClair, K. P., M. M. Bridgett, F. J. Dunont, R. G. E. Palfree, U. Hammerling, and A. L. M. Bothwell. 1989. Kinetic analysis of Ly-6 gene induction in a T lymphoma by interferons and interleukin 1, and demonstration of Ly-6 inducibility in diverse cell types. Eur. J. Immunol. 19:1233-1239. 16. LeClair, K. P., R. G. E. Palfree, P. M. Flood, U. Hammerling, and A. Bothwell. 1986. Isolation of a murine Ly-6 cDNA reveals a new multigene family. EMBO J. 5:3227-3234. 17. Maity, S. N., P. T. Golumbek, G. Karsenty, and B. D. Crombrugghe. 1988. Selective activation of transcription by a novel CCAAT binding factor. Science 241:582-585. 18. Malek, T. R., G. Ortega, C. Chan, R. A. Kroczek, and E. M. Shevach. 1986. Role of Ly-6 in lymphocyte activation. II. Induction of T cell activation by monoclonal anti-Ly-6 antibodies. J. Exp. Med. 164:709-722. 19. Palfree, R. G. E., K. P. LeClair, A. Bothwell, and U. Hammerling. 1987. cDNA characterization of an Ly-6.2 gene expressed in BW5147 tumor cells. Immunogenetics 26:389-391. 20. Porter, A. C. G., Y. Chernajosky, T. C. Dale, C. S. Gilbert, G. R. Stark, and I. M. Kerr. 1988. Interferon response element of the human gene 6-16. EMBO J. 7:85-92. 21. Reiser, H., J. Coligan, E. Palmer, B. Benacerraf, and K. L. Rock. 1988. Cloning and expression of a cDNA for the T cell activating protein (TAP). Proc. Natl. Acad. Sci. USA 85:22552259. 22. Rijn, M. V. D., S. Heimfeld, G. J. Spangrude, and I. L. Weissman. 1989. Mouse hematopoietic stem-cell antigen is a member of the Ly-6 antigen family. Proc. Natl. Acad. Sci. USA 86:4634-4638. 23. Rock, K. L., H. Reiser, A. Bamezai, J. McGrew, and B. Benacerraf. 1989. The Ly-6 locus: a mutligene family encoding phosphatidylinositol-anchored membrane proteins concerned with T cell activation. Immunol. Rev. 111:195-224. 24. Spangrude, G. J., S. Heimfeld, and I. L. Weissman. 1988. Purification and characterization of mouse hematopoietic stem cells. Science 241:58-62. 25. Su, B., and A. L. M. Bothwell. 1989. Biosynthesis of a phosphatidylinositol-glycan linked membrane protein: signals for posttranslational processing of the Ly6E antigen. Mol. Cell. Biol. 9:3369-3376. 26. Tite, J. R., and C. A. Janeway, Jr. 1984. Antigen-dependent selection of B lymphoma cells varying in Ia density by cloned antigen-specific L3T4+ T cells: a possible in vitro model for B cell adaptive differentiation. J. Mol. Cell. Immunol. 1:253-265. 27. Tsang, S. Y., M. Nakanishi, and B. M. Peterlin. 1988. B-cell specific and interferon-y-inducible regulation of the HLA-DRa gene. Proc. Natl. Acad. Sci. USA 85:8598-8602. 28. Yeh, E. T. H., H. Reiser, A. Bamezai, and K. L. Rock. 1988. TAP transcription and phosphatidylinositol linkage mutants are defective in activation through the T cell receptor. Cell 52:665-674.