THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 266, No. 26. Issue of September 15, pp. 17531-17536, 1991 Printed in U.S.A.

The Gene Encoding the Act-2 Cytokine GENOMIC STRUCTURE, HTLV-I/Tax RESPONSIVENESS OF 5’ UPSTREAM SEQUENCES, AND CHROMOSOMAL LOCALIZATION*

(Received for publication, October 18, 1990)

Monica Napolitano, William S. Modi$§, Stanley J. CevariolI, James R. Gnarra, Hector N. Seuanezn 11, and Warren J. Leonard** From the Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, the $Biological Carcinogenesis and Development Program, Program Resources, Incorporated, Frederick Cancer Research Facility, Frederick, Maryland 21701, and the (Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland 21 701

Act-2 is a cytokine that belongs to a superfamily of structurally related proteins. Act-2 expression is rap- idly induced in T cells, B cells, and monocytes upon mitogenic stimulation. The Act-2 genomic locus is on chromosome 17q. The exons and exonlintron splice junctions have been sequenced, as have the sequences upstream of exon 1. A classical TATA box is located immediately upstream of the transcription initiation site. The upstream sequences possess promoter activity and can be functionally activated after treatment of Jurkat T cells with phythohemagglutinin plus phorbol myristrate acetate. In addition, Act-2 promoter chlor- amphenicol acetyltransferase constructs are expressed in human T cell lymphotropic virus type I (HTLV-1)- infected MT-2 cells and in Jurkat cells which can be induced to express the transactivator gene (tux) prod- uct of HTLV-I.

Act-2 belongs to a superfamily of structurally related pro- teins, some of which are mediators of inflammation (1-4). Many of the proteins in this superfamily are small (approxi- mately 70 amino acids), and all conserve 4 cysteine residues. They can be divided into two groups on the basis of whether the first 2 cysteines are adjacent (first group) or are separated by a single amino acid (second group) (reviewed in Ref. 4). Act-2 is a member of the first group of proteins. I t is note- worthy that Act-2, a human protein, is highly homologous to murine macrophage inflammatory protein-1 (MIP-1)’ a and

*This work was supported in part by a National Institutes of Health Intramural AIDS grant (to W. J. L.) and by an AIDS fellow- ship from Minister0 della Sanita (to M. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) M69201-M69203.

§ Supported in part by the Department of Health and Human Services under Contract N01-CO-74102 with Program Resources, Inc.

11 On leave from the Genetics Section, Instituto, Nacional do Can- cer (RJ)/Department of Genetics, Federal University of Rio de Ja- neiro, Brazil.

tion, OD, IRP, NHLBI, NIH, Bldg. 18T, Rm. 101, 9000 Rockville ** Current address: Pulmonary and Molecular Immunology Sec-

Pike, Bethesda, MD 20892. ’ The abbreviations used are: MIP, macrophage inflammatory pro-

tein; PHA, phytohemagglutinin; PMA, phorbol myristate acetate; CAT, chloramphenicol acetyltransferase; HTLV, human T cell lym- photropic virus; CRE, CAMP responsive element; kb, kilobase pair(s); bp, base pair(s); SDS, sodium dodecyl sulfate; PIPES, 1,4-piperazine- diethanesulfonic acid PBL, peripheral blood lymphocyte(s).

p (71 and 77% identity at the amino acid level, respectively). Native MIP-1 has been reported to be chemokinetic for neu- trophils, a prostaglandin-independent endogenous pyrogen, and to be able to enhance the myelopoietic activity of granu- locyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor on bone marrow progenitors (4). Recombinant MIP- la is a reversible inhibitor of stem cell proliferation ( 5 ) .

Act-2 was originally identified as an activation cDNA in differential screening of a cDNA library prepared from human PBL stimulated with PHA plus PMA (1). Act-2 mRNA is not expressed in unstimulated cells, but is rapidly induced in T cells, B cells, and monocytes following appropriate mitogenic stimuli. However, Act-2 mRNA is not detected in all rapidly proliferating cells, as demonstrated by its nonexpression by certain cell lines and by serum-stimulated fibroblasts (1). In T and B cells, Act-2 mRNA is detected within 15 min of mitogenic stimulation, levels peak in 4 h, and then decline graduately over the next 24-48 h (1). Interestingly, Act-2 mRNA contains AU-rich regions in its 3”untranslated se- quences which are found in a variety of protooncogene and cytokine mRNAs with short half-lives (6, 7).

Because Act-2 mRNA is rapidly and strongly induced, it is an interesting gene for regulation studies. We now further characterize the genomic locus for the Act-2 gene and dem- onstrate that 5’ upstream sequences possess promoter activity as a first step to elucidating the regulation of expression of this gene.

MATERIALS AND METHODS

Cells and Cell Culture-Jurkat is an acute lymphocytic leukemia T cell line. MT-2 is an HTLV-I-infected T cell line. JPX/M are Jurkat cells stably transfected with a control plasmid pMAXneo/M; JPX-9 are Jurkat cells stably transfected with pMAXRHneo-1, which con- tains the coding region for the tax gene of HTLV-I driven by the murine metallothionein promoter, which can be induced by zinc or cadmium (8, 9). Both JPX/M and JPX-9 were generously provided by Dr. Kazuo Sugamura (Tohoku University).

Genomic Phage Libraries-Two different genomic phage libraries were used. These were a library constructed as a HaeIII/AluI partial digest of human fetal liver of undetermined sex, cloned in Charon 4A (prepared in the laboratory of Dr. Thomas Maniatis, Harvard Uni- versity and generously provided by Dr. Edward Fritsch, Genetics Institute) and a library constructed as a partial MboI digest of DNA derived from a patient with heavy chain disease, cloned in the BamHI site in Charon 28 (Ref. 10, prepared and generously provided by Dr. Ajay Bakhshi, Georgetown University). Approximately lo6 phage clones of each library were plated using Escherichia coli LE392, transferred to duplicate nitrocellulose filters, and screened using a full-length Act-2 cDNA probe which was labeled by the method of random priming (11). Duplicating positive clones were selected and

17531

17532 The Gene Encoding the Act-2 Cytokine plaque-purified by sequential platings.

DNA Sequencing-Exon-containing fragments of Act-2 genomic clones were identified by Southern blot hybridization with the Act-2 cDNA as a probe. The 7-kb EcoRI/BamHI and the 3.5-kb BamHI/ EcoRI fragments (Fig. 1B) were subcloned into pGEM vectors (Pro- mega Biotec) and DNA sequencing performed by the dideoxy chain termination method using Sequenase 2.0 (U. S. Biochemical Corp.).

Plasmid Construction, Transfection, and CATAssays-The 402-bp BalIIHindIII (-378/+24) putative promoter fragment was subcloned in pGEM 52. This was then excised as an XbaIIHindIII fragment and in the 5’ to 3’ orientation subcloned into the chloramphenicol ace- tyltransferase (CAT) vector JOL6 (Jymcat-0 (Ref. 12) containing a polylinker), 5’ to the CAT gene. The -320/+24 construct was derived by Ba131 exonuclease digestion of the -378/+24 fragment. All plas- mids were banded on two sequential cesium chloride gradients and then transfected into mammalian cell lines using the DEAE-dextran method as previously described (12). Where indicated, PHA was added at 1 pglml, and PMA at 50 ng/ml. When JPX/M or JPX-9 cells were used, 16-24 h after the transfection the cells were treated with 120 M ZnClp and 1 mM sodium butyrate. The cells were harvested 24 h later for the CAT assay. Sodium butyrate increased the absolute levels of CAT activity but not the degree of inducibility by ZnClz (data not shown), consistent with the ability of sodium butyrate to enhance the expression of transiently transfected genes (13). Each tranfection was performed at least two independent times with similar results.

Electrophoretic Mobility Shift Assays-Nuclear extract were pre- pared by the method of Dignam et al. (14). Nuclear proteins were bound to end-labeled double-stranded DNA probes, and electropho- retic mobility shift assays were performed as previously described (15). The oligonucleotides 5’-GATCCGGCTGACTCATCA-3’and 5’- AGCTTGATGAGTCAGCCG-3’ were annealed to generate a canon- ical AP-1 binding site (underlined) with 5’ overhangs to facilitate fill- in labeling with Klenow. For the Act-2 probe used in Fig. 3, a double- stranded oligonucleotide, corresponding to -101 to -94 and spanning the CAMP responsive element (CRE)-like binding site, was used.

Chromosomal Localization by Southern Blot Hybridization of So- matic Cell Hybrid DNA-The 402-bp BalIIHindIII fragment (-378/ +24) of the Act-2 gene (see Fig. 1B) was radiolabeled with [32P]dCTP and hybridized to nylon filters (Gelman Biotrace) containing BamHI- cleaved DNA from 45 human-rodent cell hybrids (16 human-mouse and 29 human-hamster). Each cell hybrid had been previously char- acterized, both biochemically and karyotypically, for the presence of human biochemical markers and chromosomes (16). Filter hybridi- zations were carried out at 37 “C for 48 h in 50 mM PIPES, 10 mM EDTA, 1 M NaCl, 200 pg/ml denatured salmon sperm DNA, 5 X Denhardt’s solution, 50% formamide, 1% SDS. Following hybridiza- tion, filters were washed with 1 x SSC, 1% SDS at 37 “C for 1 h, 1 X SSC, 1% SDS at 50 “C for 1 h, and 0.5 X SSC, 1% SDS at 50 “C for 1 h.

In Situ Hybridizations-These were performed as previously de- scribed (17). Briefly, the 402-bp BalIIHindIII fragment (-378/+24) was tritiated (specific activity of 3.2 x 107cpm/pg) and hybridized to bromodeoxyuridine-substituted human chromosomes in a solution containing 50% formamide, 2 X SSC, 10% dextran sulfate, and 20 mM sodium phosphate. Washes were carried out at 40 “C in 50% formamide in 2 X SSC followed by 2 X SSC alone three times for 10 min each.

RESULTS

As a preliminary step to being able to study Act-2 gene regulation, we first determined the exon-intron structure of the Act-2 gene, sequenced the splice junctions, and identified the 5”regulatory sequences necessary for promoter activity. We screened the Maniatis fetal liver genomic library (see “Materials and Methods”) and by plaque hybridization iden- tified six independent Act-2 genomic clones. DNA was ex- tracted from plaque-purified clones and digested, and South- ern blotting used to localize the 5’ and 3’ ends of the clones and to identify all fragments containing exons. Exon-contain- ing fragments were subcloned into pGEM vectors, and the exons and exon/intron splice junctions were sequenced (the middle line of sequence in Fig. L4). Because the sequence revealed a non-canonical splice acceptor sequence at the 3’ end of intron 2 (GG instead of AG, middle sequence boxed in

Fig. L4) from two independent genomic clones, we were concerned that the gene isolated might have represented a pseudogene. We therefore identified and characterized ge- nomic clones from a second independent genomic library (see “Materials and Methods’’ and Ref. 10). One of these clones was sequenced, and the nucleotide sequences of all donor and acceptor splice junctions were shown to be canonical (top sequence, Fig. L4). The sequences from both of the genomic libraries predicted identical amino acids to each other (except in the signal peptide, amino acids 1-23), but differed from our original cDNA sequence (1) at amino acid 70, encoding a glycine in the genomic in place of a serine in the cDNA sequence. The Act-2 gene was shown to consist of three exons spanning approximately 2 kb. The exons were isolated on a 7-kb EcoRI/BumHI fragment which contained exons 1 and 2 and a 3.5-kb BumHIIEcoRI fragment which contained exon 3 (Fig. 1B). The first exon contains the translation start site. Examination of the sequences upstream of the 5’ end of the Act-2 cDNA revealed a single TATA box at -28/-23, which corresponds to the single transcription initiation site at +1 determined by primer extension (3). It is unclear whether the clone from the first library represents a functional Act-2 gene or a pseudogene with a defective splice acceptor site.

In order to demonstrate that the upstream sequences pos- sessed promoter activity, we subcloned the 402-bp (-3781 +24) BulI/HindIII and 344-bp (-320/+24) Act-2 putative promoter fragments into a CAT expression vector (see “Ma- terials and Methods”) and transfected various cell lines. Act- 2 is expressed by Jurkat cells when stimulated by PHA and PMA (1); therefore, we first evaluated if the promoter frag- ment -378/+24 was active in stimulated Jurkat cells and found it to be responsive to mitogenic stimulation (Fig. 2.4, lane 2 uersus lane 1). Because Act-2 is expressed in HTLV-I- infected cell lines (l), we next tested if Act-2 promoter con- structs were active in MT-2 cells, and this was indeed the case (Fig. 2B, lanes 2 and 3 versus lane 1 ), but only the -320/ +24 construct had significant activity. We next asked whether the Tax protein of HTLV-I could activate expression of the Act-2 promoter. To address this question, we utilized a Jurkat cell line stably transfected with an expression vector not containing (JPX/M, as a control) or containing (JPX-9) the tux cDNA under control of the metallothionein promoter (8). Therefore in JPX-9 but not in JPX-M cells, tux is expressed when the cells are cultured in the presence of zinc or cadmium (8). As shown in Fig. 2C, the Act-2 promoter constructs with 5’ endpoints of -378 and -320 were active in tux expressing JPX-9 cells, but not JPX-M cells (compare lunes 2 and 4 with lunes I and 3 ) . Again, corresponding to the results of panel B , the -320 construct had a more potent activity. Neither con- struct was expressed in JPX-9 cells in the absence of zinc (data not shown), thus confirming that the CAT activity induced in JPX-9 with ZnClp is indeed due to tux expression. These data confirmed that both tux and PHA/PMA were capable of activating the Act-2 promoter.

PMA activation of various genes can be mediated by a variety of DNA-binding proteins including NF-KB and AP-1 (18). Tax induction can occur through NF-KB (28) and AP-1/ CRE-like motifs (HTLV-I long terminal repeat, Ref. 19, and transforming growth factor-p promoter, Ref. 20). Although canonical consensus binding sites for these proteins were not found in the Act-2 promoter, one imperfect AP-1/CRE-like motif (TGACATCA) at -101/-94 was noted.

The CRE (CAMP responsive element), whose consensus sequence TGACGTCA (21), is related to the AP-1 consensus motif TGACTCA, is able to bind the AP-1 protein but with a lower affinity than the AP-1 motif (22). We therefore hypoth-

The Gene Encoding the Act-2 Cytokine 17533

FIG. 1. Panel A, sequences of the Act- 2 gene exons and exon/intron splice junctions from the Bakhshi heavy chain disease genomic library (top sequence), the Maniatis fetal liver library (middle sequence), and the Act-2 cDNA sequence (bottom sequence, only in areas corre- sponding to exons, from Ref. 1). Hyphens indicate identity. Mismatches are indi- cated by the nucleotide. The canonical (top sequence) and imperfect (bottom se- quence) splice junctions at the end of intron 2 are boxed. Sequences upstream of the transcription initiation site are shown appended to the first exon. The sequences corresponding to the coding region are shaded. Locations of the tran- scription initiation site (underlined, fl), TATA box (boxed). Numbers at the right are the DNA sequence relative to the initiation site. Every 10th amino acid is numbered below the amino acid. Amino acids 1-23 correspond to the signal pep- tide. The DNA sequence corresponding to amino acids 40-45 (underlined) rep- resents a perfect palindrome 18 nucleo- tides in length of yet unknown signifi- cance. Amino acid 70 is the only differ- ence in the mature Act-2 protein (excluding the signal peptide), between the genomic sequence (glycine, itali- cized) and the previously determined cDNA sequence (serine, Ref. 1). AP-2 (-242/-235) (26) and AP-1/CRE (-lOl/ -94)-like sequences are boxed. Sequences were prepared for publication using the DNAdraw computer program (27). Panel E , genomic structure of the Act-2 gene which consists of 3 exons spanning ap- proximately 2 kb. Open box, 5"untrans- lated region; cross-hatched box, 3'-un- translated region.

C G A G T C A A G G T G T A G G C A G A G T C C T T T T T T C T A T T C A T G G C T G G C A A A C A G T G G G A T C T G G G G A T G G G A C ......................................................................

A A A A G G """

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A T T A T T T A T A T T A G T T T A G C C A A A

6

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1 Kb - exon1 exon2 exon3

esized that the imperfect AP-1/CRE-like motif in the Act-2 promoter might be able to bind AP-1. We tested the ability of the -1011-94 sequence of the Act-2 promoter to bind nuclear factors from HeLa- and PMA-stimulated Jurkat cell extracts. Using an electrophoretic mobility shift assay, this sequence appeared to bind AP-1, since an AP-1 oligonucleo- tide can compete, in a dose-dependent manner, for binding of nuclear extracts to the Act-2 probe (Fig. 3). Formal mutagen- esis and deletion studies will be needed to elucidate the role that this and other sequences may play in PMA- and/or Tax- induced expression of the Act-2 gene.

We had previously suggested that Act-2 was encoded by a single or low copy number gene based on comparatively simple

patterns of restriction endonuclease digestion and genomic Southern blot hybridization (1). We have now determined the chromosomal localization of the Act-2 gene using somatic cell hybrids and the -378/+24 probe. Hybrid cell DNA was iso- lated, digested with BumHI, and subjected to Southern blot analysis with an Act-2 probe. A representative experiment is shown in Fig. 44. Control filter hybridizations were also carried out with human (lunes 1, 8, and 13), mouse (lunes 2 and 7), and hamster (lune 12) genomic DNA cleaved with BurnHI. Filter hybridization was considered to be "positive" whenever a human pattern was observed in the DNA of the cell hybrids (lunes 3,4, and 11 ) and "negative" when a human pattern was not seen (lunes 5, 6, 9, and 10). A statistical

17534 The Gene Encoding the Act-2 Cytokine

A B C

1 2 1 2 3 4 1 2 3 4 FIG. 2. The Act-2 promoter is active in MT-2 cells and Jurkat cells stimulated with PHA and PMA

or Jurkat cells expressing tax. Each panel shows a representative experiment. Panel A, Act-2 promoter CAT construct is active in Jurkat cells stimulated with PHA plus PMA (lanes I and 2). Percentage CAT conversion f S.E.M.: lane I , Jymcat-0, 0.67 f 0.1; lane 2, -378/+24, 2.7 f 0.3. Panel B, Act-2 promoter is active in MT-2 cells, an HTLV-I-infected T cell line (lanes 1-4). Percentage CAT conversions f S.E.M.: lane I , Jymcat-O,O.46 f 0.16; lane 2, -320/+24,2.93 f 0.38; lane 3, -378/+24,0.62 f 0.06; lane 4, RSV-CAT, >90. Panel C, the Act-2 promoter CAT constructs (-320/+24 and -378/+24) are activated by ZnC12 treatment of JPX-9 (lanes 2 and 4, respectively), a cell line stably transfected with a construct in which tar gene expression is driven by the metallothionein promoter, but not in JPX-M cells (lanes 1 and 3, respectively). 1 mM sodium butyrate was used in these transfections. Percentage CAT conversion S.E.M.: lane I , -320/+24, 0.4 f 0.07 (JPX-M cells +.ZnCl,); lane 2, -320/+24, 2.16 2 1 (JPX-9 cells + ZnC12); lane 3, -378/+24, 0.36 f 0.06 (JPX-M cells + ZnClJ; lane 4, -378/ +24, 1.05 f 0.3 (JPX-9 cells + ZnC12).

1 2 3 4 5 6 FIG. 3. AP-1 appears to bind to the Act-2 promoter. Lane I ,

AP-1 probe; lane 2, AP-1 probe + HeLa nuclear extracts; lane 3, Act- 2 probe; lane 4, Act-2 probe + HeLa nuclear extracts; lane 5, Act-2 probe + HeLa extracts preincubated with 20-fold molar excess of AP- 1 probe; lane 6, Act-2 probe + HeLa extracts + 1000-fold molar excess of AP-1 probe.

analysis showed that the discordancy between molecular hy- bridization of the probe and the presence of chromosome 17 in the cell panel was approximately 8.9% ( p < .001), whereas the discordancy values with the other chromosomes were above 18% (Fig. 4B). This strongly suggested that the Act-2 gene is located on chromosome 17 since only discordancy values of less than 10% are highly significant. Surprisingly, the -378/+24 probe, which was expected to give only one band in Southern blots using BamHI-digested DNA since it contains no internal BamHI sites, instead yielded two bands of approximately 12 and 17 kb which uniformly cosegregated (Fig. 4A). To further investigate this finding, DNAs from 36 individuals were digested with BamHI, blotted, and hybridized

(Fig. 4C). In each case, both bands were identified. These data suggest the existence of two cross-hybridizing sequences on chromosome 17. Because of their detection in all samples, these are unlikely to represent a polymorphism but rather are most consistent with a gene duplication event. The two bands detected correspond to the largest two bands seen on the genomic Southern blot in Ref. 1, in which the full-length Act- 2 cDNA was used as a probe. Although both bands are always seen, it is interesting that their relative intensities varied to some degree. DNA from one individual (lane 33) contained an additional larger band. This might represent a polymor- phism or an additional duplicated sequence.

In order to more precisely map the location of the Act-2 gene, we next performed in situ hybridization studies. A total of 396 autoradiographic silver grains were observed over 133 metaphase cells (Fig. 5A) . Of these, 31 (7.82%) were found in the region of 17q21-23 (Fig. 5B) . This result confirmed and extended the result from the hybrid Southern blot analyses. A moderate number of grains were found on chromosome 19; however, the high discordancy value of approximately 25% in the somatic cell hybrid data essentially excluded this as a locus for the Act-2 gene. These mapping studies are in close agreement to the findings of Irving et al. (23) who mapped the Act-2 locus to 17qll-21 and who have identified that the Act-2 gene (PAT744.1 in their nomenclature) is closely linked to the LD78 gene (PAT464.1 in their nomenclature).

DISCUSSION

Act-2 is a cytokine member of a superfamily of small secreted proteins. Like other cytokines, the Act-2 gene is not expressed in resting lymphocytes, while is strongly expressed shortly after mitogenic induction in T cells, B cells, and monocytes. Similarly, the Act-2 receptor is expressed by PHA/PMA activated PBLs but not by resting PBLs (24). The genomic locus for Act-2 is on chromosome 17q. This locus is noteworthy in that it represents the approximate location for the erb-2 and erbA oncogenes and for homeobox region 2, and is implicated in acute promyelocytic leukemia and von Recklinghausen’s disease. I t is of interest that other

The Gene Encoding the Act-2 Cytokine

B

17535

C

A

Chromosome 1 2 3 4 5 6 7

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 8 9 10 11 12 13 B 14 15 16 17 18 19 20 21 22 X

A

+/+ +/- %Discordancy 1 6 3 3 4 20.5

2 5 7 3 1 1 6 2 3 5 1s.2

26.7 3 4 14 22 11.9 1 6 4 3 4 4 3 10 28

22.2 28.9

3 4 7 3 1 25.0

3 4 5 3 3 24.4

3 3 5 3 1 20.0

3 4 14 23 19.0 40.9

2 5 7 3 0 27.3 1 6 11 25 3 4 16 20

39.5 46.5

1 6 12 24 2 5 8 3 0

41.9 28.9

2 5 14 24 8.9

42.2 2 5 6 3 1 25.0 1 3 IO 27 4 2 14 24

31.7

2 4 12 26 36.4

6 1 3 6 1 36.4 S4.1

3 4 7 3 0

3 4 0 3 8

1 2 3 4 5 6 7 8 9101112131415161718

FIG. 4. Panel A, Southern blot of DNA from human Q221 (lanes I and I3), human Q220 (lane 81, mouse MlOO and M98 DNA (lanes 2 and 7), hamster H91 DNA (lane 12), mouse-human hybrids (lanes 3-6). and hamster- human hybrids (lanes 9-11), Panel B, percent discordancy based on Southern hybridization of the Act-2 cDNA to 45 human-rodent hybrids. This allowed the assignment of the Act-2 gene to chromosome 17, the only chromosome with a discordancy value of less than 10%. Panel C, polymorphism blot. DNA from 36 donors were digested with BamHI and analyzed by Southern blot analysis as in panel A.

Chromosome Number

B

.. . . ) O

1 7

FIG. 5. Panel A, histogram showing the distribution of 396 silver grains recorded over 133 metaphase cells after in situ hybridization with the largest peak on chromosome 17. Panel B, idiogram of chromosome 17 illustrating autoradiographic grain labeling in the q21-q23 region.

members (RANTES and LD78/PAT464) of the superfamily of small secreted proteins has been mapped to chromosome 17q (23, 25). Because of the significant sequence similarity between these two proteins, this represents added evidence that they may have arisen as the result of a gene duplication. I t is possible that the extra hybridizing band detected by Southern blot analysis corresponds to a closely related mem- ber of the superfamily.

By sequencing the exons and exon/intron splice junctions

and by restriction mapping, we have determined that the Act- 2 gene is comprised of three exons spanning approximately 2 kb. We have identified the 5’-flanking sequences for the Act- 2 gene and demonstrated that they possess promoter activity in response to different activation stimuli. Because the Act-2 gene is known to be constitutively expressed in HTLV-I- transformed HUT-102B2 cells (1) and because a number of cytokines and inducible genes have been shown to be activated by Tax even in the absence of other stimuli (12, 20), we investigated whether the Tax gene product of HTLV-I could transactivate the expression of the Act-2 gene using promoter CAT constructs and this was indeed the case. Thus, we here report that 5”flanking regions of the Act-2 gene are respon- sive to mitogenic and Tax stimulation.

The Act-2 promoter has a classical TATA box 28 base pairs upstream of the transcription initiation site. 5’-Flanking se- quences contain a site a t -101194 that is similar to the consensus binding site for AP-1/CRE; nuclear factor binding to the Act-2 sequence can be competed by an AP-1 oligonu- cleotide. Further studies are required to determine whether binding of nuclear factors to this sequence mediate at least in part the Act-2 inducibility in response to PHA/PMA and/or Tax. Further analysis and dissection of promoter regulatory sequences could serve as an important model of gene regula- tion in T cell activation.

Acknowledgments-We graciously thank Dr. Kazuo Sugamura of Tohoku University for providing the JPX-9 and JPX/M cell lines, Dr. Michel Toledano and Jian-Xin Lin for helpful discussion, Marvin Shapiro for valuable help with the DNAdraw computer program, and Michael Erdos for technical assistance.

1.

2.

3.

4. 5.

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17536 The Gene Encoding the Act-2 Cytokine

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