©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Human gp39 Promoter
TWO DISTINCT NUCLEAR FACTORS OF ACTIVATED T CELL PROTEIN-BINDING ELEMENTS CONTRIBUTE INDEPENDENTLY TO TRANSCRIPTIONAL ACTIVATION (*)

(Received for publication, October 19, 1995)

Lisa A. Schubert (1)(§) Gordon King(§) (2) Randy Q. Cron (1) David B. Lewis (1)(¶) Alejandro Aruffo (2) Diane Hollenbaugh (2)

From the  (1)Departments of Pediatrics and Immunology, University of Washington, Seattle, Washington 98195 and the (2)Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

gp39, a cytokine expressed on the surface of activated T cells, is essential for T cell-dependent antibody responses in vivo. We cloned and sequenced 1.2 kilobases of the 5` flank region of the human gp39 gene promoter and determined its transcription start site. When used in reporter gene assays, this DNA segment conferred promoter activity in response to T cell activation. gp39 promoter function in transfectants was inhibited by cyclosporin A, as is expression of the endogenous gp39 gene in T-lineage cells. At least 0.5 kilobase of the 5` flank region was required for promoter activity. Two putative binding sites for the NF-AT family of transcriptional activator proteins were identified at -259 to -265 and -62 to -69 with respect to the transcription start site. Both sites contributed significantly and independently to promoter activity in response to T cell activation. Additionally, when incubated in vitro with nuclear protein purified from activated human CD4 T cells, both of these sites preferentially bound the NF-AT family member, NF-ATp. These results suggest that NF-ATp, via binding to at least two cis-elements, is essential for the induction of gp39 gene expression in response to T cell activation.


INTRODUCTION

gp39(1) , also known as CD40 ligand(2) , T-BAM(3) , or TRAP(4) , is a 33-kDa protein encoded in the human chromosome Xq26-27 region and is a member of the TNF (^1)superfamily of cytokines(5, 6) . It is expressed on activated T cells, primarily those of the CD4 subset(1, 6) . Engagement of CD40 on B cells by gp39 is an essential signal for B cell activation and isotype switching(5) . The importance of this interaction has been illustrated dramatically by the identification of defects in the human gp39 gene as the cause of the X-linked hyper-IgM syndrome (reviewed in (7) ). This primary inmmunodeficiency is characterized by impaired responsiveness to T cell-dependent antigens, as evidenced by poor specific antibody production and absent isotype class switching(7) . A similar immunologic phenotype has been observed in mice in which either gp39 or CD40 has been functionally inactivated by selective gene targeting(8, 9) .

In normal T cells, most gp39 surface expression and transcript accumulation occurs after T cell activation(10) . These events can be effectively blocked in vitro or in vivo by the immunosuppressant, CsA(11) . The CsA sensitivity of gp39 gene expression suggests that gp39 gene transcription might require NF-AT proteins, a family of CsA-sensitive transcriptional activators(12, 13) . NF-AT proteins have been implicated in the CsA-sensitive transcriptional activation of a number of cytokines expressed by activated T cells, including IL-2, IL-3, IL-4, granulocyte macrophage-colony-stimulating factor, and TNF-alpha(13) . Here we report that the 5` flank region of the human gp39 gene confers transcriptional activity in response to T cell activation, that induction of gp39 promoter activity is CsA-sensitive, and that two NF-AT-binding cis-elements are both essential for optimal promoter activity. Analysis of binding to these sites in vitro suggests that a particular NF-AT protein family member, NF-ATp, may play a central role in transcriptional activation of gp39 in human peripheral CD4 T cells.


MATERIALS AND METHODS

Cloning of the 5` Flank Region of the Human gp39 Gene

A genomic fragment containing the human gp39 was isolated by screening a -phage library (Stratagene), using a P-labeled HindIII fragment of the human gp39 cDNA clone(1) , and sequenced independently on both strands. The RNA initiation site of cDNA, generated from total RNA isolated from activated human T cells, was determined by the 5` rapid amplification of cDNA ends method as described previously(14) .

Reporter Gene Constructs

A 1294-bp element of the gp39 promoter region (from -1227 to +67, just 5` of the ATG codon; numbers are given with respect to the transcription start site) was PCR-amplified from the genomic clone, using primers containing 5` HindIII sites to facilitate subcloning. The PCR product was subcloned into the HindIII site, immediately 5` of the luciferase cDNA segment of the promoterless luciferase reporter gene plasmid, pSVOAL-ADelta5` ((15), provided by G. Plowman, Bristol-Myers-Squibb, Seattle, WA) to create gp39-luc. The same approach was used to produce the pSVOAL-ADelta5`-based luciferase reporter constructs gp39.D1-luc (-495 bp to +67) and gp39.D2-luc (-94 to +67), containing the portions of gp39 gene promoter segments indicated in parentheses. PCR by overlap extension (16) was used to mutate the 1294-bp gp39-luc promoter segment at the putative distal NF-AT-binding site at -259 to -265 from the sequence GGAAAA to AAAAAA (the coding strand is shown, with mutated residues in boldface). This insert was subcloned into pSVOAL-ADelta5` to create gp39.M1-luc. A similar approach was used to create gp39.M2-luc, in which the putative proximal NF-AT binding site at -62 to -69 of the 1294-bp gp39 promoter segment was mutated from the sequence GGAAAA to AAAAAA. The absence of secondary mutations introduced by PCR was confirmed for all constructs by DNA sequencing and comparison to the sequence of the original genomic clone. RSV-luc (provided by G. Plowman, Bristol-Myers-Squibb), in which the RSV promoter was subcloned into pSVOAL-ADelta5`, has been described (15) .

Transfection of Reporter Gene Plasmids

RPMI 1640 complete medium (17) was used for cell culture and transfection unless indicated otherwise. Jurkat thymoma cells (12 times 10^6/ml), (provided by Christopher Wilson, University of Washington, Seattle, WA) were transfected with 10 µg of CsCl-purified reporter plasmid by electroporation at 260 V and 1050 microfarads using an Electro Cell Manipulator 600 electroporator (BTX). This Jurkat subline expressed gp39 mRNA, as assessed by reverse transcriptase PCR (data not shown). Cells were incubated for 6 h at 37 °C, then divided in half, and either stimulated with 6.25 µg/ml ConA (Pharmacia Biotech Inc.) and 10 ng/ml PMA (Sigma) or left untreated for an additional 18 h. Cells were harvested, washed in phosphate-buffered saline, and lysed in 100 µl of reporter lysis buffer (Promega). Twenty-five µl of each lysate was assayed for luminescence using a Monolight 1500 luminometer (Analytical Luminescence Laboratories) and luciferase assay buffer, following the manufacturer's protocol (Promega). Adult peripheral blood human CD4 T cells were isolated by negative selection using monoclonal antibodies and complement (18) and were primed by culturing in medium with ConA (3.125 µg/ml), PMA (1 ng/ml), and 10 units/ml purified human IL-2 (Boehringer Mannheim) for 10-28 days prior to use. The electroporation protocol for primed CD4 T cells was generously provided by Dr. Chris Hughes, Yale University. (^2)In brief, resting primed CD4 T cells were cultured with equal numbers of irradiated (3300 rads) allogeneic whole peripheral blood mononuclear cells (3 times 10^6/ml) and phytohemagglutinin (1 µg/ml) (Sigma) for 19.5 h. Viable CD4 T cells were obtained by Ficoll-Hypaque centrifugation, washed in Hank's balanced salt solution, and 5 times 10^6 cells were electroporated with 15 µg of plasmid in 250 µl at 250 V and 960 microfarads using a Genepulser electroporator (Bio-Rad). After incubation at 37 °C for 2 h, cells (1 times 10^6/ml) were stimulated with PMA (25 ng/ml) and ionomycin (1.5 µm) (Calbiochem) for 20 h and harvested. Lysates (40 µl for each determination) were analyzed in duplicate for luciferase activity. Where indicated, CsA (500 ng/ml) (Sandoz) was added 1 h prior to stimulation with ConA and PMA (Jurkat cells) or with ionomycin and PMA (primed adult CD4 T cells).

EMSA

Oligonucleotides were synthesized using an Oligo 1000 DNA synthesizer (Beckman). Sequences of the double-stranded oligonucleotides used as probes and unlabeled competitors are described in the legend to Fig. 3. T cells previously primed in vitro for 2-3 weeks were stimulated for 2 h with ConA (10 µg/ml) and PMA (25 ng/ml), and nuclear proteins were extracted as described(17) . Nuclear protein extracts (3-4 µg) were preincubated at room temperature with binding buffer(17) , supplemented with 1 µg of poly(dI-dC) (Pharmacia) and oligonucleotide competitors, at 50-fold molar excess, for 10 min. Probe (2 times 10^4 cpm), end-labeled with [gamma]-P]ATP using T4 kinase, was then added to a final volume of 25 µl, and the incubation was continued for an additional 15 min at room temperature. In some reactions, nuclear protein was incubated with 1 µl of rabbit antisera raised against NF-ATp peptide 67.1 (19) for 30 min at 4 °C prior to the addition of probe. Where indicated, this antiserum was first incubated with 0.5 µl (500 ng) of either peptide 67.1 or, as a negative control, NF-ATp peptide 48(20) , for 30 min at 4 °C prior to the addition of nuclear protein. NF-ATp-specific antiserum and peptides were generously provided by Dr. Anjana Rao, Dana Farber Cancer Research Center, Boston, MA. All reactions were analyzed by polyacrylamide gel electrophoresis using 4% gels in 0.027 M Tris borate, 0.6 mM EDTA buffer.


Figure 3: Electrophoretic mobility shift assays demonstrate similar binding of nuclear proteins contained in activated T cells to oligonucleotides derived from either the human gp39 or human IL-4 promoters. Nuclear protein extracts were prepared from in vitro-primed adult CD4 T cells activated for 2 h with ConA and PMA. A, a specific complex is formed with the proximal NF-AT site that has a binding specificity similar to the distal gp39 promoter and proximal human IL-4 promoter NF-AT-binding sites. The proximal NF-AT oligonucleotide, 5`TCCTGGAAAATGTGCTT (noncoding strand), was used as the probe. Oligonucleotides that were added at a 50-fold molar excess relative to the probe included: none (lane 1), self (lane 2), the gp39 distal NF-AT site, 5`AGTGATAGGAAAATACT (coding strand) (lane 3), the human IL-4 promoter P1 site, 5`ATTGGAAATTTTCGTTA (noncoding) (lane 4), and the mutant IL-4 promoter P1 site, 5`ATTTAAAATTTTCGTTA (noncoding strand) (lane 5). B, the gp39 proximal NF-AT site and the IL-4 promoter P1 sites preferentially bind NF-ATp. Oligonucleotides containing the gp39 promoter-proximal NF-AT site (lanes 1-5) or the human IL-4 promoter P1 site (lanes 6-10) were used as probes. Antiserum to the NF-ATp peptide 67.1 was added either alone (lanes 3 and 8) or in the presence of NF-ATp peptide 67.1 (lanes 4 and 9) or an irrelevant peptide, NF-ATp peptide 48 (lanes 5 and 10). Putative or previously published NF-AT binding sites are shown in italics. Residues mutated from wild-type sequences are indicated in bold.




RESULTS

A genomic clone of the human gp39 gene that included 1.2 kb of its 5` flank region was isolated and sequenced (Fig. 1). The predominant transcription start site of the gp39 gene was identified at 68 bp upstream from the initiator ATG codon (Fig. 1). To assess the capacity of this region to confer transcriptional activity in T-lineage cells, a 1.2-kb segment (Fig. 1) was subcloned into the promoterless luciferase reporter plasmid, pSVOAL-ADelta5`. Transient transfection of gp39-luc into a subline of Jurkat thymoma cells that expresses gp39 mRNA yielded basal luciferase activity approximately 10-fold higher than that observed using the promoterless luciferase plasmid (Fig. 2A). Activation of Jurkat T cells with ConA and PMA, a potent stimulus for the induction of cytokine gene expression in T cells(21) , significantly enhanced luciferase expression approximately 100-fold above those observed with pSVOAL-ADelta5`, but did not increase luciferase activity mediated by pSVOAL-ADelta5`.


Figure 1: Sequence of the wild-type human gp39 gene promoter and gp39 promoter/reporter gene constructs. The sequence of the 1.2 kb of the 5` flank segment contained in gp39-luc is shown. The transcription start site (+1), determined experimentally as described under ``Materials and Methods,'' is indicated with an asterisk. The initiator codon (+68) is italicized. Brackets denote the 5` end of the deletion constructs, gp39.D1-luc and gp39.D2-luc, and are indicated above the sequence. Two purine-rich sequences at -259 to -265 (distal) and -62 to -69 (proximal) with homology to the IL-4 promoter P element sequences are in bold and underlined. Sequence changes made for the site-specific mutants gp39.M1-luc (distal) and gp39.M2-luc (proximal) are shown above the putative NF-AT sites in boldfaced italics.




Figure 2: Luciferase activity in T-lineage cells transiently transfected with gp39 promoter-driven reporter gene constructs. Jurkat (A and B) or primed CD4 T cells (C and D) were electroporated as indicated under ``Materials and Methods.'' Constructs used for each transfection are denoted on the abcissa. For each transfection, lysates were analyzed in duplicate for luciferase activity. The mean value of activity ± S.E. is shown on the ordinate. Activity in transfected Jurkat cells: reactions were carried out both with and without ConA and PMA stimulation. A, the data shown are representative of five independent transfections. B, reactions treated with CsA are indicated. The data shown are indicative of two independent transfections. Activity in transfected primed human CD4 T cells: all transfectants were stimulated with ionomycin and PMA. C, the data shown are representative of three independent experiments. D, reactions treated with CsA are indicated. The data shown are indicative of three independent experiments.



The amount of 5` flank region sufficient to confer gp39 gene promoter activity in response to T cell activation was ascertained using the 5` flank region deletion constructs gp39.D1-luc and gp39.D2-luc. Transfection of Jurkat with gp39.D1-luc yielded reporter gene activity comparable to that obtained with the full-length construct, gp39-luc (Fig. 2A). In contrast, cells transfected with gp39.D2-luc had markedly reduced luciferase activity in response to T cell activation.

gp39 expression by activated T cells is largely inhibited by CsA(11) , and a variety of evidence suggests that NF-AT transcriptional activator proteins are an important target for the inhibition of cytokine gene transcription by CsA(12, 13) . Treatment of Jurkat cells with CsA prior to activation completely ablated the increase in reporter gene activity of gp39-luc observed with ConA and PMA treatment (Fig. 2B). This suggested that NF-AT-binding transcriptional elements might contribute to gp39 promoter activity conferred by the immediate 5` flank region. Using GGAAAA as a minimum consensus motif (13, 22) , two putative NF-AT binding sites, termed the proximal (-62 to -69) and the distal (-258 to -265) NF-AT sites, were identified in the 500-bp region immediately upstream of the transcription start site of the gp39 gene (Fig. 1). Alteration of the GG dinucleotide sequence in NF-AT-binding sites of other cytokine gene promoters, such as those of the IL-4 and IL-2 genes, has been shown to markedly reduce the increase in promoter activity observed with T cell activation and to ablate the in vitro binding of NF-AT proteins(23, 24) . To test the importance of these NF-AT binding sites in the induction of gp39 promoter activity, these residues were mutated at either the distal or proximal NF-AT sites of gp39-luc, creating gp39.M1-luc and gp39.M2-luc, respectively. Transfection of Jurkat (Fig. 2A) with either mutant construct reduced activation-induced luciferase activity by more than 90%, as compared with gp39-luc.

To address the possibility that requirements for gp39 promoter activity in transformed T-lineage cells might differ from those of normal T cells, additional experiments were performed in which these constructs were transfected into adult CD4 peripheral blood T cells that had been previously primed in vitro. This cell population expresses high levels of gp39 mRNA and surface protein in response to T cell activation(25) . The luciferase activity induced by ionomycin and PMA stimulation of gp39-luc-transfected primed CD4 T cells was markedly higher than that observed upon transfection with promoterless luciferase plasmid (Fig. 2C). This induction was inhibited by CsA treatment (Fig. 2D). In the absence of secondary stimulation, gp39-luc transfectants had promoter activity that was only approximately one-fourth that induced by ionomycin and PMA treatment (data not shown). This induction of luciferase expression without activation was not surprising, since in vitro priming alone has been shown to up-regulate gp39 expression(25) . As with Jurkat cells, transfection of primed CD4 T cells with either gp39.M1-luc or gp39.M2-luc resulted in luciferase activity that was less than 10% of that observed with the wild-type gp39 promoter plasmid (Fig. 2C). In the experiments using primed T cells, the absolute amount of luciferase activity for a particular transfection condition varied from experiment to experiment as much as 10-fold (compare gp39-luc activity in Fig. 2, C and D). This may reflect donor-to-donor variation in the cell preparations used, with regard to responsiveness to phytohemagglutinin treatment and transfection efficiency. Nonetheless, in each experiment, the relative amounts of luciferase activity obtained for a set of transfection conditions was highly replicable (data not shown).

EMSAs were used to examine the in vitro binding of nuclear protein, isolated from activated human CD4 T cell lines, to oligonucleotides containing the gp39 promoter putative NF-AT-binding sites. A complex was formed when an oligonucleotide probe containing the proximal NF-AT site (Fig. 3A, lane 1) was used. The mobility of the complex formed was indistinguishable from that formed when the human IL-4 P1 element was used as a probe (Fig. 3B, compare lanes 1 and 6). A 50-fold molar excess of unlabeled oligonucleotides, containing either the proximal gp39 NF-AT site (lane 2), the distal gp39 NF-AT site (lane 3), or the IL-4 P1 element (lane 4), specifically competed for binding of this complex. In contrast, this complex was not competed by a mutant form of the IL-4 P1 element (lane 5). Additionally, the same series of unlabeled oligonucleotides sequences competed for binding in an identical manner using either the IL-4 P1 element or distal gp39 oligonucleotides as probes (data not shown).

Previous work suggests that NF-ATp, present in nuclear extracts isolated from activated murine T cell clones, preferentially binds to the murine IL-4 P1 element in vitro(23) . The mobility and specificity of the complexes formed by nuclear protein from activated human CD4 T cells with the gp39 proximal NF-AT or the human IL-4 P1 element oligonucleotides, were indistinguishable. Therefore, we used NF-ATp-specific serum to determine if NF-ATp was a major component of the T cell-derived complexes observed by EMSA. With the gp39 proximal NF-AT probe, addition of NF-ATp-specific antiserum resulted in further retardation (supershifting) of most of the specific complex (Fig. 3B, lane 3). This effect was specific, in that inclusion of the peptide to which the NF-ATp antiserum was originally raised (67.1) ablated this supershift, while inclusion of an irrelevant NF-ATp peptide (48) did not (Fig. 3B, lanes 4 and 5). Furthermore, in lanes in which NF-ATp peptide 67.1 was included, the specific complex was resolved into two distinct bands. The basis of this finding and its biologic significance, if any, are unclear. Virtually identical results were obtained with these reagents when the human IL-4 P1 element (Fig. 3B, lanes 6-10) or the gp39 distal NF-AT site (data not shown) was used as a probe.


DISCUSSION

A DNA segment containing 1.2 kb of the gp39 gene's immediate 5` flank was sufficient to confer activation-dependent transcription of reporter gene constructs in either a transformed T-lineage cell line or normal peripheral blood T cells. As little as 0.5 kb of the immediate 5` flank was sufficient for substantial activity in these assays, suggesting that most or all of the key transcriptional activator elements for gp39 gene expression are contained in this region. CsA treatment ablated activation-induced reporter gene expression mediated by this promoter segment. CsA inhibition of cytokine gene transcription in T cells appears mediated, at least in part, by blockade of calcineurin-dependent entry of NF-AT transcription factors into the nucleus ((26, 27), reviewed in (12) ). Consistent with the CsA sensitivity of gp39 gene expression, at least two NF-AT binding sites in the 0.5-kb 5` flank region of the gp39 gene appear to contribute substantially and independently to promoter function in activated T cells. We confirmed these results in normal peripheral blood T cells, suggesting that the independent contribution of these sites to promoter activity is likely to be physiologically important.

In other cytokine gene promoters, NF-AT binding sites occur adjacent to sites for AP-1 proteins or other bZip transcription factors(28) . Surprisingly, we did not identify any AP-1 binding sites adjacent to the proximal or distal NF-AT sites or elsewhere, when T(G/T)ANT(A/C)A was used as a minimal consensus sequence(29) . However, in vitro binding of NF-AT and AP-1 proteins to adjacent sites on DNA is cooperative(24, 30) . Such cooperativitiy may permit considerable degeneracy of either or both of these sites without compromising protein binding or, presumably, transcriptional activation(24) . Therefore, our study cannot exclude the possibility that the gp39 promoter may contain other NF-AT binding sites that are of functional importance, and that bZip protein-binding sites may cooperate with at least some NF-AT sites in transcriptional activation. Further studies, such as in vitro footprinting of the gp39 promoter region with recombinant NF-AT and AP-1 proteins, may reveal these sites.

Four human NF-AT family members have been cloned, including NF-ATc, NF-ATp, NF-AT3, and NF-AT4(19, 31, 32, 33) . Analysis of binding to the human IL-4 P1 element site indicates that NF-AT proteins bind as monomers(31) . Assuming that NF-AT proteins also bind either gp39 NF-AT site with a stoichiometry of 1:1, our supershift results suggest that NF-ATp is the predominant NF-AT protein binding to these (gp39 and IL-4 gene) NF-AT binding sites in primed T cells. Preferential binding of NF-ATp could reflect the presence of a greater quantity of NF-ATp and/or of NF-ATp possessing a superior ability to bind to NF-AT cis-elements in the nucleus of activated T cells, as compared with other translocating NF-AT family members. Regardless of the mechanism, our results raise the interesting possibility that NF-ATp, and not other NF-AT family members, may play a critical role in the transcriptional activation of gp39 and other cytokines, such as IL-4, in previously primed T cells.

Importantly, identification of the human gp39 promoter segment necessary and sufficient to induce activation-dependent transcription in T cells may be useful for correcting immunodeficiencies, by gene therapy, in which there are qualitative or quantitative defects in the expression of genes by activated T cells. Patients with X-linked hyper-IgM syndrome are prime candidates for this application.


FOOTNOTES

*
This work was supported by grants from the National Science Foundation (to L. A. S.), National Institutes of Health Grant AI-26940-07 (to D. B. L.), the March of Dimes (to D. B. L.), and the Bristol-Myers Squibb Pharmaceutical Research Institute (to G. K., A. A., and D. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 GenBank(TM)/EMBL Data Bank with accession number(s) L47983[GenBank].

§
The first two authors contributed equally to this study.

To whom correspondence should be addressed: Dept. of Pediatrics, Box 356320, University of Washington, Seattle, WA 98195.

(^1)
The abbreviations used are: TNF, tumor necrosis factor; IgM, immunoglobulin M; CsA, cyclosporin A; NF-AT, nuclear factor of activated T cells; IL, interleukin; NF-ATp, NF-AT (preformed); PCR, polymerase chain reaction; bp, base pair(s); ConA, concanavalin A; PMA, phorbol 12-myristate 13-acetate; EMSA, electrophoretic mobility shift assay; kb, kilobase pair(s).

(^2)
C. Hughes and J. Pober, Yale University, submitted for publication.


ACKNOWLEDGEMENTS

We are indebted to the Sequencing Group at Bristol-Myers Squibb for DNA sequencing, to Greg Plowman for providing plasmids and advice, to Anjana Rao and Christopher Wilson for generously providing us with reagents, to Chris Hughes for sharing the T cell electroporation protocol, and to Seymour Klebanoff for allowing us to analyze transfectants in his laboratory.


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