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The Journal of Immunology, 1998, 161: 277-285.
Copyright © 1998 by The American Association of Immunologists

An NFAT-Dependent Enhancer Is Necessary for Anti-IgM-Mediated Induction of Murine CD5 Expression in Primary Splenic B Cells1

Robert Berland2 and Henry H. Wortis

Department of Pathology and Program in Immunology, Tufts University School of Medicine and Sackler School of Graduate Biomedical Sciences, Boston, MA 02111


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD5 is a 67-kDa membrane glycoprotein the expression of which in murine splenic B cells is induced by surface IgM cross-linking. To analyze this induction, we transiently transfected primary splenic B cells with luciferase reporter constructs driven by various wild-type and mutated CD5 5'-flanking sequences. The transfected cells were subsequently cultured in medium with or without F(ab')2 anti-IgM (anti-IgM), and luciferase expression was assayed. Using this approach, we identified a 122-bp enhancer element necessary for anti-IgM-mediated induction of the CD5 promoter. Electrophoretic mobility shift assays indicated that four inducible and four constitutive complexes form on the enhancer fragment in nuclear extracts of primary B cells. Supershift assays revealed that two of the inducible complexes contained NFATc. Point mutations that abolished NFAT binding severely impaired enhancer function. Thus, CD5 is a target of NFAT in B cells. A third inducible complex required an intact H4TF-1 site. One of several constitutive complexes required an intact Ebox site while a second required an intact putative ets binding site. Mutation of the H4TF-1, Ebox, and Ets sites, in the presence of wild-type NFAT sites, significantly reduced the activity of the enhancer. Therefore, the induction of B cell CD5 expression requires NFAT binding and binding to at least one of three additional sites in the CD5 enhancer.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Much progress has been made in dissecting the membrane-proximal components of B cell antigen receptor (BCR)3 signaling (1, 2, 3). Ligation of the surface Ig/Ig{alpha}/Igß complex results in the activation of the src-family protein tyrosine kinases Lyn, Blk, Lck and of the Syk protein tyrosine kinase (reviewed in Refs. 1 and 3). These activated kinases can then initiate various signaling cascades including the phosphoinositide hydrolysis cascade, the ras signaling pathway, and the PI-3 kinase pathway (reviewed in Refs. 1 and 2). Less is known about how expression of specific genes is regulated downstream of these signaling events. Although the activities of several transcription factors, including CREB, Ets-1, NFAT, NF-{kappa}B, AP-1, Egr-1 (reviewed in Ref. 1), STAT-1 (4), STAT-3 (5), STAT-5, and STAT-6 (6), have been shown to be induced downstream of the BCR, there are few analyses of their interactions with specific gene-regulatory regions. As a model system for the study of BCR-mediated transcriptional regulation of genes, we have chosen to examine the induction of CD5 gene expression by anti-IgM.

CD5 is a 67-kDa membrane protein that, in addition to being induced on conventional splenic B cells by Ag-receptor cross-linking (7, 8, 9, 10), is constitutively expressed on T cells (11) and on a subset of B cells termed B-1a (12). An increased frequency of CD5-positive B cells is associated with two pathologic conditions: chronic lymphatic leukemia, in which there is a clonal expansion of CD5-positive B cells (13); and cases of polysystem autoimmune disease, such as rheumatoid arthritis in humans (14) and NZB (15)- or motheaten (16)-related diseases in the mouse. Evidence for a physiologic function for CD5 has recently emerged. In human T cells and CD5-expressing chronic lymphocytic leukemia B cell lines, CD5 can associate with the Ag-receptor complex and become tyrosine phosphorylated on Ag-receptor ligation (17, 18, 19), suggesting it plays a role in the regulation of BCR and TCR-mediated signaling. Indeed, CD5 was found to negatively regulate Ag-receptor-mediated signals in both CD4+CD8+ thymocytes (20) and B-1a B cells (21).

We have previously shown that surface expression of CD5 as well as steady-state CD5 mRNA levels are induced by treatment of splenic B cells with F(ab')2 anti-IgM (anti-IgM) (7, 8, 9, 10). In contrast, induction is not seen in response to LPS or CD40 ligation (9), at least at the level of surface protein expression. Thus, CD5 induction is dependent specifically on signals downstream of the BCR. In addition, this induction is inhibited by the immunosuppressive drugs cyclosporin and FK506, suggesting that the transcription factor NFAT may be required for CD5 induction (10).

To further understand how BCR ligation induces CD5 in B cells, we have used transient transfection into primary B cells to identify the CD5 5'-flanking sequences necessary for induction by sIgM cross-linking. We then went on to characterize the factors from induced and noninduced primary murine splenic B cells that bound to several of these sequences.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation and transfection of B cells

B cells were prepared as previously described (10) from spleens of >8-wk-old BALB/cByJ mice obtained from The Jackson Laboratory (Bar Harbor, ME). Cells were cultured in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% heat-inactivated fetal calf serum, 20 mM HEPES (pH 7.0), 2 mM glutamine, 50 mM 2-mercaptoethanol, 1 mM sodium pyruvate, 0.1 mM MEM nonessential amino acids (Life Technologies), 100 U/ml penicillin, and 100 mg/ml streptomycin sulfate.

Transfections were performed essentially as described by McMahon et al. (22). Primary B cells were cultured for 3 days with 50 µg/ml LPS from Salmonella typhosa (Sigma, St. Louis, MO, cat. no. L-6386) prior to transfection. Transfection reactions contained 1 x 107 cells/ml, 3 µg/ml pRLTK DNA (Promega, Madison, WI) as an internal control, 3 µg/ml DNA of the reporter construct of interest, and 0.5 mg/ml DEAE-dextran, molecular mass ~500 kDa (Pharmacia, Newark, NJ) in STBS (25 mM Tris-Cl (pH 7.4), 137 mM NaCl, 5 mM KCl, 0.6 mM Na2HPO4, 0.7 mM CaCl2, 0.5 mM MgCl2). After transfection, cells were cultured for ~24 h in supplemented RPMI and then induced (or not) for 15 to 20 h with 15 µg/ml F(ab')2 fragment of goat anti-mouse IgM, µ-chain specific (anti-IgM) (Jackson Immunoresearch, West Grove, PA, or Cappel, Malvern, PA). DNA used in transfections was prepared with a kit purchased from Qiagen (Chatsworth, CA) following the manufacturer’s instructions.

Dual-luciferase assays

In all experiments, cells were cotransfected with the construct of interest (a firefly luciferase reporter construct) and, as an internal control, pRLTK (Promega) in which the HSV-tk promoter drives expression of a Renilla luciferase reporter gene. Cells were harvested and washed once in PBS (Ca2+, Mg2+ free), all residual PBS was carefully removed from the pellet, and 107 cells were lysed in 100 ml of passive lysis buffer (Promega). Dual-luciferase assays were performed using the dual-luciferase assay kit (Promega) following the manufacturer’s instructions.

Reporter constructs

Standard methods were used for all recombinant DNA work (23). After all PCR steps, PCR products were TA cloned into pCR2.1 (Invitrogen, San Diego, CA) before proceeding. All constructs used in transfections or for EMSA probes were checked by sequencing at the Tufts University Sequencing Facility (Boston, MA).

5'-Deletion mutants (-2200Luc, -2040Luc, -1965Luc, -1943Luc, and -277Luc). We obtained pGL2Ly1 from Drs. James Tung and Leonore Herzenberg (Stanford University, Stanford, CA) (24). This construct contains the murine CD5 5'-flanking sequence from -6 bp relative to the ATG to about -3000, inserted into the polylinker of pGL2Basic (Promega) between the KpnI and BglII sites. The BglII site was destroyed by this insertion. We removed the CD5 flank by digestion with HindIII and recloned it into the HindIII site of pGL3Basic (Promega).

Bal31 deletion mutants (-2200Luc, -2040Luc, and -1943Luc) were made from this construct as follows. It was digested with KpnI and then digested for varying times with Bal31. The Bal31 ends were then blunted by treatment with T4 DNA polymerase and the luciferase gene plus the 5'-deleted CD5 flanks isolated by digestion with BamHI and gel purification using Glassmilk (Bio101, La Jolla, CA). These fragments were then cloned into SmaI and BamHI cut pGL3Basic. The 5'-deletion endpoints were determined by automated DNA sequencing at the Tufts University Sequencing Facility. We were not able to sequence in either direction through a GT repeat located between approximately -1585 and -1421. Therefore, for constructs with 5'-endpoints that fall 5' of this region, we assigned an endpoint based on the endpoint of the full length construct which was taken to be -3000 (based on the sizes of restriction fragments).

A construct deleted to -1965 (-1965Luc) was made by PCR using -2040Luc as template and primers 5'-237 and StuR (see Fig. 1Go for sequences of primers used in construct making). The PCR product was digested with KpnI and StuI and cloned into the -2040 deletion construct which had been cut with KpnI and partially digested with StuI such that only the promoter-distal StuI site (at about -1608) was cut.



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  		FIGURE 1. PCR primers used for making various constructs. Sequences are written 5' to 3'. The sequences in brackets correspond to CD5 sequences and the numbers represent either the position in the vector pGL3Basic (for RV-L) or to the position in the CD5 flank (for the others). The numbering for the CD5 sequences is based on the assignment of -3000 for our full length construct based on restriction fragment sizes and therefore the absolute number is not precise. Sequences outside of brackets are present in the primer but not in the CD5-flanking sequence. For mutated primers nucleotide changes are indicated in capitals. The sequence of the wild-type enhancer is shown in Figure 3Go.

 
A construct, p{Delta}283, was not used in any experiments reported here but was used in the construction of several constructs that were, and also as the source of an electrophoretic mobility shift assay (EMSA) probe. It contains an internal deletion of CD5 sequences between -1919 and -900. It was made as follows. The 122-bp enhancer fragment (-1919 to -2040) was amplified by PCR using the RV-L and 3'-283 as primers and -2040Luc as template. The 3'-283 primer introduces a BglII site at the 3'-end of the fragment. The PCR product was cut with KpnI and BglII and cloned into the vector-containing fragment of the -2040Luc digested with KpnI and BglII.

The -277 deletion mutant (-277Luc) was made by digestion of p{Delta}283 with BstXI (which cuts at -277 of the CD5 gene relative to the ATG (25) and KpnI, blunting with T4 DNA polymerase, and religating.

pCD5Luc122R and derivatives. The construct containing the wild-type enhancer 3' of the luciferase gene (pCD5Luc122R) was made by amplifying the 122-bp enhancer fragment using p{Delta}283 as template and the primers 5'-2040 and 3'-283. These primers introduce a BamHI site at the 5'-end and a BglII site at the 3'-end of the fragment. The PCR product was digested with BamHI and BglII and cloned into BamHI cut -277Luc.

Derivatives of pCD5Luc122R with point mutations in the enhancer were made by PCR using as template several constructs not otherwise used for this study. These constructs contained point mutations introduced into the Ebox, Ets, or both Ebox and Ets sites of the enhancer in the context of the -2040Luc construct (designated -2040mEbox, -2040mEts, and -2040mm, respectively). They were made as follows. Point mutations were introduced into the enhancer fragment from -2040 to -1945 by PCR using RV-L as the 5'-primer and either mEbox, mEts, or mm as the 3'-primer and -2040Luc as the template. An overlapping fragment from -1965 to -1584 was amplified by PCR using 5'-237 as the 5'-fragment, StuR as the 3'-fragment and -2040Luc as template. A fragment from -2040 to -1584 containing point mutations was then amplified by PCR using the -1965 to -1564 product mixed with one of the mutated -2040 to -1945 products as templates. The 5'-primer was RV-L and the 3'-primer was StuR. The products of these reactions were cut with KpnI and StuI and cloned into -2040Luc cut with KpnI and partially digested with StuI as described above for the construction of -1965Luc.

Versions of pCD5Luc122R with point mutations in the Ebox (122RmEbox), Ets (122RmEts), or both Ebox and Ets (122Rmm) sites were made the same way as pCD5Luc122R (see above) except that the templates for PCR were -2040mEbox, -2040mEts, and -2040mm, respectively. A version with a mutated H4TF-1 site was made similarly except the 5'-primer was C2M. Versions with H4TF-1 and one other site mutated were made using C2M and 3'-283 as primers and either -2040mEbox or -2040mEts as template. A version with all three of these sites mutated were made using C2M and 3'-283 as primers and -2040mm as template. A version with the distal NFAT site mutated (122RmNFAT) was made by setting up two PCR reactions to generate two overlapping fragments using for the 5'-fragment primers 5'-2040 and mNFAT-AS and for the 3'-fragment mNFAT-S and 3'-283. The products of the two reactions were mixed and used as template for a PCR with primers 5'-2040 and 3'-283. The product of this reaction was cloned into the Bam site of -277Luc. A construct with both NFAT sites mutated (122Rm2NFAT) was made by PCR using as template 122RmNFAT and as primers 5'-2040 and mNFAT2.

Footprinting probe. The probe used for copper-phenanthroline footprinting (TA-1) contained the 5'-89 nucleotides of the CD5 enhancer. It was obtained by PCR using p{Delta}283 as template and the primers RV-L and 3'-89. The PCR product was cloned into pCR2.1 (Invitrogen).

Electrophoretic mobility shift assays and footprinting analysis

For preparation of nuclear extracts B cells were isolated and cultured overnight with or without anti-IgM. Viable cells were isolated by centrifugation through a cushion of Lympholyte-M (Cedarlane, Hornby, Ontario, Canada) followed by two washes in ice cold PBS (Ca2+ and Mg2+ free). Cells were lysed by incubation for 20 min at 4°C in Buffer A (10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 10 mM iodoacetamide, 1 mM PMSF, 1 µg/ml pepstatin A, 1 µg/ml aprotinin) plus 0.05% Nonidet P-40. Nuclei were washed twice in buffer A and salt extracted for 40 min on ice in ~2 vol of buffer C (20 mM HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 10 mM iodoacetamide, 1 mM PMSF, 1 µg/ml pepstatin A, 1 µg/ml aprotinin). Insoluble material was removed by centrifugation for 10 min at 16,000 rpm in a microfuge, and the supernatants were mixed with an equal volume of Buffer D (20 mM HEPES (pH 7.9), 0.2 mM EDTA, 0.5 mM DTT, 20% glycerol, 10 mM iodoacetamide, 1 mM PMSF, 1 µg/ml pepstatin A, 1 µg/ml aprotinin). Extracts were distributed into small aliquots, quick frozen in liquid N2, and stored at -80°C. Aliquots were used once and discarded. Protein was quantified by the method of Bradford (Bio-Rad, Hercules, CA).

Full-length enhancer probes were prepared either by digesting the construct p{Delta}283 with KpnI and BglII or digesting a pCD5Luc122R-based construct with ClaI and BamHI and isolating the enhancer-containing fragment from a nondenaturing polyacrylamide gel by the "crush and soak" method (23). The fragment isolated from p{Delta}283 contains the 122-bp CD5 enhancer plus 24 nucleotides of pGL3Basic vector sequences at its 5'-end consisting of polylinker between the KpnI and SmaI sites. The fragment isolated from p120R-based constructs contains at its 3'-end seven nucleotides of pGL3Basic vector sequence consisting of sequences between the BamHI site and the ClaI site at vector sequence 1997. In EMSAs, the p{Delta}283-derived probe gives rise to one more complex than the pCD5Luc122R-derived fragment. Fragments were end-labeled by standard methods using the Klenow fragment of Escherichia coli polymerase I. Unincorporated nucleotides were removed by passage through a nick column (Pharmacia).

Binding reactions for EMSA contained about 1 ng of labeled probe, 10 to 15 µg of protein from nuclear extracts, 5 µg of poly(dI-dC)-poly(dI-dC) (Pharmacia), 21.5 mM HEPES (pH 7.9), 84 mM NaCl, 1 mM EDTA, 1.2 mM DTT, 14% (v/v) glycerol, 300 µg/ml BSA, and cold competitor oligomers or supershifting Ab as indicated all in a final volume of 20 µl. Reactions were incubated at room temperature for 30 min and run at room temperature on prerun native 5% acrylamide gels cast in 0.5x TBE in 0.5x TBE tank buffer. Oligomers were prepared at the Tufts University Oligonucleotide Synthesizing Facility (Boston, MA). mAbs against NFATp (G1-D10) and NFATc (7A6) were generously provided as unpurified ascites by Gerald Crabtree (26) (Stanford University, Stanford, CA). Undiluted G1-D10 (1 µl) and 1 µl of a 1:10 dilution of 7A6 were used per reaction as per instructions from the Crabtree laboratory.

The probe used for copper-phenanthroline footprinting was prepared by purifying the insert of TA-1 by digestion with Acc65I and BstXI followed by electrophoresis through a nondenaturing polyacrylamide gel and purification by the "crush and soak" method. The fragment was 32P labeled on the bottom strand only using the Klenow fragment of E. coli DNA polymerase (this labels only the Acc65I site since the BstXI site has a 3'-overhang). A preparative EMSA was performed, and the complexes were localized by exposing the wet gel to a phosphorimaging screen overnight following which the entire gel was treated with 1,10-phenanthroline-copper for 15 min at room temperature according to the method of Kuwabara and Sigman (27). The uncomplexed probe and complex S1 were purified by electroelution onto DEAE-cellulose and run on a 6% DNA sequencing gel.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A 122-bp enhancer element located at about -2 kb is necessary for anti-IgM-induced activity of the CD5 promoters

To establish a system for studying CD5 gene regulation we wished to identify cells suitable for transfection of reporter gene constructs. Primary murine splenic B cells treated with LPS have been shown to be transfectable (28). MacMahon et al. (22, 29) reported that if the LPS is washed out, these cells can subsequently respond normally to activation by anti-IgM. In preliminary experiments, we demonstrated by RT-PCR that primary B cells mock-transfected according to the protocol of McMahon et al. could be induced by anti-IgM to express their endogenous CD5 gene. A clear induction was apparent after 15 h of anti-IgM treatment (data not shown). We next transfected primary B cells with a series of 5'-deletion constructs containing various extents of CD5 5'-flank fused to a luciferase reporter gene. The transfected cells were subsequently cultured for about 18 h in either medium alone (uninduced) or medium plus anti-IgM (induced). Figure 2GoA shows results of one such experiment. Constructs with 5'-deletions to -2200 and -2040 were induced about 12- and 7-fold, respectively, by anti-IgM treatment. Further deletion to -1965 essentially abolished activity. This indicated that sequences necessary for anti-IgM-induced activity reside between -2040 and -1965.



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  		FIGURE 2. An enhancer necessary for anti-IgM induction resides between -2040 and -1919 of the CD5 5'-flank. A, Five constructs with different extents of 5'-flank of the CD5 gene driving the firefly luciferase gene were cotransfected with pRLTK into primary B cells. After 24 h, the cells were either induced overnight with 15 µg/ml anti-IgM (filled bars) or left in medium (open bars). The relative luciferase activity is (firefly luciferase activity/Renilla luciferase activity) x 100. The results of one experiment, done in triplicate, are shown. Error bars are standard errors. The 5'-deletion endpoints are shown. The endpoint of the -277 construct is numbered with respect to the ATG. The endpoints of the other deletion constructs are precise relative to one another but not with respect to the ATG. The no promoter construct is pGL3Basic. B, Results of transfections with constructs indicated schematically on left. This figure summarizes the results with these constructs in all the transfection experiments presented in this paper (n = 12, each experiment performed in duplicate or triplicate). The mean relative activity (defined as in A) was calculated for each experiment and the mean of these ± SE is presented. Filled bars are from anti-IgM-induced cells; open bars are from uninduced cells.

 
To narrow our search for anti-IgM-responsive regulatory elements, we next tested a construct, pCD5Luc122R, in which CD5 promoter-proximal sequences (-6 to -277 relative to the ATG) were placed upstream of the luciferase gene, and CD5 promoter-distal sequences (-1919 to -2040) placed downstream of the luciferase gene and in the reverse orientation. As can be seen in Figure 2GoB, this construct was induced about 12-fold by anti-IgM treatment. The identical construct lacking the CD5-distal element was induced no more than twofold. Thus, CD5 sequences from -1919 to -2040 are necessary and sufficient to confer anti-IgM inducibility on the CD5 promoter. Because of its ability to act in a position- and orientation-independent fashion, this element is an enhancer.

The CD5 enhancer binds to NFAT in extracts from primary B cells induced with anti-IgM

Figure 3Go shows the nucleotide sequence of the CD5 enhancer as well as potential transcription factor binding sites based on sequence homologies. To determine which of these (or other) sites can bind transcription factors, we performed EMSA analysis with the entire 122-bp enhancer fragment as probe and nuclear extracts prepared from induced and uninduced primary splenic B cells. For EMSA analyses, unlike transfections, cells were not treated with LPS. Figure 4GoA shows a typical result. Lane 1 shows the probe alone, and lanes 2 and 5 show the probe incubated with extracts from uninduced and induced cells, respectively. Four complexes, labeled C1 to C4 in the figure, were formed in extracts from uninduced cells. (The unlabeled complex running just above C2 is an artifact seen only when the probe is prepared such that it includes 24 bp of vector sequence at its 5'-end. A probe lacking these 24 bp does not form this complex (see Fig. 6Go).) When extracts from induced cells were used, four additional complexes were formed (Fig. 4GoA, lane 5; S1 to S4). In addition, the amounts of constitutive complexes C3 and C4 decreased. All the complexes were competed by an excess of the unlabeled 122-bp enhancer fragment (Fig. 6Go, lane 5).



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  		FIGURE 3. Sequences of the CD5 enhancer and oligomers and probes used in EMSAs. The sequence of the CD5 enhancer is shown. Candidate transcription factor-binding sites are boxed. The mutations introduced into the H4TF-1 and NFAT sites are indicated above the sequence with the altered bases in capital letters. Sequences of Oligo1 and Oligo2 that were used as competitors in EMSAs are shown below the enhancer sequence. Changes made in Oligo-2 to mutate the Ebox (m1) and Ets site (m2) are indicated. The base changes indicated here are the same as those introduced into the various mutant reporter constructs.

 


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  		FIGURE 4. NFATc binds to the CD5 enhancer. A, EMSA performed in the presence or absence of supershifting anti-NFATp (G1-D10) or anti-NFATc (7A6) Ab as indicated using 12 µg of extracts from induced or anti-IgM-treated cells. Lane 1 is with no extract. The probe is derived from p{Delta}283 and consists of the 122-bp enhancer fragment plus 24 bp of pGL3Basic vector sequence at its 5'-end. Constitutive complexes are indicated on the left, induced complexes on the right, and supershifted complexes with asterisks. B, Only the top portion of an EMSA is shown in which the same probe as in A was incubated with anti-NFATc antibody and 12 µg of extracts from either untreated (lane 1) or anti-IgM treated (lanes 2–4) B cells. In lane 3, an excess of an oligomer containing the wild-type distal NFAT site of the murine IL-2 promoter (-293-cccaaagaggaaaatttgtttcatacag, NFAT site underlined) was included in the reaction. In lane 4, an excess of the same oligomer containing a mutated NFAT site (-293-cccaaagaCCTTaatttgtttcatacag mutated bases in capitals) was included. C, The top portion of an EMSA is shown in which 12 µg of extract from untreated (lane 1) or anti-IgM-treated (lane 2) B cells were incubated with a CD5 enhancer probe containing point mutations in both NFAT sites (see Fig. 3Go). In addition to the 122-bp CD5 enhancer sequence, this probe contains seven nucleotides of pGL3Basic vector sequence at its 3'-end.

 


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  		FIGURE 6. An intact Ebox and an intact Ets-binding site are required for formation of complexes C1 and C2. An EMSA of a CD5 enhancer probe and 10 µg of nuclear extract from anti-IgM-treated B cells is shown. In lane 1, there is no competitor oligomer, in lanes 2 to 4 an excess of competitor Oligo-2 containing either wild-type sequences (lane 2), a mutated Ebox (m1, lane 3), or a mutated Ets-site (m2, lane 4) was included. In lane 5, an excess of the wild-type 122 bp enhancer was included in the reaction. The probe contains the 122-bp enhancer and seven vector-derived nucleotides at the 3'-end. See Figure 3Go for the sequences of the competitor oligomers. Asterisks indicate the induced complexes S1 to S4 reading from top to bottom.

 
We had previously shown that the induction of CD5 protein by sIgM cross-linking is sensitive to cyclosporin and FK506 (10), suggesting a possible role of NFAT in CD5 induction. We therefore asked whether any of the complexes induced by anti-IgM contain NFAT. Extracts and probe were incubated in the presence of Abs to either NFATp (NFAT1) or NFATc (NFAT2), the NFAT family members expressed at high levels in peripheral lymphoid tissue. In Figure 4GoA, lanes 3 and 4, binding reactions contained uninduced extracts and anti-NFATp (lane 3) or anti-NFATc (lane 4). The reactions run in lanes 6 and 7 contained induced extracts and anti-NFATp or anti-NFATc, respectively. From lane 7, it is clear that in the presence of anti-NFATc complexes S3 and S4 did not form, instead, a slower migrating, presumably supershifted, doublet appeared (indicated by asterisks). Only a very small amount of this complex was apparent with extracts from uninduced cells (lane 4). The failure of anti-NFATp to supershift any complexes formed with activated extracts (lanes 3 and 6) is most likely due to the fact that we performed 18-h inductions. In anti-CD3-stimulated T cells, NFATp binding activity peaks by 2 h and has largely disappeared by 5 h after induction, at which time NFATc binding is present (30).

To confirm that the supershifted complexes contain NFAT, supershift assays were performed in the presence of an excess of an unlabeled oligomer containing either a wild-type or mutated NFAT site from the murine IL-2 promoter (Fig. 4GoB). As can be seen, the wild-type oligomer effectively competed for formation of the supershifted complexes (lane 3). In the presence of the mutated oligomer, formation of the supershifted complexes is not inhibited (lane 4). Also, as expected, in the absence of supershifting Ab the wild-type oligomer competes for formation of complexes S3 and S4 (Fig. 5, lane 3). From these experiments, we conclude that NFATc is a component of complexes S3 and S4.

Finally, we wished to determine whether the NFAT sites that we identified in the CD5 enhancer based on nucleotide sequence (Fig. 2GoA) are in fact involved in the NFAT binding that we observe by EMSA. We therefore did an EMSA experiment using a CD5 enhancer probe containing mutations in both potential NFAT sites (see Fig. 3Go). As can be seen in Figure 4GoC, complexes S3 and S4 did not form with induced extracts when this probe was used. Thus, at least one of the NFAT sites identified in Figure 3Go is necessary for NFAT binding to the CD5 enhancer probe. The induced complexes S1 and S2 were still able to form with the mutant-NFAT probe, indicating that these complexes do not contain NFAT, consistent with the failure of anti-NFAT Ab to supershift them (Fig. 4GoB, lanes 6 and 7) and with the failure of the wild-type NFAT oligomer to compete for their formation (Fig. 4GoC, lane 3).

The formation of complex S1 requires an intact H4TF-1 site

To identify the site bound in complex S-1, we did copper-phenanthroline footprinting of this shifted band (27). Figure 5GoA shows the footprint obtained with the labeled bottom strand. Although no footprint was identifiable, there was a clear hypersensitive site at -1928. This is within a sequence that is identical with that bound by the transcription factor H4TF-1 (31). To confirm that this sequence is involved in formation of S1, an EMSA was performed with the full enhancer probe in the presence of excess competitor oligomer-1 (see Fig. 3Go). This oligomer contains the intact H4TF-1 site. The result is shown in Figure 5GoB. Comparing lane 4 with lanes 2 and 3, it is apparent that the oligomer competed with the enhancer probe for formation of complex S1. We also performed an EMSA using as probe the CD5 enhancer with four base changes introduced into the H4TF-1 site (see Fig. 3Go). Figure 5GoB, lane 5, shows that this probe failed to form complex S1 with extracts from induced B cells. Thus, an intact H4TF-1 site is necessary for complex S1 formation.



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  		FIGURE 5. Complex S1 requires an intact H4TF-1 site. A, Copper-phenanthroline footprinting of complex S1. The bands corresponding to complex S1 (lane 3) and free probe (lane 2) were excised from a preperative EMSA gel after treatment of the entire gel with copper-phenanthroline. The cleaved products of the reaction were run on a 6% sequencing gel. The probe consisted of the 5'-89 nucleotides of the CD5 enhancer labeled on the bottom strand. A copper-phenanthroline hypersensitive site is indicated. A G + A ladder of the probe was run in lane 1 to allow localization of the hypersensitive site. The nucleotide sequence in the vicinity of the hypersensitive site is shown with the base at which enhanced cleavage occurs in outline. B, An EMSA is shown in which 12 µg of extract from untreated (lane 1) or anti-IgM-induced (lanes 2–5) B cells were incubated with a wild-type enhancer probe (lanes 1–4) or an enhancer probe in which point mutations were made in the H4TF-1 sequence (lane 5, see Fig. 3Go for mutations introduced into the probe). In lane 4, an excess of the oligomer Oligo1 (see Fig. 3Go), which contains the H4TF-1 site, was included in the binding reaction.

 
H4TF-1 was originally identified in HeLa cell extracts as a factor that bound to a sequence in the histone H4 promoter necessary for expression of the histone H4 gene (32). We have not attempted to determine whether the factor binding to the H4TF-1 site in extracts from anti-IgM-treated B cells is in fact H4TF-1.

The formation of constitutive complexes C1 and C2 requires intact Ebox and ets sites, respectively

On the basis of sequence homologies, we identified a potential Ebox- and a potential Ets-binding site in the CD5 enhancer (Fig. 3Go). Members of both transcription factor families have been shown to be important in B cell gene expression (33, 34, 35, 36). We therefore wished to see whether any of the complexes that we detected by EMSA involved these sites. An EMSA was performed in which the enhancer probe was incubated with extracts from anti-IgM-treated B cells in the presence or absence of the competitor oligomer Oligo2 which contains both the Ebox and Ets sites (Fig. 6Go). The wild-type oligomer competed for formation of complexes C1 and C2 (Fig. 6Go, lane 2). With an oligomer containing point mutations in the Ebox (Oligo2, m1, see Fig. 3Go) C1, but not C2, was able to form (Fig. 6Go, lane 3). This suggests that C1 contains an Ebox-binding protein. Conversely, when the competitor contained point mutations in the Ets site (Oligo2, m2, see Fig. 3Go), C2 but not C1 is formed, suggesting that C2 contains an Ets-site binding protein (Fig. 6Go, lane 4). Consistent with the results of the competition experiments, when an EMSA was performed using an enhancer probe containing point mutations in both the Ebox and Ets sites, complexes C1 and C2 failed to form (data not shown).

Inducible activity of the CD5 enhancer requires both NFAT sites and either the H4TF-1, Ebox, or Ets site. To determine whether the DNA-protein complexes identified in the EMSA analyses have functional significance, we made reporter constructs in which mutations were introduced into factor-binding sites in the context of pCD5Luc122R. Figure 7GoA shows the results of a transfection experiment in which only the distal or both proximal and distal NFAT sites were mutated. The induced activity of the singly mutated construct was 49% that of the wild-type construct. The induced activity of the doubly mutated construct was 18% that of the wild-type construct. Thus, both NFAT sites appear to play a role in the response of the CD5 enhancer to anti-IgM stimulation.



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  		FIGURE 7. Both NFAT sites and at least one of the other identified sites are necessary for full CD5 enhancer activity. A, The wild-type p122RLuc construct or derivatives with point mutations in either the distal NFAT site or both proximal and distal NFAT sites were cotransfected with pRLTK into primary B cells. After 24 h, the cells were either treated with anti-IgM overnight (filled bars) or left untreated (open bars). For each construct, at least four experiments in duplicate or triplicate were performed. All experiments included the enhancerless construct (-277 of Fig. 2GoA) and wild-type enhancer controls. In each experiment, the mean relative luciferase activity was calculated for each construct as in Figure 1Go, and this value was calculated as a percentage of the mean relative luciferase activity of p122RLuc (percent relative luciferase activity). These values were averaged over all the experiments and are presented ± SE. Mutations in binding sites are as indicated in Figure 3Go. B, Experiment was performed as in A with the mutated enhancers indicated. Three experiments in duplicate or triplicate were performed with the doubly-mutated constructs. Five experiments in duplicate or triplicate were performed with the other constructs. Mutations in binding sites are as indicated in Figure 3Go.

 
To assess the role of the other identified factor-binding sites, a construct was made with mutations introduced into all three remaining sites, H4TF-1, Ebox, and Ets. Constructs were also made with pairwise combinations of these mutant sites. Figure 7GoB shows the results of a transfection experiment using these constructs. Mutation of all three sites lowered the induced activity of the enhancer to 24% of wild-type. Mutation of the H4TF-1 site together with either the Ets or the Ebox site had only a modest effect on enhancer activity, lowering it to about 50 to 75% of wild-type activity. Mutation of both the Ebox and Ets sites had essentially no effect on activity. These results indicate that the presence of any one of these three sites is able to contribute to the inducible activity of the enhancer.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A final point of integration of signals from the BCR occurs at the cis-regulatory regions of genes induced by BCR ligation. To date, few anti-IgM-inducible regulatory regions have been characterized in detail. In this study, we have begun to characterize one such region, that controlling expression of the CD5 gene.

Using transient transfection into primary murine B cells, we have shown that an enhancer sequence located at about -1919 to -2040 is necessary for induction of CD5 promoter activity. The CD5 promoter alone (-6 to -277 relative to the ATG) is induced at most twofold by anti-IgM. In the presence of the enhancer sequence, basal activity appears to increase slightly (about 2 fold), and this activity is induced about 10-fold in response to anti-IgM treatment. Since we have performed detailed studies only on constructs in which the CD5 enhancer is driving the homologous CD5 promoter, we cannot rule out an important role for promoter-proximal sequences in enhancer activity.

In an earlier study, Weichert and Schwartz (25) used transient transfection into B, T, and fibroblast cell lines to determine the sequences necessary for constitutive, lymphocyte-specific CD5 expression. They found that a construct containing CD5 sequences from +3 to -131 relative to the ATG was sufficient for essentially maximal activity in the M12 B cell line. They did not examine sequences beyond -1706 and therefore could not have detected the enhancer identified here.

Transient transfection of primary B cells necessitated their preactivation with LPS, and it is possible that this affected the regulation of CD5 expression. However, EMSA analyses were performed with extracts from cells not previously LPS treated, and all five protein-binding sites that were identified by EMSA that were later tested for function in transfections were found to contribute to enhancer activity. Still, we cannot rule out the possibility that activity in the transfections was mediated at least in part by LPS-dependent complexes not seen in our EMSAs.

A major conclusion of the present study is that NFAT plays an indispensable role in the activity of the CD5 enhancer. This is based on a combination of EMSA analysis, which established that NFAT binds to the CD5 enhancer in vitro, and transient transfection analysis, in which we showed that point mutations that abolish the binding of NFAT to the enhancer in vitro, severely impair enhancer function in vivo.

NFAT is a family of transcription factors with four known members, NFATp, NFATc, NFAT3, and NFAT4 (reviewed in Ref. 37). All family members appear to have similar DNA-binding specificities (38), although differences in site selectivity are seen under certain conditions (39). In addition, only NFATp has been found to bind to the atypical k3 site of the TNF-{alpha} promoter (40). Both NFATp and NFATc have been found to be expressed at high levels in peripheral lymphoid tissue. However, at least in T cells, they differ in the kinetics of their induction in response to TCR ligation. NFATp DNA binding activity increases rapidly after anti-CD3 treatment but by 5 h of treatment has returned to basal levels. NFATc DNA-binding activity is induced more slowly and is high by 5 h of activation (30). It was therefore not surprising that in our EMSAs using extracts from B cells treated for 18 h with anti-IgM, all NFAT activity was supershifted by an anti-NFATc Ab and none by an anti-NFATp Ab. This by itself does not rule out a crucial role for NFATp in the initiation of CD5 induction. However, B cells from NFATp knockout mice (41), like wild-type B cells, could be induced to express surface CD5 by 2.5 days of anti-IgM treatment (data not shown). Therefore, NFATp is dispensable for CD5 induction, at least in the long term.

On the basis of nucleotide sequence, we identified two candidate NFAT sites in the CD5 enhancer, located at -1994 and -1954. Mutation of both sites abolished formation of NFAT-containing complexes in vitro, indicating that one or both of these sites are necessary for the assembly of these complexes. In transient transfections, mutation of the -1994 site alone reduced reporter activity to ~50% of wild-type, indicating that this site plays a role in induction in vivo. Mutation of both NFAT sites further reduced activity to about 20% of wild-type, demonstrating a role for the -1954 site as well.

NFAT was originally described as an inducible activity binding to the IL-2 promoter (42). It was found to contain both AP-1 and an NFAT family member, which bound cooperatively to a composite site containing an AP-1 site immediately downstream of a 9-bp sequence containing a core GGAAA NFAT recognition site (43, 44, 45, 46). This arrangement has now been found to be a common feature of many NFAT sites including sites in the regulatory regions of IL-3, IL-4, IL-5, CD40 ligand, and granulocyte-macrophage-CSF genes (37). Other arrangements are observed, however. For example, in the IL-3 enhancer, there is an NFAT site that overlaps an Octamer site to form an element the activity of which is the result of the synergistic interaction of NFAT with an Octamer-binding factor (47). There are also examples of NFAT sites that are not immediately adjacent to any known transcription factor-binding sites, and both CD5 enhancer sites fall into this category.

In addition to the two NFAT-containing complexes, we examined three others that form with the CD5 enhancer probe in B cell extracts. One of these complexes (S1) forms only in extracts from induced cells. Copper-phenanthroline footprinting of this complex revealed a hypersensitive site at -2029. An oligomer consisting of nucleotides -2040 to -2014 competed for formation of the complex in EMSAs. Furthermore, an enhancer probe with four base changes at positions -2030, -2029, -2028, and -2025 did not form the complex. These nucleotides fall within a sequence that is identical with the H4TF-1-binding site (CCCTCCCCC) (31). This site is bound by two polypeptides of 110 and 105 kDa in HeLa cell nuclear extracts and is essential for maximal activity of the histone H4 promoter in in vitro transcription assays using HeLa cell nuclear extracts (31). We do not know whether either of these proteins is involved in binding to the CD5 enhancer. Further work will be required to resolve this question.

The other two complexes that we studied (C1 and C2) contain factors present in both uninduced and induced extracts. Formation of the slower migrating complex requires that the Ebox (consensus sequence CANNTG) located at -1973 be intact. Formation of the faster migrating complex requires an intact putative ets-binding site at -1966. Although we cannot be sure that basic helix-loop-helix and ets family factors are present in the respective complexes, members of both transcription factor families have been shown to be involved in B cell gene expression (33, 34, 35, 36). In addition, several ets family members have been shown to be phosphorylated downstream of kinase pathways known to be activated in B cells by BCR ligation and thus are candidates for transducers of sIgM signaling (reviewed in Ref. 36).

Regardless of the composition of these three non-NFAT-containing complexes, reporter expression experiments demonstrate that they represent functional DNA-protein interactions. Mutation of all three sites such that the complexes do not form in vitro also decreases enhancer activity in vivo to ~25% of wild-type. Mutation of any two of these sites has only a modest effect on enhancer activity (from 50 to 90% of wild-type activity depending on the pair mutated). Thus, at least one of these sites must be intact for significant enhancer activity.

Two additional constitutive complexes, C3 and C4, were also identified. These complexes were present at higher levels in extracts from uninduced than in extracts from induced cells. We have not further characterized the elements or proteins involved in their formation, nor have we determined their functional significance.

An important issue, not addressed in detail in this study, is how factors bound to the different identified sites interact with each other (and with factors bound to promoter-proximal elements) to generate enhancer activity. Some insight into this question can be obtained from results of transfection experiments with mutated enhancer constructs. Mutation of both NFAT sites reduced enhancer activity to 20% of wild-type, suggesting that the NFAT sites contribute 80% of enhancer activity. However, mutation of the H4TF-1, Ebox, and Ets sites in the presence of wild-type NFAT sites reduced enhancer activity to 25% of wild-type. Thus, the mutation of either set of sites reduced activity to a greater extent than would be expected if the activities of the sites were simply additive. This suggests that NFAT acts synergistically with factors bound at the other three sites. This synergy could be at the level of DNA binding, transactivation, or both.

The demonstration that CD5 is a target of NFAT makes it only the second such target identified in B cells. The other known target of NFAT in B cells, based on studies with the A20 B cell line, is TNF-{alpha} (48). In these cells, NFAT binds independently to a site at -76 of the TNFa promoter. However, its activity is dependent on the binding of an ATF-2/Jun dimer to a CRE element at -102 (48). Thus the CD5 enhancer and the TNF-{alpha} promoter, although similarly dependent on NFAT for activity, appear to utilize NFAT in the context of different factors. This may reflect the fact that these two genes are regulated differently. TNF-{alpha} is induced by either BCR or CD40 ligation. CD5, at least at the level of surface protein expression, is induced only by BCR ligation (9). Since NFAT is activated by either CD40 ligation or BCR ligation (49, 50), the CD5 enhancer may require additional regulatory mechanisms to achieve BCR specificity. It will be interesting to compare CD5 enhancer function in CD40 ligand and anti-IgM-treated cells. This may provide insight into differences in signaling between these two receptors.

In addition to being induced in B cells by sIgM cross-linking, CD5 gene expression is induced in some CD8{alpha}ß intestinal intraepithelial lymphocyte T cells as well as on a subset of CD4+CD8+ thymocytes by TCR ligation (51, 52). It would be interesting to know whether the same DNA sequences and transcription factors implicated here are also involved in CD5 induction in T cells.

CD5 is constitutively expressed in all peripheral T cells (except for some intestinal intraepithelial lymphocytes) and on the B-1a subset of B cells. Intensive study of NFAT in T cells has failed to show that it is active in resting T cells. Therefore, it is unlikely that constitutive expression in these cells is NFAT dependent. NFAT has not been examined in B-1a cells, and it remains possible that in these cells NFAT is constitutively active. Recently, STAT-3, another activation-dependent transcription factor, was reported to be constitutively active in B1-a cells (5). We are currently characterizing NFAT in these cells.


   Acknowledgments
 
We thank Drs. James Tung, Leonard Herzenberg, and Leonore Herzenberg for pGL2Ly1 and for communicating unpublished results to us. We thank Bryan Hurley for construction of pCD5Luc122R. We thank Dr. Anjana Rao for providing us with NFATp knockout mice and Dr. Gerald Crabtree for Abs. We also thank Dr. Brent Cochran for useful discussions and Drs. Paul McClean, Ananda Roy, Amy Yee, Ranjan Sen, and Tom Rothstein for useful discussions and critical reading of the manuscript.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grant AI15803. Back

2 Address correspondence and reprint requests to Dr. Robert Berland, Department of Pathology, Tufts University School of Medicine, Boston, MA 02111. E-mail address: Back

3 Abbreviations used in this paper: BCR, B cell antigen receptor; anti-IgM, F(ab')2 anti-IgM; EMSA, electrophoretic mobility shift assay; sIgM, surface immunoglobulin M. Back

Received for publication November 20, 1997. Accepted for publication February 26, 1998.


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 Introduction
 Materials and Methods
 Results
 Discussion
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Blood, October 15, 2005; 106(8): 2781 - 2789.
[Abstract] [Full Text] [PDF]

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D. N. Samanta, A. Palmetshofer, D. Marinkovic, T. Wirth, E. Serfling, and L. Nitschke
B Cell Hyperresponsiveness and Expansion of Mature Follicular B Cells but Not of Marginal Zone B Cells in NFATc2/c3 Double-Deficient Mice
J. Immunol., April 15, 2005; 174(8): 4797 - 4802.
[Abstract] [Full Text] [PDF]

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M. Arman, J. Calvo, M. E. Trojanowska, P. N. Cockerill, M. Santana, M. Lopez-Cabrera, J. Vives, and F. Lozano
Transcriptional Regulation of Human CD5: Important Role of Ets Transcription Factors in CD5 Expression in T Cells
J. Immunol., June 15, 2004; 172(12): 7519 - 7529.
[Abstract] [Full Text] [PDF]