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The Journal of Immunology, 2001, 166: 6156-6163.
Copyright © 2001 by The American Association of Immunologists

Multiple Transcription Factors Regulate the Inducible Expression of the Human Complement Receptor 2 Promoter1

Lyudmila A. Vereshchagina*, Mate Tolnay*,{dagger} and George C. Tsokos2,*,{dagger}

* Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, MD 20910; and {dagger} Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Complement receptor 2 (CR2) is regulated at the transcriptional level, but the promoter elements and the transcription factors that bind to them and contribute to its regulation are unknown. After documenting that PMA and cAMP induced the activity of the CR2 promoter by 10-fold, we conducted promoter truncation and mutagenesis experiments, in conjunction with shift assays, to determine the functionally important regions of the promoter and the proteins that bind to them. We identified two regions, separated by ~900 nucleotides, which together were responsible for inducible promoter activity. Mutagenesis of single promoter elements demonstrated a functional upstream stimulatory factor/E box in the TATA box-proximal region and three equally important, closely spaced, CREB/AP-1 half-sites in the upstream promoter region. The cAMP response element-binding protein (CREB)/AP-1 half-sites bound in vitro Jun and CREB that are induced by protein kinases A and/or C. The 900-nucleotide segment stretching between the above two regions had no functional impact on the induced transcription, and its deletion increased the promoter activity. Finally, a region upstream of the distal site had a repressor activity on CR2 transcription. Moreover, IL-4 induced binding of CREB and AP-1 to the upstream promoter elements and resulted in increased CR2 surface protein expression. These studies have characterized regions of the CR2 promoter and the transcription factors that bind to them and are crucial to induced CR2 expression. Our studies may provide insights to novel approaches to modulate B cell function by regulating CR2 gene transcription.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Complement receptor 2 (CR2,3 CD21) binds the C3d, C3dg, and iC3b fragments of C3 and the EBV envelope protein gp 350/220 (1, 2). Cross-linking of CR2 alone or simultaneously with surface Ig enhances and sustains the B cell response (3, 4, 5, 6). In addition, mice treated with anti-CR2/CR1 Abs (7) and CR2/CR1-deficient mice have defective responses to T-dependent Ag (8, 9).

CR2 is expressed on mature B lymphocytes and B cell lines but not on early (pre-B cell) and late (plasma cell) developmental stages (10). Human splenic marginal zone B cells express higher levels of CR2 than other B cell subpopulations; thus, they may facilitate the primary immune response (11). Several studies indicate that the level of CR2 expression affects the intensity of the B cell response. In vitro, human spleen B cells with high levels of CR2 expression respond better to Ag (12), and B cells from knockout mice heterozygous for the inactivated Cr2 locus express ~50% of the normal levels of CR2 and respond to a T-dependent Ag less efficiently than B cells from wild-type animals (8, 9).

The 5'-promoter region of the human CR2 gene is essential for CR2 expression (13, 14). Moreover, a silencing element located in the first intron regulates cell-specific expression of CR2 in humans (15) and mice (16). Transcription of the murine CD21 gene is positively regulated by histone acetylation, suggesting an important role for the accessibility of DNA by transcription factors (17). However, the exact transcriptional mechanisms that are responsible for the regulation of CR2 expression on mature B cells are unknown. Recent studies in a B cell line have shown that expression of the CR2 gene can be induced by a cell-permeable cAMP analogue, and a protein kinase A (PKA)- and a protein kinase C (PKC)-responsive heterogeneous nuclear ribonucleoprotein (hnRNP D) was shown to bind to a novel element in the promoter region of the CR2 gene (18, 19, 20).

We undertook this study to dissect systematically the transcriptional regulation of the CR2 promoter by truncation and mutation analysis, and EMSA. We have identified two regions at -57/+75 and -1017/-955, which bind a number of proteins, to be involved in enhancing CR2 promoter activity. In addition, we show that the region -954/-58, which binds a number of well-established transcription factors, is not involved in induced CR2 promoter activity, whereas the -1252/-1018 upstream region is involved in the suppression of the transcriptional process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures

IM-9 B lymphoblastoid cells (American Type Culture Collection, Manassas, VA) were cultured in RPMI 1640 (Life Technologies, Gaithersburg, MD) containing 10% FBS (Life Technologies), 2 µM L-glutamine (Life Technologies), 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies) and supplemented with 25 mM HEPES buffer.

Construction of truncated and mutated CR2 promoter constructs

The SP65 chloramphenicol acetyltransferase (CAT) plasmid containing the human CR2 promoter from -1252 to +75 was the gift of Dr. Michael Holers, Denver, CO. Truncated CR2 promoter constructs were generated by using PCR. The PCR fragments flanked with NotI and ApaI termini were inserted into the SP65 CAT vector plasmid, which had been predigested with NotI and ApaI (New England Biolabs, Beverly, MA). Deletion constructs {Delta}-951/-65 and {Delta}-951/-71 were generated using PCR primers to introduce two NdeI restriction sites into the CR2 promoter at -953 and -66 or -72, followed by digestion with NdeI (New England Biolabs), removal of the insert, and religation.

The Quick-Change Mutagenesis Kit (Stratagene, La Jolla, CA) and PCR primers containing 3–7 mutated nucleotides were used to introduce mutations in potential transcription factor-binding sites (Table IGo). All plasmids were purified using the Qiagen Plasmid Maxi Kit (Qiagen, Valencia, CA). The sequences of the clones were confirmed by DNA sequence analysis.


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  		Table I. Site-directed mutagenesis of the human CR2 promoter1

 
DNA transfection and reporter gene analysis

Plasmid DNA (5 µg/107 cells) was introduced into IM-9 cells by the DEAE-dextran method as described (21). After transfection, 5 x 106 cells/sample were cultured for 18–24 h in 5 ml RPMI 1640 with 10% FBS medium. Cells were treated with 1 mM dibutyryl cAMP (dbcAMP; Sigma, St. Louis, MO) and/or 100 ng/ml PMA (Sigma) for the next 18–23 h or as indicated and harvested, and freeze-thaw lysates were assayed for CAT activity by separating the acetylated forms of [14C]chloramphenicol on TLC plates, as described (22). Data were analyzed using a Molecular Imager FX System and Quantity One software (Bio-Rad Laboratories, Hercules, CA).

EMSA

Five million IM-9 cells in 5 ml RPMI 1640 with 10% FBS were incubated overnight, then stimulated with 1 mM dbcAMP and 100 ng/ml PMA for 10 min or with 200 U/ml IL-4 (Sigma) and/or 0.1 µg/ml anti-CD40 mAb (Oxford Biotechnology, Raleigh, NC) for 10–30 min, or left untreated. After stimulation, the nuclear proteins were prepared as described (23). The protein concentrations of all nuclear extracts were determined using the Bio-Rad protein assay. Complementary single-stranded oligonucleotides were annealed, then end-labeled with [{gamma}-32P]ATP using a kit from Life Technologies, purified on Centri-Sep columns (Princeton Separations, Adelphia, NJ), and used as probes in EMSA.

EMSAs were performed as described (18). In supershift experiments, 1.5–3 µg purified anti-Jun/AP-1 (recognizes c-Jun, Jun B, Jun D) and anti-activating transcription factor (ATF)-1 (recognizes ATF-1, cAMP response element-binding protein (CREB)-1, cAMP response element modulator (CREM)-1) Ab (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-RFX5 Ab (Rockland, Gilbertsville, PA) were added to nuclear extracts just before the addition of labeled oligonucleotide and then incubated for 40 min at 25°C. Anti-hnRNP D Ab (gift of Dr. Gary Brewer, Winston-Salem, NC) was added after preincubation of the nuclear extracts with labeled oligonucleotide, samples were then incubated for 10 min on ice. Data were analyzed using a Molecular Imager FX System and Quantity One software.

Flow cytometry

IM-9 cells were either unstimulated or stimulated with 200 U/ml IL-4 (Sigma) and/or 0.1 µg/ml anti-CD40 mAb (Oxford Biotechnology) for 48 h. Expression of CR2 on the IM-9 cell surface was evaluated after staining with the PE-labeled anti-human mCD21 Ab (PharMingen, San Diego, CA) or the PE-labeled mouse IgG1 (Sigma) that we used as an isotypic control. Samples were analyzed using a fluorescence-activated cell sorter (FACScan) flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). We acquired 10,000 cell-gated events and analyzed them with CellQuest software (Becton Dickinson). The number of surface CR2 was estimated by quantitative calibration using a QuantiBRITE PE Fluorescence Quantitation Kit (Becton Dickinson).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
dbcAMP and PMA induce the expression of a reporter gene driven by the 5'-human CR2 promoter

To determine potential transcription factors that may be involved in transcriptional regulation of the CR2 gene expression, we examined its promoter region for consensus transcription factor binding sites using MatInspector software (24). The analysis revealed several sites that could bind known transcription factors (Fig. 1Go). Many of these factors are up-regulated by PKA and/or PKC. We have reported previously that hnRNP D binds specifically to the CR2 promoter 490–471 nucleotides upstream of the transcriptional initiation site and is optimally induced by cAMP and PMA cotreatment of the cells (18, 19). To determine whether PKA and PKC-mediated signaling pathways are involved in the transcriptional regulation of the CR2 promoter, we treated B cells with dbcAMP and PMA and evaluated the expression of a CAT reporter gene driven by the -1252/+75 CR2 promoter in IM-9 B lymphoblastoid cells. We chose IM-9 cells, after testing several mature B cell lines (data not shown), because the promoter is less active in these cells and its expression can be further induced. Treatment of cells with dbcAMP or PMA alone for 23 h increased the expression of the CAT gene by <2-fold in IM-9 cells (Fig. 2GoA), whereas treatment with both of them induced CAT gene expression by 10-fold, indicating that the PKA and PKC signaling pathways act synergistically to induce expression of the CR2 promoter. The empty CAT vector could not be stimulated (data not shown). To study the kinetics of induced expression, we performed time-response experiments (Fig. 2GoB). We detected significantly increased CAT expression after 7 h stimulation. The expression levels at later time points did not significantly differ from that at 7 h.



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  		FIGURE 1. Potential transcription factor-binding sites in the promoter region of the CR2 gene. MatInspector software (24 ) was used to determine putative CR2 promoter-defined regulatory elements. hnRNP D binds specifically to the -490/-471 region of the CR2 promoter (18 ). Numbers indicate positions of the first base in a consensus sequence. All CREB/AP-1 sites are half-sites.

 


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  		FIGURE 2. dbcAMP (dcAMP) and PMA increase the expression of the CAT reporter gene driven by the CR2 promoter. IM-9 cells were transiently transfected with the CAT plasmid containing the human CR2 promoter from -1252 to +75. After transfection, cells were divided and cultured for 18 h. Cells were treated with 1 mM dbcAMP and/or 100 ng/ml PMA for 23 h (A) or as indicated (B). Freeze-thaw lysates were assayed for CAT activity, and acetylated products of [14C]chloramphenicol were separated on TLC plates. Means ± SEM are shown (n = 3–11). **, p < 0.01 as compared with unstimulated cells using an one-way ANOVA followed by the Dunnett post test. B, Values represent percentage of CAT activity, considering the activity of the empty vector to be zero and the activity of the -1252/+75 wild-type CR2 promoter to be 100%.

 
Sequences affecting inducibility of the human CR2 promoter lie in the -1017/-955 and -57/+75 regions

To determine the regions of the CR2 promoter that are responsible for its dbcAMP and PMA-induced transcriptional activation, we generated sequential truncated versions of the CR2 promoter, transfected them into IM-9 cells and determined their ability to drive the transcription of a CAT reporter gene. We calculated the degree of inducibility of the truncated promoter constructs by setting the activity of the empty CAT vector at zero and the activity of the full-length (-1252/+75) CR2 promoter at 100%. Deletion of the -1252/-1018 region of the CR2 promoter (construct -1017/+75) increased the dbcAMP- and PMA-induced transcriptional activity to 220%, indicating that a repressor element is present in this region (Fig. 3GoA). However, deletion of additional 63 nucleotides (construct -954/+75) decreased the dbcAMP- and PMA-induced transcriptional activity of the CR2 promoter to ~10%. This finding clearly indicates that critical positive regulatory transcription factors bind to the -1017/-955 region. This region contains potential CREB/AP-1-binding half-sites. Deletion of additional sequences up to -57 did not decrease further the induced transcriptional activity. However, regulatory elements that bind to the -57/+75 region of the CR2 promoter (potential CREB/AP-1-binding half-site, X box, and upstream stimulatory factor (USF)/E box regulatory elements) were sufficient to provide 30% transcriptional activity after stimulation with dbcAMP and PMA.



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  		FIGURE 3. Deletion and site-directed mutation analysis of the human CR2 promoter reveals functionally important motifs. IM-9 cells were transiently transfected with the empty CAT plasmid (vector) or CAT plasmids driven by truncated CR2 promoters (A and B) or the full-length promoter with individual promoter elements mutagenized (C). After transfection, cells were cultured for 18 h, then treated with 1 mM dbcAMP and 100 ng/ml PMA for 23 h, and analyzed for CAT activity. A not-to-scale draft of the CR2 promoter denotes potential transcription factor-binding sites. A, Numbers on the left refer to the first and last nucleotides of the truncated promoter, where +1 is the transcription initiation site. B, -951/-65 and -951/-71 regions of the CR2 promoter were deleted. C, Numbered mutations are listed in Table IGo. Values represent percentage of CAT activity, considering the activity of the empty vector to be zero and the activity of the -1252/+75 wild-type (WT) CR2 promoter to be 100%. Statistical treatment of the data is as described in the legend to Fig. 2Go. Samples are compared with the full-length CR2 promoter (n = 2–5).

 
To address the possibility that the -1017/-955 and -57/+75 regions of the CR2 promoter are sufficient for the dbcAMP and PMA-induced transcriptional activation of the CR2 promoter, we deleted nucleotides -951/-65 or -951/-71. In these two constructs, the lengths of the spacers differ by six nucleotides, which correspond to about a half-turn of the DNA helix. We found in reporter gene experiments that removal of nearly 900 nucleotides greatly increased the inducible promoter activity. Specifically, deletion of the -951/-65 region (construct {Delta}-951/-65) or the -951/-71 region (construct {Delta}-951/-71) of the CR2 promoter increased the dbcAMP and PMA-induced transcriptional activity by 12- or 8-fold, respectively, as compared with the intact promoter (Fig. 3GoB). In addition, there was a statistically significant difference between the activity of the {Delta}-951/-65 and {Delta}-951/-71 constructs, suggesting that proper phasing of DNA-binding proteins is important. These data further support our finding that the -1017/-955 and -57/+75 regions are responsible for inducibility of the CR2 promoter and demonstrate that the long stretch between them is dispensable.

Identification of the regulatory elements controlling inducibility of the human CR2 promoter by mutagenesis

To define which specific regulatory elements in the -1017/-955 and -57/+75 regions are responsible for the dbcAMP- and PMA-induced transcriptional activation of the CR2 promoter, we performed site directed mutagenesis of these two regions. Mutation of any of three potential CREB/AP-1 half-sites at -1017/-1014, -987/-983, and -970/-964 (MUT 1–3, Table IGo) reduced the inducible promoter activity to ~30% (Fig. 3GoC).

In the -57/+75 region, mutation of a potential CREB/AP-1 half-site at -56/-53 (MUT 8) had no effect on induced transcriptional activity of the promoter. Mutation of the potential overlapping X box and USF/E box regulatory elements at -45/-41 (MUT 9) reduced activity to ~45%. To assess potential interaction of the CREB/AP-1 and X box/USF/E box regulatory elements, we tested a construct with both elements mutated (MUT 10). We did not observe any change in the decreased transcriptional activity that was already observed with MUT 9, indicating that the CREB/AP-1 half-site at -56/-53 does not play a role in induced promoter activity.

The above promoter truncation experiments showed that the -954/-58 region does not participate in the induced transcriptional regulation of the CR2 promoter (Fig. 3GoA). To verify these results, we mutated the consensus NF-{kappa}B, AP-2, and hnRNP D sites that had been predicted to exist in this region of the promoter (Fig. 1Go). Mutations of these sites alone or in combination (MUT 4–7) did not have any effect on induced transcriptional activity of the promoter (Fig. 3GoC). It should be mentioned that oligonucleotides, defining these binding sites and surrounding sequences, bound the predicted transcription factors in a specific manner (not shown).

Identification of transcription factors that bind to functionally relevant motifs of the promoter

We performed EMSA using synthetic oligonucleotides to identify proteins that bind to promoter elements that we found to be functionally important in the above mutagenesis experiments. For each binding site, we performed experiments using either wild-type or mutated oligonucleotides with flanking sequences defined by the CR2 promoter (Table IIGo) and nuclear proteins extracted from dbcAMP and PMA costimulated or unstimulated IM-9 cells. Abs were also used in shift assays to detect the presence of possible protein candidates. The mutations were similar to those used in the reporter experiments.


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  		Table II. Sequences of all oligonucleotides used in EMSA1

 
Incubation of nuclear extracts form IM-9 cells with the -1028/-997 oligonucleotide produced two shifted bands (Fig. 4GoA). Mutation of nucleotides -1017/-1014 did abolish the formation of band 1 but allowed formation of band 2, indicating that only the protein present in band 1 can be responsible for the diminished promoter activity following mutagenesis (Fig. 3GoC). Addition of either anti-Jun/AP-1 Ab or an Ab that recognizes ATF-1 p35, CREB-1, and CREM-1 (referred to as anti-CREB Ab) decreased the intensity of band 1, indicating that Jun and CREB/ATF family members can bind to the -1028/-997 oligonucleotide.



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  		FIGURE 4. EMSA analysis of the functionally important promoter elements demonstrates binding of several transcription factors after dbcAMP (dcAMP) and PMA treatment. A–D, Nuclear extracts (A, 8 µg; B and C, 4 µg; D, 2 µg/sample) of IM-9 cells that had been treated for 10 min with 1 mM dbcAMP and 100 ng/ml PMA, or left untreated, were incubated with labeled oligonucleotides or mutated (Mut) oligonucleotides, as indicated (Table IIGo), and analyzed by EMSA. In supershift assays, anti-Jun/AP-1 Ab (specific for c-Jun, Jun B, Jun D), an Ab that recognizes ATF1, CREB1, and CREM-1 (labeled as CREB); anti-hnRNP D; or anti-RFX5 Ab were included in the binding reaction. Arrows indicate the positions of the shifted bands. E, Summary of EMSA experiments depicting the CR2 promoter with DNA elements and interacting proteins that we found important for induced expression.

 
Incubation of nuclear extracts from IM-9 cells with the -996/-976 oligonucleotide produced four shifted bands. Three of the bands were barely detectable in nuclear extracts of unstimulated cells (Fig. 4GoB). Simultaneous treatment of cells with dbcAMP and PMA for 5–30 min strongly increased the intensity of all four bands (Fig. 4GoB, 10 min activation is shown). Treatment for 5–10 min with dbcAMP or PMA alone increased protein-DNA binding that returned to baseline by 30 min (data not shown). Mutation of nucleotides -987/-983 inhibited or diminished the formation of all bands, indicating that proteins present in all bands are likely to be important in the regulation of the promoter activity. Addition of anti-Jun/AP-1 Ab inhibited the formation of bands 1 and 2, indicating that Jun is present in the complex and the Ab interfered with recognition of the oligonucleotide. Addition of anti-CREB Ab induced a strong supershifted band and simultaneously caused the disappearance of band 1 and reduced the intensity of band 2 indicating that band 1 and possibly band 2 contain CREB/ATF proteins. Together these data suggest that bands 1 and 2 contain both Jun and CREB/ATF proteins. The -996/-976 oligonucleotide displays extensive sequence similarity to an hnRNP D motif (18). Addition of anti-hnRNP D Ab induced a supershifted band and selectively decreased the intensity of band 3, indicating that band 3 contains hnRNP D. We have not yet identified the protein present in band 4.

Incubation of nuclear extracts with the -975/-953 oligonucleotide produced one strong shifted band (band 1) (Fig. 4GoC). dbcAMP and PMA treatment for 10 min increased protein-DNA binding. Mutation of nucleotides -970/-964 inhibited the formation of band 1, indicating that the protein present in band 1 may be functionally important. A different complex was formed in the presence of the mutated oligonucleotide (band 2). Addition of either anti-Jun/AP-1 or anti-CREB Ab decreased intensity of band 1, indicating that Jun and CREB/ATF family members are both present in the complex.

Incubation of nuclear extracts with the -54/-32 oligonucleotide produced two shifted bands (Fig. 4GoD). dbcAMP and PMA treatment for 10 min significantly increased the DNA binding of the protein present in band 1. Mutation of nucleotides -45/-41 inhibited the formation of band 1, but not band 2, indicating that the protein present in band 1 may be functionally relevant. Addition of an Ab specific for RFX5, which was shown to bind X-boxes in promoters of MHC class II genes (25), had no effect on the intensity of the bands. Finally, Abs against Jun/AP-1 or CREB did not alter the appearance of the bands.

Stimulation of IM-9 cells with IL-4 and anti-CD40 mAb increases binding of nuclear proteins to the CR2 promoter and enhances the expression of surface CR2

To verify the physiological importance of identified transcription factor-binding sites we performed EMSA using oligonucleotides with flanking sequences defined by the CR2 promoter (Table IIGo) and nuclear proteins extracted from IL-4 and/or anti-CD40 mAb stimulated or unstimulated IM-9 cells. Abs were also used in shift assays to detect the presence of possible protein candidates. Activation of the IL-4 and CD40 signaling pathways has been shown to induce cAMP and inositol trisphosphate second messenger pathways in human B lymphocytes and to increase the cell surface density of CD21 on human B cells (26, 27).

Incubation of nuclear extracts from IM-9 cells with the -996/-976 oligonucleotide produced three shifted bands (Fig. 5Go, A and C). Treatment for 30 min with anti-CD40 mAb alone slightly increased protein-DNA binding, whereas treatment with IL-4 caused a very strong increase in protein-DNA binding (Fig. 5GoA). Simultaneous treatment of cells with IL-4 and anti-CD40 mAb for 30 min did not further increase the intensity of the bands over that already observed after treatment with IL-4 alone; therefore, for supershift experiments with specific Abs, we used nuclear extracts from cells treated with IL-4 alone for 30 min. Similarly to our observations shown in Fig. 4GoB, addition of anti-Jun/AP-1 Ab inhibited the formation of bands 1 and 2 indicating that Jun is present in the complex. Addition of anti-CREB Ab induced a supershifted band and simultaneously caused the disappearance of band 1 and reduced the intensity of band 2, indicating that bands 1 and 2 contain CREB/ATF proteins. Addition of anti-hnRNP D Ab decreased the intensity of all shifted bands, indicating that the protein complex contains hnRNP D.



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  		FIGURE 5. EMSA analysis of the functionally important promoter elements demonstrates binding of several transcription factors after IL-4 treatment. A, Nuclear extracts (4 µg/sample) of IM-9 cells that had been treated for 10 or 30 min with 200 U/ml IL-4 and/or 0.1 µg/ml anti-CD40 mAb were incubated with labeled oligonucleotide, as indicated (Table IIGo), and analyzed by EMSA. B–E, Nuclear extracts (B, 8 µg; C, D, and E, 4 µg/sample) of IM-9 cells that had been treated for 30 min with 200 U/ml IL-4 were incubated with labeled oligonucleotides, as indicated (Table IIGo), and analyzed by EMSA. In supershift assays, anti-Jun/AP-1 Ab (specific for c-Jun, Jun B, Jun D); an Ab that recognizes ATF1, CREB1, and CREM-1 (labeled as CREB); anti-hnRNP D; or anti-RFX5 Ab were included in the binding reaction. Arrows indicate the positions of the shifted bands.

 
Incubation of nuclear extracts from IL-4-treated IM-9 cells with the -1028/-997 oligonucleotide (Fig. 5GoB), the -975/-953 oligonucleotide (Fig. 5GoD), or the -54/-32 oligonucleotide (Fig. 5GoE) produced the same protein-DNA band pattern as with nuclear extracts from dbcAMP- and PMA-treated cells (Fig. 4Go, A, C, and D, respectively), further supporting the physiological significance of the identified transcription factor binding sites.

In addition, we performed flow cytometry analysis of the CR2 expression on the surface of IL-4 and/or anti-CD40 mAb-stimulated or unstimulated IM-9 cells to determine the physiological relevance of the identified functional promoter elements in the up-regulation of the surface CR2. All stimuli caused an increase of 20–30% in the surface CR2 expression (Table IIIGo). We have also observed similar increase after IL-4 and/or anti-CD40 mAb stimulation of human peripheral blood B lymphocytes (data not shown).


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  		Table III. CR2 expression on IM-9 cells1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CR2 expression on the surface of B cells is important in the regulation of the normal immune (5, 28) and autoimmune response (29, 30). Thus, we have reasoned that the CR2 gene transcription must be tightly controlled by transcription factors that bind to its promoter region. Recent studies have shown that hnRNP D, a PKA- and PKC-regulated transcription factor, binds to a newly identified enhancer element in the promoter region of the CR2 gene (18, 20) and have suggested the importance of PKA- and PKC-mediated signaling pathways in the expression of the CR2 gene. Moreover, we found that dbcAMP, a PKA stimulator, caused an early but transient increase in the CR2 mRNA and surface protein expression in IM-9 B lymphoblastoid cells that returned to baseline 4 h later (18). In the present study, we show that dbcAMP and PMA synergistically induce the -1252/+75 CR2 promoter by >10-fold in functional reporter gene assays (Fig. 2AGo). Stimulation with either agent alone resulted in <2-fold increase of the promoter activity. Yet, addition of PMA did not increase further the CR2 mRNA and surface protein expression over that observed with dbcAMP stimulation alone (data not shown). IL-4 or anti-CD40 Ab increased the expression of CR2 surface protein in IM-9 cells (Table IIIGo) and normal B cells (data not shown), but with a delayed kinetics as compared with dbcAMP stimulus. Our results are in agreement with previously published data, which had shown that stimulation of human B cells with IL-4 and an anti-CD40 mAb increased the cell surface density of CR2 (27). This information indicates that the endogenous CR2 gene is under distinctly different regulatory control than the 5'-promoter in the context of a reporter construct. Because IL-4 signals in part through cAMP and inositol trisphosphate (26), PKA/PKC-induced transcription factors could stimulate the 5'-CR2 promoter, but additional IL-4-initiated signals are required to induce the transcriptional activity of the CR2 gene in a delayed manner.

First, we defined functionally relevant regions of the promoter by gradually deleting segments from the 5'-end. By using this approach, we found that two short segments of the promoter at -1017/-955 and -57/+75 were responsible for induced promoter activity. We do not know whether there is any cross-talk between proteins binding to these two distantly positioned regions. However, deletion of the nearly 900 nucleotides between the two regions dramatically (8- to 12-fold) increased the dbcAMP- and PMA-induced transcriptional activity. The greatly increased inducibility of the deletion construct could be due to the closer proximity of the -1017/-955 region to the TATA box and the more efficient interaction with proteins binding at -57/+75. Indeed, our data suggest that proper spatial arrangement of DNA-binding proteins is important, because positioning motifs onto the opposite side of the DNA helix by introducing a half-turn of helix (6 nucleotides) significantly affected promoter activity. These data also indicate that the 900 nucleotides between the two critical regions are not required for dbcAMP- and PMA-induced transcriptional activation. Moreover, we detected a >2-fold increase in inducibility by removing the -1252/-1018 region, suggesting binding of transcriptional repressors to elements defined by this region. A possible repressor-binding site at -1223/-1217 may bind the transcription factor YY1, which can interact with AP-1/Jun and CREB/ATF proteins and act as a repressor (31). No other region of the promoter influenced the induced promoter activity, although the binding of transcription factors to these regions may regulate the basal promoter activity. The -954/-58 region contains consensus NF-{kappa}B, hnRNP D and AP-2 sites, which interact with the corresponding proteins in EMSA in an inducible manner (data not shown), yet removal or mutagenesis of these elements does not affect promoter inducibility. These findings underscore the importance of performing assays that monitor the promoter activity in the context of the entire promoter rather than relying on EMSA findings.

Next, we analyzed the -1017/-955 and -57/+75 regions in detail by mutagenizing 4- to 7-nucleotide-long segments corresponding to core elements of predicted transcription factor-binding sites. It is important that the functional effect of these mutations was evaluated in the context of the entire 1.3-kb CR2 promoter without affecting potentially critical phasing of interacting transcription factors. The TATA box proximal -57/+75 region contains a predicted CREB/AP-1 half-site at -56/-53 and a site at -45/-41 homologous to an overlapping X box and USF/E box. Mutagenesis of the X box/USF/E box resulted in a 55% reduction in the inducible activity of the promoter, suggesting the functional importance of this site. We have found by EMSA that an inducible protein binds specifically to this site. In supershift experiments using a specific Ab, we excluded that this protein is RFX5, one of several proteins that were shown to bind X boxes in promoters of MHC class II genes (25). The -45/-41 site is identical with a consensus E box and may serve as a binding site for the USF (32). USF proteins interact with the TATA-binding protein (33) and CREB/AP-1 (34, 35). Mutagenesis of the CREB/AP-1 half-site did not affect the inducibility of the promoter activity, suggesting that this site is not involved in the expression of the CR2 promoter.

By analyzing the upstream -1017/-955 segment, we have identified three functional CREB/AP-1 half-sites in close proximity to each other at -1017/-1014, -987/-983, and -970/-964. Mutation of either of these sites in the context of the entire promoter reduced inducibility of the promoter to ~30% of that of the wild type promoter. In EMSA using short synthetic oligonucleotides, members of the CREB/ATF family, hnRNP D and an unidentified protein recognized the central site in addition to Jun. Stimulation of IM-9 cells with dbcAMP and PMA or IL-4 led to strongly increased DNA binding of all identified transcription factors, supporting their role in the induced expression of the CR2 promoter. A CREB/Jun heterodimer seems to bind the two flanking sites and is moderately induced by dbcAMP and PMA treatment. It is well documented that CREB/ATF proteins and AP-1/Jun proteins bind as homo- or heterodimers to very similar promoter elements (36). Binding of hnRNP D to the central site is not surprising because of extensive sequence similarity to the only previously characterized site at -490/-471 (18). There is a shared TNGNGNNTNCNTCAAAAT motif (67% identity) between these two sites that should help in identifying hnRNP D-binding sites in other promoters. Our observation that mutation of any of the three promoter elements reduced inducibility to the same degree suggests that proteins binding to different elements are likely to cooperate with each other and function as a multiprotein unit where each element is indispensable. The size of the entire unit is one-half of that of a nucleosome hinting that it may indeed be regulated as a unit. It is tempting to speculate that the three closely spaced CREB/AP-1 half-sites are bridged by CREB/Jun dimers in a complex fashion with both Jun (37, 38) and hnRNP D (19) facilitating alignment of interacting proteins. hnRNP D interacts with the TATA-binding protein (19), whereas CREB and AP1 recruit CREB-binding protein (CBP)/p300 proteins (39, 40) with histone acetyltransferase activity (41), thus providing multiple links to the transcription initiation complex. PKA-dependent phosphorylation of CREB is required for its interaction with CBP/p300 (39, 41), whereas AP-1/Jun is regulated by PKC (42) and hnRNP D is regulated by both PKA and PKC (18, 20). In lymphocytes, CREB was regulated by PKC (43). Furthermore, CREB/ATF sites are targets of the IL-4- and anti-CD40-initiated signaling pathways in primary B cells (44).

The present study demonstrates that the human CR2 promoter is subject to regulation by PKA and PKC signaling pathways in a mature B cell line. In addition, we show that the IL-4 and anti-CD40 Ab-initiated signaling pathways participate in the regulation of the CR2 promoter and enhance the number of CR2 molecules on the cell surface (26, 44). We have delineated the molecular mechanisms that are responsible for the induced promoter activity and have uncovered a novel and complex interplay of several transcription factors. We conclude that the endogenous CR2 gene is under stricter regulatory control than the upstream promoter is; thus, we suspect additional layers of complexity in the regulation of the CR2 gene. Nevertheless, various physiological stimuli of B cells activate the PKA and PKC signaling pathways (43, 45, 46, 47); thus, they may be involved in the regulation of CR2 expression, in addition to IL-4 and CD40L. Because CR2 is crucial in determining the magnitude and duration of the B cell response, transcriptional fine-tuning of its expression is important.


   Acknowledgments
 
We thank Dr. Michael Holers for providing the SP65 CAT plasmid containing the human CR2 promoter, Dr. Gary Brewer for the hnRNP D-specific Ab, and Dr. Alan Hubbs for his help with sequencing.


   Footnotes
 
1 This work was supported by U.S. Public Health Service Grant RO1-AI42782. Back

2 Address correspondence and reprint requests to Dr. George C. Tsokos, Department of Cellular Injury, Walter Reed Army Institute of Research, 503 Robert Grant Road, Building 503, Room 1A32, Silver Spring, MD 20910-7500. Back

3 Abbreviations used in this paper: CR2, complement receptor type 2; ATF, activating transcription factor; CAT, chloramphenicol acetyltransferase; CBP, CREB binding protein; CREB, cAMP response element-binding protein; CREM, cAMP response element-modulator; dbcAMP, dibutyryl cAMP; hnRNP, heterogeneous nuclear ribonucleoprotein; PKA, protein kinase A; PKC, protein kinase C; USF, upstream stimulatory factor. Back

Received for publication August 3, 2000. Accepted for publication March 7, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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