CR2/CD21) Promoter: Characterization of Basal Transcriptional Mechanisms This Information Is Current As of September 25, 2021

Functional Analysis of the Human Complement Receptor 2 (CR2/CD21) Promoter: Characterization of Basal Transcriptional Mechanisms This information is current as of September 25, 2021. Daniela Ulgiati, Christine Pham and V. Michael Holers J Immunol 2002; 168:6279-6285; ; doi: 10.4049/jimmunol.168.12.6279 http://www.jimmunol.org/content/168/12/6279 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Functional Analysis of the Human Complement Receptor 2 (CR2/CD21) Promoter: Characterization of Basal Transcriptional Mechanisms1

Daniela Ulgiati,* Christine Pham,† and V. Michael Holers2*

Human complement receptor (CR) type 2 (CR2/CD21) is a 145-kDa membrane protein encoded within the regulators of complement activation gene cluster localized on human chromosome 1q32. Understanding the mechanisms that regulate CR2 expression is important because CR2 is expressed during speciﬁc stages of B cell development, and several lines of evidence suggest a role for altered CR2 function or expression in a number of autoimmune diseases. Additionally, even modest changes in CR2 expression are likely to affect relative B cell responses. In this study we have delineated the transcriptional requirements of the human CR2 gene. We have studied the human CR2 proximal promoter and identiﬁed sites important for controlling the level of transcription in CR2-expressing cells. We have determined that four functionally relevant sites lie within very close proximity to the transcrip- Downloaded from tional initiation site. These sites bind the transcription factors USF1, an AP-2-like transcription factor, and Sp1. The Journal of Immunology, 2002, 168: 6279–6285.

uman complement receptor (CR)3 type 2 (CR2/CD21) is binding and receptor-mediated signaling, suggesting a role for this a 145-kDa protein encoded within the regulators of com- Cr2 allele as a lupus susceptibility gene. H plement activation gene cluster localized on human Further analysis of the biological effects of CR2 has shown the http://www.jimmunol.org/ chromosome 1q32 (1). CR2 is the receptor for complement acti- importance of CR2 expression in the maintenance of B cell toler- vation fragments of C3, specifically iC3b, C3dg, and C3d (2, 3). ance and anergy (12). These studies demonstrated that combining CR2 plays a central role in the generation of normal B cell re- mice that are genetically deficient in CR2 and CR1 with B6/lpr sponses to T-dependent Ags. Cr2Ϫ/Ϫ mice demonstrate a defect in mice resulted in exacerbation of lupus disease activity. Addition- the Ag-specific response to T-dependent Ags, manifested primarily ally, B cells from soluble hen egg lysozyme anti-egg lysozyme-Ig by the lack of a robust switched IgG response (4Ð6). double transgenic mice that are deficient in CR2 failed to be ap- The significance of mechanisms that regulate CR2 expression is propriately anergized in response to self Ag (12). Furthermore, a apparent by studies of human B cell expression in a number of recent study has also reported a marked decrease in CR2 expres- autoimmune and rheumatic diseases. It has been shown that pa- sion on B cells that was associated with a breakdown of tolerance by guest on September 25, 2021 tients with systemic lupus erythematosus (SLE) have abnormali- in anergic mice and with induction of an SLE-like syndrome in ties in the expression of CR2 on B cells (ϳ50% of normal levels) graft-vs-host-induced mice (13). that may correlate with disease activity (7Ð9). Studies of lupus- Therefore, several lines of evidence exist suggesting that prone mice have also found an early decrease in CR2 expression. marked down-regulation of CR2 may play roles both in driving a This decrease was progressive and initially detectable before any breakdown in tolerance and in the pathogenesis of autoimmunity. major clinical manifestations (10). A recent study using congenic As modest changes in levels of CR2 are likely to affect relative B mice containing the major murine SLE susceptibility locus dem- cell responses (4), understanding CR2 regulation is imperative. onstrated a defect within the Cr2 gene (11). This Cr2 gene con- Our laboratory has discovered several critical elements involved tained a single-nucleotide polymorphism that introduced a novel in cell type-specific silencing or repression of human CR2. We glycosylation site. Moreover, this polymorphism was located have shown that the cell- and stage-specific expression of human within the C3dg binding domain and was shown to reduce ligand CR2 is controlled by an intronic transcriptional silencer, designated the CR2 silencer. Use of a stable transfection system and transgenic mice has shown that the CR2 silencer element in con- junction with the CR2 proximal promoter is able to repress tran- *Departments of Immunology and Medicine, Division of Rheumatology, University of Colorado Health Sciences Center, Denver, CO 80262; and †Department of Med- scription in CR2-negative cell lines and tissues (14, 15). Recently, icine, Division of Rheumatology, Washington University School of Medicine, St. we have also demonstrated the existence of a cell type-specific Louis, MO 63110 repressor element within the CR2 proximal promoter that is critical Received for publication October 30, 2001. Accepted for publication April 1, 2002. for inhibiting expression in CR2 nonexpressing cell lines (16). In The costs of publication of this article were defrayed in part by the payment of page this study, we extend the understanding of the transcriptional reg- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ulation of the human CR2 gene by characterizing the requirements for basal transcription in a human CR2-expressing cell line. 1 This work was supported by the Smyth Professorship (to V.M.H.), National Insti- tutes of Health Grant R0-1 AI31105 (to V.M.H.), and an Arthritis Foundation post- doctoral fellowship grant (to D.U.). Materials and Methods 2 Address correspondence and reprint requests to Dr. V. Michael Holers, Division of Cell lines and culture conditions Rheumatology, University of Colorado Health Sciences Center, Campus Box B-115, 4200 East Ninth Avenue, Denver, CO 80262. E-mail address: michael.holers@ The human Burkitt’s lymphoma cell line Raji (CCL-86) was obtained from uchsc.edu the American Type Culture Collection (Manassas, VA). The cell line was 3 Abbreviations used in this paper: CR, complement receptor; SLE, systemic lupus maintained at 37¡C with 5% CO2 in RPMI 1640 with L-glutamine supple- erythematosus; HLH; helix-loop-helix. mented with 10% FBS, 100 ␮g/ml streptomycin, and 100 IU/ml penicillin.

Construction of CR2 promoter deletion and mutant luciferase on ice before loading onto a 6% polyacrylamide gel. The gel was then fusion constructs electrophoresed at 150 V using 0.25ϫ Tris-taurin-EDTA (TE buffer) as the running buffer. EMSA gels were dried under vacuum and exposed to x-ray A NheI/XhoI fragment of the CR2 promoter containing either Ϫ315/ϩ75 film. All double-stranded oligonucleotides were end labeled using or Ϫ1250/ϩ75 of the CR2 promoter was cloned into the luciferase reporter [32P]ATP and T4 polynucleotide kinase. pGL3-basic vector (Clontech Laboratories, Palo Alto, CA). Site-directed mutagenesis was performed using the Quickchange mutagenesis kit (Strat- Methylation interference assays agene, La Jolla, CA), which enabled the incorporation of MluI restriction DNA probes (1Ð2 ϫ 106 cpm) were labeled on the sense or antisense strand sites extending 3Ј from the positions Ϫ220 (site 4), Ϫ140 (Sp1), Ϫ120 ␬ Ϫ Ϫ Ϫ Ϫ Ϫ and methylated with DMSO (Sigma-Aldrich, St. Louis, MO) for 1Ð2 min. (NF- B), 93 (site 3), 90 (AP-1), 81 (AP-2), 65 (E box 2) and 47 The DNA was ethanol precipitated three times and resuspended in TE. (E box 1). Deletion constructs were then prepared using the newly incor- Protein-DNA binding was performed for 20Ð30 min at room temperature porated MluI sites together with the MluI site situated in the pGL3-basic in a binding buffer containing 10 mM Tris (pH 7.5), 40 mM NaCl, 1 mM vector polylinker. Restriction enzyme digestion of the mutant plasmids EDTA, 1 mM 2-ME, and 4% glycerol. Following electrophoresis, wet gels with MluI resulted in varying lengths of upstream CR2 promoter sequence Ϫ ϩ were exposed to film at 4¡C and autoradiographs were developed. Bands of being deleted from the full-length 315/ 75 construct. All constructs interest were cut out and then electroeluted and phenol:chloroform ex- made were confirmed by both restriction enzyme digestion and nucleotide tracted, precipitated, and dried. The pellet was resuspended in 100 ␮lofa sequence analysis. 1/10 dilution of piperidine and heated at 90¡C for 40 min. Following ly- Transfection and quantitation of promoter activity ophilization, samples were then analyzed by electrophoresis on 6 or 8% acrylamide plus urea gels in Tris-borate buffer. Before each transfection, Raji cells were split and grown in log phase to DNase I footprint analysis 5 ϫ 105 cells/ml. Cells were then transfected using the Qiagen Superfect

transfection reagent with plasmid DNA prepared using Qiagen Maxiprep- DNase I footprint analysis was performed according to Dynan and Tijan Downloaded from 500 columns (Qiagen, Valencia, CA). Briefly, 10 ␮g of plasmid DNA and (18). Probes (20,000 cpm) were labeled on the sense or antisense strand, 300 ng of pRL-TK control vector was complexed together with Superfect incubated with increasing amounts of nuclear extracts (25Ð75 ␮g), and reagent for 10 min at room temperature. The transfection complexes were incubated on ice for 30 min in a buffer containing 2 mM HEPES (pH 7.8), ␮ then added dropwise to the cells, which had been plated in 5 ml of medium 12.5 mM MgCl2, 1 mM DTT, 10 M ZnSO4, 20% glycerol, and 0.1% in a six-well tray at a concentration of 5 ϫ 105Ð1 ϫ 106 cells/ml. The cells Nonidet P-40, with 2 or 4 ␮g of polyd(I-C). Samples were then digested were then incubated at 37¡C for 48 h following transfection. Cell lysates with 2 ␮l of increasing concentrations of DNase I at room temperature for from the transfected cells were prepared and assayed for both firefly and 1 min. Reactions were stopped by addition of 90 ␮l of a solution containing control renilla luciferase according to the manufacturer’s instructions (Pro- 20 mM EDTA, 1% SDS, 0.2 M NaCl, and carrier RNA. Samples were then http://www.jimmunol.org/ mega, Madison, WI). All transfection data are representative of between phenol:chloroform extracted, ethanol precipitated, dried, and resuspended three and 10 independent transfections using at least two independent prepa- in 90% formamide loading buffer. The samples were then analyzed on 6 or rations of both DNA and plasmid clones. Promoter activity is expressed as 8% acrylamide-urea gels. relative firefly luciferase activity normalized to Renilla luciferase activity. Results EMSA High basal activity is achieved by the Ϫ315/ϩ75 proximal Approximately 8 ϫ 107 cells were used to make nuclear extracts according promoter to a standard method (17). Extracts were frozen in liquid nitrogen and stored at Ϫ80¡C. Determination of protein concentration was performed To determine which regions of the proximal promoter contributed using a protein assay kit (Bio-Rad, Hercules, CA). For EMSA, nuclear to basal CR2 expression, Ϫ1250/ϩ75 and Ϫ315/ϩ75 bp of prox- by guest on September 25, 2021 extracts were preincubated (10Ð20 ␮g) on ice for 10 min together with 1 imal promoter sequence was cloned upstream of a luciferase re- ␮g poly(dI-dC) in a binding buffer consisting of 4% Ficoll (Amersham porter gene. The Ϫ1250/ϩ75 luciferase construct was shown to be Biosciences, Piscataway, NJ), 20 mM HEPES (pH 7.9), 1 mM EDTA, 1 highly active in Raji cells (Fig. 1, construct 1). Deletion of the mM DTT, and 50 mM KCl. When required, competitor oligonucleotides or Ϫ Ϫ supershift Abs (Santa Cruz Biotechnology, Santa Cruz, CA) were then proximal promoter sequence from 1250 to 315 resulted in no incubated with the nuclear extract for 30 min on ice. The nuclear extract significant difference in promoter activity, indicating that all ele- was then incubated with 80 fmol of 32P-labeled oligonucleotide for 30 min ments required for basal transcriptional activity are localized to the

FIGURE 1. Deletion analysis of the CR2 proximal promoter reveals that a high level of basal transcriptional activity is maintained by the Ϫ315/ϩ75 sequence in Raji cells and that several additional regions are critical for basal promoter activity. Normalized transcriptional activity of the CR2 5Ј promoter deletions is shown. Results are shown as mean normalized transcriptional activity vs 315 wild-type construct Ϯ SEM (n ϭ 5Ð10). The Journal of Immunology 6281

Ϫ315/ϩ75 sequence (Fig. 1, construct 2). Further truncation of the signed and analyzed. DNase I footprinting using this probe and CR2 promoter had very little effect on promoter activity (Fig. 1, increasing amounts of Raji nuclear extract revealed a strongly pro- constructs 3Ð5). Deletion of the CR2 promoter sequence up to Ϫ93 tected area over a 10-bp sequence from Ϫ47 to Ϫ38 (site 1). Addi- resulted in a 50% decrease in promoter activity (Fig. 1, construct tionally, a DNase I hypersensitive site was seen at the G residue at 6). Further truncation to Ϫ83 and Ϫ75 resulted in a small decrease position Ϫ34 (Fig. 2Bi). A second sequence, located at Ϫ67/Ϫ60, in promoter activity and, interestingly, deletion from Ϫ75 to Ϫ60 was also shown to be protected, albeit weakly (Fig. 2Bi, site 2). (Fig. 1, construct 9) resulted in a 1.3- to 1.7-fold induction in Methylation interference assays on both the sense (Fig. 2Bii) transcriptional activity. However, truncation of the promoter se- and the antisense (Fig. 2Biii) strand demonstrate methylation of quence from Ϫ60 to Ϫ47 resulted in a marked decrease in tran- several G residues. Methylation of residues Ϫ46, Ϫ42, Ϫ40, and scriptional activity (ϳ60%). This truncation results in a construct Ϫ38 on the sense strand and methylation of G residues Ϫ45 and containing only a small amount of upstream sequence plus the Ϫ43 on the antisense strand was shown to interfere with protein TATA box at position Ϫ29. These results show that these latter binding. Results are summarized in graphical form (Fig. 2A). Se- sequences are responsible for start site selection and mediate low- quence analysis of these two sites shows homology to two classes level basal activity, but that high-level basal transcription is me- of E box motif shown to bind the helix-loop-helix (HLH) family of diated by elements within the entire Ϫ315/ϩ75 sequence. transcription factors (19). These elements share a motif consisting of a core hexanucleotide sequence, CANNTG, and bind multiple DNase I footprinting and methylation interference analysis classes of HLH transcription factors, including E12, E47, c-Myc, across the functionally relevant sites reveal several protected USF, and Mad/Max. regions and methylated nucleotides Deletion analysis also revealed a strong activator domain local- Downloaded from Transcriptional analysis revealed important roles for sequences ized between nucleotides Ϫ120 and Ϫ93 within Raji cells (Fig. 1). spanning nucleotides Ϫ120 to Ϫ93 and Ϫ75 to Ϫ47 in the regu- To examine NF binding to this region, a second probe was de- lation of CR2 expression (Fig. 1). To examine these regions further signed and used in DNase I footprinting. The probe was an 80-bp and identify candidate regulatory sites, DNase I footprinting and fragment spanning nucleotides Ϫ149 to Ϫ69 (Fig. 3). Surprisingly, methylation interference analyses were preformed. First, a 165-bp a large protected region was seen over 40 bp of the probe from probe encompassing sequence from Ϫ90 to ϩ75 (Fig. 2) was de- Ϫ131 to Ϫ92 (Fig. 3B). Sequence analysis of the footprinted re- http://www.jimmunol.org/ gion demonstrates a cytosine-rich sequence that includes many Sp1 sites and several CACC boxes. Additionally, there are AP-1- like and AP-2-like elements within this region between Ϫ90 and Ϫ69; however, as was also seen with the probe in Fig. 2 (Ϫ90/ ϩ75), we could not demonstrate a clear footprint of these two elements (data not shown). by guest on September 25, 2021

FIGURE 2. A, DNase I footprinting reveals two protected sequences from Ϫ70 to Ϫ30, designated sites 1 and 2. Protected sequences are boxed. Methylated G residues as shown by interference assays are shown by stars. The arrow indicates a hypersensitive site. B, Footprint and methylation interference assays from Ϫ90 to ϩ75. i, DNase I cleavage was performed with no extract or with 25, 50, or 75 ␮g of Raji extract. A Maxam-Gilbert GϩA sequence reaction is shown in the left lane. The arrow indicates a FIGURE 3. A, Summary of DNase I footprint analysis of the Ϫ149 to DNase I hypersensitive site. Positions of the TATA box, AP-1, and AP-2 Ϫ69 region. The protected sequence designated as site 3 is shown as the sites are shown. ii and iii, Methylation interference assay is shown for the boxed sequence. B, DNase I footprint assay. A Maxam-Gilbert GϩA se- sense and antisense strands. Methylated G residues interfering with protein quence reaction is shown in the left lane. DNase I cleavage was performed binding are shown as stars. F and B represent the free and bound probes, with no extract or increasing amounts of Raji extract. The protected area is respectively. designated site 3. 6282 BASAL TRANSCRIPTIONAL MECHANISMS OF HUMAN CR2

FIGURE 4. Summary of sites localized within the CR2 proximal promoter region by DNase I footprint and sequence analysis. The boxed sequences indicate protected sequences. Sequence analysis of these sites indicate the presence of consensus binding sites for known transcription factors. The transcriptional initiation site is designated by an arrow. The TATA box is underlined. Oligonucleotides used in EMSA in Figs. 6, 7, and 8 are shown as dotted lines. Downloaded from

Deletion analysis as well as DNase I footprinting and methyl- series of site-speciﬁc mutations were made and then used in tran- ation interference analysis revealed several potentially important sient transfections of CR2-expressing Raji cells. Introduction of a sites within the CR2 promoter involved in basal transcriptional 6-bp mutation into the putative Sp1 site localized within the site 3 http://www.jimmunol.org/ control in the CR2-expressing Raji cell line. Sequence analysis of footprinted region had a modest but reproducible effect on tran- these regions using TFSEARCH (version 1.3) database search re- scriptional activity (Fig. 5, construct 3). Site-speciﬁc mutation of vealed several consensus transcription factor binding sites within the consensus AP-1 site at Ϫ90 had no effect on promoter activity the functional and DNase I footprinted regions (Fig. 4). These sites as compared with the Ϫ315/ϩ75 wild-type construct. Interest- include two adjacent E box motifs within the footprinted se- ingly, introduction of the 6-bp mutation into both the consensus quences at positions Ϫ47 to Ϫ38 and Ϫ67 to Ϫ60 and are desig- AP-2 sequence at Ϫ81 (Fig. 5, construct 5) and the E box site 2 nated E box sites 1 and 2, respectively (Fig. 4). The large protected motif at Ϫ60 (Fig. 5, construct 6) resulted in a 1.3- to 1.7-fold sequence located at position Ϫ132 to Ϫ92 contained many SP1- induction of transcriptional activity. Deletion of the AP-2 site and like sites and CACC boxes, and is designated site 3. upstream sequence (Fig. 1, construct 7) did not show a similar by guest on September 25, 2021 induction in transcriptional activity. This is most likely due to the Mutation analysis of the CR2 promoter reveals important roles interaction of this AP-2 element with sequences upstream. Muta- Ϫ Ϫ for the 81 AP-2 sequence, the 60 E box site 2 motif, and the tion of the E box site 1 motif located at Ϫ47 resulted in a marked Ϫ 47 E box site 1 motif in regulating basal expression of CR2 80% decrease in transcriptional activity, indicating a particularly To further assess whether the consensus sequences shown by foot- important role for this site in maintenance of CR2 basal printing and methylation interference (Fig. 4) were functional, a transcription.

FIGURE 5. Transcriptional activity of the site-speciﬁc mutation constructs compared with wild-type controls in Raji cells. Shown are the consensus transcription factor binding sites. Crosses (ϫ) indicated the position of the introduced 6-bp mutation. Results are shown as mean normalized transcriptional activity vs Ϫ315/ϩ75 construct Ϯ SEM (n ϭ 5Ð10). The Journal of Immunology 6283

Characterization of the factors binding the functionally relevant sites within the CR2 proximal promoter Sequence analysis of the DNase I footprinted regions revealed consensus binding sequences for known transcription factors (Fig. 4). Additionally, transcriptional assays revealed important roles for the two adjacent E box motifs at Ϫ47 and Ϫ60 as well as the AP-2 site located at Ϫ81, together with the entire site 3 motif spanning nucleotides Ϫ130 to Ϫ90. To determine whether these functionally important sequences do indeed bind the known transcription factors within their consensus binding sites, EMSA was performed using nuclear extracts prepared from Raji cells. EMSA spanning the entire site 3 DNase I footprinted region from Ϫ130 to Ϫ90 resulted in the presence of two major protein- DNA complexes (Fig. 6A, complexes A and D) and two minor protein-DNA complexes (Fig. 6A, complexes B and C). Addition of increasing amounts of cold self oligonucleotide resulted in competition of all complexes, indicating the existence of highly spe- ciﬁc complexes. Sequence analysis of the site 3 region revealed a Downloaded from match for a Sp1 sequence. Competition using a Sp1 consensus oligonucleotide resulted in abolishment of the major protein-DNA complex A as well as the minor complex C (Fig. 6A, Sp1). To FIGURE 7. EMSA using an oligonucleotide spanning Ϫ90 to Ϫ69 en- further elucidate the presence of Sp1 binding to the site 3 motif, compassing the AP-2 consensus sequence was performed using Raji nu- supershift analysis was performed (Fig. 6B). Addition of the Sp1 clear extracts. A, One major protein-DNA complex was seen and is shown ϫ Ab to the binding reaction resulted in a supershift of protein-DNA by an arrow. Fold molar excess ( ) of unlabeled competitor is indicated.

Competition analysis using a consensus AP-2 oligonucleotide shows that http://www.jimmunol.org/ complex A (Fig. 6B, A*). These results indicate that the transcrip- this transcription factor is involved. B, Supershift analysis using an AP-2␣ tion factor contained within complex A is a member of the Sp1 Ab demonstrates that the protein within complex A is serologically distinct family. Protein-DNA complexes B and D contain as-yet-uniden- from AP-2␣. tified proteins. EMSA spanning the AP-2 consensus binding site from Ϫ90 to Ϫ69 comprised one specific protein-DNA complex (Fig. 7A, com- indicating the presence of highly specific binding. Competition plex A). Cold competition using self oligonucleotide indicated assays using a consensus oligonucleotide for AP-2 also revealed competition of protein-DNA complex A at a 50-fold molar excess, competition of complex A, indicating the potential presence of AP-2 in this complex. The AP-2 consensus oligonucleotide used in by guest on September 25, 2021 these experiments is a binding site for the transcription factor AP- 2␣. However, to further investigate the role of AP-2␣ in binding this site, supershift analysis (Fig. 7B) using an Ab recognizing the AP-2␣ transcription factor was performed. Addition of this Ab to the binding reaction did not result in competition of complex A, indicating that the protein within complex A shares DNA binding specificity with AP-2 but is serologically distinct from AP-2␣. Functional assays revealed a major role for maintenance of transcriptional activity for the E box site 1 motif. This motif is a member of the E box family of transcription factors and has been shown to bind several proteins of the basic HLH-leucine zipper class, including USF1, TFE3, Max, and c-myc. EMSA spanning the E box site 1 motif from Ϫ63 to Ϫ40 resulted in the presence of three protein-DNA complexes (Fig. 8A, complexes AÐC). Addition of increasing amounts of cold self competitor oligonucleotide revealed the competition of complexes AÐC, indicating that these complexes were of high affinity. Supershift analysis (Fig. 8B) using an Ab directed against USF1 resulted in the abolishment of protein-DNA complex A and decreases of complexes B and C. As a negative control, an Ab directed to a different class of HLH protein (E2A) was used (Fig. 8B). As expected, addition of this Ab had no effect on the binding of the specific proteins to the E box site 1 motif.

FIGURE 6. EMSA using an oligonucleotide spanning the site 3 pro- Discussion tected region from Ϫ130 to Ϫ90 using Raji nuclear extracts. A, Competi- tion using increasing fold molar excess of cold self oligonucleotide dem- This study has investigated further the requirements for basal tran- onstrates the existence of two major protein-DNA complexes, designated scription of the human CR2 gene. Early studies (20, 21) on the by arrows (A and D). Fold molar excess (ϫ) of unlabeled competitor is CR2 promoter demonstrated that the transcriptional initiation site indicated. B, Competition and supershift analysis indicates that the tran- was localized to 92Ð94 bp upstream of the ATG codon. Addition- scription factor within complex A is a member of the Sp1 family. ally, in transfection experiments the 5Ј promoter sequence was 6284 BASAL TRANSCRIPTIONAL MECHANISMS OF HUMAN CR2

ever, recently it has become clear Sp1 is only one of many transcription factors belonging to a family characterized by a highly conserved DNA-binding domain consisting of three zinc fingers that bind to these sites. Site-specific mutagenesis of one of many consensus Sp1 motifs within the footprinted region had only a modest effect on transcriptional activity of the CR2 promoter. This is not surprising, as the large footprinted region could indicate a complex pattern of NF binding, which was uninterrupted by the mutation introduced. EMSA using an oligonucleotide spanning the footprinted region from Ϫ130 to Ϫ90 resulted in the presence of two major protein-DNA complexes. Competition using both a Sp1 consensus oligonucleotide and a Sp1 supershift Ab indicated the presence of this protein in binding the large footprinted region. However, other as-yet-unidentified proteins, perhaps one of the newly characterized Sp1-like zinc finger transcription factors, are also likely involved in binding the site 3 motif. Mutational analysis also revealed an important role for the AP-2 consensus sequence localized at position Ϫ81. Mutation of several base pairs spanning this motif resulted in a 1.5- to 1.8-fold induc- Downloaded from tion in promoter activity, suggesting the presence of a repressor element at this site. EMSA and supershift analysis using an oli- FIGURE 8. EMSA of the E box site 1 motif. A, An oligonucleotide gonucleotide spanning Ϫ90 to Ϫ69 encompassing the AP-2 con- spanning Ϫ63 to Ϫ40 of proximal promoter sequence shows the presence sensus sequence within the CR2 promoter showed that this site of three protein-DNA complexes (AÐC). Fold molar excess (ϫ) of unla- appeared to bind a protein that shared DNA specificity with AP-2 beled competitor is indicated. B, Supershift analysis using an Ab directed but was serologically distinct from AP-2␣. The AP transcription http://www.jimmunol.org/ against USF1 demonstrates the presence of USF1 within complex A. factor family was first isolated from HeLa cells and was initially named for its transcriptional activation function (24, 25). It has found to be active, but the specific requirements for basal tran- been shown to be an activator in regulating many genes, including ␤ scription have not been further characterized until now. We cloned HIV type 1 (26), type IV collagenase (27), and the dopamine -hy- various lengths of the upstream promoter sequence in front of a droxylase gene (28). However, the AP-2 site within the human luciferase reporter and found several regions involved in basal CR2 gene acts as a transcriptional repressor and recently more data transcription. Promoter constructs containing either Ϫ1250/ϩ75 or have been assembled suggesting that this well-known activating

Ϫ315/ϩ75 of upstream sequence had the same high level of basal transcription factor may act as a transcriptional repressor. For ex- by guest on September 25, 2021 transcriptional activity when transfected into the CR2-expressing ample, an AP-2 site functions as a repressor and contributes to the Raji cell line. These results indicated that all of the necessary ma- liver-specific expression of the serum amyloid A1 gene (25). Ad- chinery for driving basal CR2 expression was localized in this ditionally, an AP-2 site within the T cell-restricted CD2 gene has Ϫ315/ϩ75 sequence. Interestingly, a recent report (22) has shown been shown to be acting as a repressor; however, the factor binding that further upstream regions are responsible for PMA- and cAMP- to this site was shown to be serologically distinct from AP-2 (29), inducible expression of the CR2 promoter. similar to that seen in the CR2 gene. Deletion analysis of the Ϫ315/ϩ75 sequence revealed several Finally, a third region within the CR2 proximal promoter was regions within the promoter important to function. The first ele- determined to be functionally significant using both mutant and ment was localized upon deletion of the promoter sequence from deletion analysis. An E box motif located at Ϫ47 was found to be Ϫ120 to Ϫ93. This deletion resulted in a 50% decrease in tran- particularly important in maintenance of basal transcriptional con- scriptional activity, indicating the presence of a activator motif trol of the CR2 promoter. Interestingly, a recent paper by Veresh- within this sequence. DNase I footprinting across this region re- chagina et al. (22) has also shown a role for this site in induction vealed a large protected sequence from Ϫ133 to Ϫ90 of the up- of CR2 promoter activity by cAMP and PMA. Deletion and mu- stream promoter. This sequence contains a number of GC and GT tation analysis of E box site 1 at Ϫ47 demonstrated the existence boxes. It has been demonstrated previously (23) that G-rich ele- of a strong activator motif within this sequence. DNase I footprint- ments and GT/CACC boxes such as the ones seen in this protected ing demonstrated clearly protected sequences across this site. Ad- footprint are important elements in housekeeping as well as many ditionally, methylation interference assays revealed several nucle- tissue-specific genes. It was previously thought that the ubiquitous otides that were methylated across E box site 1. EMSA and transcription factor Sp1 acts through these GC/GT boxes; how- supershift analysis demonstrated the binding of USF1 to this site.

Table I. Summary of the functionally relevant sites within the CR2 proximal promotera

Nucleotide Position (bp) Footprint Methylation State Functional Role Proteins Bound

Ϫ47 ϩϩActivator USF1 and unknown Ϫ63 ϩϪCell type-speciﬁc E2A proteins (16) repressor Ϫ81 Ϫ ND Repressor AP-2-like Ϫ120/Ϫ93 ϩϪActivator Sp1 and unknown

a Shown are the nucleotide positions of the localized elements and the functional role for each motif. Also shown are the proteins that were shown to bind these sites and the footprint and methylation status of each functional motif. The Journal of Immunology 6285

USF1 is a member of the basic HLH/leucine zipper family of tran- tion of the complement cascade on B lymphocytes from patients with systemic scription factors (30, 31). USF transcription factors are involved in lupus erythematosus (SLE). Clin. Exp. Immunol. 101:60. 10. Takahashi, K., Y. Kozono, T. J. Waldschmidt, D. Berthiaume, R. J. Quigg, the regulation of many E box-containing genes, including murine A. Baron, and V. M. Holers. 1997. Mouse complement receptors type 1 (CR1; metallothionen I (32), murine p53 (33), human CD2 (34), and hu- CD35) and type 2 (CR2; CD21): expression on normal B cell subpopulations and ␤ decreased levels during the development of autoimmunity in MRL/lpr mice. man -globin gene (35). Interestingly, there is also evidence to J. Immunol. 159:1557. suggest that USF functionally interacts with basal transcriptional 11. Boackle, S. A., V. M. Holers, X. Chen, G. Szakonyi, D. R. Karp, E. K. Wakeland, machinery such as TFIID (36). USF has also been shown to bind and L. Morel. 2001. Cr2, a candidate gene in the murine Sle1c lupus susceptibility locus, encodes a dysfunctional protein. Immunity 15:775. cooperatively with TFII-I and sequences close to the initiation of 12. Prodeus, A. P., S. Georg, L. M. Shen, O. O. Pozdnyakova, L. Chu, E. M. Alicot, transcription (37, 38). It is interesting to speculate that, due to the C. C. Goodnow, and M. C. Carroll. 1998. A critical role for complement in proximity of the E box site 1 motif to the transcriptional initiation maintenance of self-tolerance. Immunity 9:721. 13. Feuerstein, N., F. Chen, M. Madaio, M. Maldonado, and R. A. Eisenberg. 1999. site (Ϫ47), USF1 binding to this motif may interact with the basal Induction of autoimmunity in a transgenic model of B cell receptor peripheral transcriptional machinery to activate transcription. This may ex- tolerance: changes in coreceptors and B cell receptor-induced tyrosine-phospho- proteins. J. Immunol. 163:5287. plain the importance of this site in maintenance of basal CR2 tran- 14. Makar, K. W., C. T. N. Pham, M. H. Dehoff, S. M. O’Connor, S. M. Jacobi, and scription as demonstrated by a dramatic loss in promoter activity V. M. Holers. 1998. An intronic silencer regulates B lymphocyte cell- and stage- when this site was mutated within the CR2 proximal promoter. specific expression of the human complement receptor type 2 (CR2, CD21) gene. J. Immunol. 160:1268. In conclusion, several sites within the proximal promoter were 15. Makar, K. W., D. Ulgiati, J. Hagman, and V. M. Holers. 2001. A site in the shown to be important in the transcriptional control of human CR2 complement receptor 2 (CR2/CD21) silencer is necessary for lineage specific in Raji cells, a CR2-expressing cell line (Table I). These sites bind transcriptional regulation. Int. Immunol. 13:657. 16. Ulgiati, D., and V. M. Holers. 2001. CR2/CD21 proximal promoter activity is the transcription factors USF1, an AP-2-like factor, and Sp1 as critically dependent on a cell type-specific repressor. J. Immunol. 167:6912. Downloaded from well as other as-yet-unknown proteins. Recently, we have also 17. Li, Y. C., J. Ross, J. A. Scheppler, and B. R. Franza, Jr. 1991. An in vitro transcription analysis of early responses of the human immunodeficiency virus type 1 long ter- discovered the presence of a cell type-specific repressor localized minal repeat to different transcriptional activators. Mol. Cell. Biol. 11:1883. to the E box 2 motif at position Ϫ63 of the proximal promoter (16). 18. Dynan, W. S., and R. Tjian. 1983. The promoter-specific transcription factor Sp1 The primary control of basal transcription in CR2-expressing cells binds to upstream sequences in the SV40 early promoter. Cell 35:79. 19. Massari, M. E., and C. Murre. 2000. Helix-loop-helix proteins: regulators of appears to lie in very close proximity to the TATA box; however, transcription in eucaryotic organisms. Mol. Cell. Biol. 20:429. we cannot rule out the possibility of other as-yet-undetected ele- 20. Rayhel, E. J., M. H. Dehoff, and V. M. Holers. 1991. Characterization of the http://www.jimmunol.org/ ments further upstream, or even within other areas of the CR2 gene human complement receptor 2 (CR2, CD21) promoter reveals sequences shared with regulatory regions of other developmentally restricted B cell proteins. J. Im- itself. The four main motifs involved in basal transcriptional con- munol. 146:2021. trol lie within 120 bp of upstream sequence (Table I). Moreover, 21. Yang, L., M. Behrens, and J. J. Weis. 1991. Identification of 5Ј regions affecting the expression of the human CR2 gene. J. Immunol. 147:2404. three of the four functionally relevant sites are localized to within 22. Vereshchagina, L. A., M. Tolnay, and G. C. Tsokos. 2001. Multiple transcription 80 bp of the transcriptional initiation site and are located very close factors regulate the inducible expression of the human complement receptor 2 together, within ϳ30 bp. How these elements interplay with one promoter. J. Immunol. 166:6156. 23. Philipsen, S., and G. Suske. 1999. A tale of three fingers: the family of mam- another to control CR2 transcription and how they may interact malian Sp/XKLF transcription factors. Nucleic Acids Res. 27:2991. with the intronic silencer that controls cell- and stage-specific ex- 24. Hilger-Eversheim, K., M. Moser, H. Schorle, and R. Buettner. 2000. Regulatory pression is yet to be determined but is currently under roles of AP-2 transcription factors in vertebrate development, apoptosis and cell- by guest on September 25, 2021 cycle control. Gene 260:1. investigation. 25. Ren, Y., and W. S. L. Liao. 2001. 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