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The Journal of Immunology, 2000, 165: 4437-4445.
Copyright © 2000 by The American Association of Immunologists

Cell-Specific Expression of the Murine CD21 Gene Depends on Accessibility of Promoter and Intronic Elements1

Mark D. Zabel, Burke L. Byrne, Janis J. Weis and John H. Weis2

Division of Cell Biology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84132


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The murine complement receptor type 2 gene (Cr2/CD21) is transcriptionally active in murine B and follicular dendritic cells, but not in murine T cells. We have previously shown that altering chromatin structure via histone deacetylase inhibitors results in CD21 expression in murine T cells, and that the minimal CD21 promoter provided appropriate cell-specific expression of luciferase reporter constructs only in the presence of the first third of intron 1, fragment A. We extend this work by showing that replacing the CD21 gene promoter with the SV40 promoter resulted in the loss of this cell-specific control. Further delineation of intronic regulatory elements by fragmentation also resulted in the loss of cell-specific gene expression, suggesting that multiple CD21 promoter and intronic elements interact for appropriate CD21 gene expression. To assess this model, we performed EMSAs to define protein binding sites within promoter and intronic regions and DNase I hypersensitivity assays to determine chromatin accessibility. Multiple DNA binding factors were shown to be present in B and T cell extracts; a minority demonstrated B cell specificity. However, the DNase I sensitivity of T cell CD21 regulatory elements was not comparable to that of B cells until the histone acetylation status of the gene was altered. Taken together, these data suggest that chromatin remodeling facilitates cell-specific CD21 gene expression by modulating access of transcription factors to regulatory elements in the promoter and intron.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The murine complement receptor type 2 (Cr2, CD21) is a receptor that binds the C3 cleavage products C3b and C3dg of the complement cascade. Normally, these complement components bind covalently to an immune complex during an infection, thus allowing for cellular uptake of such complexes via CD21 binding. CD21 forms a complex with CDl9 and CD81 on the surface of B cells to transduce signals upon ligand binding that lower the activation threshold of the cells on which they reside (1). The murine CD21 gene, a member of the regulators of complement activation gene locus (2), encodes two alternatively spliced RNA transcripts that generate two distinct proteins (3, 4). B cells express CD21 in a stage-specific manner, initiating expression when they mature and express IgM, and terminating expression when they are activated and switch Ig isotypes. Murine follicular dendritic cells found in the spleen are the only other cell type reported to express CD21 (5). Murine T cells do not express CD21 (6).

Two different regions of the CD21 gene have been identified as important for its appropriate expression. Two kilobases of the sequence located 5' of the transcriptional start site of the murine CD21 gene was shown to provide expression of reporter constructs. This minimal promoter, however, demonstrated gene expression equally well in B and T cells (7). Inclusion of up to 10 kb of 5' sequence failed to restore cell specificity. Similar results were seen with experiments involving the human CD21 gene, in which 5 kb of 5' promoter sequence failed to confer cell-specific gene expression (8).

Analysis of CD4 gene expression provided insight into the analysis of CD21 gene expression. Reporter constructs containing up to 13 kb of murine CD4 5' sequences expressed equally well in both CD4+ and CD8+ (CD4-) T cells (9). However, when CD4 intronic sequences were introduced into these constructs, gene expression was repressed in CD4- cells, thus revealing the existence of a silencer in the first intron (10). An intronic silencer was later identified in the human CD4 gene as well (11).

Using the transcriptional regulation of the CD4 gene as a model, we identified a region of the first intron of murine CD21 that conferred cell-specific expression of reporter constructs (7). This region, termed fragment A, comprised the first 1.6 kb of the intron and appeared to include both enhancer and suppressor elements. An intronic silencer was also found in the first intron of the human CD21 gene (8), implying the existence of an evolutionarily conserved regulatory mechanism for CD21 gene expression.

To exert control over gene expression by the promoter, an intronic regulatory element must somehow contact or otherwise interact with sequences 5' of the gene. The most likely scenario to facilitate an interaction would involve manipulation of the local chromosomal architecture. Chromatin remodeling has long been suggested as a regulatory mechanism for gene expression (12, 13). One mechanism for chromatin remodeling is histone acetylation. Acetylating lysine residues on the N-terminal tails of core histones neutralizes the positively charged residues, thereby weakening local interactions with the negatively charged phosphate backbone of DNA. The resulting chromatin decondensation presumably facilitates access of transcription factors to their cognate DNA binding sites. The mechanistic relationship between chromatin structure and transcription regulation has been elucidated mainly by studying telomeric and mating type loci in yeast (14, 15). A direct link was established when the p55 gene, encoding the catalytic subunit of a tetrahymena acetyltransferase, was cloned and identified as a homologue of GCN5p, a yeast transcriptional adapter protein (16). We have demonstrated that murine T cells can be induced to express CD21 by altering the conformation of the chromatin (17). Treatment with the histone deacetylase inhibitors sodium butyrate and trichostatin A (TSA)3 induced murine T cells to express CD21, suggesting that histone positioning contributes to the control of CD21 expression, and that certain regulatory elements that have been mapped within the gene may function via this pathway.

We have previously identified the minimal CD21 promoter and intronic regulatory elements within fragment A that control the cell-specific expression of the murine CD21 gene. In this manuscript we demonstrate the dependence of these intronic elements on the endogenous CD21 promoter for cell-specific gene expression. In addition, we show that segmentation of fragment A results in the loss of cell-specific CD21 gene expression. Taken together, these data indicate that multiple regulatory elements in fragment A interact with the endogenous promoter to confer cell-specific expression on the murine CD21 gene.

Two different models can be proposed to explain these data. The first involves regulating CD21 gene expression by controlling the expression of transcription factors that interact with promoter and intronic regulatory elements. A second model involves regulating the access of these factors to their binding sites. Because T cells can be induced to express CD21 by hyperacetylating chromatin in the presence of a protein synthesis inhibitor (17), the required transcription factors must be present in T cells, but cannot access CD21 regulatory elements. To further evaluate these models, we demonstrate that T cell nuclear extract possesses numerous proteins that bind CD21 promoter and intronic regulatory elements. However, DNase I hypersensitivity analysis of these regions suggests that accessibility of sites to these factors is limited in native T cells, but can be enhanced by altering chromatin structure via histone acetylation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and culture conditions

Mouse 2PK3 cells, CD21-expressing B lymphomas, and EL4 cells, CD21-nonexpressing T lymphomas, were obtained from American Type Culture Collection (Manassas, VA). 2PK3 cells were maintained at 37°C in 5% CO2 in DMEM (Life Technologies, Gaithersburg, MD) with 10% FCS (HyClone, Logan, UT) and 1% penicillin-streptomycin (Pen-Strep, Life Technologies). EL4 cells were maintained at 37°C in 5% CO2 in RPMI 1640 (Life Technologies) with 10% FCS and 1% Pen-Strep. Where indicated, cells were incubated with 100 nM of the deacetylase inhibitor TSA (Sigma, St. Louis, MO) overnight.

Luciferase reporter constructs

pCD21Basic, pCD21basicIntron 1, pCD21BasicA, pCD21basicAa, and pCD21BasicAb luciferase reporter plasmids were generated as previously described (7). The same CD21 fragments used to produce these plasmids were inserted into the BamHI site of pSV40Control firefly luciferase reporter plasmid (Promega, Madison, WI) 3' of the SV40 enhancer to generate pSV40Control intron 1, pSV40ControlA, pSV40ControlAa, and pSV40ControlAb. CD21 fragment A segments were generated by PCR using the following primers: i1 was amplified using primers 1248 (5'-AGC TCA AAT ACG GAA TTC CAC C-3') and 1249 (5'-TGG TGA TAA TCC ATC TCC CAG A-3'); i2 was amplified using primers 1254 (CCT TTG GAG ATC CCT CTG TCT C-3') and 1255 (5'-GAC TGG AAT CCA GGG AGA AGA G-3'); i3 was amplified using primers 818 (5'-CCA CAT GTC TGC TCT CTT CTC-3') and 819 (5'-CGG GAT CCT GAG ACC CAC ATT G-3'); i5 was amplified using primers 1250 (5'-CTG ATT CTC CAA TGT GGG TCT C-3') and 1251 (5'-CCT GAT CCC TTC ACT ACC AGA A-3'); i6 was amplified using primers 1246 (5'-GAG TGG ATT TTG GCA GTC TCA G-3') and 1247 (5'-CCT TAG CTG CTA ACG GCT ACC T-3'); i7 was amplified using primers 866 (5'-CAG CTA AGG ATG GAT CTG GTT-3') and 806 (5'-GGC TAT CTG ATG GAT ATG GAT G-3'); i8 was amplified using primers 805 (5'-CAT CCA TAT CCA TCA GAT AGC C-3') and 867 (5'-CTA GGA TCT GGA GTG AAG ATC G-3'); and i9 was amplified using primers 865 (5'-CGA TCT TCA CTC CAG ATC CTA G-3') and 741 (5'-CTA TGA GTT GTCT TCT GAC CTC C-3'). These PCR products were inserted into pCD21Basic 3' of the firefly luciferase gene to generate pCD21Basic i1–9. All plasmid insertions were confirmed by restriction digest and sequencing in both directions using the chain termination method (18).

Transient transfection and assay of luciferase reporter constructs

Transfections were performed as previously described (7). Briefly, 5 x 106 2PK3 or EL4 cells were resuspended in 0.65 ml of DMEM with 10% FCS and 1% Pen-Strep. Equimolar amounts of plasmid were added relative to 10 µg of pGL3Basic luciferase reporter construct (Promega). One microgram of pRL-TK Renilla luciferase reporter plasmid (Promega) was added to control for transfection efficiency. Cells were incubated with plasmids for 5 min in 0.4-cm electrode gap cuvettes (Invitrogen, Carlsbad, CA) and electroporated at room temperature using the Gene Pulser (Bio-Rad, Hercules, CA) set at 250 V and 960 µF for 2PK3 cells and at 280 V and 960 µF for EL4 cells. Cells were incubated for 5 min at room temperature, then transferred to 100 x 20-mm tissue culture dishes containing 10 ml of DMEM with 10% FCS and 1% Pen-Strep and incubated at 37°C for 48 h. Luciferase assays were performed using the Dual Luciferase Reporter Assay System (Promega). Briefly, cells were harvested, centrifuged at 200 x g for 5 min, washed twice in PBS, resuspended in 200 µl of 1x lysis buffer (Promega), and incubated at room temperature for 15 min. Cell debris was pelleted by centrifugation at 13,000 x g for 5 min, and 180 µl of lysate was removed. Cell lysate (10 µl) was loaded into the well of a white opaque microtiter plate, and the dual luciferase assay was performed automatically using the MLX microtiter plate luminometer (Dynex, Chantilly, VA). Serial injection of substrates and monitoring of light emission for 10 s were performed for both firefly and Renilla luciferase. Computer software (Dynex) automatically subtracted background and normalized raw data by calculating the ratio of firefly:Renilla light emission values. Normalized data were relativized to luciferase activity seen for pCD21Basic or pSV40Control. Statistical analysis was performed using Microsoft Excel (Microsoft, Seattle, WA); error bars were determined by SD.

Electrophoretic mobility shift assays

The protocol was performed as previously described (19) with minor modifications. Nuclear extracts were prepared from 2PK3 and EL4 cells, and all steps were performed at 4°C. Briefly, 1.8 x 107 cells were harvested, centrifuged, washed twice in ice-cold PBS, and lysed in 5 ml buffer A (10 mM HEPES (pH 7.9), 0.5% Nonidet P-40, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, and 1 mM DTT). Nuclei were pelleted by centrifugation at 13,000 x g for 10 min, washed twice with buffer A lacking Nonidet P-40, and incubated in 100 µl buffer B (10 mM HEPES (pH 7.9), 0.4 M NaCl, 25% glycerol, 50 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, and 1 mM DTT) for 30 min. Nuclei were pelleted by centrifugation at 13,000 x g for 10 min, and nuclear extract was removed and quantified by the Bradford assay (20). DNA fragments were prepared by PCR using primers specific for each segment of fragment A and the promoter. Primers used for amplifying fragment A segments i1–i9 are the same as those used in cloning the luciferase reporter constructs. In addition, i4 was amplified using primers 1250 (5'-CTG ATT CTC CAA TGT GGG TCT C-3') and 1251 (5'-CCT GAT CCC TTC ACT ACC AGA A-3'). For the promoter segments, p6 was amplified using primers 1213 (5'-GCA AGC ATA AGG GCT TGA ATT C-3') and 1160 (5'-GGT AAG GTA GTT TTT CAT AAT TTT TTG C-3'). p5 was amplified using primers 1159 (5'-GCA CAC ACA CAC ACC TCT ACT CCT-3') and 1162 (5'-GTG TTA TGT AAT TGT TAT TTC TGA CTT). p4 was amplified using primers 1163 (5'-TGT CTG TAA TTT ATT TAA AAA GTC AGA A-3') and 1164 (5'-TAA AGT GGA AAT TCA GAA ATA ACA CTG C-3'). p3 was amplified using primers 1165 (5'-AAA CCC CTT TAC AGT TAT ACA GAA A-3') and 1166 (5'-ACA GTA TTG TAT GCT TTA AAC CTG T-3'). p2 was amplified using primers 1167 (5'-GGT TTG AAA CTG AAT TAA CTG G-3') and 1168 (5'-GGA AGT CTG CTT TTG GTT CAG G-3'). p1 was amplified using primers 1169 (5'-GCC TGA ACC AAA AGC AGA CTT CAA-3') and 1170 (5'-GTA GCA AGG AGA GGA AGA GGA C-3'). PCR products were digested with EcoRII (p2–4, i2, and i8), Sau3AI (p5–6, i1, i3–7, and i9), or MspI (p1). Digested PCR products were purified by 4% PAGE and radioactively labeled via a fill-in reaction with [{alpha}-32P]dCTP, d(A,G,T)TPs, and Klenow enzyme. All binding reactions were performed at room temperature for 30 min in a 30-µl volume containing 0–5 µg nuclear extract, 10,000–30,000 cpm fragment, and 2 µg of poly(dI-dC) in binding buffer (3 mM Tris (pH 7.9), 5 mM MgCl2, 0.5 mM DTT, 0.5 mM EDTA, and 3% glycerol). Reactions were subjected to 4% PAGE for 2 h at 250 V. Gels were dried and exposed to BXR medical x-ray film (Western X-ray, Salt Lake City, UT) for 3–16 h.

DNase I hypersensitivity PCR (DNase I HS PCR)

This assay is based on modifications of previous protocols (21, 22). Briefly, 107 2PK3 and EL4 cells were harvested, centrifuged, and washed twice with PBS. Cells were resuspended in 1 ml of permeabilizing buffer (15 mM Tris (pH 7.5), 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.5 mM EGTA, 300 mM sucrose, and 0.5 mM 2-ME) supplemented with 0, 500, 2,000, or 10,000 U/ml of DNase I (Roche, Indianapolis, IN). An equal volume of permeabilizing buffer supplemented with 0.1% lysolecithin (Sigma) was added, and the reaction was incubated at room temperature for 4 min. Genomic DNA (gDNA) was isolated using the DNEasy Tissue Kit (Qiagen, Valencia, CA). PCR was performed in 10-µl reactions using 10 ng of genomic DNA (gDNA), 70 pM of each primer, 0.72 U of AmpliTaq DNA polymerase (Life Technologies), 0.8 mM dNTPs, 1x Taq buffer (50 mM Tris (pH 8.3), 3 mM MgCl2. 20 mM KCl, and 500 mg/ml BSA), and 2.5 mCi [{alpha}-32P]dCTP. Samples were loaded into capillary tubes and incubated in an air thermocycler (Idaho Technologies, Idaho Falls, ID) for denaturing at 94°C for 1 s, for annealing at 60°C for 1 s, and for extension at 72°C for 5 s. This cycle was repeated 25 times for amplifying ß-actin sequences from 2PK3 and EL4 gDNA with primers 62 (5'-ATT GAA CAT GGC ATT GTT AC-3') and 339 (5'-CTC TAT CGT GGG CCG CTC TAG-3'). The cycle was repeated 28 times for amplifying promoter and fragment A segments from 2PK3 and EL4 gDNA with the same primers as those used to generate EMSA probes. For gDNA isolated from TSA-treated EL4 cells, the cycle was repeated 28 times for amplifying ß-actin sequence and 31 times for amplifying promoter and fragment A segments. After amplification, 5 µl of stop solution (U.S. Biochemical, Cleveland, OH) was added to the reactions, and 5 µl was resolved on a 6% acrylamide sequencing gel, which was then dried and exposed to x-ray film (Western X-ray) overnight. PCR bands visualized on x-ray film were subsequently visualized using a Bio-Rad GS-250 phosphorimager and were quantified using Bio-Rad Molecular Analyst software version 1.1. Quantified band intensity values were normalized by ß-actin values and relativized to band intensity values for PCR using genomic DNA without DNase I treatment. Statistical analysis was performed using Microsoft Excel.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell specificity of CD21 intronic regulatory elements is promoter dependent

We have previously shown that the cell-specific expression of murine CD21 (B cells>T cells) is not regulated by 5' promoter sequences (7), and sequences have been described within the first third of intron 1, termed fragment A (Fig. 1Go), that do confer this cell-specific expression. Fragment A, with the CD21 promoter, increased the expression of a luciferase reporter construct in B cells, but not in T cells. The 5' half of fragment A, termed Aa, repressed luciferase reporter activity in both B and T cells, while the 3' half of fragment A, termed Ab, enhanced luciferase reporter activity. Taken together, these data suggest that fragment A contains multiple sites that can positively and negatively affect CD21 transcription, neither of which recapitulates full fragment A function. Of particular interest in this region was segment i8 of fragment Ab, which contains the MHI sequence that possesses high homology to the human CD21 intron 1 (Fig. 1Go). Sequence conservation between the mouse and human in a noncoding region of CD21 implies an important regulatory role for the i8 segment.



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  		FIGURE 1. Schematic of murine CD21 promoter and intron 1 elements. The promoter is shown divided into six segments of ~200 bp each (p6–p1). The positions of the mouse/human homology sequence (MHP), the octamer binding site (Oct), and the TATA box relative to the segments are indicated. The first third of intron 1 (fragment A) is shown divided into two pieces of ~800 bp (Aa and Ab). Both of these are furthered subdivided into segments of ~200 bp each (i1–i9). The relative position of the mouse/human homology sequence is shown within i8.

 
To further elucidate the function of these intronic elements, we examined whether fragment A required the endogenous CD21 promoter to confer cell-specific expression, or whether this intronic element would confer similar control with an exogenous promoter. We cloned CD21 intronic elements into a luciferase reporter construct driven by an SV40 promoter with an SV40 enhancer sequence 3' of the firefly luciferase gene. The murine 2PK3 B cell and the murine EL4 T cell lines were transiently transfected with equimolar amounts of the CD21-promoted constructs used previously as well as with the SV40 constructs and assayed for luciferase activity (Fig. 2Go). Fig. 2GoA recapitulates previous data that demonstrated cell-specific expression of luciferase in cells transfected with CD21 promoter reporter plasmids containing CD21 intronic elements. Luciferase activity in 2PK3 cells containing pCD21Basic intron 1, pCD21BasicA, or pCD21BasicAb increased slightly compared with that seen for 2PK3 cells transfected with the positive control pCD21Basic. Luciferase activity in 2PK3 cells transfected with pCD21BasicAa decreased compared with that in cells transfected with pCD21Basic. In contrast, luciferase activity was significantly decreased in EL4 cells transfected with pCD21BasicAa, pCD21Basic intron 1, and pCD21BasicA compared with that in cells transfected with pCD21Basic. Interestingly, luciferase activity in EL4 cells transfected with pCD21BasicAb more than doubled that in cells transfected with pCD21Basic.



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  		FIGURE 2. Luciferase assays of murine B and T cells transiently transfected with reporter constructs containing CD21 intron 1 and fragments thereof. 2PK3, B cells, and EL4, T cells, were transiently transfected with equimolar amounts of reporter plasmid. A, Relative luciferase activities of reporter constructs driven by the CD21 promoter (CD21Basic) with the entire CD21 intron 1 or its fragments (A, Aa, and Ab) inserted 3' of the firefly luciferase gene. B, Relative luciferase activities of constructs driven by an SV40 promoter and enhanced by an SV40 element 3' of the firefly luciferase gene (SV40Control), followed by the entire CD21 intron 1 or its fragments (A, Aa, and Ab). Data are averages of at least three experiments.

 
Fig. 2GoB shows luciferase activity of 2PK3 and EL4 cells transiently transfected with reporter plasmids containing the SV40 promoter and enhancer elements with the CD21 intronic sequences. When this heterologous promoter was used with the CD21 intronic elements, cell-specific luciferase expression was lost. Increased luciferase activity was seen with pSV40Control intron 1, pSV40ControlA, and pSV40ControlAb compared with pSV40Control in both 2PK3 and EL4 cells. Both cell lines containing pSV40ControlAa continued to exhibit the decreased luciferase activity similar to that seen with pCD21BasicAa. These data suggest that the CD21 promoter and intronic sequences interact together to control cell-specific gene expression.

Cell-specific CD21 gene expression is lost when fragment A is segmented

The previous experiments revealed that the cell-specific expression conferred by fragment A appears to be adversely affected when it is fragmented. Thus, while Aa and Ab appear to have negative and positive regulatory elements, respectively, neither fragment confers the cell specificity that fragment A does. It was possible that hidden effects might be displayed by further reducing the size of the fragment tested. Therefore, we expanded this analysis with reporter constructs using nine different overlapping segments (i1–i9) of ~200 bp each (Fig. 1Go) derived from fragment A. Each of these segments was amplified from intron 1 by PCR and cloned into pCD21Basic. These constructs were then transiently transfected into 2PK3 and EL4 cells, and luciferase assays were performed (Fig. 3Go). Segments i1–i5 (together constituting the Aa fragment) did not demonstrate the repressor activity that was clearly evident with the full-length Aa fragment. Segment i4 proved refractory to several cloning attempts due to a repetitive motif. However, pCD21Basic i3–i5, which contained the i4 sequence, was cloned and transfected into 2PK3 and EL4 cells. Luciferase assay results were similar to those seen for the other Aa segments (data not shown). Similarly, segments i6–i9 (together constituting the Ab fragment) did not exhibit the enhancer activity that was clearly evident from full-length Ab. In fact, i7 and, to a lesser extent, i8, strongly repressed luciferase expression. These data suggest that fragment A represents a cell-specific regulatory unit that contains many sequences that coordinately are required for its proper function. If one or more of these sequences is altered, then so is the cell-specific expression of the CD21 gene.



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  		FIGURE 3. Luciferase assays of B and T cells transiently transfected with reporter constructs containing subfragments of CD21 intron 1.2PK3 (B cells) and EL4 (T cells) were transiently transfected as previously described. The graph shows the relative luciferase activities of CD21 promoter driven constructs (CD21Basic) with fragments of Aa (i1–i5) and Ab (i6–i9) inserted 3' of the firefly luciferase gene. Data are averages of five independent experiments.

 
CD21 fragment A contains many nuclear protein binding sites

The previous experiments demonstrated that functional CD21 regulatory elements exist in fragment A. To determine the existence and approximate locations of NF binding sites that may constitute these regulatory elements, we performed EMSA analysis with fragment A sequences (Fig. 4Go). The same segments of fragment A used in the functional assays (i1–i9) were incubated with nuclear extracts from 2PK3 (B cell) and EL4 (T cells), and binding was assayed by the ability of the nuclear extract to retard the electrophoretic movement of the DNA segment through a 4% polyacrylamide gel. Nuclear extracts from both 2PK3 and EL4 cells resulted in an electrophoretic shift for many of the segments. Most of these complexes, including those produced with i1–i4 and i7, appeared identical for both nuclear extracts, although the band shifts i1a, i2b, and i3a appeared stronger with the 2PK3 extracts than with the EL4 extracts. Some of these complexes, however, were unique to the nuclear extract used. Band shifts i5a, i8a, and i8b were produced only with the 2PK3 cell extracts. The complexes with i8 are particularly interesting because it includes the MHI region (Fig. 1Go). Both nuclear extracts failed to form complexes with segments i6 and i9 (data not shown). We did not observe any T cell-specific complexes with any of the segments. These data in combination with the reporter assays suggest that fragment A of intron 1 regulates the cell-specific expression of CD21 by binding factors present in both T and B cells as well as those uniquely present in B cells.



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  		FIGURE 4. EMSA analysis of CD21 fragment A. A total of 20,000 cpm of each labeled fragment (i1–i9) was incubated with or without 1–5 µg of nuclear extract from 2PK3 or EL4 cells for 15 min. The lettered arrows indicate shifts for each fragment. Fragments i6 and i9 produced no shift (data not shown).

 
Differential CD21 promoter EMSA patterns with B and T cell nuclear extracts

The data presented in Fig. 2Go indicated that the CD21 promoter is required for cell-specific regulation by fragment A sequences, suggesting that B cell-specific NF binding sites may be present within this promoter. To determine whether the CD21 promoter does possess differences in B and T cell nuclear extract binding, we performed EMSA analysis on the proximal 1200-bp promoter sequence. We generated six overlapping segments (p6–p1) from the CD21 promoter, each ~200 bp (Fig. 1Go). Potentially important sites for transcriptional control include an octamer binding site as well as a sequence with high homology to the human CD21 promoter, termed MHP, in p2. In addition to the TATA box, an SpI site is located in p1. Identical banding patterns were observed for both 2PK3 and EL4 nuclear extracts with segments p5 and p4, although the 2PK3 nuclear extracts appeared to produce a much stronger p4b complex than the EL4 nuclear extract (Fig. 5Go). Band shift complexes p3b, p2b, p1a, p1c, and p1d were also identical with both extracts. However, three other complexes appeared to be unique to the B cell nuclear extract: p3a, p2a, and p1b. Complex p2a confirms previous data in which the B cell nuclear extract, which contains both Oct-1 (ubiquitous) and Oct-2 (B cell-specific) transcription factors, produces two band shifts when it binds the octamer binding site located in p2 of the CD21 promoter (23). These data suggest that the CD21 promoter possesses regulatory elements capable of binding lymphoid and B cell -pecific transcription factors that may cooperate with fragment A regulatory elements to control cell-specific CD21 gene expression.



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  		FIGURE 5. EMSA analysis of the CD21 promoter. Binding reactions were performed as previously described. The lettered arrows indicate shifts for each fragment. Fragment p6 produced no shift (data not shown).

 
Differential DNase I susceptibility of CD21 regulatory elements in T and B cells: alteration by histone acetylation

We have previously shown that murine T cells can be induced to express CD21 by increasing the acetylation state of core histones (17). CD21 transcript and protein were detected in and on T cells, respectively, treated with the deacetylase inhibitor TSA. Others have shown that gene activation via histone hyperacetylation correlates with the structural remodeling of key regulatory elements. Disruption of a nucleosome positioned in the HIV-1 promoter by treatment of infected cells with TSA resulted in increased accessibility of the promoter to transcription factors, as measured by DNase I hypersensitivity (24). We used similar assays to determine whether the putative regulatory sites within the promoter and fragment A sequences of the CD21 gene were equally accessible in B and T cell nuclei and, if not, whether accessibility could be enhanced by histone acetylation.

We devised a novel assay based on previous protocols that combined in vivo DNase I treatment of genomic DNA via membrane permeabilization with PCR using primers to CD21 promoter and fragment A segments (DNase I HS PCR, Fig. 1Go) (21, 22). An increasing DNase I concentration correlated to a decreasing PCR product as the template was degraded. The PCR product was measured quantitatively using phosphorimaging techniques. The purpose of following this strategy was to define hypersensitive regions within 200-bp genomic fragments. This assay is faster, easier, and more sensitive than conventional DNase I HS protocols and requires 10,000 times less gDNA for analysis. Because of this increased sensitivity, this assay is capable of identifying hypersensitivity sites that were previously undetectable using more conventional assays (22).

Primers specific for ß-actin were used to control for gDNA quantity and as a negative control for TSA treatment (Fig. 6Go). Primers specific for exons encoding the third and fourth short consensus repeats (SCR3–4) were used to demonstrate the lack of DNase I hypersensitivity in a nonregulatory region of the CD21 gene. In both cases, PCR product was made from gDNA treated with as much as 10,000 U/ml of DNase I. To ensure the validity of the assay, we analyzed known B and T cell enhancer regions for DNase I sensitivity. The MHCIIA{alpha} enhancer controls the B cell-specific expression of the MHCIIA{alpha} gene and was shown to be hypersensitive to DNase I treatment in B cells (25). The TCRß enhancer controls the T cell-specific expression of the TCRß gene and was shown to be hypersensitive to DNase I treatment in T cells (26). In our assay the MHCIIA{alpha} enhancer was sensitive in B cells after treatment with 2000 U/ml of DNase I, but was still detected in T cells treated with as much as 10,000 U/ml of DNase I. Conversely, the TCRß enhancer was not sensitive in B cells treated with as much as 10,000 U/ml of DNase I, but was sensitive in T cells after treatment with as little as 2,000 U/ml of DNase I. Any DNA segment that was sensitive with the 2,000 U/ml DNase I treatment was termed ultrasensitive, and those sensitive with 10,000 U/ml were termed hypersensitive. Thus in Fig. 6Go, the p1 segment of the CD21 promoter is ultrasensitive to DNase I within the B cell nucleus, resistant in the T cell nucleus, and hypersensitive in the nuclei of T cells treated with TSA. Interestingly, the MHCIIA{alpha} enhancer became hypersensitive to DNase I treatment in T cells treated with TSA, suggesting that the MHCIIA{alpha} gene may also be positively regulated by histone hyperacetylation. Taken together, these data indicate that the assay is sensitive enough to assess DNase I accessibility to DNA regulatory elements.



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  		FIGURE 6. CD21 promoter and fragment A DNase I HS PCR. EL4 T cells were incubated with 100 nM TSA for 16 h. These cells and untreated 2PK3 B cells and EL4 cells were then permeabilized with 0.1% lysolecithin supplemented with increasing concentrations of DNase I (0, 500, 2,000, or 10,000 U/ml) and incubated for 4 min at room temperature. The cells were lysed, and genomic DNA was isolated from them and used as template for PCR with primers specific for the following sequences: ß-actin, the exons encoding the third and fourth short consensus repeat of murine CD21 gene (SCR3–4), the hypersensitive enhancer sequence responsible for cell-specific expression of the murine MHCIIA{alpha} gene in B cells, the hypersensitive enhancer region responsible for the cell-specific expression of the TCRß gene in T cells, and individual fragments of the CD21promoter (p1–p6) and fragment A (i1–i9). Data from p1 DNase I HS PCR are shown as an example of typical results from a hypersensitive region of CD21.

 
We further assessed the DNase I hypersensitivity of all CD21 promoter and fragment A segments, quantifying PCR band intensities by phosphorimage analysis to more accurately interpret the results. Most of the CD21 promoter was hypersensitive in B cells, where CD21 is transcriptionally active (Fig. 7Go). In B cells p6–p3 were hypersensitive, while p1, the sequence closest to the transcriptional start site, was ultrasensitive in B cells. Conversely, none of the CD21 promoters was hypersensitive in T cells. However, upon treatment of T cells with TSA, p6, p5, p3, and p1 all became hypersensitive. This trend continued when we analyzed CD21 fragment A segments; i2–i4, i7, and i9 were hypersensitive, while i8 was ultrasensitive. Interestingly, i9 was the only hypersensitive site identified in T cells. When T cells were treated with TSA, i2, i4, and i6–i8 became hypersensitive, while i9 was no longer hypersensitive (Fig. 8Go). Thus, the hypersensitivity profile of the CD21 gene in T cells changes dramatically when chromatin is remodeled, closely matching the B cell profile. These data suggest that chromatin structure tightly regulates accessibility to CD21 regulatory elements in a cell-specific manner, allowing for, in a permissive state, transcriptional induction.



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  		FIGURE 7. Phosphorimage analysis of CD21 promoter DNase I HS PCR. A photoreactive plate was exposed to radioactive PCR bands separated by PAGE, scanned, and analyzed using Bio-Rad phosphorimaging software. Quantified band intensity values were normalized by ß-actin values and relativized to band intensities for PCR using genomic DNA without DNase I treatment. Fragments that demonstrated identical hypersensitivity are shown as one plot. SD error bars are shown for only the first fragment listed for any given set. Data are the averages of at least three independent experiments.

 


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  		FIGURE 8. Phosphorimage analysis of CD21 fragment A DNase I HS PCR. Analyses were performed as previously described. Fragments that demonstrated identical hypersensitivity are shown as one plot. SD error bars are shown for only the first fragment listed for any given set. Data are the averages of at least three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We and others have previously shown that intronic elements control the cell-specific expression of both murine and human CD21 genes (7, 8). These data are similar to those for human and mouse CD4, which also demonstrated intronic control of cell-specific expression (10, 11). In this study we present functional data that expand our understanding of intronic and promoter regulatory components, and structural data that define factor binding sites and the accessibility of CD21 sequences in the nuclei of expressing and nonexpressing lymphocytes.

Previous analyses of CD21 intronic control were conducted only with the CD21 promoter (7). Enhancer function is simplistically defined as promoter independent, in that a defined enhancer element can increase the transcriptional activity of a variety of different promoters. Thus, the question was raised: can the CD21 intronic elements regulate the activities of other promoters or is the cell-specific expression control specific for the CD21 promoter? As shown in Fig. 2Go, fragment A, the first 1.6 kb of intron 1 that bestowed cell-specific expression of CD21 reporter constructs failed to direct appropriate expression of the reporter gene when controlled by an SV40 promoter. Only when the CD21 promoter was used in these constructs was the cell-specific expression restored, suggesting that specific protein/sequence associations must occur between the promoter and intronic elements for appropriate cell-specific control. Of future interest will be the creation of mutations within the CD21 promoter that maintain transcriptional activity in B cells but do not show suppression in T cells in the presence of the intronic control elements.

Separate enhancer and repressor functions have been identified within the two halves of fragment A. Neither of these segments alone retained the cell specificity of full-length fragment A. When the sequences that contain the CD21 intronic enhancer and silencer elements were further segmented, their respective functions were altered, suggesting that multiple enhancer and suppressor elements exist that cooperate for appropriate transcriptional control. Intronic sequences containing both enhancer and silencer elements have been described for many genes, including the murine keratin 18 gene (27) and the ß3 tubulin gene in Drosophila (28). In both these cases, appropriate gene expression required the combined activity of all positive and negative intronic regulatory elements as well as the endogenous promoter. The murine CD21 gene appears to require similar cooperation among its many regulatory sequences.

To further elucidate the mechanism by which intronic and promoter elements control the cell-specific expression of the murine CD21 gene, we mapped nuclear protein binding sites. EMSA analysis with T and B cell nuclear extracts revealed many factor binding sites. Additionally, there are multiple sites unique for B cell factor binding, but none specific for T cell binding (Fig. 9Go). While any or all of these binding sites may be important for modulating cell-specific gene regulation, those that demonstrate B cell-specific binding are of particular interest. In the intron, two of these specific band shifts (i8a and i8b) reside in an area containing ~80 bp of sequence homologous to human intron 1 that repressed reporter gene expression (Fig. 4Go). This region is found in the same relative location in both mouse and human first introns and has been shown to be part of a silencer in the human CD21 gene (8). Paradoxically, the entire Ab fragment, of which i8 is only a part, does not demonstrate suppression in similar reporter assays (Fig. 2Go). These data suggest that additional enhancer or suppressor inhibitory elements exist within this sequence.



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  		FIGURE 9. Summary of EMSA and DNase I HS PCR results. Highlighted regions adjacent to CD21 promoter (p1–p6) and fragment A (i1–i9) segments indicate regions of hypersensitivity as determined by DNase I HS PCR and phosphorimage analysis for 2PK3 and EL4 cells with or without TSA treatment. A dark-lined highlight indicates an ultrasensitive region. Lower case letters represent DNA/protein complexes identified by EMSA and correspond to the lettered arrows in Figs. 4Go and 5Go.

 
In the CD21 promoter region, three B cell-specific nuclear protein band shifts were identified, including one in the region containing the transcriptional start site (p1b, Fig. 5Go). A 200-bp region 5' to the TATA containing region also produced a B cell-specific complex (p3b). Between these two regions another B cell-specific band shift (p2b) was produced in a region in which we had previously identified an octamer binding site (23). This site, however, was not capable of binding the B cell-specific OcaB transcription factor and, therefore, would not alone confer B cell-specific CD21 gene expression (29, 30, 31, 32). A small region of homology to the human CD21 promoter, MHP, is located in p2, but no additional bands were seen that would suggest binding of additional NFs to this site.

Data collected from EMSAs reveal significant differences in the binding of components of B and T cell nuclear extracts to the CD21 promoter and intronic elements that may explain the cell-specific regulation of the gene. However, EMSAs only reveal the capability of nuclear proteins to bind naked DNA fragments and retard their movement, which may not be physiologically relevant. Performing an in vivo DNase I-hypersensitive PCR allowed us to investigate the accessibility of NFs to putative CD21 promoter and intronic regulatory elements identified by EMSAs. This assay proved to be sensitive enough to classify hypersensitivity to different degrees, which were arbitrarily defined based on the amount of DNase I treatment that prevented PCR amplification.

A strikingly different hypersensitivity profile was found in the CD21-expressing B cell compared with that of the nonexpressing T cell. Most of the promoter and fragment A was hypersensitive in B, but not T, cells. When we induced a hyperacetylated state in T cells, however, the hypersensitivity profile changed to one nearly identical with that seen in the B cell. DNase I hypersensitivity PCR data can be compared with the EMSA data to discriminate between potentially relevant and irrelevant bindings (Fig. 9Go). Those segments that displayed both binding by B cell-specific NFs and B cell DNase I hypersensitivity, including p1, p3, and i8, are of greatest interest because they contain sites that could potentially modulate the cell-specific expression of CD21. The lack of hypersensitivity in hyperacetylated T cells at p4 and i3 suggests that binding of NFs to these sites may not be required for CD21 expression. However, because hyperacetylated T cells express only about 50% of CD21 that B cells do, these binding events may serve a marginal enhancer function.

We have shown previously that inducing hyperacetylation in nonexpressing murine fibroblasts failed to induce CD21 expression, unlike the induction of T cell to express CD21 with the same stimulus (17). This finding suggested that B and T cells, which share the same lymphoid lineage, possess the necessary transcription factors to express CD21, while nonlymphoid cells do not. This represents one level of CD21 transcriptional control in which cell- or lineage-specific transcription factors are required for CD21 expression. In a parallel system, Romanow and colleagues recently demonstrated transcription-induced rearrangement of Ig genes in nonlymphoid cells following introduction of the V(D)J recombinase and E2A/EBF transcription factors (33). Thus, identification and introduction into fibroblasts of factors controlling CD21 expression may allow for CD21 transcription, especially when histone deacetylase inhibitors are present.

Another level of CD21 transcriptional control must involve regulating access to transcription factor binding sites by modulating chromatin structure. This could be accomplished by recruiting sequence-specific deacetylases to CD21 regulatory regions. A model system of this kind has been described for the mammalian corepressor mSin3A, which recruits histone deacetylases to regulatory elements in promoters (34, 35, 36, 37, 38, 39). Recently, the B cell maturation regulator Blimp-1 and the lymphoid differentiation regulator Ikaros were both shown to repress transcription by recruiting deacetylases to promoters (40, 41). Blimp-1 and Ikaros are zinc finger NFs that bind specific DNA regulatory sites in distinct complexes. Ikaros has been shown to interact with mSin3 in vivo and in vitro, while Blimp-1 interacts directly with a histone deacetylase. Because we have shown that murine T cells express CD21 when their core histones are hyperacetylated (17), a key question to address in the future is whether any of the proteins demonstrated to bind to CD21 regulatory sites recruit or are themselves proteins that could alter the state of histone acetylation.

Finally, murine CD21 shares an expression profile similar to those of other B cell-specific proteins, including CD19, with which it forms a signal transduction complex, CD20 and CD22 (42). The MHC class II A{alpha} gene also exhibits a similar expression pattern as CD21, in that it is expressed on B, but not T, cells (25). We used the cell-specific enhancer of MHC class II A{alpha} as a positive control for DNase I hypersensitivity (Fig. 6Go). This enhancer has been previously shown to be hypersensitive in B, but not T, cells. Interestingly, when a hyperacetylated state was induced in murine T cells, the enhancer became hypersensitive. These data suggest a conserved regulatory mechanism for at least two and perhaps other genes with similar spatial and temporal expression patterns. The identification of specific transcription factors that modulate the chromatin structure in CD21, therefore, may illuminate a conserved function for those proteins in the regulation of other B cell-specific genes and provide insight into the role that transcriptional regulation plays in the biological function of the B cell.


   Acknowledgments
 
We thank the members of the Weis laboratories for insightful discussions. We appreciate the assistance of Heping Hu and Christina Gibson with this work. We thank Laurie Glimcher for pointing out the DNase-hypersensitive site within the MHCIIA{alpha} gene.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI24158 and AI42032 (to J.H.W.), an award from the American Lung Association (to J.H.W.), National Institutes of Health Grants AI32223 and AR43521 (to J.W.), funds from the Center of Excellence in Hematology (Grant DK-49219), the Huntsman Cancer Institute, and National Cancer Institute Grant 5P30CA-42014. Back

2 Address correspondence and reprint requests to Dr. John H. Weis, 50 North Medical Drive, Room 5B128SOM, Salt Lake City, UT 84060. Back

3 Abbreviations used in this paper: TSA, trichostatin A; HS, hypersensitivity; gDNA, genomic DNA. Back

Received for publication May 31, 2000. Accepted for publication July 24, 2000.


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 Introduction
 Materials and Methods
 Results
 Discussion
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