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Lymphoid Transcription of the Murine CD21 Gene Is Positively Regulated by Histone Acetylation¹

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

	Abstract

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Intronic transcriptional control sequences influence the cell- and tissue-specific expression of the CD21 gene. The interactions of such intronic control sequences, which are physically separated from the gene’s promoters, suggest that factors that alter chromatin structure might be influential in this process. Accordingly, we analyzed the effect of histone acetylation on the expression of CD21 in nonexpressing T and B lymphocytes, respectively. The acetylase inhibitors sodium butyrate and trichostatin A were used to create hyperacetylated histones. The CD21 gene was specifically activated in the previously transcriptionally quiescent cells in a time- and dose-dependent fashion: the expression of a number of other genes was not influenced. These data suggest a model of cell-type-specific deacetylase activity that serves to repress gene transcription when present and active.

	Introduction

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The murine complement receptor type 2 (Cr2/CD21) gene, located near the telomere of chromosome 1, is a member of the regulators of complement activation gene locus (1). It encodes two alternatively spliced mRNA that produce two distinct protein products (2, 3). The gene is transcriptionally active in maturing murine B and follicular dendritic cells (FDC)³ (4), but inactive in nearly all other cells, including murine T cells (5).

Two distinct domains of the murine CD21 gene have been defined to be important for cell- and tissue-specific expression. Analysis of the 5' promoter sequence of murine CD21 has identified multiple transcription factor binding sites, including octamer, AP-1,2, and an IgE enhancer sequence (6). In addition, a sequence within the promoter, homologous to the human CD21 gene, has been identified. While these promoter sequences do drive transcription, they do not confer tissue specificity. Fusion of either the murine or human CD21 promoter sequences 5' of reporter constructs provides for virtually equivalent expression of the reporter in T and B cells (7, 8, 9).

Analyses of the expression of the murine and human CD4 genes demonstrated a similar expression pattern. In that case, the gene’s promoter and enhancer sequences were shown to be critical for transcription, but did not confer cell specificity (10). Expression constructs possessing CD4 5' sequences were equally effective at promoting transcriptional activity in both CD4⁺ T cells and CD8⁺ (CD4^-) T cells. Only when constructs that contained intronic sequences (a putative transcriptional silencer) were analyzed was the correct tissue-specific expression pattern observed (11, 12, 13)

Based upon the CD4 model system, we utilized the intronic sequences of the murine CD21 gene within reporter constructs and found specific regions in the first intron that conferred tissue specificity (8). This effect was localized to a small region of the intron, and apparently consisted of both enhancer as well as suppressor elements. Similar data was also generated for the human CD21 gene, suggesting that the mechanism of expression control has been conserved during mammalian evolution (9). These data have raised the mechanistic question of how can two or more distinct genetic elements separated in the gene by >1000 bp be brought together to coordinately control transcriptional activation. Presumably, such an interaction would require an alteration in the structure of the chromatin.

Recent work has focused on the effect of chromatin structure on gene transcription. Chromatin remodeling has long been suggested as a mechanism by which transcription can be regulated (reviewed in Refs. 14, 15). Strict nucleosomal positioning regulates chromatin structure. Acetylation states of core histones H3 and H4 directly influence nucleosomal positioning. Acetylating lysine residues on the N termini of core histones negatively charges the lysine residues, neutralizing positive charges, thus weakening interactions with the phosphate backbone of DNA. This diminishes chromatin associations and presumably facilitates access of transcription factors to the "open" DNA. The best-characterized examples of the relationship between chromatin structure and transcriptional regulation is found in telomeric and mating type loci silencing in yeast (16, 17). A direct mechanistic correlation between histone acetylation and gene expression was established when the gene for p55, the catalytic subunit of a tetrahymena histone acetyltransferase, was cloned and identified as a homologue of Gcn5p, a yeast adapter protein that is required for full activity of a group of transcription factors (18).

If an alteration in chromatin structure is required for the appropriate expression of CD21 in developing B cells and FDCs, this may be facilitated by the state of histone acetylation. To determine the effect of acetylation on CD21 transcription, we used the deacetylase inhibitors sodium butyrate (19, 20) and trichostatin A (TSA) (21), a Streptomyces-derived fungal antibiotic with very potent and specific deacetylase inhibitor activity, to create a hyperacetylation state in murine T cells. CD21 expression in such treated cells was then analyzed via FACS and RT-PCR. Murine T cells with hyperacetylated histones expressed the CD21 gene in a time- and dose-dependent manner. This induction was specific for CD21 in T cells and was not observed in nonlymphocyte cell lines. These data suggest that intronic elements may play a role in regulating gene expression by a generalized function involving nucleosomal positioning.

	Materials and Methods

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Cell lines and culture conditions

Mouse 2PK3 and A20 cells, CD21-expressing B lymphomas, were obtained from the American Type Culture Collection (ATCC; Manassas, VA) and maintained at 37°C, 5%CO₂ in DMEM (Life Technologies, Bethesda Research Laboratories, Gaithersburg, MD) with 10% FCS (HyClone Laboratories, Logan, UT) and Pen-Strep (Life Technologies). Mouse EL4 and TK.1 cells, CD21-nonexpressing lymphomas (ATCC), were grown in RPMI 1640 (Life Technologies) with 10% FCS and Pen-Strep. Mouse L cell, a fibroblast, was grown in RPMI 1640 with 10% FCS and Pen-Strep. Splenocytes and thymocytes were isolated from adult NIH outbred mice and maintained in single cell suspensions with DMEM with 10% FCS and Pen-Strep. Where indicated, cells were treated with 10, 50, 100, and 500 nM concentrations of TSA (Sigma, St. Louis, MO) and 200 µg/ml cyclohexamide in media; or with 0.01, 0.1, 1, 10, or 100 mM concentrations of sodium butyrate (Sigma) in media for 0.5, 1, 1.5, 2, 4, 8, 10, 12, 16, or 20 h.

FACS analysis

The following mAbs were prepared at the University of Utah core facility: FITC-conjugated rat anti-mouse B220, PE-conjugated rat anti-mouse CD3, FITC-conjugated rat anti-mouse CD4, and FITC-conjugated rat anti-mouse IgG2b. FITC-conjugated CD21 7G6, a rat anti-mouse CD21 mAb, was purchased from PharMingen (San Diego, CA). Abs were incubated with cells at 4°C for 1 h in the presence of 1/50 dilution of mouse serum. Incubations were followed by two washes in the staining buffer (0.1% BSA in 10% PBS). Samples were analyzed with a FACScan flow cytometer, and data were analyzed using CellQuest software (Becton Dickinson, Mountain View, CA).

RNA preparation and cDNA synthesis

Total RNA from cells was isolated using the CsCl guanidine method (22). Briefly, cells were resuspended in 4 M guanidinium solution and disrupted in a tissuemizer homogenizer. RNA was isolated by ultracentrifugation at 30,000 rpm over a 5.7 M cesium chloride gradient overnight. RNA was resuspended in water and quantified by measuring A₂₆₀ absorbance. cDNA was synthesized by mixing 5 µg of RNA, 10 µl of 5x first strand buffer, 5 µl of 5 mM dNTP, 5 µl of 0.1 M DTT, 2 µl of 1.25 mM random primers (Life Technologies), 2 µl of Maloney murine leukemia virus reverse transcriptase and water to final volume of 50 µl. The reaction mixture was incubated at 37°C for 1 h. A total of 2 µl of DNase-free RNase (1 mg/ml) was then added, and the reaction mixture was incubated for an additional 5 min followed by cDNA isolation using the Qiaquick PCR purification kit (Qiagen, Chatsworth, CA)

PCR

PCR using radioactive detection was performed with 10 µl reactions containing 200 ng cDNA, 70 pM of each primer, 0.72 U Amplitaq DNA polymerase (Life Technologies), 0.8 mM dNTP, 1x Taq buffer (50 mM Tris (pH 8.3), 3 mM MgCl₂, 20 mM KCl, and 500 mg/ml BSA), and 2.5 mCi [³²P]dCTP. Samples were loaded into capillary tubes and incubated in an air thermocycler (Idaho Technology, Idaho Falls, ID) for denaturing at 94°C for 1 s, for annealing at 59°C for 1 s, and for extension at 72°C for 3 s (23). This cycle was repeated 18 times for amplification of ß-actin transcripts and 22 times for amplification of CD21 transcripts. After amplification, 5 µl of stop solution (United States Biochemical, Cleveland, OH) was added to the reactions, and 5 µl was resolved in a 6% acrylamide sequencing gel, which was then dried and exposed to BXR medical x-ray film (Western X-Ray, Salt Lake City, UT) overnight.

PCR using fluorescence detection was performed with 10 µl reactions containing 20 ng cDNA, 5 µM of each primer, 0.5 U Amplitaq DNA polymerase, 110 ng TaqStart Ab (Clontech Laboratories, Palo Alto, CA), 0.8 mM dNTP, 1x light cycler buffer (3 mM MgCl₂, 50 mM Tris-HCl (pH 8.3), 500 mg/ml BSA), and 1:30,000 dilution of SYBR Green I (Molecular Probes, Eugene, OR). Samples were loaded into capillary tubes and incubated in a fluorescence thermocycler (LightCycler LC24; Idaho Technology) for denaturing at 94°C for 0 s, for annealing at 62°C for 0 s, and for extension and fluorescence detection at 72°C for 3 s. This cycle was repeated 40 times, preceded by an initial incubation at 94°C for 60 s and followed by a melting curve acquired by cooling to 60°C and slowly heating at 0.2°C/s to 94°C, with fluorescence data collected at 0.2°C intervals (24).

CD21 transcript were amplified using primers 43 (5'-ATG GGA TCC TTG GGT TCG CTC-3') and 45 (5'-GCT AGG TGA ACA AGT GTA CCT-3'). CD19 transcripts were amplified using primers 945 (5'-GCT ACC ATG CCA TCT CCT CTC C-3') and 947 (5'-ATC CTC CAC GTT CAC TGT CCA G-3'). CD3 transcripts were amplified using primers 949 (5'-GGG TTT GAC GTC TCC TTT GCT C-3') and 950 (5'-GGC CTC TTC CTG GTG ATC TCT C-3'. ß-actin transcripts were amplified using primers 62 (5'-ATT GAA CAT GGC ATT GTT AC-3') and 339 (5'-CTC TAT CGT GGG CCG CTC TAG-3').

Relative transcript level calculation

Light cycler PCR data were analyzed using LightCycler Data software (Idaho Technology). The software first normalizes each sample by background subtraction of initial cycles. A fluorescence threshold is then set at 5% full scale, and the software determines the cycle number at which each sample reached this threshold. The fluorescence threshold cycle number correlates inversely with the log of initial template concentration. ß-actin transcript levels were used to normalize the amount of cDNA in each sample, and CD21 transcript levels were reported relative to levels found in 2PK3 B cells. Since amplification occurs logarithmically, a difference in cycle threshold of one correlates to an 2-fold difference in relative transcript level. Melting curve profiles were used to confirm amplification of specific transcripts.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD21 expression induction in T cells by deacetylase inhibitors

It has previously been shown that expression of a small proportion of cellular genes can be influenced by acetylation states of core histones (25). We therefore investigated whether the expression of the murine CD21 gene could be influenced (i.e., up-regulated) in nonexpressing T cells by altering the acetylation state of histones. Treatment of various cell lines with as little as 33 nM of the deacetylase inhibitor TSA has been shown to significantly hyperacetylate all core histones (21). This concentration is five orders of magnitude lower than the similarly effective dose reported for the deacetylase inhibitor sodium butyrate (21). Toxicity of both inhibitors relates to G₁ and G₂ cell cycle arrest at concentrations starting at 70–100 nM for TSA and 10 mM for sodium butyrate.

We treated the murine T cell line EL4 with 100 nM TSA and 10 mM Sodium Butyrate and stained for CD21 expression on the cell surface (Fig. 1A). The murine B cell line 2PK3 expresses CD21 on its surface without treatment, while the EL4 cell line does not. Upon treatment with Sodium Butyrate and TSA, EL4 cells express CD21. Therefore, hyperacetylation of histones resulted in the cell surface expression of CD21 in T cells.

View larger version (33K): [in this window] [in a new window]   FIGURE 1. Effects of deacetylase inhibitors on CD21 expression. A, FACS analysis of CD21 expression. Panels 1 and 2, Untreated 2PK3 and EL4 cells, respectively. Panels 3 and 4, EL4 cells treated for 16 h with 100 nM TSA and 10 mM sodium butyrate, respectively. Cells were stained with anti-IgG2b (filled peak), anti-CD21 7G6 (thick-lined peak), anti-B220 (thin-lined peak for 2PK3 cells), and anti-CD3 (thin-lined peak for EL4 cells) Abs, and FACS analyzed. B, RT-PCR of mRNA from hyperacetylated cells. 2PK3 and EL4 cells were incubated for 4 or 20 h with or without 100 or 500 nM TSA. RNA from these cells was reverse transcribed using random primers, and the cDNA was analyzed for CD21 transcripts with specific primers and rapidly amplified by Taq polymerase in capillary tubes using an air thermocycler (Idaho Technologies). ß-actin primers were used to control for cDNA quantity. 32P-labeled PCR reactions were visualized by PAGE and overnight exposure to x-ray film. C, RT-PCR of mRNA from cells treated in hyperacetylation time course. EL4 cells were incubated for the indicated time points with 100 nM TSA. RT-PCR was performed as previously described. Untreated 2PK3 and EL4 cells were used as controls. Data in this figure are representative of three distinct experiments.

Detection of CD21 transcript using continuous monitoring PCR

CD21 transcript was detected in the previous experiments by RT-PCR using radiolabeled nucleotide and autoradiography (23). In the following experiments, we used a variation of this technique in which a fluorescent dye that binds dsDNA is used instead of the radiolabeled nucleotide (24). This method continually analyzes the product during the cycle set and provides more sensitive detection of low levels of product than the radioactive protocol.

EL4 cells were treated with TSA, and total cellular RNA was isolated and reverse transcribed as in previous experiments. PCR products were detected and quantified by continuous monitoring PCR using detection threshold as an indication of relative starting transcript numbers. As shown (Fig. 2A), fluorescence increases as the cDNA is amplified. The cycle number at which fluorescence can be detected was used to determine the amount of transcript present in each sample relative to that found in 2PK3 cells. Plotting the log fluorescence vs cycle number more clearly defines this cycle threshold. In this panel, 2PK3 cells, EL-4 cells, and TSA-treated EL-4 cells had equivalent levels of ß-actin transcripts. Virtually identical amplification profiles for CD21 transcripts were generated for 2PK3 and TSA-treated EL-4 cells. No CD21 transcripts were detected in the untreated EL-4 sample in the 40 cycle profile.

View larger version (26K): [in this window] [in a new window]   FIGURE 2. Continuous monitoring PCR analysis of transcript levels in hyperacetylated cells. 2PK3 and EL4 cells were treated with 100 nM TSA for 16 h, mRNA isolated, and reverse transcribed, as previously described. cDNA transcripts were amplified in the presence of Sybergreen (Molecular Probes) and continuously monitored using an air cycler with fluorescence detection (Idaho Technologies). A, Log fluorescence vs cycle number, which defines cycle threshold. Curves showing cycle thresholds of 16 represent ß-actin transcripts amplified from cDNA from EL4 cells, TSA-treated EL4 cells, and 2PK3 cells. Curves showing cycle thresholds of 25 and 26 represent CD21 transcripts amplified from cDNA from 2PK3 cells and TSA-treated EL4 cells, respectively. No CD21 transcript was amplified from cDNA from untreated EL4 cells. B, ß-actin-normalized values for CD21 transcript levels in a repeat of the time course assay represented by Fig. 1C. Data are representative of three experiments.

To ensure that induction of the CD21 gene in murine T cells was not due to the TSA-induced expression of a transcription factor required for CD21 transcription, we incubated EL4 cells with 100 nM TSA and 200 µg/ml cyclohexamide, which blocks de novo protein synthesis. We also performed a shorter time course to more clearly define the point of CD21 gene induction by TSA. Addition of cyclohexamide had no effect on the induction of CD21 transcription in EL4 cells, which produced transcript in as little as 90 min after TSA treatment (Fig. 3).

View larger version (28K): [in this window] [in a new window]   FIGURE 3. Effect of protein synthesis inhibition on TSA induced CD21 transcription. EL4 cells were incubated with 100 nM TSA for the indicated time points with or without 200 µg/ml cyclohexamide. Continuous monitor RT-PCR was performed as previously described. Untreated 2PK3 and EL4 cells were used as controls. Data are representative of three analyses.

The preceding experiment demonstrated that continuous PCR monitoring can efficiently and effectively determine relative CD21 transcript levels. TSA was previously shown to induce CD21 transcription in EL4 cells in a time-dependent manner. As shown, deacetylase inhibitors induced CD21 transcription in a dose-dependent fashion (Fig. 4). TSA induced transcription of CD21 by EL4 cells starting at a concentration of 10 nM, with maximal induction at 500 nM (Fig. 4A). Sodium butyrate induction of CD21 transcription by EL4 cells was seen at 10 nM, with maximal induction at 100 mM (Fig. 4B). These data suggest that the extent of histone hyperacetylation influences the degree to which the CD21 gene is transcribed in T cells.

View larger version (24K): [in this window] [in a new window]   FIGURE 4. Dose effect of histone acetylation on transcript levels. EL4 cells were incubated overnight with the indicated concentrations of TSA (A) and sodium butyrate (B). Continuous monitor RT-PCR was performed as previously described. Untreated 2PK3 and EL4 cells were used as controls. Data are representative of three experiments.

The induction of CD21 transcription by deacetylase inhibitors was shown in the previous experiments to be time- and dose-dependent. We were concerned as to the specificity of this induction. First, was this induction specific for the CD21 gene in T cells, or were other quiescent genes also activated? Second, could other, nonlymphocytic cells be induced to express CD21 after histone hyperacetylation? To address these questions, a variety of cell types were treated with TSA, and continuous monitoring PCR was performed using primer sets for additional genes. TSA treatment of EL4 cells did not induce the expression of CD19, a gene expressed specifically in B cells (Fig. 5A). Expression of CD3, a gene specifically expressed in T cells, was also unaffected. Additionally, TSA treatment induced CD21 transcription in TK1 cells, a T cell line, but not in L cells, a fibroblast cell line (Fig. 5B). Finally, TSA treatment increased CD21 expression 4-fold in A20 cells, a pre-B cell line. These data demonstrated that induction of CD21 gene expression by histone hyperacetylation was highly selective for the CD21 gene in lymphoid cell lines.

View larger version (42K): [in this window] [in a new window]   FIGURE 5. Gene- and cell-specific induction of CD21. A, The effect of hyperacetylation on T and B cell-specific transcription. Cells were treated for 16 h with 100 nM TSA. Relative transcript levels were determined by continuous monitor PCR, as previously described. CD21 and CD19 are B cell-specific transcripts. CD3 is a T cell-specific transcript. B, The effect of hyperacetylation on CD21 expression in different cell types. Relative transcript levels were determined by continuous monitor PCR, as previously described. 2PK3 cells are mature B cells. A20 cells are pre-B cells. EL4 and TK1 cells are T cells. L cells are fibroblasts. Data are representative of three experiments.

The data presented thus far have been generated using transformed cell lines that display characteristics of immature cells. We were interested to observe the effects of TSA on CD21 gene expression in splenocytes and thymocytes. Accordingly, single cell suspensions were prepared from spleens and thymuses isolated from mice. Splenocytes and thymocytes were treated with TSA and analyzed for protein and transcript. No increase in CD21 cell surface expression was observed in either splenocytes or thymocytes (Fig. 6A). However, CD21 transcript levels did increase significantly in thymocytes after treatment (Fig. 6B). Paradoxically, CD21 transcript levels decreased significantly in splenocytes after treatment. While the induction of CD21 transcription in thymocytes would be expected from the previous experiments, the suppression of transcription in splenocytes was not. One difference between the spleen and thymus is that the spleen has a large percentage of mature, end-stage cells, while the thymus does not. It may be that maturing lymphocytes are more sensitive to the effects of histone acetylation than mature, committed cells. The 4-fold increase of CD21 transcription by the pre-B cell line A20 (Fig. 5) after TSA treatment supports this view.

View larger version (39K): [in this window] [in a new window]   FIGURE 6. Effect of hyperacetylation on CD21 expression in primary cell culture. Splenocytes and thymocytes were treated for 16 h with 100 nM TSA and assayed for protein and transcript, as previously described. A, CD21 expression on splenocytes (counterstained with B220) and thymocytes (counterstained with CD3) by FACS. B, CD21 transcription by continuous monitor PCR. Data are averages of six experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The transcriptional activation of the CD21 gene in T cells is specific for this gene in that other, transcriptionally silent genes were not similarly activated. These data do not suggest, however, that the CD21 gene is the only gene within these cells that is activated by such treatment. Previous screening analyses have found that up to 2% of cellular genes can be similarly activated via histone hyperacetylation (25). Thus, the CD21 gene must be a member of a select subset of genes whose transcriptional control is thus influenced by hyperacetylation.

The induction of CD21 expression was shown to be dependent upon treatment of the correct cell type. T and B cells share many common lymphoid transcriptional control proteins, which are absent in other cell lines, such as fibroblasts. The lack of CD21 activation in fibroblasts following histone hyperacetylation suggests that both T and B cells possess the required transcriptional control apparatus proteins to transcribe CD21, but that such transcription is inhibited in T cells. Our previous mapping of specific transcriptional suppressor/silencing elements within the first intron of the CD21 gene supports this view (see below) (8).

The CD21 gene is expressed in a subset of cells (B cells and FDCs) and is under the transcriptional control of promoter and intronic sequences. We have found at least two silencer/suppressor sites within the murine CD21 intronic sequence (8), and Makar et al. (9) have proposed the presence of intronic silencer sites within the human CD21 first intron. These data, in total, suggest that the positioning of silencing elements within the first intron several hundred to thousands of base pairs from the promoter region, places them in a region of the gene in which they exert their effect via chromosomal alterations. The general question can thus be asked whether or not histone hyperacetylation similarly affects other genes that possess such intronic silencer regions.

The role that the state of histone acetylation has upon gene transcription has been an actively investigated area of study. Histone hyperacetylation itself (acetylation of lysine residues) has been proposed to lead to an increased level of transcription by opening up the chromatin for the insertion of transcription factors (reviewed in Ref. 26). How such hyperacetylation is accomplished falls into two contrasting models. The first is that promoter TFIID complexes recruit specific acetyl transferases (27, 28). Such transferases allow for an increased level of acetylation of histones holding the promoter region of the gene, allowing for the recruitment of additional control factors and eventual up-regulation of the gene. This model does not appear to fit that of the CD21 gene. The lack of expression of CD21 in T cells does not appear to be due to the lack of an effective transcriptional activation complex at the promoter region, but instead due to a repression of transcription via the intron control sequences.

The second model to explain histone hyperacetylation control of gene transcription is based upon the model of recruiting sequence-specific histone deacetylases. A number of systems have demonstrated that transcriptional inhibition can be mediated by the recruitment of a deacetylase via a sequence-specific DNA binding factor (29, 30, 31, 32, 33, 34, 35). Once in place, the deacetylase removes acetyl groups on the histones, thus inhibiting the entrance of proteins that bind to enhancer and promoter elements. These systems have described the effect of deacetylase recruitment within promoter sites, while the CD21 model would suggest analogous intron control.

The second model of deacetylase recruitment fits with the model system of CD21, which we previously proposed (8). We demonstrated the existence of at least one transcriptional enhancer sequence and two silencer elements within fragment A of the first intron of the CD21 gene (Fig. 7). Previously, we could not predict the nature of the proteins that would bind the silencer elements: we can now propose that they may be histone deacetylases. The binding of such deacetylases, in a T cell-specific fashion, would silence the activity of the enhancer element. This enhancer element is equally active in T and B cells (our unpublished observations). The repression gained by the deacetylase would be abrogated by the addition of TSA or sodium butyrate into the cell. The repressive effect of histone deacetylases has, in yeast systems, a limitation of only a few hundred base pairs (32, 36). Thus, the presence of an enhancer element close to the sites of suppression (i.e., deacetylase binding) would be required. The enhancer element we have described is within this functional range of chromatin. Thus, the key pertinent question to address in the future is the identification of the proteins that affect this transcriptional repression, and the determination of whether or not they are, or are linked to, a histone deacetylase.

View larger version (21K): [in this window] [in a new window]   FIGURE 7. Model for the cell-specific expression of the CD21. Abbreviations are as follows: HDAC, T cell-specific histone deacetylase; S, suppressor binding site; E, enhancer binding site; BA, B cell-specific activator; Enhancer, an intron site-specific binding factor that up-regulates CD21 transcription. A detailed description of the model is in Discussion.

	Acknowledgments

 
We thank Heping Hu for help and timely advice in the early stages of this work.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants AI-42032 and AI-24158 (to J.H.W.), AI-3223 and AR-43521 (to J.J.W.), an award from the American Lung Association (to J.H.W.), and funds from the Center of Excellence in Hematology, Grant DK-49219. This work was also supported by the Hunstman Cancer Institute and National Cancer Institute Grant 5 P30 CA-42014.

² Address correspondence and reprint requests to Dr. John H. Weis, Department of Pathology, University of Utah School of Medicine, 50 North Medical Drive, Salt Lake City, UT 84132. E-mail address:

³ Abbreviations used in this paper: FDC, follicular dendritic cell; TSA, trichostatin A.

Received for publication February 17, 1999. Accepted for publication June 21, 1999.

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