International Immunology

This Article Abstract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (15) Request Permissions Google Scholar Articles by Makar, K. W. Articles by Holers, V. M. Search for Related Content PubMed PubMed Citation Articles by Makar, K. W. Articles by Holers, V. M. Social Bookmarking          What's this?

International Immunology, Vol. 13, No. 5, 657-664, May 2001
© 2001 Japanese Society for Immunology

A site in the complement receptor 2 (CR2/CD21) silencer is necessary for lineage specific transcriptional regulation

Karen W. Makar^1,4, Daniela Ulgiati², James Hagman³ and V. Michael Holers^1,2

¹ Departments of Immunology and
² Medicine, Division of Rheumatology, University of Colorado Health Sciences Center, Denver, CO 80262, USA
³ Department of Immunology, National Jewish Medical and Research Center, Denver, CO 80206, USA

Correspondence to: V. M. Holers, Division of Rheumatology, Box B-115, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262, USA

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Expression of human complement receptor type 2 (CR2/CD21) is primarily restricted to mature B cells and follicular dendritic cells. We previously described an intronic transcriptional silencer that controls the appropriate B cell-specific and developmentally restricted expression of human CR2/CD21 in both stably transfected cell lines and transgenic mice. Here we report the identification of a nucleotide sequence within the 2.5 kb CR2 silencer (CRS) that is crucial to its silencer function. This site comprises a binding site for the transcriptional repressor CBF1 (RBP-J or RBP-J) as well as Sp1 and other as yet uncharacterized proteins. A 2-bp mutation which eliminates the binding of CBF1 and other protein(s) in vitro results in loss of silencer activity in vivo. These results demonstrate the importance of this site in regulating CR2 expression and suggest that CBF1, a component of the developmentally important Notch signaling pathway, may play a role in the control of human CR2 gene expression.

Keywords: B lymphocytes, CBF1, cellular differentiation, complement, gene regulation, transcription factors

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Complement receptor type 2 (CR2/CD21) binds the C3d fragment of complement C3. CR2 can form a complex with CD19 and CD81 in the B cell membrane to function as a co-receptor that enhances signals through the BCR (1). CR2 is an important component of the immune system because it is required for the generation of a robust immune response to T-dependent antigens (2–4). Additionally, CR2 plays a role in the survival of germinal center B cells (5).

Expression of CR2 within the B cell lineage is tightly regulated. CR2 is displayed on mature B cells at approximately the same stage as IgD and CD23 (6,7). Pre-B cells, early immature B cells and late stage plasma cells all lack CR2 expression. CR2 is one of the first proteins up-regulated after B cells escape negative selection and migrate to the periphery (8–10). Because CR2 has important immunological functions and its expression pattern is temporally regulated during B cell development, we have investigated the transcriptional regulation of the human CR2 gene.

We have previously shown that the cell type-specific and developmentally restricted pattern of human CR2 expression is controlled by an intronic transcriptional silencer that we designated the CRS (for CR2 Silencer) (11). By using reporter constructs containing the CR2 proximal promoter with or without the CRS in a stable transfection assay system as well as transgenic mice, we showed that the CRS can repress transcription driven by the CR2 proximal promoter in CR2^– cells and tissues. This repression was only seen when reporter constructs containing these elements were stably integrated into the genome. We demonstrated that transcriptional silencing via the CRS occurs in CR2^– cells of either B or non-B lineages, indicating that the CRS was responsible for mediating both the tissue-specific and the developmentally restricted expression of human CR2. In the absence of the CRS, the CR2 proximal promoter is active regardless of endogenous CR2 expression (11). In more recent studies using the complete 45 kb human CR2 gene to generate transgenic mice, we observed human CR2 expression only in the B cell lineage, further supporting a role for the CRS as a relevant in vivo mechanism for controlling lineage specificity (4).

The transcription of human CR2 can be specifically up-regulated by Epstein–Barr virus (EBV) nuclear antigen (EBNA) 2, an EBV protein important in cellular immortalization (12,13). EBNA2 does not bind directly to DNA, but is targeted to responsive promoters by a ubiquitously expressed cellular protein named CBF1 (C-promoter binding factor 1) or RBP-J. The CBF1/RBP-J protein is highly conserved through evolution and has been shown to function in the Notch signaling pathway. Upon ligand binding, the Notch receptor is cleaved and its activated intracellular portion translocates to the nucleus where it interacts with CBF1 to mediate changes in gene expression (14). Notch signaling has been shown to be important in the development of many cell lineages. In T cells, Notch influences developmental choices between ß and T cells, and also between CD4 versus CD8 T cells (15,16). Other studies have shown that Notch signaling affects earlier stages of hematopoiesis by influencing the choice between T and B cell development (17,18).

It has been proposed that EBNA2 is the functional equivalent of an activated Notch receptor. Both EBNA2 and activated Notch mediate changes in gene expression through interaction with the transcription factor CBF1 (19). EBNA2 can functionally replace an activated form of Notch1 and competes with Notch for binding to RBP-J, indicating that interaction sites on RBP-J partially overlap (20). A recent study by Strobl et al. demonstrated that an activated form of Notch induces many of the same changes as EBNA2 in Burkitt lymphoma-derived B cell lines (21). Most importantly, both activated Notch and EBNA2 up-regulated expression of the endogenous CR2 gene at the mRNA and protein level. These observations demonstrate the importance of the Notch signaling pathway in the regulation of CR2 expression.

Because Notch and EBNA2 both up-regulate human CR2 expression, and because both interact with the transcription factor CBF1, we sought to determine if CBF1 plays a role in the transcriptional regulation of CR2. We first searched for potential CBF1 binding sites within the 2.5 kb CRS, which controls the tissue-specific expression of CR2. Sequence analysis revealed a site containing a perfect heptamer match with the CBF1 consensus binding site. In this report, we now show that CBF1 and additional nuclear factors, including Sp1, bind to this site in vitro in a sequence specific manner. Importantly, when this single site within the 2.5 kb CRS is mutated and protein–DNA interactions are lost, the silencing activity of the entire CRS is eliminated in a non-B cell line. These results demonstrate a crucial role for this site in the transcriptional regulation of CR2, and suggest that CBF1 and the factors that bind this site silence CR2 transcription in a tissue-specific manner.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Electrophoretic mobility shift analysis (EMSA)
Cells were grown and maintained as described (11). Nuclear extracts were prepared from human cell lines as described (22). Oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA). Probes were labeled with ³²P and purified as described (22).

For each reaction, 1–5 µg of nuclear extract protein was incubated with 15,000 c.p.m. of ³²P-labeled probe in 20 mM HEPES, 2% glycerol, 1 mM EDTA, 0.5 mM DTT, 25 mM KCl, 400 ng dI–dC, 400 ng BSA and 0.1% NP-40 in a total volume of 20 µl on ice for 30 min. Unlabeled annealed competitor oligonucleotides or antisera were preincubated with the nuclear extract on ice for 30 min before the addition of the radiolabeled probe. Samples were electrophoresed on 6% non-denaturing polyacrylamide gels at 4°C as previously described (23). Gels were dried and exposed to autoradiography film overnight.

Antibodies to Sp1 and Ying-Yang 1 (YY1) were obtained from Santa-Cruz Biotechnology (Santa Cruz, CA). Anti-CBF1 antibodies and pre-immune sera were kindly supplied by Dr John E. Coligan (NIH-NIAID, Bethesda, MD) (rabbit 4701 antisera) (24) and Dr Emery Bresnick (University of Wisconsin Medical School, Madison WI) (25). Dr Tasuku Honjo (Kyoto University) kindly supplied the anti-RBP-J mAb #K0043 (26). All antibodies were used at a final dilution of 1:50.

Reporter gene constructs
Constructs were generated using the human CR2 promoter (designated –5) (11), the CR2 intronic silencer (CRS) (11) and a human CD2 reporter gene from plasmid 4303/Transgene 8 (27) (a gift from S. Sawada and D. Littman, NYU School of Medicine, New York, NY). The –5 CD2 construct contains the human CR2 promoter from –5677 to +75, human CD2 cDNA and a polyadenylation sequence. The full-length CRS (1–2487 bp) was amplified by PCR and inserted downstream of the polyadenylation sequence in the sense orientation, creating the –5 CD2 CRS construct. The QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to mutate bp 687–689 of the CRS from GTG to CTT, eliminating CBF1 binding to the site (CRS mut). Mutation of the CBF1 site was confirmed by sequence analysis.

Stable transfection and analysis of reporter gene expression
K562 cells were stably transfected as described (11). Plasmid DNA was purified by CsCl₂ or Qiafilter maxiprep (Qiagen, Valencia, CA). Then 1 µg of purified linearized hygromycin resistance plasmid, pMON, and a 10-fold molar excess of linearized reporter construct were added to 10⁷ K562 cells and electroporated using a BTX electroporator at 225 V/600 µF. The cells were then placed in 10 ml media for 48 h in 5% CO₂ at 37°C followed by transfer to media containing 400 U/ml hygromycin. Polyclonal cell populations were analyzed after 2.5–4.5 weeks of selection in hygromycin. The harvested cells were divided for analysis by flow cytometry and for DNA isolation. DNA was isolated from stably transfected cells in parallel with each staining experiment for analysis by Southern blot as described (11). Cells were stained with a phycoerythrin (PE)-conjugated antibody to human CD2 (clone S5.2; Becton Dickinson, San Jose, CA) or a PE-conjugated isotype (IgG2a) control. Stained cells were analyzed at the University of Colorado Cancer Center Flow Cytometry Core facility using a Coulter Epics XL flow cytometer. Alignment of the machine was checked daily using Coulter flow check beads. Data was analyzed using the WinMDI program (Scripps Research Institute). The percent expression of CD2 was determined by gating on the viable cell population on a forward versus side scatter plot and then analyzing PE staining of viable cells on a histogram. Cells transfected with pMON alone were included in each experiment and were stained for hCD2–PE to serve as a negative control.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Characterization of protein binding to the CRS CBF1 site
The sequence of the 2.5-kb CRS was analyzed for potential CBF1 binding sites. This analysis revealed a perfect match at bp 687–693 for the CBF1 consensus binding site, GTGGGAA. To determine if this putative CBF1 site within the CRS could bind CBF1 or other factors, we performed gel-shift experiments with a probe (CRS CBF1) corresponding to CRS bp 672–707, which includes the CBF1 heptamer site (Table 1). Four oligonucleotides were then used as competitors in these experiments to demonstrate the sequence specificity of the protein–DNA complexes. The competitors include: unlabeled CRS CBF1; Cp CBF1, which corresponds to a sequence from the Epstein–Barr virus C-promoter (Cp) that contains a well-characterized CBF1 binding site; and mutated forms of these oligonucleotides in which positions 1 and 3 of the CBF1 heptamer site have been changed. Importantly, these mutations eliminate the ability of CBF1 to bind to this sequence (28). The oligonucleotide sequences of all probes and competitors used in these experiments are shown in Table 1.

View this table: [in this window] [in a new window]   Table 1. Sequences of oligonucleotides used in EMSA experiments

 
In gel-shift experiments using the CRS CBF1 probe, four complexes were detected using nuclear extracts from the Ramos B cell line (Fig. 1A). Three complexes (1, 3 and 4) demonstrate sequence specificity as they are detected at reduced levels in the presence of excess unlabeled CRS CBF1 oligonucleotide (Fig. 1A, lanes 1–5). When Cp CBF1 is used as a competitor, only complex 1 is decreased (Fig. 1A, lanes 6–10). This result indicates that complex 1 is the only complex that is absolutely dependent upon the CBF1 heptamer binding site, which is the only sequence common to both the CRS CBF1 and Cp CBF1 probes. A competitor oligonucleotide containing a mutated CBF1 site is unable to compete away any of the specific CRS CBF1 oligonucleotide complexes (Fig. 1A, lanes 11–15). These results indicate that these 2 bp are essential not only for the formation of the CBF1 containing-complex (1) but also for complexes 3 and 4.

View larger version (68K): [in this window] [in a new window]   Fig. 1. The CBF1 site within the CRS binds multiple proteins with different, yet overlapping sequence specificities. (A) EMSA analysis using nuclear extracts from Ramos cells (CR2+ human B cell line) with radiolabeled CRS CBF1 oligonucleotide and increasing amounts of unlabeled competitors to show the sequence specificity of the complexes formed. (B) EMSA analysis using nuclear extracts from Jiyoye cells (CR2+ human B cell line) to show the sequence requirements of each complex and the formation of these complexes using different radiolabeled probes. Competitor oligonucleotides were used at 400x. *Non-specific band.

 
To better define these interactions, we performed further gel-shift competition experiments using three different labeled probes: CRS CBF1, Cp CBF1 and CRS CBF1 mut (Fig. 1B). The Cp CBF1 probe (Fig. 1B, lanes 1–5) forms one complex that can be competed away by unlabeled Cp CBF1. This complex demonstrates the same mobility as complex 1 formed on the CRS CBF1 probe (Fig. 1B, lanes 6–10). Both complexes demonstrate identical nucleotide sequence requirements in that they are competed by unlabeled probes containing intact CBF1 heptamer binding sites (Fig. 1B, lanes 2, 5, 7 and 10), but not by oligonucleotides containing mutated CBF1 sites (Fig. 1B, lanes 3, 4, 8 and 9). These results support the conclusion that the complex formed with the Cp CBF1 probe and complex 1 formed with the CRS CBF1 probe are assembled with the same proteins. However, proteins that assemble on CRS CBF1 and Cp CBF1 do so with different affinities, which may be due to the influence of sequences that flank their respective CBF1 binding sites (28).

In addition to complex 1, the CRS CBF1 probe forms two other complexes (complexes 3 and 4). These two additional complexes are competed by unlabeled CRS CBF1 oligonucleotide (Fig. 1B cf,. lanes 6 and 10); however, they are not competed by Cp CBF1 or CRS CBF1 mut competitor oligonucleotides (Fig. 1B, lanes 7 and 9). This indicates that proteins in these complexes have a different DNA binding specificity than those in complex 1. Nucleotide sequences including at least part of the CBF1 heptamer site are required for the formation of complexes 3 and 4, though, as the CRS CBF1 mut oligonucleotide is an ineffective competitor. In addition, labeled CRS CBF1 mut oligonucleotide does not form complexes 1, 3 and 4 (Fig. 1B, lanes 11–15), indicating that the formation of these complexes requires the 2 bp within the CBF1 binding site. One potential explanation for these observations is that the proteins within complexes 3 and 4 have binding site(s) that include part of the CBF1 site and flanking sequences. Complex 2 is not competed significantly by any of the unlabeled oligonucleotides and represents a very low affinity complex.

Antibodies to transcription factors were used to determine if the complexes formed on the CRS CBF1 probe contain known proteins (Fig. 2). An antibody that recognizes the transcription factor Sp1 retarded the mobility of complex 4, indicating its presence within this complex (Fig. 2, lane 2). Complex 2, a minor low-affinity complex, was eliminated by the addition of an antibody recognizing the transcription factor YY1 (Fig. 2, lane 4). YY1 is known to act as a transcriptional repressor but its significance is unclear as it binds the probe with extremely low affinity. Complex 3 was not affected by any of the antibodies used in these experiments. Importantly, three different and independently generated antibodies to CBF1 interfered with the formation of complex 1 (Fig. 2, lanes 6–8), whereas pre-immune antisera did not (Fig. 2, lane 9). Additionally, the CBF1 antibodies did not disturb any of the other complexes formed, suggesting that complex 1 is the only complex containing CBF1. Based on these results and the lack of detection of this complex in the presence of Cp CBF1 competitor oligonucleotides (Fig. 1B), we conclude that complex 1 contains the transcription factor CBF1.

View larger version (55K): [in this window] [in a new window]   Fig. 2. Identification of proteins that bind to the CRS CBF1 probe. (A) EMSA analysis using Ramos nuclear extracts and the CRS CBF1 probe. The antibodies specific for CBF1 were as follows: A, anti-RBP-J rabbit antisera (24); B, anti-RBP-J mAb #K0043 (25); C, anti-CBF1 and pre-immune antisera (26). (B) The CRS CBF1 probe is used in EMSA analysis of nuclear extracts from Reh (CR2– human pre-B cell), SKW-1 (CR2– human Ig-secreting B cell), CEM (CR2– human T cell) or K562 (CR2– human erythroleukemic cell) to demonstrate that all sequence-specific complexes are detected in multiple cell types irregardless of cell type or endogenous CR2 expression. Competitor oligonucleotides were used at 400x. *Non-specific band.

 
To determine if the CRS CBF1 oligonucleotide binds cell type-specific complexes, nuclear extracts from cell lines representing different cell lineages and stages of B cell development were compared (Fig. 2B). Using the CRS CBF1 probe, all four complexes were detected using each nuclear extract, demonstrating that the factors present in these complexes are found in many cell types. This result is consistent with the reported detection of CBF1 (29) and Sp1 (30) in many cell types, and suggests that the complexes are generated by non-tissue-specific factors.

Analysis of the functional role of the CRS CBF1 site
Although the presence of these complexes in CR2⁺ and CR2^– cells may seem inconsistent with a role in the tissue-specific expression of CR2, there are many examples of non-tissue-specific factors participating in tissue-specific gene regulation (31). In particular, CBF1 is a ubiquitously expressed factor associated with changes in gene expression whose effects are mediated by interactions with other proteins such as EBNA2 or activated Notch. In addition, detection of complexes using in vitro binding assays may not reflect binding of specific factors to DNA in vivo, where other factors such as chromatin structure play important regulatory roles. The most relevant test of whether a particular DNA sequence is important in the transcriptional regulation of a gene is to analyze that DNA element in a functional assay.

Therefore, to determine if the CBF1 heptamer site in the CRS is important in CRS silencer function, we generated constructs containing the CR2 promoter, a human CD2 cDNA reporter gene and modified versions of the CRS (Fig. 3A). In one construct, we mutated positions 1 and 3 of the CBF1 heptamer site within the CRS to a sequence identical to the CRS CBF1 mut probe. As shown in the EMSA experiments (Fig. 1), these mutations prevent the binding of CBF1, Sp1 and other protein(s) to this sequence.

View larger version (22K): [in this window] [in a new window]   Fig. 3. An intact CBF1 site is crucial to CR2 silencer function. (A) Constructs stably transfected into K562 cells. The –5 CD2 CRS CBF1 mut construct is identical to the –5 CD2 CRS construct except for two mutations introduced into the CBF1 binding site at bp 687 and 689 of the CRS. The mutations are identical to those introduced into the CRS CBF1 mut EMSA probe (see Table 1). (B) K562 cells were stably transfected with the constructs shown in (A) and the resulting polyclonal hygromycin-resistant populations were analyzed for CD2 reporter gene expression by flow cytometry. A representative histogram is shown. Each population was analyzed multiple times to generate a MFI and percent CD2+ for each individual transfection. PMON = K562 cells transfected with only the hygromycin resistance plasmid. (C) Bar graph representing the MFI for eight to 12 independent stable transfections of each construct is shown. Error bars represent the SEM. Statistical analysis was performed using a one-tailed Student's t-test with unequal variance to compare the MFI from individual transfections: pMON (n = 10) versus –5 CD2 (n = 12), P < 0.02; pMON versus –5 CD2 CRS (n = 8), P > 0.07; –5 CD2 versus –5 CD2 CRS, P < 0.03; –5 CD2 CRS versus –5 CD2 CRS CBF1 mut (n = 8), P < 0.06; –5 CD2 versus –5 CD2 CRS CBF1 mut, P > 0.28. (D) Bar graph representing the percentage of CD2+ for the eight to 12 independent stable transfections of each construct. Error bars represent the SEM. Statistical analysis was performed using a one-tailed Student's t-test with unequal variance to compare the mean percent CD2+ from individual transfections: pMON versus –5 CD2, P < 0.005; pMON versus –5 CD2 CRS, P > 0.09; –5 CD2 versus –5 CD2 CRS, P < 0.02; –5 CD2 CRS versus –5 CD2 CRS CBF1 mut, P < 0.03; –5 CD2 versus –5 CD2 CRS CBF1 mut, P > 0.29.

 
Our previous data showed that the CRS does not silence transcription in transient transfection assays; therefore, we utilized our stable transfection system to analyze silencer function (11). The reporter gene was changed to CD2 to allow quantitative flow cytometric analysis. The constructs shown in Fig. 3(A) were stably transfected into K562 cells, the CR2^– and CD2^– erythroleukemia cell line that we originally used to demonstrate that the CRS can silence the CR2 proximal promoter (11). Hygromycin-resistant polyclonal populations that arose from these stable transfection experiments were analyzed by flow cytometry both for differences in MFI and differences in the percentage of CD2⁺ cells. Figure 3(B) is a representative histogram demonstrating the levels of expression of the CD2 reporter gene in stably transfected, hygromycin-resistant K562 cells. The results of these analyses are summarized in Figs 3(C and D). As expected, and in agreement with our previous results using a neo reporter gene, K562 cells transfected with the –5 CD2 construct expressed the CD2 reporter gene, while cells transfected with the –5 CD2 CRS construct (containing the silencer) expressed only very low levels of CD2. Strikingly, the mutation of the CBF1 site in the CRS eliminated the ability of the CRS to silence the CR2 promoter, as indicated by the expression of the CD2 reporter gene in K562 cells transfected with the –5 CD2 CRS CBF1 mut construct.

The validity of these results is supported by several lines of evidence. First, these results were reproducible over multiple transfection experiments (n = 8–12). Second, the results are identical and statistically significant whether analyzed by changes in MFI (Fig. 3C) or changes in percent CD2 expression (Fig. 3D). Third, using the same methodology as we employed previously with a neo reporter (11), we confirmed the presence of the CD2 reporter construct in all the transfected cell populations (data not shown). Therefore, the absence of CD2 expression in cells transfected with –5 CD2 CRS bulk populations is not due to lack of stable integration of the construct.

These observations support the idea that, although the CRS CBF1 site can interact with factors from both CR2⁺ and CR2^– cell extracts, this site is functionally important in the tissue-specific transcriptional regulation of CR2 in the context of fully chromatinized DNA.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This report includes the first identification of a specific site within the CRS region that controls lineage-specific expression of the human CR2 gene. The tissue-specific and developmentally restricted expression of human CR2/CD21 is controlled primarily by the CRS, a 2.5 kb intronic transcriptional silencer that functions only when stably integrated into chromatin. We have localized an important regulatory feature of the CRS to a single short oligonucleotide sequence that contains a binding site for CBF1, a mediator of transcriptional silencing. A 2-bp mutation that eliminates binding of CBF1 to the CRS in vitro results in loss of silencer activity in vivo. These results demonstrate the importance of this site for regulating CR2 expression and suggest that CBF1, a component of the developmentally important Notch signaling pathway, may play a role in the control of human CR2 gene expression.

We have shown that an oligonucleotide sequence containing the consensus CBF1 binding site from the CRS is capable of assembling three sequence-specific complexes. We have investigated the sequence requirements for the formation of these complexes and have used antibody supershift assays to identify the transcriptional repressor CBF1 and the transcription factor Sp1 as two of the proteins bound to this oligonucleotide sequence. The dramatic loss of silencer function upon destruction of the binding sites for these complexes strongly indicates that some or all of the proteins within these complexes, including CBF1 and Sp1, have important roles in mediating silencer function.

While we have demonstrated the importance of the CBF1 binding site in the tissue-specific silencing mediated by the CRS, we did not observe tissue-specific differences in the proteins that bind to this site in vitro. All three sequence-specific complexes were detected with nuclear extracts from both CR2⁺ and CR2^– cells. We believe this observation does not rule out an important role for this site because transcription factors can be regulated through protein–protein interactions or post-translational modifications not readily detectable in EMSA. As mentioned earlier, both CBF1 and Sp1 are ubiquitously expressed but contribute to the developmentally restricted or tissue-specific gene expression through interactions with other proteins. Additionally, changes in DNA accessibility mediated by histone acetylation or altered chromatin structure contribute to tissue-specific or developmentally restricted gene expression. The observation that the CRS only functions when stably integrated into chromatin supports the hypothesis that chromatin structure is an important part of the mechanism by which the CRS mediates appropriate transcriptional regulation of CR2 in vivo.

Because multiple protein complexes are disrupted when the CBF1 site is mutated, we have not yet been able to determine which of the proteins that bind to this site in vitro are of primary importance in vivo. However, the known functional characteristics of CBF1 suggest that it plays an important role in CRS function. CBF1 is a member of the conserved CSL transcription factor family and is homologous to the Drosophila protein Suppressor of Hairless [Su(H)] (32). CBF1 binds DNA and functions as a transcriptional repressor as part of the Notch signaling pathway. Notch signaling causes activated forms of Notch to translocate to the nucleus where they can bind to CBF1 and mask CBF1-mediated transcriptional repression. The EBV transactivator EBNA2 uses a mechanism similar to that employed by activated Notch and is also targeted to responsive genes via CBF1. Supporting a role for CBF1 in CR2 regulation, recent studies have shown that activated Notch can up-regulate endogenous CR2 expression in B cell lines in manner similar to EBNA2 (21). In addition to up-regulating CR2 EBNA2 increases transcription of CD23 (33), which is expressed at approximately the same stage in B cell development as CR2. CD23 has a CBF1 site within its promoter and can also be up-regulated by signals through Notch (28,34). While the CD23 promoter has a CBF1 binding site within its EBNA2 response element, no such EBNA2 response element has been found in the 5' region of the CR2 gene despite extensive analysis (V. M. Holers, unpublished data). Our identification of the CBF1 site within the CRS and demonstration of its role in silencer function makes this site an excellent candidate for an EBNA2 response element and also suggests that activated Notch may act to up-regulate CR2 through the site we have identified.

Although the evidence implicating CBF1 as a protein that mediates transcriptional silencing by the CRS is compelling, our current studies do not rule out other possibilities. These include Sp1 acting as a repressor, other as yet unidentified proteins in complex 3 performing this task, or silencing as a function of multi-protein complexes containing CBF1 and/or Sp1. Because of the nature of the binding characteristics of this site, future studies must be directed at comparing the sequence requirements for binding of complexes 1, 3 and 4 to their in vivo silencing activity. However, we believe that the identification of this functional site is a significant advance that will allow us to further characterize the specific protein(s) mediating silencing and to determine how their activities are regulated during development.

CD23 and CR2 are each expressed at similar stages of B cell development; therefore, regulation by CBF1 may link CR2 and CD23 transcription. These results also suggest a possible role for Notch and its ligands at these later stages in B cell development. Thus, Notch signals could promote the survival of immature/transitional B cells as well as regulate CR2, which begins to be expressed at this stage (10). It is interesting to note that Notch2 is highly expressed in splenocytes (35) and is expressed in Raji cells, a CR2⁺ mature human B cell line (36). How signals generated by Notch and its ligands would coordinate with signals through the BCR is currently unknown. In the case of CR2 gene activation, a Notch signal induced during the differentiation of the B cell could be coupled with the inactivation of CRS silencer activity, possibly through CBF1.

Studies of the CRS have shown that the intronic silencer is unable to act on a heterologous promoter (data not shown), indicating a requirement for CR2 proximal promoter sites. Figure 4 summarizes our current understanding of human CR2 gene regulation in the context of the data presented herein. CBF1, together with as yet unknown proteins, binds to a site within the intronic silencer and represses transcription, possibly through interference with positive regulatory sites within the promoter region (Fig. 4, `1'). Activated forms of Notch or Notch-related proteins can then bind to CBF1 and mask CBF1-mediated transcriptional repression (Fig. 4, `2'), thereby allowing access of positive regulatory elements to the basal transcriptional machinery. This results in active transcription of the gene.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Diagrammatic representation of CR2 silencing mechanism. The transcriptional repressor CBF1 (together with other unknown proteins) binds a crucial site within the intronic silencer and functions to control cell type-specific silencing (`1'). We hypothesize that activated Notch may bind CBF1, thus masking the repressive effect and allowing access of positive regulatory proteins to sites in the CR2 proximal promoter (`2'). In addition, several negative (–ve) and positive (+ve) regulatory elements have been identified within the proximal promoter which are critical to the control of relative promoter activity (data not shown).

 
Finally, although we have focused on the regulation of human CR2 transcription, other studies have demonstrated that the mouse CR2 gene also contains an intronic silencer (37). Although the specific proteins which mediated silencing of mouse CR2 are unknown, recent studies have shown that histone acetylation can influence mouse CR2 transcript levels (38). In this light, CBF1 has been found in complexes with histone deacetylases (39,40). Thus, silencing through a CBF1/Notch-related mechanism may control CR2 expression in both humans and mice.

	Acknowledgments

 
This work was supported by NIH R0-1 AI31105 (V. M. H.), the Smyth Professorship in Rheumatology (V. M. H.) and the National Arthritis Foundation (D. U.). J. H. is generously supported by funds from the Monfort Family Foundation. The UCCC Flow Cytometry Core facility is supported by NIH grant 2P30CA46934-09. We would like to thank Kevin Marchbank, Joel Guthridge, Susan Boackle, Tim Nichols and other members of the Holers lab for helpful discussions and suggestions.

	Abbreviations

 

CBF1 C-promoter binding factor 1

Cp C-promoter

CR2 complement receptor type II

CRS CR2 Silencer

EBV Epstein–Barr virus

EBNA Epstein–Barr virus nuclear antigen

EMSA electrophoretic mobility shift assay

PE phycoerythrin

YY1 Ying-Yang 1

	Notes

 
⁴ Present address: Department of Immunology, Box 357650, University of Washington, Seattle, WA 98195, USA

Transmitting editor: L. H. Glimcher

Received 18 December 2000, accepted 6 February 2001.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Tedder, T. F., Zhou, L. J. and Engel, P. 1994. The CD19/CD21 signal transduction complex of B lymphocytes. Immunol. Today 15:437.[ISI][Medline]
2. Molina, H., Holers, V. M., Li, B., Fang, Y.-F., Mariathasan, S., Goellner, F., Strauss-Schoenberger, J., Karr, R. W. and Chaplin, D. D. 1996. Markedly impaired humoral immune response in mice deficient in complement receptors 1 and 2. Proc. Natl Acad. Sci. USA 93:3357.[Abstract/Free Full Text]
3. Ahearn, J. M., Fischer, M. B., Croix, D. A., Georg, S., Ma, M., Xia, J., Zhou, X., Howard, R. G., Rothstein, T. L. and Carroll, M. C. 1996. Disruption of the Cr2 locus results in a reduction in B-1a cells and in an impaired B cell response to T-dependent antigen. Immunity 4:251.[ISI][Medline]
4. Marchbank, K. J., Watson, C. C., Ritsema, D. F. and Holers, V. M. 2000. Expression of human complement receptor 2 (CR2,CD21) in Cr2^–/– mice restores humoral immune function. J. Immunol. 165:2354.[Abstract/Free Full Text]
5. Fischer, M. B., Goerg, S., Shen, L., Prodeus, A. P., Goodnow, C. C., Kelsoe, G. and Carroll, M. C. 1998. Dependence of germinal center B cells on expression of CD21/CD35 for survival. Science 280:582.[Abstract/Free Full Text]
6. Takahashi, K., Kozono, Y., Waldschmidt, T. J., Berthiaume, D., Quigg, R. J., Baron, A. and Holers, V. M. 1997. Mouse complement receptors type 1 (CR1; CD35) and type 2 (CR2; CD21): expression on normal B cell subpopulations and decreased levels during the development of autoimmunity in MRL/lpr mice. J. Immunol. 159:1557.[Abstract]
7. Tedder, T. F., Clement, L. T. and Cooper, M. D. 1984. Expression of C3d receptors during human B cell differentiation: immunofluorescence analysis with the HB-5 monoclonal antibody. J. Immunol. 133:678.[Abstract]
8. Melamed, D., Benschop, R. J., Cambier, J. C. and Nemazee, D. 1998. Developmental regulation of B lymphocyte immune tolerance compartmentalizes clonal selection from receptor selection. Cell 92:173.[ISI][Medline]
9. Melamed, D. and Nemazee, D. 1997. Self-antigen does not accelerate immature B cell apoptosis, but stimulates receptor editing as a consequence of development arrest. Proc. Natl Acad. Sci. USA 94:9267.[Abstract/Free Full Text]
10. Hartley, S. B., Cooke, M. P., Fulcher, D. A., Harris, A. W., Cory, S., Basten, A. and Goodnow, C. C. 1993. Elimination of self-reactive B lymphocytes proceeds in two stages: arrested development and cell death. Cell 72:325.[ISI][Medline]
11. Makar, K. W., Pham, C. T. N., Dehoff, M. H., O'Connor, S. M., Jacobi, S. M. and Holers, V. M. 1998. An intronic silencer regulates B lymphocyte cell- and stage-specific expression of the human complement receptor type 2 (CR2, CD21) gene. J. Immunol. 160:1268.[Abstract/Free Full Text]
12. Cordier-Bussat, M., Billaud, M., Calender, A. and Lenoir, G. M. 1993. Epstein–Barr virus (EBV) nuclear antigen 2-induced up-regulation of CD21 and CD23 molecules is dependent on a permissive cellular context. Int. J. Cancer 53:153.[ISI][Medline]
13. Cordier, M., Calender, A., Billaud, M., Zimber, U., Rousselet, G., Pavlish, O., Banchereau, J., Tursz, T., Bornkamm, G. and Lenoir, G. M. 1990. Stable transfection of Epstein–Barr virus (EBV) nuclear antigen 2 in lymphoma cells containing the EBV P3HR1 genome induces expression of B-cell activation molecules CD21 and CD23. J. Virol. 64:1002.[Abstract/Free Full Text]
14. Artavanis-Tsakonas, S., Matsuno, K. and Fortini, M. E. 1995. Notch signaling. Science 268:225.[Abstract/Free Full Text]
15. Robey, E., Chang, D., Itano, A., Cado, D., Alexander, H., Lans, D., Weinmaster, G. and Salmon, P. 1996. An activated form of Notch influences the choice between CD4 and CD8 T cell lineages. Cell 87:483.[ISI][Medline]
16. Washburn, T., Schweighoffer, E., Gridley, T., Chang, D., Fowlkes, B. J., Cado, D. and Robey, E. 1997. Notch activity influences the ß versus T cell lineage decision. Cell 88:833.[ISI][Medline]
17. Robey, E. 1999. Regulation of T cell fate by Notch. Annu. Rev. Immunol. 17:283.[ISI][Medline]
18. Pui, J. C., Allman, D., Xu, L., DeRocco, S., Karnell, F. G., Bakkour, S., Lee, J. L., Kadesch, T., Hardy, R. R., Aster, J. C. and Pear, W. S. 1999. Notch1 expression in early lymphopoiesis influences B versus T lineage determination. Immunity 11:299.[ISI][Medline]
19. Strobl, L. J., Hofelmayr, H., Stein, C., Marschall, G., Brielmeier, M., Laux, G., Bornkamm, G. W. and Zimber-Strobl, U. 1997. Both Epstein–Barr viral nuclear antigen 2 (EBNA2) and activated Notch1 transactivate genes by interacting with the cellular protein RBP-J. Immunobiology 198:299.[ISI][Medline]
20. Sakai, T., Taniguchi, Y., Tamura, K., Minoguchi, S., Fukuhara, T., Strobl, L. J., Zimber-Strobl, U., Bornkamm, G. W. and Honjo, T. 1998. Functional replacement of the intracellular region of the Notch1 receptor by Epstein–Barr virus nuclear antigen 2. J. Virol. 72:6034.[Abstract/Free Full Text]
21. Strobl, L. J., Hofelmayr, H., Marschall, G., Brielmeier, M., Bornkamm, G. W. and Zimber-Strobl, U. 2000. Activated Notch1 modulates gene expression in B cells similarly to Epstein–Barr viral nuclear antigen 2. J. Virol. 74:1727.[Abstract/Free Full Text]
22. Fitzsimmons, D., Hodsdon, W., Wheat, W., Maira, S. M., Wasylyk, B. and Hagman, J. 1996. Pax-5 (BSAP) recruits Ets proto-oncogene family proteins to form functional ternary complexes on a B-cell-specific promoter. Genes Dev. 10:2198.[Abstract/Free Full Text]
23. Hagman, J. and Grosschedl, R. 1992. An inhibitory carboxyl-terminal domain in Ets-1 and Ets-2 mediates differential binding of ETS family factors to promoter sequences of the mb-1 gene. Proc. Natl Acad. Sci. USA 89:8889.[Abstract/Free Full Text]
24. Shirakata, Y., Shuman, J. D. and Coligan, J. E. 1996. Purification of a novel MHC class I element binding activity from thymus nuclear extracts reveals that thymic RBP-J/CBF1 binds to NF-B-like elements. J. Immunol. 156:4672.[Abstract]
25. Lam, L. T. and Bresnick, E. H. 1998. Identity of the ß-globin locus control region binding protein HS2NF5 as the mammalian homolog of the Notch-regulated transcription factor Suppressor of Hairless. J. Biol. Chem. 273:24223.[Abstract/Free Full Text]
26. Sakai, T., Furukawa, T., Iwanari, H., Oka, C., Nakano, T., Kawaichi, M. and Honjo, T. 1995. Loss of immunostaining of the RBP-J transcription factor upon F9 differentiation induced by retinoic acid. J. Biochem. 118:6210.
27. Sawada, S., Scarborough, J. D., Killeen, N. and Littman, D. R. 1994. A lineage-specific transcriptional silencer regulates CD4 gene expression during T lymphocyte development. Cell 77:917.[ISI][Medline]
28. Ling, P. D., Hsieh, J. J. D., Ruf, I. K., Rawlins, D. R. and Hayward, S. D. 1994. EBNA-2 upregulation of Epstein–Barr virus latency promoters and the cellular CD23 promoter utilizes a common targeting intermediate, CBF1. J. Virol. 68:5375.[Abstract/Free Full Text]
29. Lu, F. M. and Lux, S. E. 1996. Constitutively active human Notch1 binds to the transcription factor CBF1 and stimulates transcription through a promoter containing a CBF1-responsive element. Proc. Natl Acad. Sci. USA 93:5663.[Abstract/Free Full Text]
30. Saffer, J. D., Jackson, S. P. and Annarella, M. B. 1991. Developmental expression of Sp1 in the mouse. Mol. Cell Biol. 11:2189.[Abstract/Free Full Text]
31. Henderson, A. and Calame, K. 1998. Transcriptional regulation during B cell development. Annu. Rev. Immunol. 16:163.[ISI][Medline]
32. Schweisguth, F. and Posakony, J. W. 1992. Suppressor of Hairless, the Drosophila homolog of the mouse recombination signal-binding protein gene, controls sensory organ cell fates. Cell 69:1199.[ISI][Medline]
33. Henkel, T., Ling, P. D., Hayward, S. D. and Peterson, M. G. 1994. Mediation of Epstein–Barr virus EBNA2 transactivation by recombination signal-binding protein J. Science 265:92.[Abstract/Free Full Text]
34. Bash, J., Zong, W. X., Banga, S., Rivera, A., Ballard, D. W., Ron, Y. and Gelinas, C. 1999. Rel/NF-B can trigger the Notch signaling pathway by inducing the expression of Jagged1, a ligand for Notch receptors. EMBO J. 18:2803.[ISI][Medline]
35. Weinmaster, G., Roberts, V. J. and Lemke, G. 1992. Notch2: a second mammalian Notch gene. Development 116:931.[Abstract]
36. Hsieh, J. J. D., Nofziger, D. E., Weinmaster, G. and Hayward, S. D. 1997. Epstein–Barr virus immortilization: Notch2 interacts with CBF1 and blocks differentiation. J. Virol. 71:1938.[Abstract]
37. Hu, H., Martin, B. K., Weis, J. J. and Weis, J. H. 1997. Expression of the murine CD21 gene is regulated by promoter and intronic sequences. J. Immunol. 158:4758.[Abstract]
38. Zabel, M. D., Weis, J. J. and Weis, J. H. 1999. Lymphoid transcription of the murine CD21 gene is positively regulated by histone acetylation. J. Immunol. 163:2697.[Abstract/Free Full Text]
39. Kao, H. Y., Ordentlich, P., Koyano-Nakagawa, N., Tang, Z., Downes, M., Kintner, C. R., Evans, R. M. and Kadesch, T. 1998. A histone deacetylase corepressor complex regulates the Notch signal transduction pathway. Genes Dev. 12:2269.[Abstract/Free Full Text]
40. Hsieh, J. J., Zhou, S., Chen, L., Young, D. B. and Hayward, S. D. 1999. CIR, a corepressor linking the DNA binding factor CBF1 to the histone deacetylase complex. Proc. Natl Acad. Sci. USA 96:23.[Abstract/Free Full Text]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				D. Liu, J.-Y. Zhu, and Z.-X. Niu Molecular Structure and Expression of Anthropic, Ovine, and Murine Forms of Complement Receptor Type 2 Clin. Vaccine Immunol., June 1, 2008; 15(6): 901 - 910. [Full Text] [PDF]

				I. Debnath, K. M. Roundy, J. J. Weis, and J. H. Weis Defining In Vivo Transcription Factor Complexes of the Murine CD21 and CD23 Genes J. Immunol., June 1, 2007; 178(11): 7139 - 7150. [Abstract] [Full Text] [PDF]

				H. Wu, S. A. Boackle, P. Hanvivadhanakul, D. Ulgiati, J. M. Grossman, Y. Lee, N. Shen, L. J. Abraham, T. R. Mercer, E. Park, et al. Association of a common complement receptor 2 haplotype with increased risk of systemic lupus erythematosus PNAS, March 6, 2007; 104(10): 3961 - 3966. [Abstract] [Full Text] [PDF]

				M. Sironi, G. Menozzi, G. P. Comi, R. Cagliani, N. Bresolin, and U. Pozzoli Analysis of intronic conserved elements indicates that functional complexity might represent a major source of negative selection on non-coding sequences Hum. Mol. Genet., September 1, 2005; 14(17): 2533 - 2546. [Abstract] [Full Text] [PDF]

				S. Maier, M. Santak, A. Mantik, K. Grabusic, E. Kremmer, W. Hammerschmidt, and B. Kempkes A Somatic Knockout of CBF1 in a Human B-Cell Line Reveals that Induction of CD21 and CCR7 by EBNA-2 Is Strictly CBF1 Dependent and that Downregulation of Immunoglobulin M Is Partially CBF1 Independent J. Virol., July 15, 2005; 79(14): 8784 - 8792. [Abstract] [Full Text] [PDF]

				H.-g. Hu, H. Illges, C. Gruss, and R. Knippers Distribution of the chromatin protein DEK distinguishes active and inactive CD21/CR2 gene in pre- and mature B lymphocytes Int. Immunol., June 1, 2005; 17(6): 789 - 796. [Abstract] [Full Text] [PDF]

				H. Chang, Y. Gwack, D. Kingston, J. Souvlis, X. Liang, R. E. Means, E. Cesarman, L. Hutt-Fletcher, and J. U. Jung Activation of CD21 and CD23 Gene Expression by Kaposi's Sarcoma-Associated Herpesvirus RTA J. Virol., April 15, 2005; 79(8): 4651 - 4663. [Abstract] [Full Text] [PDF]

				J. Esparza-Gordillo, E. G. d. Jorge, A. Buil, L. C. Berges, M. Lopez-Trascasa, P. Sanchez-Corral, and S. R. d. Cordoba Predisposition to atypical hemolytic uremic syndrome involves the concurrence of different susceptibility alleles in the regulators of complement activation gene cluster in 1q32 Hum. Mol. Genet., March 1, 2005; 14(5): 703 - 712. [Abstract] [Full Text] [PDF]

				D. Ulgiati, C. Pham, and V. M. Holers Functional Analysis of the Human Complement Receptor 2 (CR2/CD21) Promoter: Characterization of Basal Transcriptional Mechanisms J. Immunol., June 15, 2002; 168(12): 6279 - 6285. [Abstract] [Full Text] [PDF]

				D. Ulgiati and V. M. Holers CR2/CD21 Proximal Promoter Activity Is Critically Dependent on a Cell Type-Specific Repressor J. Immunol., December 15, 2001; 167(12): 6912 - 6919. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (15) Request Permissions Google Scholar Articles by Makar, K. W. Articles by Holers, V. M. Search for Related Content PubMed PubMed Citation Articles by Makar, K. W. Articles by Holers, V. M. Social Bookmarking