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Departments of Medicine and Immunology, Division of Rheumatology, University of Colorado Health Sciences Center, Denver, CO 80262; and
Department of Medicine, Division of Rheumatology, Washington University School of Medicine, St. Louis, MO 63110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(8). CR2 expression on human B lymphocytes was first studied by Tedder et al., and found to be limited to the mature and early activated stages during development (9). Using flow-cytometric analysis, CR2 was undetectable on normal bone marrow-derived pre-B or immature B cells, and was also lost at the plasma cell stage. Using a second anti-CR2 mAb, CR2 expression was again found to be limited to IgM+/IgD+ bone marrow cells and to be absent from IgM+/IgD- cells (10). Studies using the hen egg lysozyme:anti-hen egg lysozyme dual transgenic system have shown that mouse CR2 is among the first cell surface markers detected on bone marrow B cells after the escape of these cells from negative selection by self Ag (11), a process that occurs in the IgMlow/B220intermediate/IgD- developmental stage (reviewed in 12 . An identical CR2 expression pattern is found using the class I:anti-class I transgenic system (13) in which CR2- immature B cells undergo editing, and then transition to an IgMintermediate/B220high/IgD+/CR2+ phenotype after becoming tolerant to self Ag (11) (Y. Kozono, D. Nemazee, and V. M. Holers, unpublished observations). In addition to B cells, human CR2 is also expressed on follicular dendritic cells (14), early thymocytes (15), a small subset of CD4+ and CD8+ peripheral T lymphocytes (16, 17), and epithelial cells (18).
Some studies have reported the immortalization of human pre-B cells with EBV, possibly using CR2 as a receptor, and that CR2 is on a small subpopulation of CD19+/IgM- pre-B cells and pre-B cell malignancies (19). However, based on the analysis of CR2 expression on normal human B cells and well-characterized B cell lines (9, 10), in addition to the pattern of expression of mouse CR2 (20), which has marked structural and functional similarities with human CR2 (21, 22, 23), it is apparent that the expression of human CR2 on the great majority of developing B cells appears at a comparable point as murine CR2. That point is during the transition from IgM+/IgD- to IgM+/IgD+ cells.
CR2 plays an important role in the generation of a normal immune response. Mice lacking CR2 show greatly impaired humoral immune responses, particularly in the generation of a switched IgG response (24, 25, 26). In addition, coupling the CR2 ligand C3d directly to an Ag results in a 10,000-fold decrease in the amount of Ag necessary to generate an IgG response (27). Several other studies have demonstrated a role for human CR2 in the activation and proliferation of B lymphocytes: 1) cross-linking of CR2 leads to increased [Ca2+]i and proliferation in the presence of suboptimal amounts of phorbol esters, T cell factors, or anti-IgM (28, 29); 2) coligation of CR2 with mIgM results in substantially enhanced expression of c-fos mRNA (30); 3) CR2 is noncovalently associated with CD19 (31), a membrane glycoprotein capable of promoting B cell activation (32); 4) the interaction of CR2 with CD23 in the presence of IL-4 and CD40-ligand results in enhanced B cell production of IgE (33, 34); 5) CR2 ligation induces homotypic adhesion of tonsillar B lymphocytes and B cell lines (30, 35); and 6) EBV binding to CR2 on resting B cells increases CD23 mRNA levels independently of viral gene expression (36).
CR2 is one of a number of non-Ig B lymphocyte stage-specific markers expressed during development and activation (reviewed in Refs. 19 and 37). Other genes of this type include CD10, CD19, CD20, CD22, CD23, CD40, and HLA class II. Several transcriptional factors such as B-specific activation protein, Oct-1, and Oct-2 have been shown to play important roles in the transcriptional control of CD19, CD20, and CD22, which are three proteins whose expression is also primarily restricted to B cells (38, 39, 40). However, the regulatory elements that control cell- and stage-specific expression of these genes have not yet been clearly identified (37, 41, 42). In addition, there is little understanding of the molecular signals that lead to transcription of genes that, like CR2, are primarily activated after the negative selection processes that occur in the bone marrow.
In B cell lines, infection with EBV or transfection with constructs directing the synthesis of the EBV transactivator EBNA-24 leads to increased expression of CR2 in addition to CD23 (43, 44). Recently, EBNA-2 has been shown to transactivate CD23 and several other target genes at least in part by relieving repression mediated by the regulatory protein CBF1 (45). CBF1 binds to the CD23 promoter site previously defined as the EBNA-2 response element (46, 47). EBNA-2 also has been shown to act as a transcriptional activator by interactions with PU.1 (48) and with the general transcription factor TFIIE and a novel coactivator p100 (49). The mechanisms by which EBV or EBNA-2 increases CR2 expression, though, are not yet known. Other studies have suggested that HTLV-1 infection of T cells can also up-regulate CR2 expression (50); however, the specific mechanisms by which this occurs are also unknown.
We have previously cloned and sequenced the human CR2 promoter-containing region from -1253 bp to +75 bp (-1253/+75) (51). We demonstrated that this region contains potential binding sites for the widely expressed transcription factors SP1 and AP-1/AP-2. In addition, we showed that the (-1253/+75) region could direct transcription of a CAT reporter gene when transiently transfected into the CR2-positive Raji B cell line (51). However, neither we nor others (52) have been able to demonstrate that this particular region contains regulatory elements that control cell- or stage-specific CR2 transcription.
To utilize human CR2 as a model of B lymphocyte transcriptional regulation, we extended our analysis further 5' of the transcriptional initiation site and extensively characterized the proximal CR2 5' promoter elements. Because we found that these additional upstream gene segments still did not confer cell- and stage-specific expression, we searched for other regulatory elements throughout the CR2 gene. In this study, we report the identification of an intronic element that regulates both cell- and stage-specific CR2 transcription by utilizing a transcriptional silencing mechanism. The silencer element requires chromosomal integration for its activity, suggesting that nuclear matrix or chromatin interactions may be necessary for its effects.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Total cellular RNA was prepared from 1 x 108 cells using the Chirgwin technique (53). The RT reaction using 4 µg total RNA proceeded for 60 min at 30°C using RT with both oligo(dT) and random primers (Invitrogen Corp., San Diego, CA). The reaction mixture was then split into equal aliquots and used directly in the PCR reaction with the following oligonucleotide primers: CR2, 5'-GGCAGGAAAACTTTCTATATGG-3' and 5'-ATGAGGGGCAGGTTGGCTCC-3' (corresponding to positions 2271 to 2291 and 2787 to 2768, respectively, of the CR2 cDNA sequence (54)), and MCP, 5'-ACATACCTAACTGATGAGACCCACAGA-3' (corresponding to nucleotides 1 to 28 of the MCP cyto-1 exon (55)) and 5'-CAAGCCACATTGCAATATTAGCTAAGCCACA-3' (corresponding to nucleotides 1323 to 1293 of the MCP cDNA sequence (56)). MCP is expressed in all bone marrow-derived cell lines tested to date (55). The PCR reactions proceeded at 95°C x 1 min, 56°C x 1 min, and 72°C x 2 min for 20 cycles (Perkin-Elmer Cetus, Norwalk, CT). PCR products were electrophoresed on a 1.8% agarose gel.
Nuclear runoff analysis
Nuclear runoff analysis was performed as described (57) with the following changes. Transcription proceeded for 20 min at 30°C. RNA was harvested and hybridized to DNA slot blots with 2.5 µg of target DNA at 42°C for 72 to 96 h. Blots were washed in 2x SSC, 0.1% SDS twice for 5 min at 20°C, followed by washing in 0.2x SSC, 0.1% SDS twice for 10 min at 55°C. The previously described CR2 cDNA-containing plasmid pBSCR2–12 (54), lacking the poly(A) tail, was in the pBS (Bluescript; Stratagene, San Diego, CA) plasmid. A CR2–5' genomic DNA-containing plasmid substrate was created as a 418-bp dsDNA PCR product encoding the sequence from +1 to +418 of the CR2 gene hnRNA transcript using oligonucleotide primers 5'-ATTCGAATTCAGCTGCTTGCTGCTCCAGCCTTGCC-3' and 5'-ATTCGAATTCAGAAACTTTCCTGCCGCACGCTCCA-3'. The PCR product was subcloned into pBS using the flanking EcoRI oligonucleotide sites. To assure the quantity and quality of RNA transcripts, ß-actin and Alu (57) probes were also used as targets. Autoradiographs were developed using Kodak XAR-5 film. Densitometric analysis was performed using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Cloning of CR2 regulatory elements
Phage genomic subclones of a previously described regulators
of complement activation-2 yeast artificial chromosome clone containing
the CR2 gene (58) were created using the Lambda DASH II
BamHI cloning vector (Stratagene, La Jolla, CA) (library
gift of Dr. Dennis Hourcade, Washington University School of Medicine,
St. Louis, MO). The library was screened using genomic clones
encompassing both the 5' regulatory region (pBSCR2gH-3; see Fig. 2
A for location of this and other subclones) (51) and a
4.4-kb BamHI-SalI subclone (pBSCR2gA8B-1)
containing the extreme 5' sequences of the previously described cosmid
A8.1 (54). This strategy ensured that any clone hybridizing with both
probes would contain genomic elements spanning the first intron of the
human CR2 gene. One such phage clone, designated CR2g1.12, was
identified and further characterized for these studies. This clone
contained approximately 18 kb of DNA spanning from approximately 9.5 kb
5' of the first CR2 exon and extending approximately 3.5 kb into the 5'
end of the A8.1 cosmid clone. Analysis of human genomic DNA paired with
this clone using informative restriction enzyme digests demonstrated
identical band sizes, indicating that the phage clone was unaltered
during yeast artificial chromosome and subsequent phage cloning and
represents authentic human genomic DNA (data not shown). Likewise,
partial DNA sequence analysis described in Results further
confirmed this conclusion. The sequence of the 2.5-kb
XbaI-SacI intronic fragment described in this
work has been entered in GenBank under accession number U34741.
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The plasmid constructs shown in Figure 3A were made
using a neomycin (neo) reporter gene (59) (generous gift of Dr. Tim
Ley, Washington University School of Medicine). The CR2(-1253/+75)-neo
and CR2(-149/+75)-neo constructs were created by subcloning DNA from
the previously described plasmid pBSCR2 (-1253/+75) (51). The
CR2(-5-kb/+75)-neo reporter construct was created by excision of the
5-kb SacI fragment (pBSCR2gS-2) of CR2g1.12 (see Fig. 2
A also) and subcloning into the unique SacI site
at -149 bp in CR2(-149/+75)-neo. Constructs containing the 2.5-kb
intronic region described in Results were created by
excision of a 2.5-kb XbaI-SacI fragment of
pBSCR2gS-1 (using a polylinker XbaI site at the 3' end) and
subcloning into a 3' XbaI site in both CR2(-5-kb/+75)-neo
and CR2(-1253/+75)-neo downstream of the SV40 polyadenylation
sequence.
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Raji cells used for transfection were maintained in suspension culture in Iscove’s medium containing 10% FCS, supplemented with 2 mM glutamine, penicillin, and streptomycin at 37°C in 5% CO2. All other cells were maintained in suspension culture in RPMI 1640 containing the same additives and 10% FCS (Reh were grown in 15% FCS). Approximately 12 to 15 h before transfection, cells were fed and resuspended in media at a density of 5 x 105 cells/ml.
For transient transfections of cells, plasmid DNA (50 µg) was transfected into 2 x 107 cells by electroporation with 125 µg of carrier salmon sperm DNA, using conditions as described below and a BTX Transfector 300 (Biotechnologies and Experimental Research, San Diego, CA). As an internal control to correct for transfection efficiency, 1 to 5 µg of RSV-neo (60) was cotransfected with neo reporter constructs.
For stable transfection studies, neo reporter constructs (15 µg)
directed by CR2 promoter regions (see also Fig. 3A)
were cotransfected into 2 x 107 cells by
electroporation using a BTX transfector with 125 µg of carrier salmon
sperm DNA and 1.5 µg of either the pMon (gift of Drs. Sandip Godambe
and David Chaplin, Washington University School of Medicine) (Jiyoye
and SKW-1 cell lines) or pRep4 (Stratagene) (Jiyoye, Reh, and K562 cell
lines) hygromycin-resistance plasmid. The following transfection
conditions were established by pilot experiments and used for transient
transfection experiments and to develop stably transfected lines: Raji,
1200 µF and 225 V; Jiyoye, 800 µF and 225 V; and SKW-1 and K562,
600 µF and 225 V. Reh cells were transfected using 1 µg plasmid DNA
and 0.1 µg pRep-4 hygromycin-resistance plasmid in addition to 10
µg DEAE-dextran (Pharmacia, Piscataway, NJ) at 950 µF and 300 V.
All cells were maintained in the absence of selective medium for
48 h after transfections and were then placed into 200 µg/ml
(Reh), 300 µg/ml (K562), or 400 µg/ml (Jiyoye and SKW-1) of
hygromycin (Calbiochem, San Diego, CA) in RPMI medium, as above. After
the initial outgrowth of a stable resistant population, RNA was
harvested as above for S1 analysis, and DNA was harvested in parallel
for Southern blot analysis.
The levels of neo mRNA and other reporter mRNA in transfection studies were determined by ImageQuant following PhosphorImager (Molecular Dynamics) analysis. The ß-actin probe described above also served as an internal control for RNA quantity and quality in transient transfections of neo reporter constructs. To determine the relative copy number of stably incorporated neo reporter plasmid equivalents, DNA from stably transfected cell lines was digested with HindIII and BamHI to release the neo-encoding cassette from the surrounding sequences of the expression plasmid. Following transfer to nitrocellulose, the blot was probed using a 2-kb BglII-BamHI neo cDNA probe. The internal control used to assure equivalent amounts of genomic DNA in each lane was a 1.8-kb EcoRI fragment of the human CGL-1 cDNA (61).
DNase I hypersensitivity analysis of nuclear chromosomal DNA
DNase I hypersensitivity analysis was performed as previously
described (62). After isolation of nuclei and treatment with increasing
amounts of DNase I, followed by overnight proteinase K digestion, DNA
was extracted with phenol:CHCl3, CHCl3
alone, and ethanol precipitated and resolubilized into TE buffer.
DNA was then digested with BamHI, electrophoresed,
transferred to nitrocellulose, and probed with a 4.4-kb probe from the
5' end of cosmid A8.1 (see Fig. 3A), whose 3'
anchored end is the BamHI site indicated. A 0.6-kb
XbaI-BamHI fragment of the A8.1 cosmid with the
same 3' end was also used as a probe. A PhosphorImager and ImageQuant
software were used to analyze the autoradiographs from the CR2-negative
cell lines.
Creation and analysis of transgenic mice
To create transgenic mice using the CR2(-5-kb/+75)-neo constructs with or without the intronic regulator described in Results, plasmid DNA was first linearized using KpnI. FVB female pups (21–28 days old) were used as embryo donors. The mice were superovulated using 5 IU pregnant mares’ serum gonadotropin. After 44 h, 5 IU human chorionic gonadotropin was injected i.p., and mice were then placed with fertile males for mating. Following this, oviducts were then dissected and flushed with 50 µg/ml bovine testis hyaluronidase, embryos were sorted, and fertile embryos were injected with a solution of MTE (10 mM Tris, pH 8, 0.25 mM EDTA, and Na salt) containing 2 ng/ml of construct. Following injection, embryos were transferred into pseudopregnant recipient females, which were then housed in a clean specific pathogen-free barrier for gestation and lactation.
To screen for potential founders in the populations of putative transgenic pups, tail DNA was purified and tested by dot blot using the BamHI-HindIII fragment encoding the neo reporter cDNA. Founders that were identified using this means were confirmed by Southern blot analysis. Confirmed founders were then subsequently backcrossed onto the B10.D2 strain and followed by tail blot analysis. F1 and F2 mice were used for the analysis of reporter gene expression presented in this study, unless otherwise noted.
To analyze neo reporter gene mRNA expression in individual murine tissues, RNA was first isolated using the Chirgwin technique (53). In some experiments, S1 analysis was performed using the CR2(-149/+75)-neo plasmid that had been linearized within neo using BglII and end labeled at that site using T4 kinase. The control for endogenous murine mRNA quantity and quality in these studies was ß2-microglobulin (63). This probe was prepared by digestion with EcoRI, followed by calf intestinal phosphatase treatment and end labeling with T4 polynucleotide kinase. In other experiments, neo reporter mRNA expression was analyzed using RT-PCR. For this analysis, 50 µg of RNA from each tissue was first treated with 10 U RNase-free DNase I (Boehringer Mannheim Corp., Indianapolis, IN) for 30 min at 37°C in 0.1 M sodium acetate, 5 mM MgCl2, pH 5, followed by ethanol precipitation. DNase I-treated RNA was resuspended in TE at 1 µg/µl, and 1 µg was utilized in each RT-PCR reaction. RT reactions were primed with a 1:1 mixture of oligo(dT) and random primers, according to manufacturer’s specifications (GeneAmp; Perkin-Elmer Cetus). Parallel samples with and without Moloney murine leukemia virus RT were used to assure that templates were mRNA and not DNA derived. To further assure that amplified bands were mRNA derived, a strategy was designed to detect spliced neo mRNA using the oligonucleotides 5'-CCTTACTTCTGTGGTGTG-3' and 5'-CCTCATTAAAGGCATTCC-3'. These oligonucleotides generate a 115-bp PCR fragment that results from splicing out 66 bp of the SV40-derived 3' untranslated sequence at positions (1568)TAAG/GTAAAT... to ...TTTTAG/ATTC(1641), a common splicing event, as previously described (64). PCR was performed using the GeneAmp RNA PCR kit (Perkin-Elmer Cetus), according to manufacturer’s directions, using 2 mM MgCl2 and amplified 35 cycles using 94°C x 1 min, 54°C x 1 min, and 72°C x 1 min. In preliminary experiments, the 115-bp mRNA-derived PCR fragment was cloned using the TA vector system (Invitrogen Corp.), and nucleotide sequence analysis of the fragment confirmed the identity and sequence of the spliced product.
The positive control for endogenous murine RNA quality and relative quantity for these studies is Crry/p65 (65), a gene expressed in all murine tissues (65, 66), which was detected using the oligonucleotides 5'-CACTGCCCAGCCCCATCAC-3', derived from short consensus repeat (SCR) 1 in the cDNA sequence, and 5'-CGAGATACACATTTGGCCAG-3', derived from SCR 5 in the cDNA sequence (65). PCR conditions were identical, except for a hybridization temperature of 58°C. This oligonucleotide pair results in the creation of a 953-bp spliced PCR product from mRNA that extends over five exon-intron junctions in the Crry gene.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous results using flow-cytometric analysis of B cell lines
have shown that CR2 surface expression is limited to the mature B
lymphocyte stage (9). To determine whether this reflects the presence
or absence of CR2 mRNA, and to establish the CR2 phenotype of both B
and non-B cell lines, we have developed a sensitive method to detect
CR2 mRNA using RT-PCR (Fig. 1A). For these studies,
we utilized B cell lines that have been phenotyped by Ig heavy and
light chain rearrangements and other non-CR2 surface markers to the
pre-B (Nalm-6, Nall-1, Reh), mature (Raji, Jiyoye), or late
Ig-secreting (SKW-1, DHL-4) stages (67, 68).
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Southern blot analysis of the PCR products using a CR2 cDNA probe also confirmed these results, except that, on prolonged exposure, the Ig-secreting cell lines SKW-1 and DHL-4 had a very faint CR2 signal (<<1% of Raji) that could be detected (data not shown). Overall, these studies have shown that the surface expression of CR2, unlike Ig heavy chain, is closely linked to the presence of mature mRNA. In addition, they have allowed us to use these cell lines as in vitro models of B lymphocyte CR2 expression.
Lineage- and stage-specific CR2 expression is controlled primarily at the level of transcriptional initiation
To determine whether the presence of mature CR2 mRNA and protein
is controlled primarily by the process of transcriptional initiation,
we performed nuclear runoff analysis using specific DNA target probes
containing either the initial 418 bp of the CR2 hnRNA (pBSCR2(+1/+418))
or a portion of the CR2 cDNA that excludes the poly(A) tail
(pBSCR2–12) (Fig. 1B). Positive controls included
ß-actin and Alu, and the pBS plasmid itself served as a negative
control in each line. Densitometric analysis of the experiment in
Figure 1B is shown in Figure 1C. Data for each
cell line are expressed as the value of the CR2-specific probe,
pBSCR2–12 or pBSCR2(+1/+418), over the value of the
pBS-negative control. Quantitation of the CR2-specific signal in the
mature B cell line, Raji, shows a twofold increase over the
pBS-negative control, and a 4.5- to 15-fold increase relative to the
CR2-specific signals seen with the other cell lines.
These results demonstrate that cell- and stage-specific CR2 mRNA expression is controlled primarily by the initiation of transcription. They also suggest that CR2 is part of the developmental program that controls B lymphocyte stage-specific gene expression first by transcriptional activation and then by inactivation of the promoter upon transition to the Ig-secreting plasma cell stage. Finally, they further validate the use of these cell lines as models in which to test transcriptional activation and silencing using CR2 promoter constructs and probes.
Two DNase I hypersensitive sites are present in the human CR2 gene
Since DNase I hypersensitive sites are often indicative of
important regulatory elements, we scanned the CR2 gene for the presence
of DNase I hypersensitive sites as a method to identify potential
regulatory regions within the CR2 gene. Using an approach able to
detect hypersensitive sites from approximately 14 kb upstream to 10 kb
downstream of the CR2 gene, we identified two hypersensitive sites
using Raji cell nuclei (Fig. 2B) and the A8B-1 probe
shown in Figure 2A. The site labeled HS1 was further mapped
using flanking subclones of pBSCR2gH-3 (see Fig. 2A)
to be within the proximal 300 bp upstream of the +1 site within the CR2
promoter, consistent with our identification of this region as
containing the transcriptional initiation site and regulatory sites
(data not shown). HS2 was mapped to be within a 2.5-kb
XbaI-SacI segment of the first intron.
Hypersensitive analysis was also performed using cell lines
representative of earlier and later stages of B cell development as
well as the non-B cell, K562. Using the A8B-1 probe and the
XbaI-BamHI fragment shown in Figure 2A, both hypersensitive sites were determined to be cell and
stage specific, as they could only be detected in the CR2+
cells (Fig. 2B, Table I).
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Identification of a transcriptional silencer near HS2 within the first intron of the CR2 gene
Since we were unable to recreate appropriate regulation of
the CR2 gene using 5' promoter constructs from +75 bp to -5 kb (see
below), our studies focused on the second cell- and stage-specific
hypersensitive site (HS2) located within the first intron of the CR2
gene. To determine the functional importance of the region containing
the HS2 site, we used a 2.5-kb XbaI-SacI fragment
(that contained the HS2 site) in a series of constructs using the CR2
promoter driving a neo reporter gene. As controls, we also created the
same constructs without the XbaI-SacI fragment
(Fig. 3A). An
additional construct contained only the -149/+75 region, but without
the intronic segment. Transient transfection of these constructs and
analysis using a probe detecting correctly initiated transcripts showed
no evidence of appropriate regulation using a panel of cell lines
representing B and non-B cells, as well as different stages of B cell
development (summarized in Table II).
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B). Appropriate expression was due to
the apparent transcriptional silencing by an element (designated from
here as CRS (CR2 Silencer)) in the 2.5-kb intronic region that acts on
the (-5-kb/+75) promoter region in non-CR2-expressing cells.
Importantly, in this analysis, comparable genomic equivalents of neo
reporter DNA were present in each matched pair of transfected cells
(see figure legend). The experiments shown in Figure 3B are
also representative of at least two to three experiments with each line
and, because of the method used, represent pools of many clones of
stably transfected cells.
In these studies, the CRS-containing segment is cloned both
downstream of the polyadenylation sequences in the neo reporter gene
and in the antisense orientation. The finding of neo reporter mRNA
expression in many cell types transiently transfected with this
construct (Table II), in stably transfected Jiyoye cells (Fig. 3B), and in lymphoid tissues in transgenic animals
(see below) essentially rules out trivial explanations for our
findings, such as the intronic segment interfering nonspecifically with
neo reporter mRNA in Reh, SKW-1, and/or K562 cells. These results
demonstrate the presence of a transcriptional silencer that is active
in non-CR2-expressing cell lines of pre-B and Ig-secreting B cell
stages, in addition to non-B lineage cell types represented by K562
cells. Interestingly, this silencing effect is only seen following
stable transfection of the construct that contains it, implying that
nuclear matrix and/or chromatin interactions may be necessary for its
regulatory activity. In addition, this silencing can act at a distance
and in the antisense orientation.
Tissue-specific silencing by the CRS element in transgenic mice
To confirm the silencing effect of the CR2 intronic segment, we made transgenic mice using the CR2(-5-kb/+75)-neo reporter construct with and without the 2.5-kb intronic sequence. As discussed above, mouse CR2 is expressed on B lymphocytes in a stage-specific manner similar, if not identical, to human CR2. Mouse CR2 is also expressed on follicular dendritic cells, as is human CR2. One difference between the two species is the lack of mouse CR2 expression on T cells or thymocytes (70), whereas human CR2 is found on a subpopulation of peripheral T cells and early thymocytes. On the other hand, like human CR2, mouse CR2 is not expressed in the kidney, heart, liver parenchyma, or other nonlymphoid tissue types. Thus, with some minor differences, the expression of mouse CR2 largely parallels that of human CR2.
We performed parallel pronuclear injections using linearized
CR2(-5-kb/+75)-neo in addition to the CR2(-5-kb/+75)-neo-CRS
constructs. Four founders were identified by tail DNA dot-blot
analysis, and further confirmed by Southern blot, from mice injected
with the CRS-containing construct, and three founders with the
non-CRS-containing construct. Table III
summarizes the characteristics of each founder.
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A demonstrates the
result of an S1 analysis of RNA derived from progeny of founder 1419,
which contains the CRS element. A neo mRNA signal essentially identical
to the signal from the transfected positive control Jiyoye cells is
found only in RNA from spleen and bone marrow, but not liver, kidney,
or heart. Shown for comparison also is spleen and liver RNA from a
nontransgenic mouse, which, as expected, has no neo mRNA-derived S1
signal, but does from the internal control,
ß2-microglobulin.
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Figure 4B shows the results of an RT-PCR analysis of RNA
from transgenic mice. As can be seen, a neo mRNA-derived band in the
CRS-containing 1424 founder line is seen in the lymphoid tissues:
spleen, thymus, and bone marrow, but not liver, kidney, or heart. In
contrast, the 1515 founder line, generated with the (-5
kb/+75) CR2 promoter region without the CRS, demonstrated a neo
mRNA-derived RT-PCR signal in all tissues tested. In this analysis,
mouse Crry/p65, which is expressed in all cell types, serves as a
positive control, confirming the quality of the RNA in which a neo
mRNA-derived signal is not seen.
Table IV summarizes results of identical
studies of neo mRNA expression in all transgenic founder lines. In this
analysis of seven different tissue types, neo mRNA expression in the
1419 and 1424 founder lines is only detected in the lymphoid tissues,
while neo mRNA expression was found in every tissue tested in the 1515
founder line. Additionally, the 1515 founder line expressed neo mRNA in
muscle tissue, while the CRS-containing founders did not (data not
shown). These results, which agree with the data generated using S1
analysis, are consistent with our results using stably transfected cell
lines, and provide strong evidence for a tissue-specific silencer
within the CRS.
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In summary, these results support the conclusion that the 2.5-kb CR2 intronic segment contains a silencing activity. Importantly, in progeny of two founders (1419 and 1424), the silencing activity allows reporter gene expression only in the thymus, spleen, and bone marrow, which are the tissues in which human CR2 is expressed. In organs such as liver, kidney, heart, and muscle, which do not express CR2 in humans or mice, neo mRNA expression is silenced. In the absence of the intronic element, neo reporter mRNA is found widely in the progeny of one founder (1515). This is identical to the results of stable transfection analyses in human cell lines, in which the promoter region in this construct (-5 kb/+75) is active in all cell types independent of endogenous CR2 expression status. Interestingly, the presence of neo mRNA in the thymus of CRS-containing founders suggests that this construct directs expression in a human pattern with regard to T cells, as mouse thymocytes do not express CR2, but early thymocytes in humans do.
The sequence of the 2.5-kb CRS intronic element is shown in Figure 5. Before sequencing of the segment, we
were concerned that it might contain one or more pseudo-exons
previously described in this general region as remnants of a primordial
CR2/CR1 gene shared with mouse (71). However, sequence analysis
demonstrated no evidence of such pseudo-exons within the CRS (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Through stable transfections and transgenic studies, our findings strongly support the hypothesis that both the tissue specificity and developmentally restricted expression of the human CR2 gene are controlled primarily by an intronic silencer that we have designated CRS. This CRS element is able to confer cell- and stage-specific expression of the CR2 proximal promoter when the reporter constructs containing these elements are stably integrated into the genome. In the absence of the CRS, or when the construct containing it is only transiently transfected into cells, the CR2 proximal promoter is active, regardless of endogenous CR2 expression.
The silencer activity we have described in this work shows functional similarities to intronic silencers identified in other eukaryotic systems. For instance, the CD4 gene is inactive early in thymocyte development, becomes active at a defined developmental point (the CD4+CD8+ double-positive stage), and then is inactivated again in CD8+ single-positive T cells. This regulated pattern of expression also appears to be controlled primarily by an intronic silencer in combination with an active promoter (72, 73, 74). Both the CD4 and CR2 intronic elements function when placed outside of the coding region of the reporter gene and in the reverse orientation.
In a recent report, Hu and colleagues identified intronic sequences in the murine CR2 gene that partially silence the 5' proximal promoter of CR2 in T cells, but not in B cells (75). Analysis of subfragments of this intronic region indicates that the tissue-specific expression is most likely controlled by both positive and negative regulatory elements, although the specific factors have not yet been identified and it is unknown whether this region can mediate developmentally restricted expression of murine CR2 in B cells.
One notable difference between the silencer characterized in this
report and the murine CR2 silencer is the necessity for the CRS to be
integrated stably into the chromatin to mediate its effects. This
strongly suggests that nuclear matrix or chromatin interactions are
necessary for functional silencing of human CR2. This result is
reminiscent of several observations demonstrating important roles for
nuclear matrix interactions in the control of the Ig µ heavy chain
enhancer activity (76) and 
gene expression in transgenic mice (77).
In addition, developmentally regulated alterations in chromatin
structure have been suggested to play a role in Ig heavy chain allelic
exclusion (78).
The possibility that CR2 genetic elements may interact with matrix is
supported by the observation that both the CRS region and the 5'
promoter contain AT-rich sequences with structural features of matrix
attachment regions (79). Additionally, the CRS contains a 9- of 11-bp
match with the consensus sequence for a Drosophila
topoisomerase II binding site (Fig. 5) (80) as well as a DNase I
hypersensitive site. Both of these sites are characteristics of matrix
attachment regions and may indicate that the silencer activity within
the CRS is related to interactions between this intronic region and
nuclear matrix. Several potential mechanisms, such as the necessity of
matrix to localize specific protein complexes with silencing activity
to the gene, or to control the access of such factors to genetic
elements, are consistent with our results to date. Furthermore,
specific silencing mechanisms, such as the binding of transcriptional
repressors, competition for transactivator factor binding sites, and
interference with protein-protein interactions necessary for
promoter/enhancer activity, could work in concert with developmental
changes in chromatin structure or accessibility to modulate the
expression of CR2.
Our results showing that four of seven transgenic founders are nonexpressors of neo reporter mRNA suggest the surrounding chromatin can have profound effects on the expression of our transgenes. It is likely that other CR2 genomic elements, such as a locus control region or insulator, are required to achieve copy number dependence and site of integration independence in transgenic animals. Observations in other transgenic settings have shown that such regulatory elements are necessary to obtain high levels of expression, but do not necessarily control the specificity of expression (Ref. 69 and references therein).
In the founder lines that did express neo mRNA, the CRS appears to restrict expression to lymphoid tissues in transgenic mice. However, the pattern of expression in this transgenic setting could be more human-like, with T cell and thymocyte expression, or more mouse-like, with no such expression. The finding of neo reporter mRNA expression in the thymus indicates the former possibility is more likely.
CR2 is a gene that is activated at an important point during B lymphocyte development. It has been proposed that the addition of CR2 and other genes at this point arms the B cell with receptors that are capable of interacting with environmental signals such as activated complement C3 (11). Whether the activation of genes at this developmental point is a stochastic process, is regulated by cytokines or cell-cell interactions, or is possibly controlled by a combination of mechanisms, is currently unknown. Further dissection and identification of specific nucleotide target sites within the CRS element may provide an appropriate assay system in which to test putative signaling and transcriptional regulatory mechanisms that are active at this point.
Of interest is the presence at positions 687 to 693 of a perfect match
with the heptamer binding site for CBF1, GTGGGAA (Fig. 5) (46). CBF1 is
a ubiquitously expressed protein that acts as a repressor within the
CD23 gene (45). EBNA-2, which increases expression of both CD23 and
CR2, has been shown to interact with CBF1 and relieve CBF1-mediated
repression of CD23 in addition to the EBNA-2-responsive EBV latency
promoter (Cp) and the promoter for the EBV protein LMP-1. Unlike CD23,
the CR2 5' proximal promoter from (-1253/+75) could not be
transactivated significantly by EBNA-2 in coexpression experiments (C.
Pham and V. M. Holers, unpublished observations), and does not
contain an identical match to a consensus CBF1 binding site. If the
putative CBF1 site within the CR2 intronic segment is found to act as a
functional silencer and an EBNA-2 response element, this will link CR2
and CD23 transcription to a common mechanism. In this regard, it is of
some theoretic interest that CR2 is a receptor for CD23. Coordination
of expression by a common regulatory mechanism would be an elegant way
to help link activities of these two proteins.
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Acknowledgments |
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Footnotes |
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2 The first two authors contributed equally to these studies.
3 Address correspondence and reprint requests to Dr. V. Michael Holers, Division of Rheumatology Box B-115, University of Colorado Health Sciences Center, 4200 East Ninth Ave., Denver, CO 80262. E-mail address:
4 Abbreviations used in this paper: EBNA, Epstein-Barr virus nuclear Ag; CAT, chloramphenicol transferase; CBF, c-promoter binding factor; CRS, complement receptor 2 silencer; hnRNA, heterogeneous nuclear RNA; HS, hypersensitive site; MCP, membrane cofactor protein; neo, neomycin; SCR, short consensus repeat.
Received for publication July 7, 1997. Accepted for publication October 22, 1997.
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X174 DNA-HaeIII
digest fragments. B, Nuclear runoff analysis using the CR2
cDNA plasmid pBSCR2–12, a plasmid (pBSCR2 (+1/+418))
containing the first 418 bp of CR2 hnRNA, negative control pBS, and
positive controls ß 12-actin and Alu. Nuclear transcription
demonstrated by specific hybridization of the nascent RNA to pBSCR2–12
and pBSCR2(+1/+418), as compared with pBS alone, is seen in Raji only.
C, Densitometric analysis of the experiment shown in
B. Data are expressed as the ratio of pBSCR2 over pBS for
each cell line to normalize for background differences. A specific CR2
RNA signal is indicated by a ratio greater than one. The pre-B (Nall-1,
Reh), Ig-secreting (SKW-1, DHL-4), and T cell (CEM) lines did not show
a specific signal with either CR2 probe, while the pBSCR2–12 signal is
increased 7 (Reh)-, 5.5 (Nall-1)-, 5.8 (SKW-1)-, 4.5 (DHL-4)-, and 5.7
(Cem)-fold, and the pBSCR2(+1/+418) signal is increased 14.9 (Reh)-,
7.5 (Nall-1)-, 5.2 (SKW-1)-, 4.6 (DHL-4)-, and 11.2 (Cem)-fold in Raji
cells relative to the other cell lines. Data were generated using
ImageQuant software (Molecular Dynamics).
