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Departments of Immunology and Medicine, and
Division of Rheumatology, University of Colorado Health Sciences Center, Denver, CO 80262
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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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(7). Human CR2 is primarily expressed during later stages of B cell ontogeny (8); however, it is also expressed on follicular dendritic cells (9), epithelial cells (10), some thymocytes (11), and a small subset of CD4+ and CD8+ peripheral T cells (12, 13). Within the B cell lineage, CR2 is only found on immature and mature B cells, and its expression begins at approximately the same stage as IgD and CD23 (8, 14). It has been shown that CR2 is up-regulated after B cells escape negative selection and migrate to the periphery (15, 16, 17).
Previously, we have shown that cell- and stage-specific expression of human CR2 is controlled by an intronic transcriptional silencer, designated the CRS (CR2 silencer). The use of a stable transfection system and transgenic mice has shown that the CRS element, in conjunction with the CR2 proximal promoter, is able to repress transcription in CR2-negative cell lines and tissues (18). Recent studies have further defined the CRS element and have shown a sequence within the silencer crucial to its function. This sequence binds the transcriptional repressor C-promoter binding factor 1 (CBF1), a member of the developmentally important Notch signaling pathway. Mutation of this site results in loss of function of the silencer and strongly suggests that CBF1 plays a role in controlling human CR2 expression (19). Furthermore, it was shown that the silencer was unable to repress a heterologous promoter, suggesting specificity for proximal promoter sites. Similarly, in the mouse, CR2 expression is regulated by an intronic silencer (20) that also requires CR2 proximal promoter sites for appropriate function (21).
In the studies reported herein, we have further analyzed the human CR2 proximal promoter to identify cell type-specific elements that could act as putative interaction sites for the CR2 intronic silencer. We demonstrate the presence of a cell type-specific repressor that shows broad lineage- and stage-specific utilization.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All human cell lines used in these experiments were obtained from American Type Culture Collection (Manassas, VA). Cells lines were maintained at 37°C with 5% CO2 in RPMI 1640 with L-glutamine supplemented with 10% FBS, 100 µg/ml streptomycin, and 100 IU/ml penicillin.
Creation and confirmation of mutant CR2 promoter/luciferase fusion constructs
An NheI/XhoI fragment of the CR2 promoter containing nt -315/+75 was cloned into the luciferase reporter pGL3-basic vector (CLONTECH Laboratories, Palo Alto, CA). Site-directed mutagenesis was performed using the Quickchange mutagenesis kit (Stratagene, La Jolla, CA), which enabled the incorporation of MluI restriction sites extending 3' from positions -140 (Sp1), -90 (AP1), -81 (AP2), -60 (E box 2) and -47 (E box 1). The accuracy of all constructs created was assured by both restriction enzyme digestion and nucleotide sequence analysis.
Creation and analysis of mutant CR2 linker scanning constructs
Linker scanning mutagenesis was performed using the Quickchange
mutagenesis kit (Stratagene). Incorporation of MluI
restriction sites was made across the E box 2 motif at intervals of
2 bp. An internal deletion construct was also made by use of a
primer that lacked bp -67 to -61 of the CR2 promoter. All constructs
made were confirmed by nucleotide sequence analysis.
Transfection and measurement of promoter/reporter gene activity
Before each transfection, cells were split and grown in log
phase to 5 x 105 cells/ml. Cells were
then transfected using the Qiagen Superfect transfection reagent
according to the manufacturer’s specifications with plasmid DNA
prepared using Qiagen Maxiprep-500 columns (Qiagen, Valencia, CA).
Briefly, 10 µg of plasmid DNA and 300 ng of pRL-thymidine kinase
control vector were complexed in combination with Superfect reagent for
10 min at room temperature. The transfection complexes were then added
dropwise to the cells that had been plated in 5 ml of medium in a
six-well tray at a concentration of 5 x
105–1 x 106
cells/ml. The cells were then incubated at 37°C for 48 h
following transfection. Cell lysates from the transfected cells were
prepared and assayed for both firefly and Renilla luciferase
according to the manufacturer’s instructions (Promega, Madison, WI).
All transfection data shown are the mean of 3–10 independent
transfections, with n values shown in each experiment.
Additionally, multiple preparations of DNA were used and yielded
essentially identical results. Promoter activity is expressed as
relative firefly luciferase activity normalized against
Renilla luciferase activity.
EMSA
Approximately 8 x 107 cells were used to make nuclear extracts according to a standard method (22). Extracts were frozen in liquid nitrogen and stored at -80°C. Determination of protein concentration was performed using the Bio-Rad protein assay kit (Hercules, CA). For EMSA, nuclear extracts were preincubated (10–20 µg) on ice for 10 min along with 1 µg of poly(dI-dC) in a binding buffer consisting of 4% Ficoll, 20 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM DTT, and 50 mM KCl. When required, competitor oligonucleotides or supershift Abs (Santa Cruz Biotechnology, Santa Cruz, CA) were incubated with the nuclear extract for 30 min on ice. The nuclear extract was then incubated with 80 fmol of 32P-labeled oligonucleotide for 30 min on ice before loading onto a 6% polyacrylamide gel. The gel was electrophoresed at 150 V using 0.25x Tris-taurin-EDTA as the running buffer. EMSA gels were dried under vacuum and exposed to x-ray film. All double-stranded oligonucleotides were end-labeled using [32P]ATP and T4 polynucleotide kinase.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether the CR2 proximal promoter demonstrates a cell
type-specific component to its activity, the -315/+75 luciferase
construct was transiently transfected into informative cell lines that
either did or did not express CR2. The -315/+75 construct was used in
these studies, as previous experiments revealed that the
transcriptional activity of this construct was comparable to that of a
longer construct containing -1250 to +75 of upstream promoter sequence
(data not shown). In mature B cell lines (Fig. 1
; Daudi and Raji), which express CR2 at
a high level, the mean normalized transcriptional activity (vs
Renilla internal control) was 5.272 ± 1.374
(n = 10) for the Daudi cell line and 5.065 ±
0.271 for the Raji (n = 5) cell line. This is in
contrast to two non-B cell lines (that do not express CR2), K562 and
U937, which demonstrated much lower promoter activity (0.627 ±
0.157 (n = 11) and 0.281 ± 0.065
(n = 4), respectively; Fig. 1).
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, Reh). These results also demonstrated a low relative level of
transcription in this cell line (0.563 ± 0.115). Overall, these
results show that even though the CR2-proximal promoter is active in
all cell lines tested compared with the pGL3 vector alone (0.0538
± 0.005; n = 15), the relative CR2 promoter activity
strongly correlates with CR2 expression status. Cell lines that express
CR2 possess a markedly higher basal promoter activity compared with
cell lines that do not express CR2. Functional analysis of the CR2 proximal promoter using site-directed mutagenesis
Because the -315/+75 luciferase construct possessed a cell
type-specific level of activity, mutant constructs were made and
transiently transfected into K562 (CR2-negative) or Raji (CR2-positive)
cell lines. The mutants were constructed by introduction of a
MluI site into previously identified transcription factor
binding sites (Fig. 2). The results were
analyzed to determine the presence of either a cell type-specific
enhancer element that activated transcription in the CR2-expressing
cell lines or, alternatively, a cell type-specific repressor element
that dampened transcriptional activity in CR2-negative lines.
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, construct 2) and the consensus AP1
site at -90 (Fig. 3, construct 3) resulted in no significant change in
transcriptional activity compared with the -315/+75 wild-type
construct. This result was seen in both cell lines tested.
Site-specific mutation of the consensus AP2 site at
position -81 (Fig. 3, construct 4) resulted in a modest 1.7-fold
increase in transcriptional activity in K562 cells and a 1.6-fold
increase in activity in Raji cells. These results indicate the possible
presence of a repressor element at this site; however, this element
does not appear to be cell type specific. Mutation of the E box 1 motif
located at position -47 resulted in an 80% reduction of
transcriptional activity in Raji cells. This result was also seen in
K562 cells, as this mutation resulted in a marked (55%) reduction in
promoter activity (Fig. 3, construct 6). These results demonstrate the
presence of an activating motif at this site; however, again, its
activity is not cell type specific.
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, construct 5) at
position -60 was mutated, cell type-specific activity was identified.
Mutation of this site and transient transfection into Raji cells
resulted in a modest 1.5-fold increase in promoter activity (Fig. 3, construct 5). However, in K562 cells the same mutant demonstrated an
almost 6-fold increase in transcriptional activity compared with the
-315/+75 wild-type construct. These results reveal the presence of a
cell type-specific repressor located at position -60 of the
CR2-proximal promoter. Broad lineage utilization of the cell type-specific repressor
To determine whether this cell type-specific repression was
merely limited to a unique effect in either Raji or K562 cells or was
due to a true broadly used repressor element, the E box 2 mutant
construct was transiently transfected into other informative cell
lines. When the mutant construct was transfected into two mature B cell
lines (CR2-positive), Raji and Daudi, only a modest increase in
transcriptional activity was seen (1.561 ± 0.139
(n = 5) and 1.128 ± 0.149 (n =
5), respectively; Fig. 4). This was in
contrast to the two non-B cell lines tested, neither of which expresses
CR2. In the K562 cells, the mutation resulted in a 5.699 ±
0.0745-fold (n = 7) increase in transcriptional
activity compared with the wild-type construct. Similarly, in U937
cells, a 5.758 ± 0.396-fold (n = 7) increase in
promoter activity was seen (Fig. 4). To determine whether the repressor
element was also active in cells of the B cell lineage that did not
express CR2, the E box 2 mutant was transiently transfected into Reh
cells, a pre-B cell line. A similar result was observed in this cell
line as in the non-B cells. The mutant construct resulted in a
4.636 ± 0.938-fold increase in transcriptional activity compared
with the -315/+75 wild-type construct. These results indicate the
presence of a cell type-specific repressor that is primarily active in
cell lines that do not express CR2.
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The reporter gene analysis of the E box 2 motif demonstrated the
importance of this site as a cell type-specific repressor. To determine
the nature of the transcription factors binding to this site, EMSA was
performed using double-stranded oligonucleotides corresponding to -73
to -52 region of the proximal promoter encompassing the E box 2 motif
(Fig. 5, E box 2). The EMSA pattern
observed when the oligonucleotide was labeled and bound to K562 nuclear
extracts was complex, with the presence of three protein-DNA complexes
(Fig. 5A, complexes A–C) as well as complexes with slower
mobility (Fig. 5A, higher order complex). All protein-DNA
complexes were shown to be specific, as addition of increasing amounts
of cold self-competitor resulted in abolishment of all complexes (Fig. 5A).
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, Mutant). The mutant
oligonucleotide corresponded to the same nucleotides as the wild type
(-73 to -52); however, positions -66 to -61 were changed to
nucleotides, ACGCGT, corresponding to the MluI restriction
sequence used in the transcriptional assays in Figs. 3 and 4. The
wild-type and mutant oligonucleotides were labeled and bound to K562
nuclear extract, and the EMSA profiles were compared (Fig. 5B). The mutant oligonucleotide was no longer able to bind
protein-DNA complexes A and C and the higher order complexes compared
with the wild-type oligonucleotide. These results indicate that the
most relevant proteins required for the functional activity of this
repressor are in these latter complexes. Linker-scanning mutagenesis reveals critical nucleotides for repressor function
It was apparent from the functional studies that the CR2 promoter
possesses a repressor activity and that loss of this activity
correlates with loss of specific protein-DNA complexes in EMSA. To
determine which specific nucleotides were critical for repressor
function, linker-scanning mutagenesis was performed. Each
linker-scanning mutant was made by introduction of an
MluI restriction site across the sequence separated by two
nucleotides (Fig. 6A).
Additionally, to eliminate the possibility that introduction of the
MluI site artificially introduced a new transcription factor
binding site potentially containing activation functions, an
internal deletion construct was made. This construct had nucleotides
-67 to -61 deleted from an otherwise intact -315/+75 sequence (Fig. 6A).
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B; n =
4–10), again indicating that this repressor is not active in
CR2-expressing cells. These results were in contrast to those seen in
K562 cells. Parallel transfection of linker scanning (LS)
mutants (-62/-57), LS (-60/-55), and the internal deletion
possessed similar fold increase over the -315/+75 wild-type construct
as the original E box 2 mutant (Fig. 6B; n =
4–10). Thus, the nucleotides that are critical for the function of
this cell type repressor are situated from nucleotides -67 to -59 of
the proximal promoter. The results of the analysis using the internal
deletion construct showed that no unanticipated activation sequence was
introduced. Cross-competition EMSA reveals protein-DNA complexes important to repressor function
Linker-scanning mutagenesis revealed nucleotides critical for
functioning of the CR2 repressor element. EMSA oligonucleotides were
made that comprised -73 to -52 of promoter sequence (Fig. 7A, wild type). Mutant
oligonucleotides were designed spanning the same region and included
MluI restriction sequences substituted at the same positions
as those used in the functional assays. The MluI sequence
(ACGCGT) was substituted in the E box 2 mutant at position -65 to
-61, the LS (-62/-57) at position -62 to -57, and the LS
(-60/-55) at position -60 to -55; the LS Internal deletion (LS Int
Del) had nt -67 to -61 deleted (Fig. 7A).
Cross-competition EMSA was performed using labeled wild-type
oligonucleotide competed with a 250-fold molar excess of cold self,
cold E box 2 mutant, cold LS (-62/-57), cold LS (-60/-55), or cold
LS Int Del, competitor. The proteins unaffected by the specific
mutations will bind the mutant oligonucleotides, leaving behind only
proteins still able to bind the wild-type probe on the gel. This
enables identification of each protein-DNA complex critical for
function.
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B, Self). Competition using the E box 2
mutant probe resulted in binding of complex A, complex C, and the
higher order complex to the wild-type probe. Similar results were seen
using the internal deletion oligonucleotide. These results indicate
that deletion of positions -67 to -61 or mutation of -65 to -61
results in the inability of these complexes to bind (Fig. 7B, LS Int Del, E box 2 Mut). Competition using LS
(-62/-57) resulted in abolishment of the higher order complexes and
complex C, but not complex A, indicating that nucleotides -62 to -57
are critical for binding of complex A (Fig. 7B, LS
(-62/-57)). Interestingly, the opposite was true for LS (-60/-55).
Competition of wild type with this probe resulted in loss of complexes
A and C, but not the higher order complexes. These results demonstrate
that nucleotides -60 to -55 are critical for formation of the higher
order complexes (Fig. 7B, LS (-60/-55)). As all mutations
used in the EMSA experiments corresponded to mutations resulting in
functional activity, these results suggest that protein-DNA complexes A
and C and the higher order complexes are all necessary for functioning
of the repressor. Mutations of nucleotides that result in abolishment
of protein-DNA complex A alone (Fig. 7B, LS (-62/-57)),
the higher order complex alone (Fig. 7B, LS
(-60/-55), or their combination (Fig. 7B, E box 2 mut, LS
Int Del) all result in loss of repressor function in the transfection
assays, as observed by an increase in transcriptional activity in K562
cells. Supershift assays reveal binding of the basic helix-loop-helix (bHLH) protein E2A to the repressor element
To further characterize the proteins required for repressor
activity, supershift assays were performed. The EMSA oligonucleotide
used in these experiments corresponded to the oligonucleotide used in
the linker-scanning mutagenesis experiments and comprised -73 to -52
of promoter sequence (Fig. 7A, wild type). Supershift assays
were performed using labeled wild-type oligonucleotide (Fig. 8, wild type). Various Abs were then
added. Addition of an Ab directed against the bHLH protein E2A resulted
in abolishment of protein-DNA complex A (Fig. 8, E2A).
Cross-competition experiments using mutant EMSA oligonucleotides
(Fig. 7) have indicated the importance of protein-DNA
complex A to the function of the repressor element. Addition of an Ab
directed against E47 alone resulted in abolishment of one of the higher
order complexes, also shown by cross-competition experiments to be
involved in repressor function. Protein-DNA complex C, a functionally
relevant complex, was not effected by either Ab, indicating that an as
yet unidentified protein is also involved in the binding of this
repressor motif. Addition of Abs directed against two other bHLH
proteins, namely, USF1 and TFE3 (Fig. 8), did not have an effect on
complex formation. These results strongly suggest that the E2A proteins
E12/E47 are functionally relevant proteins in the repressor activity
associated with the E box 2 motif.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have extended the analysis of the transcriptional requirements for human CR2 and have analyzed the proximal promoter region for candidate sites involved in cell type-specific regulation of CR2. Data obtained from transfection of a -315/+75 proximal promoter sequence upstream of a luciferase reporter demonstrated several informative results. This construct, although active in all cell lines tested compared with empty vector control, clearly demonstrated a cell type-specific component to its activity. These results could indicate the presence of an activator element within the proximal promoter that elevated transcription in CR2-expressing cells or, alternatively, a repressor element within this sequence could be present that dampened transcription in CR2-nonexpressing cells. The use of several site-directed mutants revealed the presence of a repressor element within CR2-negative cell lines that inhibited CR2 transcription, demonstrating that the latter mechanism is operating.
Data collected from linker-scanning mutagenesis revealed that the critical nucleotides for repressor function matched an E box motif that is known to bind the HLH family of transcription factors (23). The HLH family of proteins plays a major role in multiple developmental processes. To date, >240 HLH proteins have been identified in many different organisms (24). E box elements and HLH proteins have been identified in many promoter and enhancer elements that regulate muscle (25), pancreas (26), neuron (27), and B cell-specific gene expression (28, 29). Further characterization of the proteins binding the CR2 E box repressor element using Abs directed against many members of the HLH family of transcription factors was undertaken. Supershift analysis revealed competition of functional protein-DNA complexes by Abs directed against E2A and E47, indicating that both E12 and E47 are able to bind the CR2 E box repressor element. The E12 and E47 proteins arise by alternative splicing of the E2A gene (30, 31) and are known as class I HLH proteins.
Due to the large number of HLH proteins within this family, tissue distribution, dimerization capabilities, and DNA binding specificity were used to devise a classification code (32). Class I HLH proteins are also known as E proteins and include the following; E12, E47, HEB, E2-2, and Daughterless. All of these proteins are widely expressed and are able to form either homodimers in B cells or heterodimers with tissue-specific class II HLH proteins in other cell types (23). E proteins, in particular E2A gene products, have been shown to be involved in cell differentiation, lineage commitment, and B lineage-specific gene expression (32). Interestingly, E2A knockout mice lack pre-B and mature B cells and have reduced numbers of B220+CD43+ B cell progenitors, indicating a critical role for E2A in B cell development (33, 34).
As human CR2 is tightly regulated during B cell development, E2A may also play a role in the expression of CR2. However, E proteins have generally been shown to play a role in activating B cell-specific genes (23). In the case of the CR2-proximal promoter, E2A proteins are involved in repression. To date, very little is known about the role of E2A proteins in repressing transcriptional activity. However, recent data suggest that mitogen-activated protein-activated protein kinases interact with E47 and are able to phosphorylate this protein, resulting in repression of the transcriptional activity of E47 on an E box-containing promoter (35). Additionally, E47 phosphorylation inhibits binding of E47 homodimers, but allows E47 heterodimer formation, suggesting a differential regulation of E proteins in B cells and non-B cells (36). Class II HLH proteins have also been shown to act as transcriptional repressors. For example, ABF-1 is able to inhibit E47-dependent activation through formation of heterodimers. E2A-ABF-1 heterodimers may function to actively repress E box-containing genes, or, alternatively, these heterodimers may be transcriptionally inactive (37). Furthermore, E proteins are able to form homodimers as well as heterodimers with other members of the bHLH family and thus differentially regulate transcription. It has been shown that Id proteins preferentially dimerize with E proteins and consequently prevent heterodimers from binding DNA and activating target genes (38). These studies suggest that while E2A proteins are, in general, transcriptional activators, several mechanisms have been discovered that could explain the presence of E2A in a repressor complex regulating CR2 promoter activity.
In conclusion, we have discovered a cell type-specific repressor element within the human CR2-proximal promoter that displays broad lineage utilization. Furthermore, this repressor appears to require HLH transcription factors, in particular, gene products of E2A to function. Whether adapter proteins such as the Id family of proteins are involved and are contributing to the repression mechanism is currently being examined.
Previous studies involving the human intronic silencer has shown critical function for CBF1 (19), a known transcriptional repressor. CBF1 is a component of the Notch signaling pathway. Interestingly, activated Notch 1 and Notch 2 are also able to inhibit E47 activity (39). Notch signaling has now been tied to two repressor elements functioning within the human CR2 regulatory regions. Data collected from our laboratory have shown that the CR2 silencer must interact with the CR2-proximal promoter for function. Therefore, it is intriguing to speculate whether theE2A-containing promoter repressor element and the CBF1 silencing element interact with one another or act in concert with Notch signaling. Alternatively, as CR2 expression is tightly regulated, several mechanisms may play a role in controlling CR2 gene regulation in different cell lineages and stages. These questions are currently under investigation.
Finally, we used the data presented herein together with previous
results to generate a model of how the human CR2 gene is regulated
(Fig. 9). Early studies (18)
have shown the lack of DNase I-hypersensitive sites within cell lines
that do not express CR2, indicating the possibility of a closed
chromatin configuration over the CR2 control elements (Fig. 9, OFF).
This is in contrast to CR2-expressing cell lines that possess two
hypersensitive sites, one across the proximal promoter and the other
within the first intron, i.e., the silencer site (18). It
is interesting to speculate that external signals such as Notch
signaling or histone deacetylases may be relevant in opening the
chromatin configuration to allow CR2 to be expressed (Fig. 9, ON).
Histone deacetylation appears critical for mouse CR2 regulation
(21), but to date has not been confirmed in the human gene
(K. Makar and V. M. Holers, unpublished observations). Two
repressor elements have been identified within the human CR2 regulatory
regions. The first is CBF1 within the intronic silencer
(19), and the second are the E2A gene products within the
proximal promoter as shown herein. Within the silenced locus (Fig. 9, OFF), CBF1 may interact with the E2A proteins to repress transcription.
Alternatively, phosphorylation of E47 or another as yet unidentified
negative regulator may interact with the promoter element to repress
transcription. Within an active locus (Fig. 9, ON), E47/E12 may no
longer be repressed by external factors. Additionally, Notch may mask
the inhibitory effect of CBF1 and, therefore, allow positive regulatory
elements within the promoter to activate CR2 transcription.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. V. Michael Holers, Division of Rheumatology, Campus Box B-115, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262. E-mail address: michael.holers{at}uchsc.edu
3 Abbreviations used in this paper: CR2, complement receptor type 2; HLH; helix-loop-helix; bHLH, basic HLH; CBF1, C-promoter binding factor 1; CRS, CR2 silencer; LS, linker scanning.
Received for publication August 15, 2001. Accepted for publication October 16, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor. EMBO J. 10:919.[Medline]
-cell insulin gene transcription factor that binds to and appears to activate cell-specific gene expression: its possible relationship to other cellular factors that bind to a common insulin gene sequence. Mol. Cell. Biol. 10:1564.2-subunit of the neuronal nicotinic acetylcholine receptor studied in vitro and in transgenic mice. Neuroscience 69:807.[Medline]
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