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From the Department of Biochemistry, University of Western Australia, Nedlands, Australia
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activity consistent
with the positive effect of CD30 on
proliferation.2,9,12-14
CD30 has been implicated in the
control of differentiation and/or selection events within the thymus.
CD30-deficient mice have been found to contain elevated numbers of
thymocytes and exhibit defects in negative selection, suggesting a role
for CD30 in the deletion of autoreactive T cells.15
These
findings have been confirmed in transgenic mice overexpressing CD30, in
which overexpression enhances the deletion of
CD4+/CD8+ thymocytes and so
may act as a costimulatory molecule in thymic negative
selection.16 CD30 was originally identified as a surface marker on Hodgkin’s and Reed-Sternberg (H-RS)3 cells, the neoplastic component of Hodgkin’s disease.17,18 Diagnosis of Hodgkin’s disease is based on the presence of a small population of these H-RS cells surrounded by a cellular infiltrate composed of normal immunoreactive lymphocytes, neutrophils, eosinophils, histiocytes, plasma, and stromal cells.10,19 The exact cellular origin of these H-RS cells remains to be determined, although all of these cell types have been implicated.20,21 Overexpression of CD30 is characteristic of Hodgkin’s disease, where the interaction of CD30 with its ligand CD153 appears to be involved in the regulation of cell-cell interactions, particularly between the H-RS cells and the surrounding lymphoid cells.22 Hence it has been suggested that loss of regulatory control may lead to the observed overexpression of CD30 and contribute to the progression of the disease state. In the later stages of Hodgkin’s disease, the membrane form of CD30 is proteolytically cleaved, and high serum levels of the soluble form of CD30 can be detected, making it a good clinical marker for disease progression.23,24 The soluble form of CD30 may function by competing with surface CD30 for the binding of CD153 (CD30 ligand), thus preventing signaling through the membrane form of CD30, blocking normal intracellular signaling processes, as seen with TNF.25 Interestingly, CD30 overexpression is also a characteristic of other lymphoproliferative disorders, such as non-Hodgkin’s lymphoma, as well as embryonal carcinoma and a number of Th2-associated diseases.26-32 Recently it has been shown that CD30 signaling limits the proliferative potential of autoreactive CD8 effector T cells and protects against autoimmune diabetes.33 It is possible that the observed alterations in the normal expression of CD30 may result in the progression of these CD30-positive diseases through autocrine or paracrine signaling mechanisms. The mechanisms involved in up-regulation of CD30 expression have yet to be investigated.
As a first step toward identifying factors involved in regulating CD30 expression we have recently described the isolation and characterization of the 5' region of the human and murine CD30 genes.34 Sequence comparison revealed significant identity between the mouse and human CD30 promoters. Both genes belong to an expanding group that lack consensus TATA and CAAT boxes, an increasingly common feature among members of the TNF receptor family.35-38 The human and murine CD30 promoters share a number of common consensus transcription factor binding motifs that may be involved in the regulation of gene expression. In particular, our previous study revealed the presence of conserved Sp1 and initiator elements,34 both of which have been implicated in positioning transcriptional machinery in genes that lack a TATA box.39-42
In this report we present the results of functional studies carried out to investigate these conserved regions in the human CD30 promoter. We have identified a number of functionally important regulatory regions, including an Sp1 element in the minimal promoter and a downstream promoter element (DPE) within the initiator, which is required for start site selection. In addition, the discovery of a polymorphic, upstream tetranucleotide repeat that binds proteins acting to repress transcriptional activity of the CD30 promoter, may implicate this region in the dysregulation of CD30 expression in neoplastic cells.
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Materials and Methods |
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Cultured cell lines were obtained from the American Type Culture Collection (Bethesda, MD). All cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum, 2 mmol/L L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2.
DNA Transfection and RNase Protection Assays
Jurkat T cells were transfected by electroporation, using a BioRad (Hercules, CA) Gene Pulser at 240 V, 960 µF. Approximately 4 x 106 cells in a final volume of 400 µl were cotransfected with 10 µg of each test construct and 300 ng of control plasmid pRL-TK. Cells were cultured for 32 hours after transfection in 5 ml complete media before preparation of cellular extracts. Cells were harvested, washed twice in phosphate-buffered saline, and lysed in 300 µl of passive lysis buffer (Promega Corp., Madison, WI). Promoter activity of constructs and controls was measured using the Dual Luciferase Assay kit (Promega), following the manufacturer’s instructions.
Total RNA was isolated from Jurkat T cells transfected with either the -336 CD30/luciferase construct or the DPE downstream mutant, using the RNasol B method (Geneworks, Adelaide, Australia). A fragment of the CD30 promoter from -83 to +167 plus 153 bp (bp) of the luciferase gene derived from the WT CD30/luciferase construct was subcloned into pGEM-3ZF+ (Promega), linearized with an enzyme distal to the promoter, and used as a template for the T7 MAXI Script in Vitro Transcription Kit (Ambion, Austin, TX) to generate an antisense riboprobe. RNase protection assays were performed on 50 µg total RNA, using the Hybspeed RPA kit (Ambion). Protected fragments were analyzed on a 6% denaturing polyacrylamide/urea gel. Gels were dried and exposed to X-ray film.
CD30 Reporter Constructs
A 3.7-kb CD30 DNA fragment spanning the region from approximately -3500 to +199 relative to the major transcription start site was generated by polymerase chain reaction (PCR) from a plasmid containing the human CD30 promoter. This fragment was directionally cloned, via SacI and HindIII sites in the primers, into the polylinker of pGL3-Basic (Promega) to generate the wild-type CD30 reporter construct. The Quick-Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) was used to introduce KpnI or SacI restriction sites at different positions within the wild-type construct to generate a series of mutant constructs. Mutated plasmids were subsequently digested with SacI or KpnI and religated to generate deletion constructs. The microsatellite region of the CD30 promoter (~400 bp) was isolated from the CD30 promoter by digestion with KpnI and cloned upstream from the SV40 promoter in the plasmid pGL3-Promoter (Promega) to generate a heterologous promoter construct. All DNA used in transfections was prepared using the Qiafilter plasmid maxi kit (Qiagen GmbH, Hilden, Germany), and all constructs were checked by sequencing.
Nuclear Extracts, DNase I Footprint Analysis, and Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared according to the method of Li et
al.43
For DNase I footprinting, a fragment of the
CD30 gene spanning the region from -311 to +152 bp was
generated by PCR and labeled with [
-32P]ATP,
using T4 DNA polymerase (Promega). Approximately 10,000 cpm of the
labeled DNA fragment was incubated with 25 µg nuclear extract or 5
µg BSA and 1 µg poly(dI-dC) for 20 minutes on ice. Samples were
treated with increasing units of DNase I (0.02–0.04 U with BSA and
2–10 U with nuclear extract) at room temperature for 2 minutes.
Samples were analyzed using a 6% denaturing polyacrylamide/urea gel.
For comparison a G/A ladder of the same end-labeled DNA fragment was
generated by Maxam-Gilbert chemistry.
Wild-type and mutant double-stranded oligonucleotides
representing -57 to -22 of the CD30 promoter or the
microsatellite region
[(CCAT)5CACCTTATGCAT(CCAT)2]
were synthesized for electrophoretic mobility shift assay (EMSA)
with a single 5' G overhang to facilitate end-labeling with
[
-32P]dCTP. Nuclear proteins were
preincubated on ice with 1 µg poly(dI-dC) in a binding buffer (4%
Ficoll, 20 mmol/L HEPES (pH 7.9), 1 mmol/L EDTA, 1 mmol/L DTT, 50
mmol/L KCl, 0.8 µg ssDNA) to give a final reaction volume of 20 µl.
For competition assays a 50-fold molar excess of specific or
nonspecific competitor, as indicated, was incubated with nuclear
extract for 10 minutes on ice before the addition of
32P-labeled oligonucleotide. Nuclear proteins
were then incubated with 32P-labeled
oligonucleotide (0.32 pmol) for 30 minutes on ice. For supershift
assays anti-Sp1 or anti-Sp3 antibody was added after 30 minutes and
incubated on ice for 1 hour. Products were analyzed by electrophoresis
through a 6% polyacrylamide gel at 150 V.
Microsatellite Typing
DNA was obtained by mouthwash from seven randomly chosen individuals essentially as described, but with the use of 10% Chelex (Biorad) instead of EDTA.44 An aliquot (1/20th of the total) of DNA was used in a 50-ml PCR reaction with 25 pmol each of the microsatellite flanking primers 30MSF (5'-ACCCATTTACCCACTCACCTGC-3') and 30MSR (5'-CAACTGGCCTAGGGAGACTGC-3'). The reactions contained 2.0 mmol/L MgCl2, 1x PCR buffer (Promega, Madison, WI), 2 mmol/L dNTPs, and 1 U Taq polymerase (Promega). Reactions were treated as follows: 94°C for 5 minutes; 29 cycles of 94°C for 30s, 60°C for 30s, 72°C for 1 minute; and 72°C for 10 minutes. PCR products were analyzed on 6% nondenaturing polyacrylamide gel electrophoresis (PAGE).
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Results |
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To identify DNA elements that control the regulation of CD30 transcription, we subcloned the 5' flanking region of the human CD30 gene from -3.5 kb to +199 bp into the luciferase expression vector pGL3-Basic. Restriction sites were introduced into the core recognition sequences contained in the full-length CD30/luciferase construct (-3.5 kb) by site-directed mutagenesis to generate mutant/deletion derivatives for transient transfection assays in Jurkat T cells. Potential transcription factor binding sites chosen for mutation included two Sp1 sites, two ETS sites, and an MZF site. The region of the CD30 promoter containing the Sp1 site (-43 to -38) has been previously shown to be highly conserved between human and murine CD30 promoter sequences. Preliminary transfection results also indicate that the region between -90 and -25 is important in driving transcription.34
Analysis of transient transfection results showed that the full-length
-3.5-kb construct was able to direct low-level expression of the
luciferase reporter in Jurkat T cells as illustrated in Figure 1
. Deletion of DNA sequences between
-3.5 kb to -1.2 kb had no effect on promoter activity. However,
deletion of the sequence between -1.2 kb and -336 resulted in a
3.5-fold increase in promoter activity (Figure 1A)
. This result
suggests that the region contained strong repressor activity. Further
sequential deletion of sequences between -336 and -90 had little
effect on promoter activity. Deletion of the region between -90 and
-40 had a dramatic effect on promoter activity and resulted in a
decrease in activity to the level of the full-length construct and
confirmed the presence of strong positive regulatory elements in the
immediate upstream region containing the Sp1 site.
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Results generated from transfection of mutant constructs in Jurkat
T cells (Figure 1B)
indicated that mutations within the upstream ETS
sites (-238 to -233 and -158 to -153) had little effect on
expression. However, mutation of the highly conserved Sp1 site at
position -43 to -38 had a profound effect on transcription (Figure 1B)
and completely abolished activity to the level seen with the
luciferase vector alone (data not shown), suggesting that proteins
binding within this region are essential for CD30 expression. This
result is consistent with data generated from analysis of the 5'
CD30 deletion series, where deletion of this potential Sp1
site essentially abolishes high-level transcription.
To define DNA-protein interactions in the region surrounding the
conserved Sp1 site, we performed in vitro DNase I footprint
assays, using a fragment from -311 to +152 of the CD30
promoter as a probe. Three major regions of protection were identified
in the presence of Jurkat T-cell nuclear extract. These regions
encompassed nucleotides -159 to -149, -142 to -128, and -44 to
-28 (Figure 2A)
. The region spanning
-44 to -28 contains the conserved Sp1 site, which appears to be
essential for high-level expression of the -3.5-kb
CD30/luciferase construct. The two upstream regions (-164
to -148 and -144 to -131) both contain sites mutated in the
transfection experiments; however, the mutations did not appear to
alter basal transcription levels (Figure 1B)
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, lane 1). All of these complexes
could be competed away by using a 50-fold molar excess of unlabeled
probe (Figure 2B
, lane 2). Complexes a and b could be completely
competed away by using a 50-fold molar excess of consensus Sp1 GC and
GT box sequences, suggesting that complexes a and b recognized
Sp1-binding sequences (Figure 2B
, lanes 3 and 4). An unrelated GC-rich
oligonucleotide sequence (NI) was able to compete for complex d and f
binding but was less efficient at competing away complexes a–c (Figure 2B
, lane 5). To determine whether members of the Sp family of
transcription factors were involved in formation of complexes a and b,
the DNA protein complexes, formed during the binding reaction, were
incubated in the presence of either anti-Sp1 or anti-Sp3 antibodies.
Results indicated that complexes a and b were supershifted with the Sp1
antibody, suggesting that Sp1 formed part of these complexes (Figure 2B
, lane 6). No shift was seen with the Sp3 antibody (lane 7),
indicating that the complexes a and b were antigenically related to Sp1
specifically.
To further characterize the nature of complexes c–e, mutated versions
of the EMSA oligonucleotide were designed and used to identify the
relative binding position of the observed complexes. Three mutated
oligonucleotides were generated (M1, M2, M3) and used as probes or
competitors in EMSAs with Jurkat T-cell extracts (Figure 3)
. As predicted, mutation of the
consensus Sp1 binding site in M2 affected binding of complexes a and b,
shown in the supershift assays to contain Sp1 (Figure 3
, lanes 3 and
6). M2 also partially effected binding of complex e. The M1 mutation
affected binding of complexes c and d and to some extent the binding of
e compared to wild type (Figure 3
, lanes 2 and 5). However, the M3
mutation completely abolished binding of complex e (Figure 3
, lanes 4
and 7). These results suggest that complexes c and d are binding to the
region upstream from the Sp1 consensus site, whereas complex e binds to
the downstream region, including all or part of the ATTCAA sequence,
which was mutated in the M3 oligonucleotide.
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The transfection results (see Figure 1
) identified a strong
repressor element in the region between -1.2 kb and -336 bp of the
CD30 promoter; transcriptional activity increased by
3.5-fold when this region was deleted. This segment of the
CD30 promoter was sequenced and shown to contain ~400 bp
of a complex microsatellite of the type
[(CCAT)2–12CCACTTATGCAT]n,
although some of the repeat elements were imperfect (Figure 4)
. Comparison of the human sequence with
the equivalent murine sequence indicated that the microsatellite was
evolutionarily conserved and arose before the rodent/primate divergence
(Figure 4)
. However, the mouse microsatellite sequence was very
degenerate compared with the human sequence, although the CCAT repeat
was evident throughout the sequence. To investigate the role of the
microsatellite in the observed repression, a construct (
MS) in which
~400 bp of human microsatellite sequence was spontaneously deleted in
Escherichia coli, was compared in transfection assays with
the -1.2 kb and -336 constructs (Figure 5)
. Sequencing of the MS construct
showed that it lacked most of the microsatellite sequence but retained
four copies of the CCAT repeat. The MS deletion resulted in a
1.6-fold increase in transcription compared with the -1.2-kb
construct. The MS deletion did not completely relieve repression as
compared to the -336 deletion construct (3.8-fold over the -1.2-kb
construct), indicating that the (CCAT)4 sequence
alone was capable of repressing transcription from the CD30
promoter. Although we have not tested the function of the murine
microsatellite sequences, we would predict that they also are
able to repress the transcription of murine CD30. Although
the mouse sequence is degenerate, five contiguous copies of CCAT are
present at least twice (see Figure 4
), and we have shown that four
copies are sufficient to repress CD30 transcription.
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, suggesting that proteins binding within the microsatellite
region strongly repress transcription from the SV40 promoter.
To define DNA-protein interactions occurring within the microsatellite
region, a double-stranded oligonucleotide probe [(CCAT)
5CACCTTATGCAT(CCAT)2] was
used in EMSA to analyze the interaction of this region with Jurkat
T-cell nuclear proteins. Four major complexes (a–d) were formed
between the probe and nuclear proteins (Figure 6B
, lane 1). Mutations in either the
central region (M2 and M3) or in the flanking tetranucleotide repeat
sequences (M1 and M3, Figure 6A
) failed to significantly alter protein
binding (Figure 6B
, lanes 2–4). In addition, use of any of the mutated
sequences as unlabeled competitors in the EMSA reactions achieved
removal of all protein binding activities (Figure 5B
, lanes 5–7).
Furthermore, competition with a 50-fold molar excess of a
(CCAT)6 competitor removed all protein binding
activities (Figure 6B
, lane 8), indicating that the CCAT sequences
alone are interacting with nuclear proteins in the EMSA. The results
indicate that repression of the CD30 promoter appears to be
mediated by CCATCCAT consensus binding protein(s).
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As our results suggested that the size of the CCAT repeat
influenced the degree of repression seen in the transcription assays,
we were interested in determining the degree of length polymorphism in
a small sample population. DNA samples from seven randomly chosen
individuals were typed for CD30 microsatellite length
variation by PCR and analysis on PAGE. The results showed a high degree
of polymorphism, with at least seven alleles present in the seven
samples, ranging in size from 390 bp to 350 bp (Figure 7A)
. This size difference corresponds to
a difference of 10 CCAT repeat units. We also tested one leukemia
(Jurkat E6-1), one hepatoma (HepG2), and four lymphoma cell lines
(U937, CA46, RAJI, HuT78), although none of these lines were derived
from Hodgkin’s disease or non-Hodgkin’s lymphoma patients. There
appeared to be at least five alleles in the six cell lines, ranging in
size from 390 bp to 350 bp. Four of the cell lines show apparent
homozygosity (Figure 7B)
.
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Previously we identified a region of extreme sequence conservation
downstream of the major transcription initiation site when comparing
the human and murine CD30 genes.34
We
investigated the functional significance of this conserved 21-bp region
by introducing 6-bp mutations at sequences reminiscent of AP1 (+8 to
+14) and downstream promoter element (DPE) (+20 to +26) sites directly
downstream from the major transcription initiation site
(TIS).45
Transient transfection analysis of the AP1 mutant
in Jurkat cells indicated that the site was not important in regulating
the expression of CD30 (Figure 8A)
.
However, mutation within the DPE element resulted in a twofold increase
in reporter gene activity. To determine the effect of the DPE mutation
on transcription and/or TIS selection, mRNA expression from the mutated
CD30/luciferase DPE construct was compared with the
equivalent wild-type construct, -336. Mutation of the DPE element
resulted in both a shortened transcript compared with the -336
construct and a possible decrease in the amount of transcript produced
(Figure 8B)
. These results suggest that mutation of the DPE element
results in altered start site selection to produce a transcript
truncated by ~70 bp (TSS at +70) compared to the wild-type
CD30 gene. Comparison of the luciferase assay results and
those obtained from the RNase protection assay suggests that although
the amount of transcript decreases, posttranscriptional events may
serve to increase expression at the protein level. For instance, the
shortened transcript may exhibit a decreased turnover rate or,
alternatively, may differentially regulate interaction with the
translational machinery.
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Discussion |
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Our results demonstrated that the Sp1 binding site at position -44 to -28 is a critical element required for initiating transcription of the CD30 gene and forms a DNA-protein complex with the Sp1 protein. Sp1 sites immediately upstream of the TIS have been shown, in other genes, to act as an anchor for the basal transcription machinery in the absence of a TATA box.39-42,46 Sp1 appears to recruit TATA binding proteins such as TBP, which are normally involved in the formation of transcription initiation complexes around the TATA consensus sequence. Hence Sp1 appears to act as a surrogate TATA motif in the CD30 gene, as in other TATA-less genes.40 The downstream element, or DPE, located at +30 has recently been described as the downstream counterpart to the TATA box.45 Positioning of the DPE with respect to the initiator element has been found to be critical in directing both the accuracy and level of transcription,45 and these functions have been confirmed in the case of the CD30 promoter. The presence of these two functional sites in the CD30 gene provides additional evidence regarding the importance of these elements in transcriptional regulation from TATA-less promoters. As the lack of a canonical TATA box appears to be a common feature in the promoters of both the TNF ligand47,48 and receptor families,35-38 it is possible that transcription of other members of the family is regulated in a similar manner and requires further investigation.
Sp1 is known to be up-regulated by viral activation, so it is possible that viral infection of lymphoid cells may up-regulate Sp1, leading to the overexpression of CD30, as seen in Hodgkin’s disease. Epstein-Barr virus (EBV) is frequently found in CD30-positive lymphomas and is known for its ability to up-regulate CD30 in EBV-positive B-lymphoma cell lines.3 A number of CD30-related receptors, such as CD40 and Fas, are also up-regulated in Hodgkin’s disease.10 Therefore, alterations in a common mechanism involving Sp1 may explain why the H-RS cells of Hodgkin’s disease display unbalanced cytokine production and deregulated expression of surface antigens.
Reporter gene transcriptional assays identified a repressor between positions -1.2 kb and -336 bp. Sequencing of this region revealed the presence of a 400-bp tetranucleotide microsatellite. This variable repeat region was found to repress luciferase gene expression when cloned upstream from the SV40 promoter in pGL3 promoter. Deletion of almost the entire variable region (excluding four copies of the CCAT repeat) relieved transcriptional repression by twofold compared with complete removal of all CCAT repeats, which relieved repression up to 3.8-fold. EMSA established that at least four nuclear proteins are able to interact with the tetranucleotide sequences. Increasing evidence in the literature suggests that simple repeats can exhibit regulatory activity. For instance, a tetranucleotide repeat located in the first intron of the tyrosine hydroxylase gene has been shown to act as a transcriptional enhancer in vitro.49 A number of dinucleotide repeats have also been shown to cause up-regulation or down-regulation of basal transcription when cloned upstream from a minimal promoter.50,51 The extent of these regulatory effects has been shown to be dependent on the length of the repeating element.51 Our microsatellite typing data suggest that allelic differences in the length of the CD30 upstream microsatellite may differentially regulate CD30 expression in vivo.
The involvement of a microsatellite sequence in the control of CD30 expression may have significant implications for the observed overexpression in lymphoma and leukemia. The generation of the spontaneous CD30 microsatellite deletion in E. coli and the degree of polymorphism seen in a small sample of randomly chosen individuals suggest that the CD30 microsatellite has the potential to be unstable. Microsatellite instability results, at least partly, from a defect in DNA mismatch repair and has been identified as a common molecular mechanism in the development of cancer. Microsatellite instability has been identified in adult T-cell leukemia, childhood T-cell acute lymphoblastic leukemia, Hodgkin’s disease, and some B-cell non-Hodgkin’s lymphomas.52-57 Whether the degenerate microsatellite within the CD30 promoter is specifically affected by microsatellite instability in vivo remains to be investigated. However, as the microsatellite in CD30 is a strong repressor, it is possible that deletion of the CD30 microsatellite may lead to relief of transcriptional repression, resulting in the overexpression of CD30 in Hodgkin’s and non-Hodgkin’s lymphomas. Further studies are required to screen for polymorphism in the CD30 microsatellite repressor in different cell lines derived from CD30-positive tumors, as well as the germline of patients from whom the cell lines are derived.
The CD30 locus occurs within a genomic region that has been shown to confer susceptibility to Lupus nephritis in (NZW x NZB) F1 mice.58,59 The observed regulatory effects of the CD30 microsatellite may have significant implications for Lupus in humans. Promoter polymorphisms in other genes, such as TNF,60 have been implicated in susceptibility to autoimmunity. Considering the high degree of polymorphism in our small sampling of healthy individuals, it is a priority to screen patients to determine whether carriage of particular alleles of the CD30 microsatellite are associated with susceptibility to systemic lupus erythematosus (SLE). Determination of the degree of repression from the disease associated alleles may provide a molecular explanation for disease susceptibility.
This work provides a first step in identifying the mechanisms involved in CD30 gene expression by identifying three key regulatory elements. Further work will be required to elucidate the exact roles that the Sp1 and CCAT microsatellite regions play in the altered cytokine and receptor expression associated with CD30+ disease states.
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Acknowledgements |
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Footnotes |
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Supported by the Arnold Yeldham and Mary Raine Medical Research Foundation, Western Australia.
Accepted for publication February 2, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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