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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 8, 2004 as doi:10.1096/fj.03-0960fje. |
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B motif drives the transition from a repressed to an activated state of gene expression 1
Department of Cancer Biology, Box 179, M.D. Anderson Cancer Center, Houston, Texas, USA
2Correspondence: Department of Cancer Biology, Box 173, M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA. E-mail: dboyd{at}mdanderson.org
SPECIFIC AIMS
The 92 kDa type IV collagenase (MMP-9) plays a major role in physiological and pathological events such as trophoblast implantation in pregnancy, bone development, and tumor progression. Regulation of MMP-9 protein levels has been largely ascribed to transcriptional activation of the gene. Considering its role in physiology and pathology, there is a need for an increased understanding of how this gene is transcriptionally controlled. In the present study, we report on the development and characterization of a novel system (which overcomes several limitations associated with studies using extrachromosomal plasmids) in which a single copy of a wild-type or mutated MMP-9 promoter fragment(s) fused to a luciferase reporter was inserted at a Flp recombinase target site (FRT) in the HT1080 cell genome.
PRINCIPAL FINDINGS
To direct MMP-9 promoter-reporter constructs to a single genomic locus, we first generated constructs bearing a MMP-9 regulatory sequence-driven luciferase cDNA and a Flp FRT site. These constructs were cotransfected with a Flp recombinase into a HT1080 clone that harbors a single FRT site. The Flp recombinase catalyzed a recombination event between the plasmid and the FRT site in the HT1080 clone genome. Thus, all MMP-9/Luc constructs were integrated at the identical genomic locus (FRT site) in cells derived from a single clone.
MMP-9 expression is reduced by growth factor-deprivation and, conversely, stimulated by refeeding. Like the endogenous gene, reporter activity in the HT1080 clone harboring the integrated 2.2 kb MMP-9 fragment-luciferase was reduced by serum withdrawal and induced by refeeding; these changes were less dramatic with the integrated 0.67 kb MMP-9 promoter fragment. These data established the responsiveness of the integrated 2.2 kb MMP-9 promoter construct to physiological cues.
Similar studies were performed with pharmacological agents. Curcumin, which has been shown to reduce MMP-9 expression, caused a dose-dependent decrease in luciferase activity in the clone harboring the integrated 2.2 kb MMP-9 promoter-reporter, with the highest concentration yielding a >90% reduction. This repression mirrored the reduction in endogenous MMP-9 activity as evident in zymography assays. Conversely, the phorbol ester PMA and trichostatin A, alone or in combination, stimulated the integrated 2.2 kb MMP-9 promoter-reporter construct in parallel with the endogenous gene.
Biochemical analysis of the integrated MMP-9 promoter fragments
The above-mentioned functional studies were complemented by biochemical approaches to compare the accessibility of the integrated transgenes and the endogenous MMP-9 gene. Digestion of intact nuclei with micrococcal nuclease coupled with quantitation of the digested DNA with real-time PCR indicated that nucleosomal positioning of the endogenous MMP-9 gene closer resembled the exogenous, integrated 2.2 kb MMP-9 fragment than the integrated 0.67 base pair fragment.
Divergent responsiveness of the integrated and nonintegrated mutated MMP-9 promoter to PMA
To investigate the possibility that genomic integration of the MMP-9 promoter modulates its regulation, we compared wild-type and mutated promoter activity in cells transiently transfected (to assess nonintegrated reporter activity) with that achieved with the FRT-integrated MMP-9 promoter-reporter constructs. While the PMA-dependent induction of luciferase activity in the HT1080 clone harboring the integrated AP-1-mutated (nucleotide position -79) construct (in context of 2.2 kb of 5' flanking sequence) was abrogated, the extrachromosomal construct was still induced albeit to a lesser extent than achieved with the transiently transfected wild-type fragment. PMA repressed the integrated NF-
B (-600) -mutated MMP-9 promoter by 40% (compared with the untreated cells). In contrast, the extrachromosomal NF-B-mutated MMP-9 reporter was still stimulated. Thus, the integrated and extrachromosomal MMP-9 promoters diverge in their responsiveness to mutation of the AP-1 motif. Further, when constrained into chromatin, the NF-B cis element drives the transition from a repressed to an activated state of gene expression.
CONCLUSIONS AND SIGNIFICANCE
We describe here a novel system to study MMP-9 transcriptional regulation. In this system, the MMP-9 promoter (wild-type and mutated) -luciferase sequence is integrated into an identical genomic locus in HT1080 cells, and the activity of the wild-type 2.2 kb flanking sequence closely mirrors expression of the endogenous gene in response to both physiological and pharmacological cues. Since clones harboring the various MMP-9 promoter fragments are all derived from a single clone and because exogenous constructs are targeted to an identical genomic locus, the current system obviates the need to generate multiple clones.
The current system is superior to transcriptional studies, which make use of transiently transfected promoter-reporter constructs, for several reasons. First, in the latter studies, there is no control over DNA copy number, thus confounding comparisons between different promoter constructs and rendering the corresponding transcription factors limiting. Second, nonintegrated constructs (as occurs with transient transfections) are poorly chromatinized. This is an important shortcoming since the chromatin environment itself alters transcription factor binding/activity, thereby modulating gene expression. Although other investigators have sought to overcome the shortcoming of poor chromatinization of transiently transfected constructs by generating genomic-integrated promoter-reporter constructs, such studies are still limited by the random integration of the constructs, varying copy number, and clonal heterogeneity of the parental cell population. These shortcomings are overcome with the system described here. Indeed, our studies also demonstrate that the genomic-integrated and extrachromosomal MMP-9 promoter-reporter constructs show marked differences in responsiveness. Notably, mutation of the NF-B motif yielded a PMA-dependent repression of the integrated promoter whereas the activity of the extrachromosomal plasmid was augmented. These findings suggest caution in interpreting data generated with extrachromosomal constructs, which are poorly chromatinized.
In summary, we have generated a novel system that allows analysis of the transcriptional regulation of the MMP-9 gene when constrained into the genome. The described system could also provide a means of screening anti-invasion/metastases drugs that interfere with the transcriptional regulation of this gene. The present strategy may be applied to the study of transcriptional regulation of other genes.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0960fje; 
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