A novel homologous recombination system to study 92 kDa type IV collagenase transcription demonstrates that the NF-kappaB motif drives the transition from a repressed to an activated state of gene expression.

Chunhong Yan, Heng Wang, Bharat Aggarwal, Douglas D. Boyd

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39 Citations (Scopus)

Abstract

The 92-kDa type IV collagenase (MMP-9) contributes to tissue remodeling in both physiology and pathology. Previous studies on the transcriptional regulation of this gene have used transiently transfected promoter-reporter constructs. However, this approach suffers from several limitations including (a) multiple copies of the plasmid and (b) the plasmid is not genomically integrated and consequently poorly chromatinized. We developed a novel system for studying MMP-9 transcription in which a single copy of a MMP-9 promoter-luciferase construct(s) is integrated at an identical genomic locus in HT1080 cells by homologous recombination. We report that the activity of a genomic-integrated 2.2 kb MMP-9 promoter sequence mirrors expression of the endogenous MMP-9 gene in response to both physiological and pharmacological (curcumin) cues. Further, when constrained into chromatin, the integrated NF-kappaB-mutated MMP-9 promoter is repressed by PMA, a situation not apparent using nonintegrated plasmids. Thus, we have developed a novel method for studying MMP-9 expression that overcomes some of the limitations associated with transient transfection approaches and which may be of utility in screening for agents that repress the expression of this gene.

Original languageEnglish (US)
Pages (from-to)540-541
Number of pages2
JournalThe FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume18
Issue number3
StatePublished - Jan 1 2004

Fingerprint

NF-kappa B
Homologous Recombination
Matrix Metalloproteinase 9
Transcription
Matrix Metalloproteinases
Gene expression
Gene Expression
Plasmids
Genes
Curcumin
Physiology
Pathology
Drive
Luciferases
Chromatin
Cues
Transfection
Screening
Pharmacology
Tissue

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

Cite this

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title = "A novel homologous recombination system to study 92 kDa type IV collagenase transcription demonstrates that the NF-kappaB motif drives the transition from a repressed to an activated state of gene expression.",
abstract = "The 92-kDa type IV collagenase (MMP-9) contributes to tissue remodeling in both physiology and pathology. Previous studies on the transcriptional regulation of this gene have used transiently transfected promoter-reporter constructs. However, this approach suffers from several limitations including (a) multiple copies of the plasmid and (b) the plasmid is not genomically integrated and consequently poorly chromatinized. We developed a novel system for studying MMP-9 transcription in which a single copy of a MMP-9 promoter-luciferase construct(s) is integrated at an identical genomic locus in HT1080 cells by homologous recombination. We report that the activity of a genomic-integrated 2.2 kb MMP-9 promoter sequence mirrors expression of the endogenous MMP-9 gene in response to both physiological and pharmacological (curcumin) cues. Further, when constrained into chromatin, the integrated NF-kappaB-mutated MMP-9 promoter is repressed by PMA, a situation not apparent using nonintegrated plasmids. Thus, we have developed a novel method for studying MMP-9 expression that overcomes some of the limitations associated with transient transfection approaches and which may be of utility in screening for agents that repress the expression of this gene.",
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T1 - A novel homologous recombination system to study 92 kDa type IV collagenase transcription demonstrates that the NF-kappaB motif drives the transition from a repressed to an activated state of gene expression.

AU - Yan, Chunhong

AU - Wang, Heng

AU - Aggarwal, Bharat

AU - Boyd, Douglas D.

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N2 - The 92-kDa type IV collagenase (MMP-9) contributes to tissue remodeling in both physiology and pathology. Previous studies on the transcriptional regulation of this gene have used transiently transfected promoter-reporter constructs. However, this approach suffers from several limitations including (a) multiple copies of the plasmid and (b) the plasmid is not genomically integrated and consequently poorly chromatinized. We developed a novel system for studying MMP-9 transcription in which a single copy of a MMP-9 promoter-luciferase construct(s) is integrated at an identical genomic locus in HT1080 cells by homologous recombination. We report that the activity of a genomic-integrated 2.2 kb MMP-9 promoter sequence mirrors expression of the endogenous MMP-9 gene in response to both physiological and pharmacological (curcumin) cues. Further, when constrained into chromatin, the integrated NF-kappaB-mutated MMP-9 promoter is repressed by PMA, a situation not apparent using nonintegrated plasmids. Thus, we have developed a novel method for studying MMP-9 expression that overcomes some of the limitations associated with transient transfection approaches and which may be of utility in screening for agents that repress the expression of this gene.

AB - The 92-kDa type IV collagenase (MMP-9) contributes to tissue remodeling in both physiology and pathology. Previous studies on the transcriptional regulation of this gene have used transiently transfected promoter-reporter constructs. However, this approach suffers from several limitations including (a) multiple copies of the plasmid and (b) the plasmid is not genomically integrated and consequently poorly chromatinized. We developed a novel system for studying MMP-9 transcription in which a single copy of a MMP-9 promoter-luciferase construct(s) is integrated at an identical genomic locus in HT1080 cells by homologous recombination. We report that the activity of a genomic-integrated 2.2 kb MMP-9 promoter sequence mirrors expression of the endogenous MMP-9 gene in response to both physiological and pharmacological (curcumin) cues. Further, when constrained into chromatin, the integrated NF-kappaB-mutated MMP-9 promoter is repressed by PMA, a situation not apparent using nonintegrated plasmids. Thus, we have developed a novel method for studying MMP-9 expression that overcomes some of the limitations associated with transient transfection approaches and which may be of utility in screening for agents that repress the expression of this gene.

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