Transcription factor AP2 is required for expression of the rat transforming growth factor-α gene

DNase I footprinting of the rat TGF alpha promoter in the presence of crude cell nuclear extract revealed three sites of protein-DNA interaction (Fp-A, Fp-B, Fp-C) in the region from -222 to +73. Mutation of specific sites within the Fp-A and Fp-B regions reduced expression of a TGF alpha promoter-r...

Full description

Saved in:

Bibliographic Details
Published in:	Oncogene Vol. 14; no. 18; pp. 2229 - 2238
Main Authors:	BERKOWITZ, E. A, HECHT, C. P, AZIZKHAN, J. C, CHEN, X, LEE, D. C
Format:	Journal Article
Language:	English
Published:	Basingstoke Nature Publishing 08-05-1997 Nature Publishing Group
Subjects:	Animals Base Sequence Binding Sites Biological and medical sciences Cell Line Cell Line, Transformed CHO Cells - metabolism Conserved sequence Cricetinae Deoxyribonuclease Deoxyribonucleic acid DNA DNA Footprinting DNA-Binding Proteins - metabolism Drosophila - genetics Drosophila - metabolism Drosophila Proteins Electrophoresis - methods Electrophoretic mobility Epithelial Cells Footprinting Fundamental and applied biological sciences. Psychology Gene expression Gene Expression Regulation Liver - cytology Molecular and cellular biology Molecular genetics Molecular Sequence Data Mutation Oligonucleotides Promoter Regions, Genetic Proteins Rats Recombinant Proteins - genetics Recombinant Proteins - metabolism Reporter gene Tata box TATA-binding protein TATA-Box Binding Protein Transcription Factor AP-2 Transcription Factor TFIIA Transcription Factors - metabolism Transcription initiation factor TFIIA Transfection Transforming Growth Factor alpha - genetics Transforming Growth Factor alpha - metabolism Transforming growth factor-a Transcription factor AP2 Rat Transcription promoter Rodentia Molecular interaction Binding site Gene expression Vertebrata Regulation(control) Mammalia Gene Regulatory sequence Transforming growth factor α
Online Access:	Get full text
Tags:	Add Tag No Tags, Be the first to tag this record!

Description
Summary:	DNase I footprinting of the rat TGF alpha promoter in the presence of crude cell nuclear extract revealed three sites of protein-DNA interaction (Fp-A, Fp-B, Fp-C) in the region from -222 to +73. Mutation of specific sites within the Fp-A and Fp-B regions reduced expression of a TGF alpha promoter-reporter gene (TGF alphaLUC) from 50-90% in transiently transfected CHO cells, indicating the importance of protein/DNA interactions at these sites. Since Fp-A contained a perfect AP2 consensus sequence (5'-GCCNNNGGC-3') as its center, we investigated the possibility that AP2 binding is important for TGF alpha promoter activity. A double-stranded oligonucleotide spanning Fp-A displayed a distinct mobility shift in the presence of nuclear extract that was inhibited by an excess of known functional AP2-binding sequence. Moreover, a similar mobility shift occurred in the presence of purified AP2 protein, and the further addition of AP2 antibody produced a supershifted complex. More refined DNase I footprinting of a smaller, oligonucleotide probe in the presence of purified AP2 protein revealed a protected region that included the putative AP2 binding site. Additionally, co-transfection of an AP2 expression vector increased TGF alphaLUC expression 25-fold in Drosophila Schneider cells. These various findings corroborate a role for AP2 in TGF alpha promoter activity. The Fp-B region contains a T5 motif that has been previously suggested to function as an atypical TATA box. An Fp-B oligonucleotide displayed a specific gel mobility shift in the presence of a TATA binding protein (TBP)-TFIIA complex, and the further addition of TBP antibody produced a supershift. These results confirm that protein binding within Fp-B is functionally important, and they also indicate that the T5 motif functions as a TBP binding site.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2
ISSN:	0950-9232 1476-5594
DOI:	10.1038/sj.onc.1201051