Identification of a novel cis-acting element for fibroblast-specific transcription of the FSP1 gene.
The FSP1 gene encodes a filament-binding S100 protein with paired EF hands that is specifically expressed in fibroblasts. This led us to look for cis-acting elements in the FSP1 promoter that might engage nuclear transcription factors unique to fibroblasts. The first exon of FSP1 is noncoding, therefore, a series of luciferase reporter minigenes were created containing varying lengths of 5'-flanking sequence, the first intron, and the noncoding region of the second exon. A position and promoter-dependent proximal element between -187 and -88 bp was shown to be active in fibroblasts but not in epithelium. Sequence in the first intron from +777 to +964 had an enhancing effect that was not cell type specific. Hsv TK reporter constructs driven by this promoter/intron cassette in transgenic mice were coexpressed appropriately with FSP1 in tissue fibroblasts. Gel mobility shift competitor assays identified a novel domain, FTS-1 (fibroblast transcription site-1; TTGAT from -177 to -173 bp), that specifically interacts with nuclear extracts from fibroblasts. The necessity of this binding site was confirmed by site-specific mutagenesis. Database searches also turned up putative FTS-1 sites in the early promoter regions of other fibroblast expressed proteins, including the alpha1 and alpha2(I), and alpha1(III) collagens and the alphaSM-actin gene. We hypothesize that the selective engagement of FTS-1 elements may contribute to the mesenchymal phenotype of fibroblasts and perhaps other dedifferentiated cells.
Penn Center for the Molecular Studies of Kidney Diseases, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6144, USA.
H
H
Okada
T M
TM
Danoff
A
A
Fischer
J M
JM
Lopez-Guisa
F
F
Strutz
E G
EG
Neilson
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D002460:Cell Line; D019175:DNA Methylation; D004847:Epithelial Cells; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D007381:Intermediate Filament Proteins / Q000096:biosynthesis / Q000235:genetics; D007438:Introns; D007684:Kidney Tubules; D007687:Kidney Tubules, Proximal; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; D013937:Thymidine Kinase / Q000096:biosynthesis; D014158:Transcription, Genetic; D014162:Transfection
D002135:Calcium-Binding Proteins; D007381:Intermediate Filament Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; C470944:S100a4 protein, mouse; D008156:Luciferases; D013937:Thymidine Kinase