Identification of a cis-acting DNA antisilencer element which modulates vimentin gene expression.
Vimentin is a tissue-specific, developmentally regulated member of the intermediate filament protein family normally expressed in cells of mesenchymal origin. Transcription factors which recognize specific cis-acting elements of the chicken gene include Sp-1 and the 95-kDa silencer protein which binds to a 40-bp silencer element at -608 (F. X. Farrell, C. M. Sax, and Z. E. Zehner, Mol. Cell. Biol. 10:2349-2358, 1990). In this study, we have identified a region upstream of the silencer element which restores gene activity. This region has been further delineated into two functional subelements of 75 and 260 bp. In transient transfection assays, the 75-bp element overrides the silencer effect of pStkCAT by 100%, while the 260-bp element is about half as active. Neither element affects gene activity when the silencer element is absent. Therefore, these elements do not function as enhancers, but they may serve only to override the silencer element and therefore can be viewed as antisilencers. In addition, the 75-bp element binds a specific 140-kDa protein, as determined by gel mobility shift assays and Southwestern (DNA-protein) blots, the binding site of which has been delineated to a 10- to 17-bp element by DNase I protection experiments. During myogenesis, a direct correlation can be made between the binding efficiency of the 140-kDa protein, the silencer protein, and gene activity in vivo. Genes known to contain a functional silencer element also contain at least one antisilencer element, as determined by sequence identity. Therefore, we have identified an antisilencer element and protein important in the developmental regulation of vimentin gene expression which may be involved in the regulation of other genes.
Stover
D M
DM
Department of Biochemistry and Molecular Biophysics, Medical College of Virginia/Virginia Commonwealth University, Richmond 23298.
Zehner
Z E
ZE
D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002645:Chickens; D002678:Chimera; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I; D015870:Gene Expression; D005809:Genes, Regulator; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014746:Vimentin / Q000235:genetics*
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D014746:Vimentin; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D003850:Deoxyribonuclease I