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Abstract
Vimentin is a component of the eukaryotic cytoskeleton belonging to the family of intermediate filament proteins. It exhibits a complex pattern of tissue- and development-specific expression. It is also a marker of the metastatic potential of many tumor cells. Previously, the human vimentin promoter was shown to contain several regions for the binding of positive and negative acting regulatory factors. Until now, the silencer element, which shuts down vimentin synthesis in selected tissues during development, was not precisely localized; nor was its binding protein known. In vivo DMS footprinting by ligation-mediated PCR delineated the position of guanine residues important to vimentin expression. Transient transfection assays in HeLa cells of various vimentin 5'-end promoter sequences and mutants thereof precisely defined two regulatory elements, a negative element and an adjoining positive acting element. Band shift assays, UV cross-linking, and Southwestern blot analysis confirm that the silencer element specifically binds a protein. Several lines of evidence show that ZBP-89, a zinc finger, Kruppel-like repressor protein is vimentin's silencer element binding factor. Co-immunoprecipitation and DNA affinity chromatography prove that Sp1 heterodimerizes with ZBP-89 when bound to the silencer element to yield a DNA-protein complex whose mobility is indistinguishable from that displayed by HeLa nuclear extract in band shift assays.
Highlights
The cytoskeleton, an important complex and dynamic cell component, is composed of three networks: the microtubules, the microfilaments, and the intermediate filaments (IFs).1 IFs
In Vivo dimethyl sulfate (DMS) Footprinting Analyses—In vivo DMS footprinting via ligation-mediated PCR (LMPCR) was used to determine which regions of the human vimentin promoter are important for gene expression
All in vitro studies on the interaction between regulatory factors and their target genes are hampered by their questionable application to genes as positioned in chromatin
Summary
64.9 a Tm ϭ 81.5 ϩ 16.6(log M) ϩ 0.41 (%GC) Ϫ (500/n), where M represents the molar salt concentration and n represents the number of bases. An example of a second class of repressors is Dr1 [25] These proteins do not bind to any specific sequence in DNA but are proposed to act directly at the level of transcription initiation by blocking the formation of the preinitiation complex. The third class of repressors involves proteins that do recognize specific sequences in DNA [22]. These proteins can be further divided into two subgroups depending on their proposed mechanism of action. We present evidence for its interaction with the transcriptional activator, Sp1 These results suggest a mechanism by which the vimentin gene is regulated
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