The zinc finger repressor, ZBP-89, binds to the silencer element of the human vimentin gene and complexes with the transcriptional activator, Sp1.

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.

A GC-box is required for expression of the human vimentin gene.

Vimentin is an intermediate filament protein normally expressed in cells of mesenchymal origin. The promoter of the human vimentin gene (-1416 to +73) was shown to contain two positive-acting regions, separated by a negative region, and at least eight GC-boxes as determined by sequence homology (Rittling, S.R., Baserga, R., 1987. Mol. Cell. Biol. 7, 3908-3915). We have analyzed the region -900 to +41 for protein binding by in vivo footprinting experiments using ligation-mediated PCR. For the various GC-boxes, we detect protein binding only to that GC-box (at position -64 and -55) closest to the transcriptional start site. Transient transfection assays of various vimentin 5'-end fragments and mutations thereof fused to the reporter gene cat indicate that this sequence is indispensable for promoter function regardless of the inclusion of upstream DNA sequences. In vitro binding studies confirm that this region binds protein specifically. We suggest that this GC-box and its binding factor are required for regulated expression of the human vimentin gene.

Two homologous enhancer elements in the chicken vimentin gene may bind a nuclear factor in common with a nearby silencer element.

Vimentin, a cytoskeletal protein belonging to the intermediate filament protein family, exhibits a complex pattern of expression. In the case of the chicken vimentin gene, several regulatory elements within the 5' region of the gene have been characterized, including an enhancer activity between -160 and -320, which may contribute to the down-regulation of vimentin expression during myogenesis. In this study, sequences within this region were examined via transient transfections of various deletion constructs, and two distinct enhancer elements were found, one on either side of a previously described silencer element. These two enhancer elements also enhanced transcription when fused separately to the basal promoter region of the chicken vimentin gene. Gel mobility shift assays, UV cross-linking experiments, and DNase I protection studies indicate that these two enhancer elements and the silencer element all contain a common binding site for the previously described 95-kDa silencer element binding protein, suggesting that this regulatory protein can act as both an activator and a repressor.