Identification of a negative element in the human vimentin promoter: modulation by the human T-cell leukemia virus type I Tax protein.

Abstract

The vimentin gene is a member of the intermediate filament multigene family and encodes a protein expressed, in vivo, in all mesenchymal derivatives and, in vitro, in cell types of various origin. We have previously demonstrated that the expression of this growth-regulated gene could be trans activated by the 40-kDa Tax protein of HTLV-I (human T-cell leukemia virus tpe I) and that responsiveness to this viral protein was mediated by the presence of an NF-KB binding site located between -241 and -210 bp upstream of the mRNA cap site (A. Lilienbaum, M. Duc Dodon, C. Alexandre, L. Gazzolo, and D. Paulin, J. Virol. 64:256-263, 1990). These previous assays, performed with deletion mutants of the vimentin promoter linked to the chloramphenicol acetyltransferase gene, also revealed the presence of an upstream negative region between -529 and -241 bp. Interestingly, the inhibitory activity exerted by this negative region was overcome after cotransfection of a Tax-expressing plasmid. In this study, we further characterize the vimentin negative element and define the effect of the Tax protein on the inhibitory activity of this element. We first demonstrate that a 187-bp domain (-424 to -237 bp) behaves as a negative region when placed upstream either of the NF-KB binding site of vimentin or of a heterologous enhancer such as that present in the desmin gene promoter. The negative effect can be further assigned to a 32-bp element which is indeed shown to repress the basal or induced activity of the NF-KB binding site. Indeed, the negative effect is still observed in HeLa cells treated with phorbol myristate acetate, an inducer of nuclear NF-KB activity. Furthermore, deletion experiments allowed identification within the NE site of 19 bp which bind in vitro a protein complex and which are essential to exert the negative effect. We further demonstrate that expression of the Tax protein is required for overcoming the down-regulation of the NF-KB binding site by the'negative element. Indeed, the negative effect can be relieved in HeLa cells cotransfected with a Tax-expressing plasmid or in a clone of HeLa cells stably producing this viral protein. Collectively, these observations give a new insight into the complex regulation of the human vimentin gene expression and suggest that the Tax protein of human T-cell leukemia virus type I up-regulates this cellular promoter through a dual mechanism consisting of the activation ofthe NF-KB binding site and the relief of the negative effect exerted by the negative element.

The Zinc Finger Repressor, ZBP-89, Binds to the Silencer Element of the Human Vimentin Gene and Complexes with the Transcriptional Activator, Sp1

“…Surprisingly, transient transfection assays also indicated the presence of a positive acting element located between Ϫ339 and Ϫ319, close to the PS site. This region was described previously as a negative acting element and referred to as ⌬19 (15). The inclusion of more upstream DNA (greater than Ϫ339) had little effect on reporter gene activity until reaching the tandem AP-1 sites at approximately Ϫ700 (14, 44).…”

“…Where Is the "True" Binding Site for the Repressor of Vimentin Gene Expression?-The presence of a negative element (⌬19) at position Ϫ339 to Ϫ319 had been reported earlier (15). However, these transient transfection assays were performed only with fragments of vimentin's 5Ј-end fused to a heterologous promoter.…”

“…It is postulated that negative regulation is involved in the cellular extinction of vimentin during embryogenesis. To date, the following elements have been identified in the promoter region of the vimentin gene: a TATA-box, several positive regulatory elements including eight GC-boxes (11), a NF-B site (12), a PEA3 site (13), and two AP-1 binding sites (14) plus silencer elements (15,16). Previously, we have shown that of the eight GC-boxes, GC-box 1 is indispensable for vimentin gene expression (17).…”

Different vimentin expression in two clones derived from a human colocarcinoma cell line (LoVo) showing different sensitivity to doxorubicin

“…The DNA sequences regulating the expression of vimentin have been cloned (Farrell et al, 1990;Hennekes et al, 1990;Stover and Zehner, 1992; de Klundert et al, 1992;Salvetti et al, 1993;Lilienbaum and Paulin, 1993) and certain proteins keep the gene silent when bound to DNA (Farrell et al, 1990; de Klundert et al, 1992;Salvetti et al, 1993), while others such as the NFKcB protein, activate its expression . The simultaneous presence of both kinds of protein seems to result in inactivation of transcription (Salvetti et al, 1993 In a human breast cancer cell line made resistant to doxorubicin there was an increase in the expression of vimentin not detected in the wild-type cell line (Sommers et al, 1992).…”

“…The simultaneous presence of both kinds of protein seems to result in inactivation of transcription (Salvetti et al, 1993 In a human breast cancer cell line made resistant to doxorubicin there was an increase in the expression of vimentin not detected in the wild-type cell line (Sommers et al, 1992).…”