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

Abstract

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.

Introduction

In recent years considerable progress has been made towards understanding the mechanisms of eukaryotic gene regulation. Much of the progress in this field has come from the purification and subsequent characterization of a various regulatory and transcription factors that bind to specific DNA sequences and modulate Pol II activity. One of the first factors characterized was Sp1, a factor that bound to GC-rich sequences and was required for activation of the early SV40 promoter (Kadonaga et al., 1987). For some time this factor seemed to be the only protein binding to the GC-motif. Later research revealed additional members of the Sp-family (Sp2, Sp3, Sp4, BTEB, BTEB2), which displayed considerable homology with Sp1 (Kingsley and Winoto, 1992, Lania et al., 1997). Like Sp1, some of these proteins (Sp2, Sp3) were ubiquitously expressed in many tissues, whereas one, Sp4, displayed a tissue-specific expression pattern. In addition, Sp1 was shown to interact with a variety of proteins involved in transcription, which include such regulatory factors as NF-κB, E2F, p53, RB, STAT-1 as well as transcription factors like TAFs or TBP itself (Chen et al., 1996, Chiang and Roeder, 1995, Emili et al., 1994, Gill et al., 1994, Gualberto and Baldwin, 1995, Karlseder et al., 1996, Lania et al., 1997, Look et al., 1995, Perkins et al., 1993). We report here that a GC-box is important in regulating expression of the vimentin gene.

Vimentin, a member of the intermediate filament protein family, exhibits a complex pattern of gene expression that can be observed at several levels. Vimentin is first expressed during development in mesoderm cells located between the primitive streak and the proximal endoderm (Duprey and Paulin, 1995). Many tissues differentiate from this origin and continue to express vimentin, whereas others, like muscle, down-regulate vimentin expression and activate the muscle-specific gene desmin (Duprey and Paulin, 1995). For the most part, vimentin expression coincides with cellular growth and is cell cycle regulated (Franke et al., 1979, Rittling and Baserga, 1987). Moreover, it has been suggested that vimentin can act as a signal transducer, relaying information from the extracellular matrix to the nucleus (Skalli and Goldman, 1991).

The human vimentin promoter is thought to contain two positive modules separated by a negative region (Rittling and Baserga, 1987). The proximal promoter region, from the transcriptional start site at +1 to −240, was shown to contain a TATA and CAAT box, plus binding sites for the regulatory factor, PEA3 (Chen et al., 1996, Rittling and Baserga, 1987). The intermediate region (from −241 to −669) contains three transcriptional elements: an NF-κB site, a negative element, and Δ19 (Lilienbaum et al., 1990, Moura-Neto et al., 1996, Salvetti et al., 1993, Wieczorek et al., 1999). The upstream region (−1114 to −669) consists of tandem AP-1 sites, plus antisilencer (ASE) and distal silencer (DS) elements (Izmailova and Zehner, 1999a, Rittling et al., 1989). At least eight sequences, which match the GC-box consensus site, were found within the vimentin promoter (Rittling and Baserga, 1987). Our in vivo footprinting experiments using ligation-mediated PCR (LMPCR) indicate that of these multiple GC-boxes only the first site, closest to the transcriptional start site, displays evidence of protein binding in HeLa and MDA-MB 231 (MDA) cells, which express vimentin. Transient transfection assays of various vimentin promoter elements containing this GC-box and mutations thereof fused to the cat gene show that this sequence is required for promoter function in HeLa cells. Band shift experiments verify that this GC-box specifically binds a protein(s) from HeLa nuclear extract.