Abstract
Transcriptional repression of the murine vascular smooth muscle alpha-actin gene in fibroblasts results from the interaction of two sequence-specific single-stranded DNA binding activities (VACssBF1 and VACssBF2) with opposite strands of an essential transcriptional enhancer factor-1 (TEF-1) element (Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995) Mol. Cell. Biol. 15, 2429-2436). Here, we identify a sequence element located within a protein-coding exon of the gene that bears structural similarity with the TEF-1 enhancer. This includes a 30-base pair region of purine-pyrimidine asymmetry encompassing a perfect 6-base pair GGAATG TEF-1 recognition motif. Unlike the enhancer, however, the exon sequence exhibits no TEF-1 binding activity nor does the pyrimidine-rich strand bind VACssBF1. However, VACssBF2 interacts equally well with the purine-rich strand of both the enhancer and the exon sequence. To test the ability of VACssBF2 to independently repress transcription, the exon sequence was placed upstream of a deletionally activated promoter containing an intact TEF-1 binding site. The exon sequence repressed promoter activity, whereas a mutant deficient in VACssBF2 binding did not. Moreover, VACssBF2 similarly repressed activator protein-1-dependent transcription of a heterologous tissue factor promoter. These results suggest that VACssBF2 possesses an intrinsic ability to disrupt enhancer function independently of the enhancer-binding proteins involved.
Highlights
The cell- and tissue-specific transcription of eukaryotic genes is regulated by cis-acting elements that are generally localized outside of the RNA/protein coding region of the gene
A recent study suggested a model in which two ssDNAbinding factors, designated VACssBF1 and VACssBF2, function to repress transcription of the mouse vascular smooth muscle (VSM) ␣-actin gene by disrupting base pairing within an essential transcriptional enhancer factor 1 (TEF-1) enhancer element [12]
These results were based upon the initial identification of a DNA element within the protein-coding region of the gene that bears intriguing sequence similarity to the negatively regulated enhancer element located in the proximal promoter (Fig. 1)
Summary
EMSA reaction mixtures typically contained 0.5–5 g of whole cell protein, 0.6 g of poly(dI-dC), 5 mM Tris-HCl (pH 7.5), 1–5 mM Hepes (pH 7.9), 0.5 mM dithiothreitol, 0.5 mM EDTA, 50 –150 mM NaCl, 2.5–5% v/v glycerol, and 15,000 –20,000 cpm (ϳ10 –100 fmol) of 32P- labeled DNA probe in a volume of 20 l. Molecular Weight Determination of Nucleic Acid-binding Proteins— Apparent molecular weights of proteins bound to [␥-32P]ATP end-labeled ssDNA oligonucleotide in EMSAs were determined by UV crosslinking and subsequent SDS-polyacrylamide gel electrophoresis as described previously [13]. Southwestern Blotting—An equivalent amount of protein from whole cell, cytosolic, and nuclear extracts was resolved by denaturing electrophoresis on 10% SDS-polyacrylamide gels according to the method of Laemmli [27] using a Bio-Rad Mini-PROTEAN II apparatus at 150 V for 90 min. Apparent molecular weights were calculated from a plot of log molecular weight versus relative mobility of pre-stained molecular weight standards (Bio-Rad) using a linear least squares regression fit
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