Characterization of three transcriptional repressor sites within the 3' untranslated region of the rat serine protease inhibitor 2.3 gene

The activity of the rat serine protease inhibitor 2.3 gene (spi 2.3) is controlled by several positive promoter elements [Simar-Blanchet, A.-E., Paul, C., Mercier, L. and Le Cam, A. (1996) Eur. J. Biochem. 236, 638-648] and a negative element located in the 3' untranslated gene region (3' UTR) [Le Cam, A. and Legraverend, C. (1996) Eur. J. Biochem. 231, 620-627]. In the present studies, we dissected the 348-bp spi 2.3 3' UTR silencer to precisely define repressor sites and look for specifically interacting proteins. Three short elements referred to as A (nucleotides 1751-1776 in the cDNA), B (nucleotides 1812-1827) and C (nucleotides 1958-1974) sites repressed transcription from the homologous spi 2.3 promoter as well as from a heterologous minimal promoter containing the spi GAGA box enhancer. All three sites harbor a (TTTC) motif whose mutation affected silencer activity that was also dependent on flanking sequences. Those sites share the (TTTC) motif and a CCAAT/enhancer-binding-protein(C/EBP)-binding site with a fatty-acid- binding-protein gene promoter element shown to interact specifically with a transcriptional repressor [He, G. P., Muise, A., Wu Li, A. and Ro, H.-S. (1995) Nature 378, 92-96]. This repressor is however unlikely to mediate spi 2.3 3' UTR silencer action since it was not detected in rat hepatocytes. In vitro footprinting of the spi 2.3 3' UTR silencer region revealed a strong interaction with liver nuclear proteins. Among the six identified footprints, three of them (F-II, FIII and F-IV) bound C/EBPs and mapped in regions harboring the repressor function. Binding of C/EBPs to all three spi 2.3 3' UTR repressor sites, although rather weak, was confirmed by electrophoretic mobility shift assays that otherwise failed to reveal specific interactions with other liver nuclear proteins in vitro. However, none of the most largely liver expressed C/EBP species (i.e. α, β and δ) activated the spi 2.3 3' UTR silencer function in NIH 3T3 cells, suggesting that binding of those transcription factors did not mediate the transcriptional repression.