CHARACTERIZATION OF 3 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) i s controlled by several positive promoter elements [Simar-Blanchet, A. -E., Paul, C., Mercier, L,. & Le Cam, A. (1996) fur: J. Biochem. 236, 638-648] and a negative element located in the 3' untranslated gene re gion (3' UTR) [Le Cam, A. & Legraverend, C. (1996) Eur J. Biochem. 231 , 620-627]. Ln the present studies, we dissected the 348-bp spi 2.3 3' UTR silencer to precisely define repressor sites and look for specifi cally interacting proteins. Three short elements referred to as A (nuc leotides 1751-1776 in the cDNA), B (nucleotides 1812-1827) and C (nucl eotides 1958-1974) sites repressed transcription from the homologous s pi 2.3 promoter as well as from a heterologous minimal promoter contai ning the spi GAGA box enhancer. All three sites harbor a (TTTC) motif whose mutation affected silencer activity that was also dependent on f lanking sequences. Those sites share the (TTTC) motif and a CCAAT/enha ncer-binding-protein(Ci EBP)-binding site with a fatty-acid-binding-pr otein gene promoter element shown to interact specifically with a tran scriptional repressor [He, G. P., Muise, A., Wu Li, A. & Ro, H.-S. (19 95) 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 hepato cytes. In vitro footprinting of the spi 2.3 3' UTR silencer region rev ealed a strong interaction with liver nuclear proteins. Among the six identified footprints, three of them (F-II, FIII and F-IV) bound C/EBP s and mapped in regions harboring the repressor function. Binding of C /EBPs to all three spi 2.3 3' UTR repressor sites, although rather wea k, was confirmed by electrophoretic mobility shift assays that other w ise failed to reveal specific interactions with other liver nuclear pr oteins in vitro. However, none of the most largely liver expressed C/E BP species (i.e. alpha, beta and delta) activated the spi 2.3 3' UTR s ilencer function in NIH 3T3 cells, suggesting that binding of those tr anscription factors did not mediate the transcriptional repression.