IDENTIFICATION OF A SILENCER MODULE WHICH SELECTIVELY REPRESSES CYCLIC AMP-RESPONSIVE ELEMENT-DEPENDENT GENE-EXPRESSION

The cyclic AMP (cAMP)-inducible promoter from the rat lactate dehydrog enase A subunit gene (LDH A) is associated with a distal negative regu latory element (LDH-NRE) that represses inherent basal and cAMP-induci ble promoter activity. The element is of dyad symmetry, consisting of a palindromic sequence with two half-sites, 5'-TCITG-3'. It represses the expression of an LDH A/chloramphenicol acetyltransferase (CAT) rep orter gene in a dose-dependent, orientation- and position-independent fashion, suggesting that it is a true silencer element. Uniquely, it s electively represses cAMP-responsive element (CRE)-dependent transcrip tion but has no effect on promoters lacking a CRE sequence. The repres sing action of LDH-NRE could be overcome by cotransfection with LDH A/ CAT vector oligonucleotides containing either the LDH-NRE or CRE seque nce. This suggests that the reversal of repression was caused by the r emoval of functional active, limiting transacting factors which associ ate,vith LDH-NRE as well as with CRE. Gel mobility shift, footprinting , and Southwestern blotting assays demonstrated the presence of a 69-k Da protein with specific binding activity for LDH-NRE. Additionally, g el supershift assays with anti-CREB and anti-Fos antibodies indicate t he presence of CREB and Fos or antigenically closely related proteins with the LDH-NRE/protein complex. We suggest that the LDH-NRE and CRE modules functionally interact to achieve negative modulation of cAMP-r esponsive LDH A transcriptional activity.