ANALYSIS OF THE RAT LACTATE-DEHYDROGENASE A-SUBUNIT GENE PROMOTER REGULATORY REGION

The rat lactate dehydrogenase (LDH) A subunit gene promoter contains a putative AP-1 binding site at -295/-289 bp, two consensus Spl binding sites at -141/-136 bp and -103/-98 bp, and a single copy of a consens us cyclic AMP-responsive element (CRE) at -48 to -41 bp upstream of th e transcription initiation site. Additionally, an as yet unidentified silencer element is located within the -1173/-830 bp 5'-flanking regio n. Transient transfection analyses of a -1173/+25 bp LDH A-chloramphen icol acetyltransferase fusion gene has indicated a complete inability of the promoter fragment to direct basal or forskolin-induced transcri ption. Deletion of the -1173/-830 bp sequence restored basal and cycli c AMP (cAMP)-inducible activity. Point mutations in the Spl binding si tes of a -830/+25 bp promoter fragment reduced basal but not the relat ive degree of cAMP-inducible activity. cAMP regulated transcriptional activity was dependent upon an 8 bp CRE, -TGACGTCA-, located at the -4 8/-41 bp upstream region. Mutations in the CRE abolished cAMP-mediated induction and reduced basal activity by about 65 %. The CRE binds a 4 7 kDa protein which has previously been identified as CRE binding prot ein (CREB)-327, an isoform of the activating transcription factor/CREB transcription factor gene family. Co-transfection of a vector that ex presses the catalytic subunit of cAMP-dependent protein kinase stimula tes LDH A subunit promoter activity suggesting that cAMP induces LDH A subunit gene expression through phosphorylative modification of CREB- 327. This study emphasizes a fundamental role of several modules inclu ding Sp1 and CREB binding sites in regulating basal and cAMP-mediated transcriptional activity of the LDH A gene.