5-Aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB)

The first and rate-controlling step of the haem biosynthetic pathway in mam mals and fungi is catalysed by the mitochondrial-matrix ix enzyme 5-aminola evulinate synthase (ALAS). The purpose of this work was to explore the mole cular mechanisms involved in the cAMP regulation of rat housekeeping ALAS g ene expression. Thus we have examined the ALAS promoter for putative transc ription-factor-binding sites that may regulate transcription in a cAMP-depe ndent protein kinase (PKA)-induced context. Applying both transient transfe ction assays with a chloramphenicol acetyltransferase reporter gene driven by progressive ALAS promoter deletions in HepG2, and electrophoresis mobili ty-shift assays we have identified two putative cAMP-response elements (CRE s) at positions -38 and -142. Functional analysis showed that both CRE-like sites were necessary for complete PKA induction, but only one for basal ex pression. Co-transfection with a CRE-binding protein (CREB) expression vect or increased PKA-mediated induction of ALAS promoter transcriptional activi ty. However, in the absence of co-transfected PKA, CREB worked as a specifi c repressor for ALAS promoter activity. A CREB mutant deficient in a PKA ph osphorylation site was unable to induce expression of the ALAS gene but cou ld inhibit non-stimulated promoter activity. Furthermore, a DNA-binding mut ant of CREB did not interfere wi th ALAS promoter basal activity. Site-dire cted-mutagenesis studies showed that only the nearest element to the transc ription start site was able to inhibit the activity of the promoter. Theref ore, we conclude that CREB, through its binding to CRE-like sites, mediates the effect of cAMP on ALAS gene expression. Moreover, we propose that CREB could also act as a repressor of ALAS transcription, but is able to revers e its role after PKA activation. Dephosphorylated CREB would interfere in a spatial-disposition-dependent manner with the transcriptional machinery dr iving inhibition of gene expression.