Retinal melatonin biosynthesis: interactions of A/T–rich regions and CRE–like sequences contribute to cAMP–dependent regulation of the chicken AANAT promoter

Abstract

Purpose:Arylalkylamine N–acetyltransferase (AANAT) is the key regulatory enzyme in the biosynthesis of melatonin. Previous studies have shown that AANAT activity and the abundance of AANAT mRNA in the chicken photoreceptor cells are regulated by cAMP–dependent mechanisms. The purpose of this study was to identify regulatory elements within the AANAT gene promoter responsible for cAMP–dependent induction. Methods: Photoreceptor–enriched retinal cell cultures were prepared from 6–day–old embryos and were incubated for 5 days. Cells were transfected with pGL3 plasmid containing the wild type and mutated AANAT promoter constructs fused to luciferase. To examine the influence of cAMP, cells were treated with 10µM forskolin and incubated for 6 h before harvesting for measurement of luciferase activity. For DNA–protein binding experiments, electrophoretic mobility shift assay (EMSA) of nuclear proteins was performed with wild type and mutated oligonucleotides. Results:Forskolin–treatment stimulated luciferase activity driven by a 4 kb reporter construct and all 5’–deletion constructs except the smallest, AANAT (–217 to +120)luc. Both the basal and forskolin–stimulated expression levels were maximal with AANAT (–484 to +120)luc, which contains an 8 x TTATT repeat (TTATT8 ) and three CRE–like sequences, designated CLS1–3. Basal expression of AANAT (–217 to +120)luc, which does not contain the TTATT8 sequence, was minimal and unresponsive to forskolin. EMSA demonstrated a number of nuclear protein complexes that bind to the TTATT8 sequence and CLS1–3. Proteins that bound to the TTATT8 sequence were displaced by unlabeled TTATT oligonucleotides, and also by an oligonucleotide containing CLS1, suggesting an interaction between these sites. Mutations of TTATT8 or CLS1 reduced protein binding and eliminated forskolin–stimulated promoter activity. Supershift EMSA using c–Fos and delta CREB antibodies identified these factors as probable components of the protein complexes that bind to the TTATT8 and CLS sites. Conclusions:Transcription driven by this promoter is enhanced by activation of the cAMP/PKA signaling cascade. This appears to be mediated by CLS elements in the promoter acting in concert with the TTATT8 motif, and that combination mediates the full cAMP–induced transcriptional response of a 484 bp segment of AANAT proximal promoter, in the absence of a consensus CRE