Cry1 circadian phase in vitro: wrapped up with an E-box.

The circadian timing of gene expression is determined by transcriptional regulation through upstream response elements present throughout the genome. Central to this regulation are the actions of a core group of transcriptional activators and repressors, which act through, and are themselves regulated by, a small set of canonical circadian response elements. Among these, the E-box (CACGTG) is crucial for daytime transcriptional activity. The mammalian Period (Per1-3) and Cryptochrome (Cry1-2) genes are E-box-regulated genes, but in peripheral tissues peak Cry1 mRNA expression is delayed by several hours relative to that of Per. It has been proposed that this delay originates from interactions between the proximal E-box and retinoic acid-related orphan receptor response elements (RORE) present in the Cry1 promoter. By using real-time luciferase reporter assays in NIH3T3 cells the authors show here that a proximal 47-bp E-box containing region of the Cry1 promoter is both necessary and sufficient to drive circadian Cry1 transcription with an appropriate phase delay (around 4 h) relative to Per2. The results therefore suggest that, at least in this in vitro model of the clock, RORE are not necessary for the appropriate circadian regulation of Cry1 expression and rather suggest that sequences surrounding the proximal E-boxes confer gene-specific circadian phasing.

Egr1 involvement in evening gene regulation by melatonin.

Seasonal photoperiodic responses in mammals depend on the pineal hormone melatonin. The pars tuberalis (PT) region of the anterior pituitary has emerged as a principal melatonin target tissue, controlling endocrine responses. Rising melatonin levels acutely influence the expression of a small cluster of genes either positively (exemplified by cryptochrome-1, cry1) or negatively (exemplified by the type 1 melatonin receptor, mt1). The purpose of this study was to characterize the pathways through which these evening actions of melatonin are mediated. In vitro experiments showed that cAMP signaling in the PT directly influences mt1 but not cry1 expression. Analysis of nuclear extracts from sheep PT tissue collected 90 min after melatonin or saline control injections highlighted the response element for the immediate early gene egr1 (EGR1-RE) as a candidate for acute melatonin-dependent transcriptional regulation. We identified putative EGR1-RE's in the proximal promoter regions of the ovine cry1 and mt1 genes, and confirmed their functionality in luciferase reporter assays. Egr1 expression is suppressed by melatonin in PT cell cultures, and is rhythmic in the ovine PT with a nadir in the early night. We propose that melatonin-dependent effects on EGR1-RE's contribute to evening gene expression profiles in this pituitary melatonin target tissue.