A new promoter element associated with daily time keeping in Drosophila.

Brandi Sharp,Akanksha Bafna,Felix Naef,Eric Paquet,Herman Wijnen

doi:10.1093/nar/gkx268

Abstract

Circadian clocks are autonomous daily timekeeping mechanisms that allow organisms to adapt to environmental rhythms as well as temporally organize biological functions. Clock-controlled timekeeping involves extensive regulation of rhythmic gene expression. To date, relatively few clock-associated promoter elements have been identified and characterized. In an unbiased search of core clock gene promoters from 12 species of Drosophila, we discovered a 29-bp consensus sequence that we designated as the Clock-Associated Transcriptional Activation Cassette or ‘CATAC’. To experimentally address the spatiotemporal expression information associated with this element, we generated constructs with four separate native CATAC elements upstream of a basal promoter driving expression of either the yeast Gal4 or firefly luciferase reporter genes. Reporter assays showed that presence of wild-type, but not mutated CATAC elements, imparted increased expression levels as well as rhythmic regulation. Part of the CATAC consensus sequence resembles the E-box binding site for the core circadian transcription factor CLOCK/CYCLE (CLK/CYC), and CATAC-mediated expression rhythms are lost in the presence of null mutations in either cyc or the gene encoding the CLK/CYC inhibitor, period (per). Nevertheless, our results indicate that CATAC's enhancer function persists in the absence of CLK/CYC. Thus, CATAC represents a novel cis-regulatory element encoding clock-controlled regulation.

Highlights

The circadian clock of higher eukaryotes is understood to be a conserved transcription/translation auto-regulatory feedback mechanism controlled via rhythmic transcriptional activation and repression
Mammals as well as insects have circadian clocks that operate via interlocking transcriptional feedback loops that rely on heterodimers of basic helix–loop–helix, Per-Arnt-Sim (PAS) domain transcription factors (TFs) for transcriptional activation [1]
In order to detect over-represented cis-acting elements associated with clock gene expression, we combined MEME [13] with a Hidden Markov Model ((12) and Materials and Methods) to analyse an alignment of promoter sequences of the core clock genes for 12 species of Drosophila

Summary

Introduction

The circadian clock of higher eukaryotes is understood to be a conserved transcription/translation auto-regulatory feedback mechanism controlled via rhythmic transcriptional activation and repression. Mammals as well as insects have circadian clocks that operate via interlocking transcriptional feedback loops that rely on heterodimers of basic helix–loop–helix (bHLH), Per-Arnt-Sim (PAS) domain transcription factors (TFs) for transcriptional activation [1]. In the clock circuit of the fruit fly, Drosophila melanogaster, a heterodimer consisting of CLOCK (CLK) and CYCLE (CYC), acts as the core transcription factor [2,3]. CLK/CYC binds DNA by associating with a canonical CACGTG E-box or E-box-like sequences [4,5,6]. E-boxes, are poor predictors of potential clock-regulated genes because the sequence motif is widespread throughout the fly genome. The nucleotides flanking the E-box and/or an arrangement of closely spaced associated motifs, on the other hand, likely contribute to clock transcriptional activity, TF specificity and increased binding affinity [7,8]

Methods

Results

Conclusion