E-box function in a period gene repressed by light

Abstract

In most organisms, light plays a key role in the synchronization of the circadian timing system with the environmental day-night cycle. Light pulses that phase-shift the circadian clock also induce the expression of period (per) genes in vertebrates. Here, we report the cloning of a zebrafish per gene, zfper4, which is remarkable in being repressed by light. We have developed an in vivo luciferase reporter assay for this gene in cells that contain a light-entrainable clock. High-definition bioluminescence traces have enabled us to accurately measure phase-shifting of the clock by light. We have also exploited this model to study how four E-box elements in the zfper4 promoter regulate expression. Mutagenesis reveals that the integrity of these four E-boxes is crucial for maintaining low basal expression together with robust rhythmicity and repression by light. Importantly, in the context of a minimal heterologous promoter, the E-box elements also direct a robust circadian rhythm of expression that is significantly phase-advanced compared with the original zfper4 promoter and lacks the light-repression property. Thus, these results reveal flexibility in the phase and light responsiveness of E-box-directed rhythmic expression, depending on the promoter context.

Circadian rhythms in Mexican blind cavefish Astyanax mexicanus in the lab and in the field

“…In this study, we have explored, under both laboratory and field conditions, how the circadian clock has evolved in this species. In the laboratory, surface fish show a robust circadian oscillation in per1 gene expression, as has been previously reported for zebrafish and other teleost species 20,31,32,34,42 . A high-amplitude per1 rhythm is also apparent in surface fish under natural conditions.…”

“…S1). Per1 represents a key element of the core clock mechanism and, in most teleosts studied to date, shows high-amplitude circadian oscillations 20,31,32 . Per1 is therefore an excellent marker of clock function.…”

“…Independent circadian clocks are found in virtually all zebrafish tissues, and these tissues are themselves directly light responsive 18,19 . Light signals have a strong impact, not only on resetting of the zebrafish circadian clock [20][21][22] , but also on numerous aspects of zebrafish cell biology, such as the activation of DNA repair processes [23][24][25][26] and the regulation of cell cycle events [27][28][29][30] . Evolving in a completely dark environment is therefore likely to impact fish physiology in a dramatic manner.…”

Real-Time Luminescence Reporting of Circadian Gene Expression in Mammals

“…The TopCount (a plate reader, Packard Instrument Co., Meriden, CT) has been used to measure circadian luminescence rhythms of fly and zebrafish at room temperature (Stanewsky et al, 1997;Vallone et al, 2004). However, because of condensation issues (it is very difficult to set the temperature inside the machine to be exactly the same as the temperature outside the machine and because the plates stay outside the machine most of the time except during luminescence reading and small temperature changes produce condensation on top of the culture sample; see earlier discussion), the TopCount is a difficult unit to use in mammalian tissue culture experiments at 36 °.…”

“…This method has faithfully monitored the rhythms of circadian genes in the fly (Brandes et al, 1996), mouse (Asai et al, 2001;Geusz et al, 1997;Wilsbacher et al, 2002;Yoo et al, 2004), rat (Yamazaki et al, 2000), and fungi (Morgan et al, 2003), as well as immortalized cell lines driven from the rat (Izumo et al, 2003;Ueda et al, 2002), zebrafish (Vallone et al, 2004), and human (Maronde and Motzkus, 2003). Using this noninvasive assay, we are able to measure real-time expression of circadian and circadian output genes, as well as the protein dynamic of the circadian genes.…”