The E3 ubiquitin ligase UBE3A is an integral component of the molecular circadian clock through regulating the BMAL1 transcription factor.

Nicole C Gossan,Hikari Yoshitane,Yoshitaka Fukada,Feng Zhang,Nick Glossop,Baoqiang Guo,Ding Jin,Qing-Jun Meng,Yong Q Zhang,Aiyu Yao

doi:10.1093/nar/gku225

Abstract

Post-translational modifications (such as ubiquitination) of clock proteins are critical in maintaining the precision and robustness of the evolutionarily conserved circadian clock. Ubiquitination of the core clock transcription factor BMAL1 (brain and muscle Arnt-like 1) has recently been reported. However, it remains unknown whether BMAL1 ubiquitination affects circadian pacemaking and what ubiquitin ligase(s) is involved. Here, we show that activating UBE3A (by expressing viral oncogenes E6/E7) disrupts circadian oscillations in mouse embryonic fibroblasts, measured using PER2::Luc dynamics, and rhythms in endogenous messenger ribonucleic acid and protein levels of BMAL1. Over-expression of E6/E7 reduced the level of BMAL1, increasing its ubiquitination and proteasomal degradation. UBE3A could bind to and degrade BMAL1 in a ubiquitin ligase-dependent manner. This occurred both in the presence and absence of E6/E7. We provide in vitro (knockdown/over-expression in mammalian cells) and in vivo (genetic manipulation in Drosophila) evidence for an endogenous role of UBE3A in regulating circadian dynamics and rhythmic locomotor behaviour. Together, our data reveal an essential and conserved role of UBE3A in the regulation of the circadian system in mammals and flies and identify a novel mechanistic link between oncogene E6/E7-mediated cell transformation and circadian (BMAL1) disruption.

Highlights

Circadian clocks drive ∼24-h rhythms in gene expression, physiology and behaviour and are strikingly well conserved in flies and mammals [1,2]
Aberrant rhythms in transformed cells were confirmed by studying BMAL1 protein and messenger Ribonucleic acid (RNA) expression following clock synchronization (Figure 1C and D)
Degradation of the clock proteins through the ubiquitin-proteasome pathway is critical for the precision and robustness of the circadian clock

Summary

Introduction

Circadian clocks drive ∼24-h rhythms in gene expression, physiology and behaviour and are strikingly well conserved in flies and mammals [1,2]. Circadian oscillations rely on transcriptional/translational feedback loops. The circadian transcription factors BMAL1 (brain and muscle Arnt-like 1) and CLOCK activate transcription of two repressors PERIODS (PERs) and CRYPTOCHROMES (CRYs) in turn feedback to periodically suppress their own transcription [1,3]. Post-translational modifications modulate protein stability to ensure robust 24-h oscillations. Degradation of the circadian clock proteins (such as PERs, CRYs and REV-ERB␣) has been shown to be essential for circadian timing [4,5,6,7,8]. It is known that BMAL1 is targeted for degradation by ubiquitination [9]. Little is known about the ubiquitin ligase(s) targeting BMAL1and the potential significance of this to circadian pacemaking

Methods

Results

Conclusion