The E 3 ubiquitin ligase UBE 3 A is an integral component of the molecular circadian clock through regulating the BMAL 1 transcription factor

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/E7mediated cell transformation and circadian (BMAL1) disruption. INTRODUCTION Circadian clocks drive ∼24-h rhythms in gene expression, physiology and behaviour and are strikingly well conserved in flies and mammals (1,2). At the molecular level, circadian oscillations rely on transcriptional/translational feedback loops. In mammals, 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. Timely degradation of the circadian clock proteins (such as PERs, CRYs and REV-ERB ) has been shown to be essential for circadian timing (4–8). Degradation by the 26S proteasome requires the transfer of ubiquitin moieties to substrate proteins, the final step of which requires a substrate specific E3 ubiquitin protein ligase. It is known that BMAL1 is targeted for degradation by ubiquitination (9). However, little is known about the ubiquitin ligase(s) targeting BMAL1and the potential significance of this to circadian pacemaking. Of the ubiquitin ligases, UBE3A (ubiquitin protein ligase E3A, also known as E6-AP, E6-associated protein) is unique in that it can be activated by the E6 oncoprotein from high risk human papillomaviruses (HPV16/18), in addition to its normal physiological regulators. HPV infections underlie several human cancers and are responsible for almost all cervical cancers (10,11). During tumorigenesis, the E6/UBE3A complex targets p53 for ubiquitination and degradation (12), a major tumour suppressor. However, additional E6 targets have been identified that often contribute to tumour formation (13,14). Core clock proteins interact with components of the cell cycle to rhythmically control cell-cycle genes, such as Wee1, Cyclin D1, C-myc *To whom correspondence should be addressed. Tel: +44 161 3068912. Fax: +44 161 2755762. Email: Qing-Jun.Meng@manchester.ac. uk Present Address: Feng Zhang, Department of Pharmacology, School of Pharmacy, Taishan Medical University, 619 Chang Cheng Road. Tai’an city, 271016, Shandong, P.R. China C © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. D ow naded rom http/academ ic.p.com /nar/article-at/42/9/5765/1260700 by gest on 10 Jauary 2019 5766 Nucleic Acids Research, 2014, Vol. 42, No. 9 and p21(Waf1/Cip1) (15–21), and PERIODs and BMAL1 have been described as tumour suppressors (16,20,21). To date, a direct link between cell transformation and circadian clock disruption has not been established. Here, we show that E6/E7-mediated cell transformation disrupts molecular clock rhythms through increasing UBE3A-dependent degradation of BMAL1. Further, we provide experimental evidence implicating UBE3A as an endogenous key regulator of the circadian pacemaking in mammals and flies, revealing the physiological role of UBE3A in the biological timing system. MATERIALS AND METHODS