9860–9873 Nucleic Acids Research, 2017, Vol. 45, No. 17 Published online 18 August 2017 doi: 10.1093/nar/gkx714 Meta-analysis of transcriptomic datasets identifies genes enriched in the mammalian circadian pacemaker Laurence A. Brown1, John Williams2, Lewis Taylor1, Ross J. Thomson1,PatrickM.Nolan2, Russell G. Foster1,* and Stuart N. Peirson1,*
1Sleep and Circadian Neuroscience Institute (SCNi), Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, OX1 3RE, UK and 2MRC Harwell Institute, Harwell Campus, Oxfordshire OX11 0RD, UK
Received September 07, 2016; Revised July 27, 2017; Editorial Decision July 30, 2017; Accepted August 04, 2017
ABSTRACT of an endogenous biological clock. In mammals, the site of the master circadian pacemaker is the suprachiasmatic The master circadian pacemaker in mammals is lo- nuclei (SCN) in the anterior hypothalamus (1). The SCN cated in the suprachiasmatic nuclei (SCN) which reg- receives light information from the retina via the retinohy- ulate physiology and behaviour, as well as coordi- pothalamic tract, synchronizing (entraining) SCN rhythms nating peripheral clocks throughout the body. In- to the external environment. SCN lesions result in loss of vestigating the function of the SCN has often fo- physiological and behavioural rhythms, and transplanta- cused on the identification of rhythmically expressed tion of foetal SCN can restore rhythmicity with a period genes. However, not all genes critical for SCN func- consistent with that of the donor tissue (2). The circa- tion are rhythmically expressed. An alternative strat- dian clock is the product of an intracellular transcriptional- egy is to characterize those genes that are selec- translational feedback loop (TTFL), comprised of a num- tively enriched in the SCN. Here, we examined the ber of so-called ‘clock genes’. Whilst clock genes are ex- pressed in tissues throughout the body, the coordinated transcriptome of the SCN and whole brain (WB) of function of the circadian system depends upon neural, hor- mice using meta-analysis of publicly deposited data monal and behavioural output of the SCN pacemaker (3– across a range of microarray platforms and RNA- 5). Seq data. A total of 79 microarrays were used (24 The identification of mammalian clock genes has crit- SCN and 55 WB samples, 4 different microarray plat- ically depended upon the application of forward and re- forms), alongside 17 RNA-Seq data files (7 SCN and verse genetics. Large scale mutagenesis projects have been 10 WB). 31 684 MGI gene symbols had data for at invaluable in identifying novel mouse mutants with circa- least one platform. Meta-analysis using a random ef- dian phenotypes. Mapping the underlying mutation has led fects model for weighting individual effect sizes (de- to the identification of a number of key clock genes, includ- rived from differential expression between relevant ing Clock (6), Fbxl3 (7), Fbxl21 (8)andZfhx3 (9). By con- SCN and WB samples) reliably detected known SCN trast, reverse genetics has been used to identify homologs of Drosophila clock genes (identified by forward genetics), in- markers. SCN-enriched transcripts identified in this cluding Period 1–3 (10–12)andCryptochrome 1–2 (13,14)as study provide novel insights into SCN function, in- well as proteins that interact with known clock components, cluding identifying genes which may play key roles such as Arntl (Bmal1, (15)). A key feature of many of the in SCN physiology or provide SCN-specific drivers. core clock genes is that they are rhythmically expressed over the 24-h cycle. Therefore, studies to identify new clock genes INTRODUCTION have often taken the approach of identifying transcripts that are rhythmically expressed in the SCN or other periph- Life on Earth has evolved under a predictably changing cy- eral clocks, under either entrained light/dark conditions or cle of light and darkness and, as a result, virtually all or- over one or more circadian cycles under constant conditions ganisms demonstrate striking changes in physiology and be- (16). These studies have been successful in identifying novel haviour over the 24-h day. These rhythms are not simply clock genes as well as genes important for SCN function a response to the changing environment, but persist under (17,18). However, not all clock genes are rhythmically ex- constant conditions––providing evidence for the existence
*To whom correspondence should be addressed. Tel: +44 0 1865 618 674; Email: [email protected] Correspondence may also be addressed to Russell G. Foster. Tel: +44 0 1865 618 661; Email: [email protected]