Circadian rhythms in the absence of the clock gene Bmal1
Authors: Utham K Valekunja
Alessandra Stangherlin
Sandipan Ray
Steven A Howell
Ambrosius P Snijders
Akhilesh B Reddy
Gopinath Damodaran
Journal: Science (2020)
Discipline: Multidisciplinary
Keywords: Circadian clocks; BMAL1; molecular oscillations; skin fibroblasts; liver slices
Affiliations: Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA., Institute for Translational Medicine and Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA., Institute of Metabolic Science, University of Cambridge, Addenbrooke’s Hospital, Cambridge CB2 0QQ, UK., The Francis Crick Institute, London NW1 1AT, UK., Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA., Chronobiology and Sleep institute (CSI), Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Abstract:  Circadian (~24 hour) clocks have a fundamental role in regulating daily physiology. The transcription factor BMAL1 is a principal driver of a molecular clock in mammals. Bmal1 deletion abolishes 24-hour activity patterning, one measure of clock output. We determined whether Bmal1 function is necessary for daily molecular oscillations in skin fibroblasts and liver slices. Unexpectedly, in Bmal1 knockout mice, both tissues exhibited 24-hour oscillations of the transcriptome, proteome, and phosphoproteome over 2 to 3 days in the absence of any exogenous drivers such as daily light or temperature cycles. This demonstrates a competent 24-hour molecular pacemaker in Bmal1 knockouts. We suggest that such oscillations might be underpinned by transcriptional regulation by the recruitment of ETS family transcription factors, and nontranscriptionally by co-opting redox oscillations. 

Retracted: No

PubPeer comments:
 Supplementary Figure 7 : Could the authors check please? More similar than expected. ![file] , 
 #1 Thank you for your comments on these blot images. Unfortunately, in the process of compiling the images, we inadvertently pasted the same image twice. We are in the process of formally correcting this mistake. The quantifications shown in the related main figure were derived from the source image for Bmal+ + Replicate 2, which we have double-checked. That image will be in the corrected version u2013 i.e. the unintentionally duplicated image will be replaced with the one actually used for quantification. , 
 Erratum for the Report u201cCircadian rhythms in the absence of the clock gene Bmal1u201d by S. Ray, U. K. Valekunja, A. Stangherlin, S. A. Howell, A. P. Snijders, G. Damodaran, A. B. Reddy  The genotypes (Bmal1+ + and Bmal1u2212 u2212) in one of the datasets (liver slice time course) were inadvertently inverted during analysis. This affects some of the panels in Figs. 1 and 3 of the Report and the associated supplementary figures and tables. No other datasets or analyses are affected. These corrections do not change the conclusions of the paper. , 
  Even after applying more stringent FDR, we identified 5098 transcripts at FDR 0.1 or 3822 at FDR 0.05 in Bmal1u2212 u2212 liver tissue (Fig. 1B). With few exceptions, if a transcript oscillated in Bmal1u2212 u2212 liver, it did not do so in Bmal1+ + tissue and vice versau2014that is, the sets of oscillating transcripts were almost mutually exclusive (Fig. 1C), with slightly different phase distribution patterns (fig. S1E).  Rhythms in WT and KO are mutually exclusive.  What might, therefore, be the underlying molecular mechanism driving circadian rhythmicity in Bmal1u2212 u2212 tissue? To establish this, we analyzed the promoter regions of rhythmically expressed transcripts (FDR 0.1), focusing on the two principal phase peaks at subjective dawn and dusk (Fig. 3A). Unbiased motif analysis indicated enrichment [q value (Benjamini) 0.05] of E26 transformation-specific (ETS) factors, for the dawn phase rhythmic transcripts in both Bmal1+ + and Bmal1u2212 u2212 mice. (Fig. 3A). We found rhythmic expression of multiple ETS transcription factors with comparable peak phases clustered around dawn (Fig. 3B). We observed rhythmic expression of 9 and 11 ETS transcription factors in Bmal1u2212 u2212 and Bmal1+ + liver tissues, respectively (FDR 0.1), with 5 overlapping candidates (fig. S6A and table S3). But we explain the rhythms by transcription factors that are equally rhythmic in WT and KO. However:  Mixing up data from WT and KO do not change the conclusions of the paper! , 
 Two new comments and analysis of the data conclude that there is insufficient evidence to support circadian transcriptional rhythms in the absence of Bmal1. The analysis concludes that the various genes detected as u201ccyclingu201d in any given study are likely false positives or experimental artifacts. https: science.sciencemag.org content 372 6539 eabf0922 https: science.sciencemag.org content 372 6539 eabe9230 , 
 #5 Although only an interested observer (only peripherally involved in circadian rhythm). For balance, you should also refer to the responses to these comments that were also published: Response to Abruzzi: https: science.sciencemag.org content 372 6539 eabf1941 Response to Ness-Cohn: https: science.sciencemag.org content 372 6539 eabf1930 These raise some critical points about the reanalyses performed by Abruzzi and Ness-Cohn, including questionable statistics and Abruzzi not referring to analyses that were performed showing robust rhythms. Contrary to their comments. , 
 https: twitter.com papersofnote status 1383480744354271232 , 
  These corrections do not change the conclusions of the paper. Figure 3: except for Elf4, results are different between the 2 versions. ![file] ,