Abstract
Evolutionarily conserved circadian clocks generate 24-hour rhythms in physiology and behaviour that adapt organisms to their daily and seasonal environments. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus is the principal co-ordinator of the cell-autonomous clocks distributed across all major tissues. The importance of robust daily rhythms is highlighted by experimental and epidemiological associations between circadian disruption and human diseases. BMAL1 (a bHLH-PAS domain-containing transcription factor) is the master positive regulator within the transcriptional-translational feedback loops (TTFLs) that cell-autonomously define circadian time. It drives transcription of the negative regulators Period and Cryptochrome alongside numerous clock output genes, and thereby powers circadian time-keeping. Because deletion of Bmal1 alone is sufficient to eliminate circadian rhythms in cells and the whole animal it has been widely used as a model for molecular disruption of circadian rhythms, revealing essential, tissue-specific roles of BMAL1 in, for example, the brain, liver and the musculoskeletal system. Moreover, BMAL1 has clock-independent functions that influence ageing and protein translation. Despite the essential role of BMAL1 in circadian time-keeping, direct measures of its intra-cellular behaviour are still lacking. To fill this knowledge-gap, we used CRISPR Cas9 to generate a mouse expressing a knock-in fluorescent fusion of endogenous BMAL1 protein (Venus::BMAL1) for quantitative live imaging in physiological settings. The Bmal1Venus mouse model enabled us to visualise and quantify the daily behaviour of this core clock factor in central (SCN) and peripheral clocks, with single-cell resolution that revealed its circadian expression, anti-phasic to negative regulators, nuclear-cytoplasmic mobility and molecular abundance.
Highlights
Circadian clocks are evolutionarily conserved time-keeping mechanisms that generate ca. 24-hour rhythms in physiology and behaviour, allowing organisms to anticipate and adapt to their daily and seasonal environments
The cellular distribution of BMAL1-immunoreactivity (-ir) was comparable between Bmal1Venus/Venus and Bmal1WT/Welcome Trust (WT) hip cartilage (Fig 1C and S1D Fig), whilst in the suprachiasmatic nucleus (SCN) Venus fluorescence was in direct spatial register with BMAL1-ir
Venus::BMAL1 was expressed in the nucleus of most vasoactive intestinal polypeptide (VIP)- and arginine vasopressin (AVP)-ir cells, whereas fewer than 20% of gastrin-releasing peptide (GRP)-ir neurons contained Venus::BMAL1 (Fig 1E and 1F and S2B Fig)
Summary
Circadian clocks are evolutionarily conserved time-keeping mechanisms that generate ca. 24-hour rhythms in physiology and behaviour, allowing organisms to anticipate and adapt to their daily and seasonal environments. The genetic decoding of circadian clockworks has been a standout success in revealing the transcriptional/translational feedback loops (TTFL) within cells that define circadian time and temporally co-ordinate the expression and activity of downstream genes and pathways to the 24-hour day [1,2,3,4]. 24 hour time: we need to put protein flesh onto the genetic scaffold of the self-sustaining TTFL. This is true for the essential transcriptional activator BMAL1 (a bHLH-PAS domain-containing transcription factor). A single gene knockout of BMAL1 in animals is sufficient to eliminate circadian rhythms in cells, tissues and at the whole organism level. Direct measures of the intra-cellular behaviour of BMAL1 in central and peripheral clocks are still lacking
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
