Quantitative live imaging of Venus::BMAL1 in a mouse model reveals complex dynamics of the master circadian clock regulator

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 Bmal1^Venus 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. Cell-autonomous circadian clocks are transcriptional/translational feedback loops that co-ordinate almost all mammalian physiology and behaviour. Although their genetic basis is well understood, we are largely ignorant of the natural behaviour of clock proteins and how they work within these loops. This is particularly true for the essential transcriptional activator BMAL1. To address this, we created and validated a mouse carrying a fully functional knock-in allele that encodes a fluorescent fusion of BMAL1 (Venus::BMAL1). Quantitative live imaging in tissue explants and cells, including the central clock of the suprachiasmatic nucleus (SCN), revealed the circadian expression, nuclear-cytoplasmic mobility, fast kinetics and surprisingly low molecular abundance of endogenous BMAL1, providing significant quantitative insights into the intracellular mechanisms of circadian timing at single-cell resolution.