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Summary: Joseph Takahashi is interested in understanding the genetic and molecular basis of circadian rhythms as well as other complex behaviors. He uses forward genetic approaches in the mouse to discover genes regulating the nervous system and behavior. The molecular mechanisms of the clock are being studied at the biochemical and genomic levels.

Circadian rhythms are 24-hour oscillations in behavior, physiology, and biochemistry that are generated by a cell-autonomous clock system found in all classes of living organisms. To understand the molecular mechanism of the circadian clock, our laboratory has used forward genetic approaches to discover genes that regulate circadian behavior in mice.

Clock Gene
Using high-efficiency N-ethyl-N-nitrosourea mutagenesis, we isolated the first single-gene mutation that affects circadian rhythms in mice. The Clock mutation lengthens circadian period by 4 hours in homozygous mutants, followed by a complete loss of circadian rhythmicity in constant conditions. Using a combined approach of positional cloning and transgenic (functional) rescue of the Clock mutation in mice, we found that the Clock gene encodes a novel member of the basic helix-loop-helix (bHLH)-PAS family of transcription factors. The CLOCK protein acts as a heterodimeric transcription factor with a partner known as BMAL1 (ARNTL).

	Figure 1: Model of circadian clock mechanism. Figure 2: Crystal structure of CLOCK:BMAL1. Figure 3: Circadian transcriptional landscape.

Molecular Mechanism of the Clock
The circadian clock in mammals is composed of an autoregulatory transcriptional network with interlocked feedback loops (Figure 1). At the core, the bHLH-PAS transcriptional activators CLOCK and BMAL1 activate the Period (Per1, Per2) and Cryptochrome (Cry1, Cry2) genes, whose transcripts and proteins slowly accumulate during the daytime. The PER and CRY proteins associate and translocate into the nucleus during the evening and physically interact with the CLOCK:BMAL1 complex to repress their own transcription. As the PER and CRY proteins are progressively phosphorylated during the night, they are targeted for ubiquitination by specific E3 ligases and are eventually degraded by the proteasome. The waxing and waning of this transcriptional feedback loop takes approximately 24 hours and represents the core mechanism of the circadian clock in mammals.

Crystal Structure of CLOCK:BMAL1
CLOCK and BMAL1 belong to the bHLH-PAS family of transcription factors, which participate in a wide array of functions including responses to environmental contaminants (aryl hydrocarbon receptor, AHR), hypoxia (hypoxia inducible factor, HIF), neurogenesis (SIM1), synaptic plasticity (NPAS4), and circadian regulation (CLOCK, NPAS2, BMAL1); most of these proteins remain poorly characterized at the structural level. In contrast, the structures of individual PAS domains and their interactions with small molecule ligands such as heme and flavin cofactors are well understood, especially in microorganisms and plants, where PAS domains serve important roles in two-component signaling and blue-light detection. We recently solved the three-dimensional crystal structure of the bHLH-PAS domains of mouse CLOCK:BMAL1 at 2.3 Å resolution (Figure 2).

The structure reveals an unusual asymmetric heterodimer with the three domains in each of the two subunits—bHLH, PAS-A, and PAS-B—tightly intertwined and involved in dimerization interactions, resulting in three distinct protein interfaces. A novel mode of PAS domain interaction was observed for the PAS-B domains in which the beta-sheet surface of BMAL1 inserts along the alpha-helical surface of CLOCK (previously PAS domain interactions were found to be mediated by the beta-sheet interfaces). Mutations that perturb the observed heterodimer interfaces affect the stability and activity of the CLOCK:BMAL1 complex as well as the periodicity of the circadian oscillator. The structure reveals the locations of mutations on CLOCK that affect repression by CRY and predict sites for CRY interaction. In addition, an interesting parallel between the PAS domains of BMAL1 and PER can be seen, and it suggests possible mechanisms for PER interactions. The structure of the CLOCK:BMAL1 complex is a starting point for understanding at the atomic level the mechanism driving the mammalian circadian clock.

Transcriptional Architecture and Chromatin Landscape of Core Circadian Clock
Although it is generally acknowledged that most of the core components of the circadian gene network are likely known and that many hundreds to thousands of transcripts have been shown to express circadian oscillations in various tissues, the genome-wide architecture of the transcriptional network regulated by the core circadian clock remains to be defined. We have interrogated the dynamics of the transcriptional architecture of the entire core circadian transcriptional regulatory loop on a genome scale and found a highly stereotyped, time-dependent pattern of core circadian transcription factor binding, RNA polymerase II (RNAPII) recruitment, RNA expression, and chromatin states. We found that the circadian transcriptional cycle of the clock consists of three distinct phases: (1) a poised state in which CLOCK:BMAL1 and CRY1 bind to E-box sites in a transcriptionally silent state associated with RNAPII-Ser5P; (2) a coordinated de novo transcriptional activation state in which RNAPII and p300 recruitment, pre-mRNA transcript expression, histone-3 lysine-9 acetylation (H3K9ac), histone-3 lysine-4 trimethylation (H3K4me3), and histone-3 lysine-27 acetylation (H3K27ac) occupancy oscillate; and (3) a repressed state in which PER1, PER2, and CRY2 occupancy dominates and displaces CLOCK:BMAL1 and PER2 is associated with CREB binding protein (CBP) (Figure 3). Only 22 percent of mRNA cycling genes are driven by de novo transcription, suggesting that transcriptional and post-transcriptional mechanisms underlie the mammalian circadian clock. Surprisingly, circadian modulation of RNAPII recruitment and chromatin remodeling occurs on a genome-wide scale far greater than that seen previously by gene expression profiling. Taken together, these results reveal that the circadian clock regulates hepatic transcriptional architecture as well as chromatin state dynamics on a genome-wide scale.

The past five years have witnessed a remarkable set of discoveries concerning the circadian clock mechanism in mammals. An important goal for our future work is to define novel regulatory pathways within the clock mechanism, to understand the biochemical mechanism of CLOCK:BMAL1 function using structural biology, and to discover how the clock regulates RNAPII and histone modifications on a genome-wide scale.

Grants from the National Institutes of Health provided partial support for these projects.

As of November 30, 2012