UC San Diego

Many aspects of mammalian physiology display circadian--or once daily--rhythms, such as heart rate, blood pressure, activity levels, metabolism, and liver regeneration. These rhythms are regulated by an entrainable, self-sustaining, cell-autonomous mechanism found in nearly every cell of the body: the circadian clock. The circadian clock itself represents a regulatory network, composed of interlocking negative feedback loops, that in turn is influenced by two other types of regulatory networks: it is impinged upon by input networks to synchronize the clock to the external environment and coordinate the timing of clocks throughout the body; and output networks by which the circadian clock governs overt rhythms in behavior and physiology. To expand our understanding of the composition of input network and gears of the clock, a genome-wide siRNA screen was performed, and identified hundreds of novel genes that can alter clock function, which represent input and novel clock gene candidates. These clock modifier genes not only display knockdown effects similar to known clock components, they also revealed a high degree of interconnectedness between the circadian clock and other functional pathways, suggesting intertwinement between the circadian system and overall cellular biology. To address the composition and nature of circadian output regulation, the respective roles of local, cell-autonomous regulation in peripheral tissues and systemic circadian regulation emanating from the central nervous system needed to be assessed. Using the MMH-D3 hepatocyte cell line, gene expression profiling revealed that cell-autonomous circadian regulation can drive rhythms in over 1,000 transcripts, indicating that cell-autonomous clock does contribute to circadian rhythms in gene expression and establishing MMH-D3 as a valid circadian cell-based model system. The protein-protein interactions of these circadian genes display organization based on co- and anti -phasic relationships, suggesting that competitive relationships may represent an organizing theme for circadian regulation, extending beyond the clock itself. Finally, circadian oscillations in polyamine synthesis were revealed at both the transcriptional and enzymatic level in MMH-D3. As polyamines are closely associated with cell proliferation and required for initiation of liver regeneration, this may represent a role for the cell- autonomous clock in circadian gating of liver regeneration