Mechanisms in the mammalian circadian clock - TITLE Mechanisms in the mammalian circadian clock ABSTRACT The circadian clock is a conserved molecular oscillator that synchronizes biological processes with the ~24-hour day-night cycle, regulating critical functions such as sleep-wake patterns, metabolism, and immune responses. Disruptions in circadian rhythms are linked to disorders like Delayed Sleep Phase Disorder (DSPD) and Advanced Sleep Phase Disorder (ASPD), as well as psychiatric conditions such as depression and anxiety. Circadian oscillations are driven by a central transcription-translation feedback loop (TTFL) involving core clock proteins— CLOCK, BMAL1, CRY1, PER2, CK1d, and GSK3b. While studies have explored individual interactions between these proteins, key aspects of their network dynamics and the molecular mechanisms governing their nucleocytoplasmic transport remain poorly understood. This proposal seeks to address these gaps through three specific aims: (1) define the full protein interactome of the human circadian clock by reconstituting core TTFL proteins and mapping their interactions using biochemical and biophysical techniques and reveal complex dynamics, stoichiometry, and isoform-specific effects; (2) elucidate the molecular basis of CRY1- and PER2- mediated transcriptional repression of CLOCK•BMAL1 by resolving their atomic structures, while assessing how isoform variations and our recently identified DSPD-linked CRY1 variant impact repression; and (3) investigate the nucleocytoplasmic transport of circadian proteins by identifying their karyopherin (KAP) transport factors through systematic interaction studies with recombinant proteins, validated using in vitro transport assays and cellular localization studies. Collectively, this work will provide a comprehensive understanding of the dynamic interactions, transcriptional repression mechanisms, and nucleocytoplasmic transport processes essential for establishing the central TTFL and sustaining circadian rhythms. These findings will advance our knowledge of circadian biology and identify potential therapeutic targets for sleep and psychiatric disorders associated with circadian dysfunction.
