UC Santa Cruz

Timing mechanisms are utilized by organisms in a variety of biological functions. From circadian rhythms to nematode development, the genetic networks that underly the keeping of time are complex and thoroughly regulated. Circadian rhythms allow organisms to anticipate daily environmental changes and thus confer an adaptive advantage and the genetic network that governs them is well-established. While much is still to be gleaned about the molecular basis of circadian timekeeping, a model of a rewired developmental timer based on conserved circadian clock orthologs C. elegans, is beginning to emerge. In this dissertation, I discuss the conservation of specific factors and provide new insights that highlight the biochemical mechanisms that regulate C. elegans development. C. elegans are a widely studied model organism, yet little is known about the molecular basis for its temporal control of development. Two intricately linked timers, the molting cycle and heterochronic pathway, coordinate cuticle regeneration and growth with stage-specific cellular events. Several circadian orthologs have established roles in regulating these processes. Chapter 2 describes the homology between nuclear hormone receptors (NHRs), retinoic acid-related nuclear receptor (RORα/β/γ) and NHR-23, transcription factors that activate the expression of a core clock component and drives the transcriptional network that governs nematode molting, respectively. We lay the groundwork for a conserved mode of ligand-binding, as well as identify a separate class of small molecules that bind to NHR-23. The interaction between PERIOD (PER) proteins and its cognate kinase, Casein Kinase 1 and ε (CK1), is integral to determining the phase and timing of circadian rhythms. PER is a stoichiometrically limiting factor in the repressive complex that provides the inhibition of circadian transcription. Stable anchoring of CK1 to PER2 mediates phosphorylation of PER that regulates its stability and abundance in the cell. This interaction is also required for CK1-dependent displacement of the core clock transcription factor from DNA. Chapter 3 demonstrates the C. elegans homologs to PER and CK1, LIN-42 and KIN-20, respectively, interact in a similar mode to regulate C. elegans development. We show that two kinase-binding motifs within the CK1-binding domain (CK1BD; CK1BD-A and CK1BD-B) are conserved enough in LIN-42 to mediate binding to CK1 in vitro. We determine that the expression of LIN-42 and KIN-20 temporally and spatially overlaps and that the CK1BD as well as KIN-20 kinase activity are required from proper molting timing. We further show that phosphorylation of LIN-42 by CK1 leads to kinase inhibition, suggesting a conserved mode of product inhibition whereby phosphoserine(s) anchor into conserved anion binding sites along the kinase active site. In chapter 4, we discuss our recent work to identify a novel regulator of the C. elegans molt cycle. Through in vivo techniques, we show that KIN-20 and a previously uncharacterized ankyrin repeat domain-containing protein (ANKRD49), are similarly expressed temporally and spatially, and interact. We show that C. elegans ANKRD49 binds to human CK1 with nanomolar affinity in vitro, and that this interaction influences kinase activity on LIN-42. An AlphaFold binding model of the complex predicts that stable binding is mediated through the ANKRD49 structured C-terminus and a flexible CK1 helix near the active site that is important for substrate recognition and processing. This model also predicts that the interaction is enhanced via binding of the ANKRD49 unstructured N-terminus to the CK1 substrate binding cleft. We show that deletion of the ANKRD49 unstructured N-terminus as well as mutations near and on the flexible CK1 helix that alter circadian period in mammals, reduce the C. elegans ANKRD49/human CK1 affinity >10-fold. Depletion of ANKRD49 in vivo leads to asynchronous and delayed molting similar to kin-20(null) phenotypes. Given the high conservation of CK1 across organisms as well as several proteome-scale studies that also identify a human ANKRD49/CK1 interaction, this work potentially has broader implications for understanding circadian rhythms and temporal regulation in diverse organisms. In summary, throughout this dissertation I have used an interdisciplinary approach of utilizing biochemistry and in vivo C. elegans genetics, to describe the molecular basis for circadian homolog function in C. elegans development. In addition to the findings discussed herein, this work provides a framework elucidating the molecular underpinnings of nematode timing mechanisms as well as an additional insight into evolutionary conservation of timekeeping mechanisms.