Merlin Lab
Department of Biology
Texas A&M University
Circadian Clock Research
The negative impact of genetic and environmentally-induced circadian dysfunction on human health (e.g., abnormal sleep-wake cycles, mental health disorders, cancer) underscores the importance of understanding how animals keep circadian time. The circadian clock that generates ~24-hr rhythms in animal physiology and behavior relies on a conserved core transcriptional loop via feedback repression of CLOCK-BMAL1. Although the PERIOD (PER) and CRYPTOCHROME (CRY) repressors were identified decades ago, the mechanisms by which they rhythmically repress CLOCK-BMAL1 to generate a ~24-hr cycle are still not well understood.
We have established the monarch as a new system for studying circadian repression because the it retains the mammalian-like CRY that was lost in Drosophila but lacks the paralog complexity observed in mammals .
As in mammals, we demonstrated that the mammalian-like cryptochrome (CRY2) functions in vivo as the main transcriptional repressor of the monarch molecular oscillator (Merlin et al, 2013 Genome Research). In collaboration with the lab of Paul Hardin and support from the National Institute of Health, we have provided the first in vivo mechanistic insights of CRY2 mode of action. Based on previous in vitro biochemical studies, the prevailing model posited that repression and regulation of circadian period by mammalian CRY1 relies primarily on competition for binding with co-activators on an α-helix located within the transactivation domain (TAD) of the BMAL1 C-terminus. Using CRISPR/Cas9-mediated monarch mutants lacking the conserved BMAL1 C-terminal α-helix, our work revealed that despite being necessary for regulating circadian phase the α-helix was not required for generating circadian rhythms in vivo, suggesting the existence of a novel TAD-independent mechanism for repression.
Clockwork mechanism in fruit fly,
monarch and mouse.
We are also investigating how PER orchestrates transcriptional repression of CLOCK-BMAL1. PER complexes with DOUBLETIME/CASEIN KINASE 1 (DBT/CK1) initiate transcriptional repression by binding CLK on DNA. This is followed by DBT/CK1 dependent phosphorylation of PER and CLK, which are thought to remove the PER-CLK complex from DNA. We discovered that a region on CLOCK known as exon 19 (CLK19r) acts as a conserved molecular hub to coordinate transcription activation and repression (Rivas et al, 2021 Curr Biol; Zhang et al, 2022 PNAS). Our lab showed that TRITHORAX (TRX), a histone methyltransferase ortholog of mammalian MLL1 known to bind CLK19r to activate transcription via histone methylation, also promotes PER-CLK binding and PER repression by directly or indirectly methylating Heat Shock Protein (HSP) 68, a member of the HSP70 family of chaperonins. This work established HSP68 as a new and critical player required for monarch CLK-BMAL1 activation and for PER-dependent repression (Zhang et al, 2022 PNAS). We are currently investigating the precise mechanisms by which HSP68 promotes PER repression.
By defining fundamental features of the clock mechanism, our work will provide a molecular foundation that has the potential to advance the diagnosis and treatment of clock disorders and to understand how clocks have evolved in different lineages.
Proposed model for TRX and HSP68 activation and repression of CLK-BMAL1 (above).
We are part of the Center for Biological Clocks Research, which provides fantastic opportunities for training in circadian biology. Applicants interested in joining a dynamic group are invited to look at our open positions HERE.