Vertebrate-like CRYPTOCHROME 2 from monarch regulates circadian transcription via independent repression of CLOCK and BMAL1 activity

Aug 24, 2017Proceedings of the National Academy of Sciences of the United States of America

Monarch butterfly's CRYPTOCHROME 2 controls daily gene activity by separately blocking CLOCK and BMAL1 proteins

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Abstract

Monarch butterfly mutants lacking the BMAL1 C terminus exhibited arrhythmic eclosion behavior.

Insect CRY2 regulates circadian repression via TAD α-helix-dependent and -independent mechanisms.
Mutants without the TAD α-helix showed delayed peak eclosion rhythms under constant darkness.
The presence of a single functional allele of dpCRY2 exacerbated phase delays in eclosion rhythms.
Circadian rhythms were abolished in mutants lacking dpCRY2.
dpCRY2 represses through both TAD-dependent and TAD-independent mechanisms involving different domains.

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Circadian repression of CLOCK-BMAL1 by PERIOD and CRYPTOCHROME (CRY) in mammals lies at the core of the circadian timekeeping mechanism. CRY repression of CLOCK-BMAL1 and regulation of circadian period are proposed to rely primarily on competition for binding with coactivators on an α-helix located within the transactivation domain (TAD) of the BMAL1 C terminus. This model has, however, not been tested in vivo. Here, we applied CRISPR/Cas9-mediated mutagenesis in the monarch butterfly (), which possesses a vertebrate-like CRY (dpCRY2) and an ortholog of BMAL1, to show that insect CRY2 regulates circadian repression through TAD α-helix-dependent and -independent mechanisms. Monarch mutants lacking the BMAL1 C terminus including the TAD exhibited arrhythmic eclosion behavior. In contrast, mutants lacking the TAD α-helix but retaining the most distal C-terminal residues exhibited robust rhythms during the first day of constant darkness (DD1), albeit with a delayed peak of eclosion. Phase delay in this mutant on DD1 was exacerbated in the presence of a single functional allele of, and rhythmicity was abolished in the absence of dpCRY2. Reporter assays inS2 cells further revealed that dpCRY2 represses through two distinct mechanisms: a TAD-dependent mechanism that involves the dpBMAL1 TAD α-helix and dpCLK W328 and a TAD-independent mechanism involving dpCLK E333. Together, our results provide evidence for independent mechanisms of vertebrate-like CRY circadian regulation on the BMAL1 C terminus and the CLK PAS-B domain and demonstrate the importance of a BMAL1 TAD-independent mechanism for generating circadian rhythms in vivo. Danaus plexippus dpCry2Drosophila

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Vertebrate-like CRYPTOCHROME 2 from monarch regulates circadian transcription via independent repression of CLOCK and BMAL1 activity

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