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

Abstract

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 (Danaus plexippus), 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 dpCry2, and rhythmicity was abolished in the absence of dpCRY2. Reporter assays in Drosophila S2 cells further revealed that dpCRY2 represses through two distinct mechanisms: a TADdependent 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.circadian clock | CRYPTOCHROME 2 | BMAL1 C terminus | CLOCK | CRISPR

The genetics and epigenetics of animal migration and orientation: birds, butterflies and beyond

“…The seasonal plasticity of monarch butterflies trans-generational migratory behaviour, on the other hand, offers opportunities to study the epigenetic basis of migration. With the ability to maintain colonies in the laboratory year-round, a fast generation time and its accessibility to in vivo genetic manipulation (Merlin et al, 2013;Markert et al, 2016;Zhang et al, 2017), the monarch is also uniquely suited to rapidly assess the function of candidate migratory genes (Fig. 3B).…”

“…Brain clocks are, however, most likely involved in the induction of the migratory traits and could impact migratory traits by regulating the transcription of clock genes and clock-controlled genes in this tissue (Hardin and Panda, 2013). With the availability of several clock gene knockouts in monarchs (Merlin et al, 2013;Markert et al, 2016;Zhang et al, 2017), functionally determining whether circadian clocks or clock genes play a role in the migratory switch should now be feasible, at least in this system.…”

Geomagnetic field intensity as a cue for the regulation of insect migration

“…The magnetic response of positive phototaxis we found here is likely to be a result of interactions between the roles of Drosophila -like Cry1 in phototaxis and magnetoreception [1, 3, 4, 40], even though Cry2 may also function in a complex with photoreceptors or downstream in magnetoreception signaling [42]. Moreover, given that Cry2 is a component of the core circadian clock and it is reported that circadian clock is sensitive to changes in magnetic field intensity [11, 43], a Cry2 function as a potential timing mechanism for migration based on GMF intensity might also exist [44].…”

Protein dynamics regulate distinct biochemical properties of cryptochromes in mammalian circadian rhythms

“…Notably, the secondary pocket in photoreceptive cryptochromes from Arabidopsis and Drosophila (Brautigam et al, 2004;Levy et al, 2013), as well as the evolutionarily related DNA repair enzymes, CPD or (6-4) photolyase (Hitomi et al, 2009;Park et al, 1995), is considerably smaller in volume, where it binds to small molecule cofactors that facilitate light harvesting (Sancar, 2003). The ability of repressor-type cryptochromes to interact with CLOCK PAS-B appears to be deeply rooted in evolution of the metazoan circadian clock, because the vertebrate-like cryptochromes that act as transcriptional repressors in insects (Zhu et al, 2005) like the monarch butterfly also depend to the same extent on multivalent interactions with CLOCK PAS-B and the BMAL1 TAD as they do in mammals (Sato et al, 2006;Xu et al, 2015;Zhang et al, 2017).…”

“…The BMAL1 TAD is the primary driver of transcriptional activation by CLOCK:BMAL1 in mammalian and insect systems with repressor-type cryptochromes; truncation or mutation of the TAD decimates CLOCK:BMAL1 activity and leads to arrhythmicity in vivo (Kiyohara et al, 2006;Park et al, 2015;Zhang et al, 2017). Sequestration of the TAD by repressor-type cryptochromes allows them to compete with coactivators at a highly conserved and overlapping binding motif (Xu et al, 2015).…”