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 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.

Keywords: BMAL1 C terminus; CLOCK; CRISPR; CRYPTOCHROME 2; circadian clock.

Fig. 1.

Monarch dpCLK:dpBMAL1 transcriptional activity requires the dpBMAL1 C-terminal domain lacking in Drosophila CYC. (A) Schematic representation of monarch dpBMAL1 and its C-terminal domain (G and TAD) conserved with mammalian BMAL1, lacking in its Drosophila ortholog dCYC. The gray star indicates the position of the single-guide RNA (sgRNA) used to introduce indels. (B, Upper Left) DpBmal1 genomic locus with the sgRNA and the primers used to amplify the 863-bp targeted region for analysis of mutagenic lesions. (Upper Right) Detection of mutagenic lesions (mut) in somatic cells of a subset of potential founder G0 butterflies using a Cas9-based in vitro cleavage assay. DC, digested control; L, ladder; UC, undigested control. The black star indicates the somatic mutant selected for backcrossing to generate a monarch dpCYC-like mutant lacking the BMAL1 G and TAD regions. DpCYC-like mutants carry a 13-bp deletion. (Lower) Partial alignment of dpBMAL1, dpCYC-like mutant, and dCYC proteins showing the position of the truncation in dpCYC-like relative to the C terminus of dCYC. (C) Profiles of adult eclosion in DD of wild-type (black bars), heterozygous (white bars), and hemizygous mutant (gray bars) siblings of the dpCyc-like mutant line (designated “m1”) entrained to 15 h light/9 h dark (LD 15:9) throughout the larval and pupal stages. Data from DD1 and DD2 are pooled and binned in 1-h intervals. The horizontal bars at the bottom of the graphs show subjective day (gray) and night (black). P < 0.0001 (one-way ANOVA); dpBmal1^+/+ vs. dpCyc-like^m1/+, P > 0.05; dpBmal1^+/+ vs. dpCyc-like^m1/W, P < 0.01; dpCyc-like^m1/+ vs. dpCyc-like^m1/W, P < 0.01 (Tukey’s post hoc test). (D) Circadian expression of period and timeless in brains of wild-type (solid black lines) and hemizygous mutant (dashed gray lines) siblings of the dpCyc-like mutant line. Values are mean ± SEM of three animals. The horizontal bars at the bottom of the graphs show subjective day (gray) and night (black). Interaction genotype × time: per, P < 0.01; tim, P < 0.05 (two-way ANOVA).

Fig. S1.

CRISPR/Cas9-mediated targeted mutagenesis of dpBMAL1 after mRNA microinjection. (A) Table summarizing results for the generation of two dpBMAL1 mutants. The percentage of live larvae corresponds to the number of larvae that survived to adulthood relative to the number of injected eggs. The percentage of somatic mutants corresponds to the number of live larvae that presented any degree of somatic mosaicism. The germline mutation rate corresponds to the percentage of progeny carrying a mutated allele from the total of progeny tested. The number of mosaic mutants crossed (Mut crossed, n) is shown. (B) Targeted mutagenesis of the dpBmal1 exon 10 for all alive potential founders (G₀) validated by PCR and the Cas9-based in vitro cleavage assay. Full cleavage of a wild-type PCR fragment by the sgRNA and Cas9 was validated, and PCR products from somatic tissue of each founder were subjected to the Cas9-based in vitro cleavage assay. The gray arrow indicates genomic amplicons carrying targeted mutations that are resistant to cleavage by Cas9, and the black arrows indicate cleaved wild-type fragments (+) subjected to and (−) not subjected to Cas9 cleavage assay. Gray stars indicate somatic mutants, and two-stacked stars indicate the individual (founder #5) used to establish the dpCyc-like mutant line. (C, Left) Genotyping of founder #5 progeny (G₁) with the Cas9-based cleavage assay. Six of ten individuals carry a mutated allele, including one heterozygote and five homozygotes. (Right) All mutants carried a single 13-bp deletion. (D) Targeted mutagenesis of the dpBmal1 exon 12 for all alive potential founders (G₀) validated by PCR and the Cas9-based in vitro cleavage assay. The red arrow indicates genomic amplicons carrying targeted mutations that are resistant to cleavage by Cas9, and the black arrow indicates the larger cleaved wild-type fragment. Red stars indicate somatic mutants, and two stacked stars indicate the individual (founder #10) used to establish the dpBmal1ΔCter mutant line. The two mutations identified in somatic tissues of this founder are shown. (E) Genotyping of founder #10 progeny (G₁) with the Cas9-based cleavage assay. Ten of nineteen individuals carried a mutated allele, either a 7-bp deletion or a 6-bp deletion. (F) Alignment of nucleotide (center) and amino acid (outward) sequences of the wild-type or mutated (7-bp deletion) dpBMAL1 C-terminal regions. The conserved most distal residues are in red.

Fig. S2.

Profiles of adult eclosion during the first 2 d of DD. (A) Profiles of wild-type (black bars), heterozygous (white bars), and hemizygous mutant (gray bars) siblings of the dpCyc-like mutant line. (B) Profiles of wild-type (black bars), heterozygous (white bars), and hemizygous mutant (red bars) siblings of the dpBmal1ΔCter mutant line. (C) Profiles of dpBmal1ΔCter mutants in a wild-type background (red bars), in a heterozygous background for dpCry2 (blue bars), and in a dpCry2-null background (brown bars). Butterflies were entrained throughout their larval and pupal stages to LD 15:9. Eclosion is binned at 1-h intervals. The horizontal bars below the graphs indicate subjective night (black) and day (gray).

Fig. 2.

The dpBMAL1 TAD helix is dispensable for the generation of circadian rhythm in vivo but regulates the phase by maintaining high activation levels. (A, Left) DpBmal1 genomic locus showing positions of the sgRNA used to generate a TAD truncation mutant (dpBMAL1ΔCter) and primers used to amplify the 1,352-bp targeted region for analysis of mutagenic lesions. (Right) Detection of mutagenic lesions (mut) in somatic cells of a subset of potential founder G0 butterflies using a Cas9-based in vitro cleavage assay. DC, digested control; L, ladder; UC, undigested control. The red star indicates the somatic mutant selected for backcrossing to generate a dpBMAL1ΔCter mutant lacking most of the TAD but retaining three of the most distal amino acids. DpBMAL1ΔCter mutants carry a 7-bp deletion. (B) Sequence alignment showing TAD regions of BMAL1 and BMAL2 proteins from mouse (m), dpBMAL1, and the dpBMAL1ΔCter mutant. The TAD helix region is shown below the alignment. (C) Profiles of adult eclosion in DD of wild-type (black bars), heterozygous (white bars), and hemizygous mutant (red bars) siblings of the dpBmal1ΔCter mutant line (designated “m2”) entrained to LD 15:9 throughout the larval and pupal stages. Data from DD1 and DD2 are pooled and binned in 1-h intervals. Horizontal bars at the bottom of the graphs indicate subjective day (gray) and night (black). (D) Distribution of eclosion during the subjective day for each genotype. Dots indicate the number of butterflies eclosed at each time interval relative to subjective lights on. P < 0.0001 (one-way ANOVA); *P < 0.05, **P < 0.01, NS, nonsignificant (Tukey’s post hoc test). (E) Circadian expression of period and timeless in brains of wild-type (solid black lines) and hemizygous mutant (dashed red lines) siblings of the dpBmal1ΔCter mutant line. Values are the mean ± SEM of three animals. Horizontal bars below the graphs show subjective day (gray) and night (black). per, P > 0.05; tim, P < 0.02 (two-way ANOVA, interaction genotype × time); *P < 0.05 (Student’s t test between each genotype at each time point).

Fig. 3.

Behavioral and molecular rhythms in dpBmal1ΔCter mutants are driven by dpCRY2 repression. (A) Profiles of adult eclosion in DD of dpBmal1ΔCter mutants (m2, containing both homozygous males and hemizygous females) in a wild-type background for dpCry2 (red bars), in a heterozygous background for dpCry2 (blue bars), and in a dpCry2-null background (brown bars). Data collected in DD1 and DD2 are pooled and binned in 1-h intervals. The horizontal bars below the graphs indicate subjective day (gray) and night (black). dpBmal1 m2; dpCry2^+/+ vs. dpBmal1 m2; dpCry2^−/−, P < 0.01; dpBmal1 m2; dpCry2^+/+ vs. dpBmal1 m2; dpCry2^+/−, P < 0.05 (one-way ANOVA followed by Tukey’s post hoc test). (B) Circadian expression of period and timeless in brains of dpBmal1ΔCter mutants in a wild-type background for dpCry2 (red lines), in a heterozygous background for dpCry2 (blue lines), and in a dpCry2-null background (brown lines). Values shown are the mean ± SEM of three animals. The horizontal bars below the graphs indicate subjective day (gray) and night (black). Interaction genotype × time, dpBmal1 m2; dpCry2^+/+ vs. dpBmal1 m2; dpCry2^−/−: tim and per, P < 0.00001; dpBmal1 m2; dpCry2^+/+ vs. dpBmal1 m2; dpCry2^+/−: per, P < 0.001; tim, P < 0.005 (two-way ANOVA).

Fig. 4.

Several domains on dpCLK and dpBMAL1 contribute to transcriptional repression by dpCRY2 in S2 cells. (A, Upper) DpCRY2 does not repress on the BMAL1 G and ΔCter mutant TAD domains. A UAS luciferase reporter (UAS_Luc; 10 ng) was used in the presence (+) or absence (−) of Gal4DBD, Gal4DBD_G+TAD, Gal4DBD_G+ΔCter, Gal4DBD_G, and Gal4DBD_TAD expression plasmids (5 ng each), and increasing doses of dpCRY2 (amounts are given in nanograms). Firefly luciferase activity was computed relative to renilla luciferase activity. Each value is the mean ± SEM of three replicates. (Lower) Western blots of V5-tagged dpCRY2 and Drosophila β-actin protein expression levels. (B) DpCRY2 inhibits dpCLK:dpBMAL1- and dpCLK:dpBMAL1ΔCter-mediated transcription. The monarch per E box luciferase reporter (dpPerEp_Luc; 10 ng) was used in the presence (+) or absence (−) of dpCLK, dpBMAL1, dpBMAL1ΔCter, dpBMAL1ΔTAD, and dpCYC-like expression plasmids (5 ng each) and increasing doses of dpCRY2 (amounts are given in nanograms). Quantification of luciferase activity, values, and Western blot analysis are shown as in A. (C) DpCRY2 dose-dependent inhibition of dpCLK:dpBMAL1 mutants fused to the VP16 transactivation domain in their N termini. The monarch per E box luciferase reporter (dpPerEp_Luc; 10 ng) was used in the presence (+) or absence (−) of dpCLK, dpBMAL1, VP16-dpBMAL1, VP16-dpBMAL1ΔCter, VP16-dpBMAL1ΔTAD, and VP16–dpCYC-like expression plasmids (5 ng each) and increasing doses of dpCRY2 (amounts are given in nanograms). Quantification of luciferase activity, values, and Western blot analysis are depicted as in A. One-way ANOVAs for dose-dependent repression by dpCRY2 on each BMAL1 variant: P < 0.0001 to P < 0.005. (D) DpCLK is required for VP16-dpBMAL1–mediated transcription. The monarch per E box luciferase reporter (dpPerEp_Luc; 10 ng) was used in the presence (+) or absence (−) of dpCLK and VP16-dpBMAL1 expression plasmids (5 ng each). Quantification of luciferase activity and values are depicted as in A. (E) DpCRY2 does not repress the VP16 activation domain. A UAS luciferase reporter (UAS_Luc; 10 ng) was used in the presence (+) or absence (−) of Gal4DBD and Gal4DBD-VP16 expression plasmids (5 ng each) and increasing doses of dpCRY2 (amounts are given in nanograms). Quantification of luciferase activity, values, and Western blot analysis are depicted as in A. P = 0.79 (one-way ANOVA).

Fig. S3.

DpCRY2-mediated inhibition of dpCLK:dpBMAL1 mutants fused to the VP16 transactivation domain in their C termini. The monarch per E box luciferase reporter (dpPerEp_Luc; 10 ng) was used in the presence (+) or absence (−) of dpCLK, dpBMAL1, dpBMAL1ΔCter_VP16, dpBMAL1ΔTAD_VP16, or dpCYC-like_VP16 expression plasmids (5 ng each) and increasing doses of dpCRY2 (amounts are given in nanograms). Firefly luciferase activity was computed relative to renilla luciferase activity. Each value is the mean ± SEM of three replicates. Western blots of V5 epitope-tagged CRY2 and Drosophila β-actin protein expression levels are shown below the graph.

Fig. S4.

Knockdown of p300 in S2 cells cotransfected with dpCLK:dpBMAL1. (A) The monarch per E box luciferase reporter (dpPerEp_Luc; 10 ng) was used in the presence (+) of dpCLK and dpBMAL1 (5 ng each) and either dsRNA against eGFP as a control (7.5 μg) or dsRNA against p300 (7.5 μg), in the absence (−) or presence (+) of increasing doses of dpCRY2 (amounts are given in nanograms). Firefly luciferase activity was computed relative to renilla luciferase activity. Each value is the mean ± SEM of three replicates. P < 0.0001 without dsRNA; P < 0.0002 with dsRNA (one-way ANOVA). (B) Normalized relative luciferase activities from A. *P < 0.05; ns, not significant; P > 0.05 (Student’s t test). (C) mRNA levels of p300 from samples subjected or not to dsRNA-mediated knockdown of p300 and of eGFP as a control. Legend is as in B. Asterisks denote significant differences at P < 0.05 between each condition treated with dsRNA against endogenous p300 and the untreated control in absence of dpCRY2; crosses denote significant differences between each condition treated with dsRNA against endogenous p300 and the untreated control in presence of 10 ng of plasmid expressing dpCRY2. ⁺P < 0.005; ⁺⁺P < 0.001; ⁺⁺⁺P < 0.0005; ns, not significant (Student’s t test).

Fig. 5.

Fusing the dpBMAL1 C terminus to Drosophila dCLK:dCYC or mutating dCLK F349W/D354E independently restores strong repression by dpCRY2 in S2 cells. (A) Evolutionary relationship of insect species representative of lepidopterans and dipterans (Left) with their respective core clock components (Right). Dipterans shown comprise the Nematoceran (mosquitoes) and the Brachyceran (flies) lineages with the melon fly and the fruit fly. The mouse is shown as a representation of vertebrate clocks and as an outgroup of the tree. (B, Top) dpCRY2 affects Drosophila dCLK:dCYC transcription by decreasing dcyc mRNA levels. The monarch per E box luciferase reporter (dpPerEp_Luc; 10 ng) was used in presence of a dpCLK expression plasmid (5 ng each) and increasing doses of dpCRY2 (amounts are given in nanograms). Firefly luciferase activity was computed relative to renilla luciferase activity. P < 0.0001 (one-way ANOVA). (Middle) Endogenous dcyc mRNA levels were quantified using qPCR. P < 0.0005 (one-way ANOVA). Each value is the mean ± SEM of three replicates. (Bottom) Western blots of V5-tagged dCLK and dpCRY2 and Drosophila β-actin protein expression levels. (C) DpCRY2 weakly represses transcription by acting directly on Drosophila dCLK:dCYC proteins. The monarch per E box luciferase reporter (dpPerEp_Luc; 10 ng) was used in the presence of dCLK with (+) or without (−) exogenous dCYC expression plasmids (5 ng each) in the presence of dsRNA against the 5′ and 3′ UTR of endogenous dcyc (7.5 μg each). Increasing doses of dpCRY2 were provided; amounts are given in nanograms. Quantification of luciferase activity and endogenous dcyc, values, and Western blot analysis are depicted as in B. (D) Fusing a dpBMAL1 C terminus to dCYC rescues dpCRY2’s strong repressive capability. The monarch per E box luciferase reporter (dpPerEp_Luc; 10 ng) was used in the presence of dCLK alone or in the presence of dCLK and dCYC-dpBMAL1 C terminus expression plasmid (5 ng each) and dsRNA against the 5′ and 3′ UTR of endogenous dcyc (7.5 μg each). Increasing doses of dpCRY2 were provided; amounts are given in nanograms. Quantification of luciferase activity and endogenous dcyc, values, and Western blot analysis are depicted as in B. (E) Alignment of partial CLK proteins from the mouse (Mus musculus), the monarch butterfly (Danaus plexippus), and the fruit fly (Drosophila melanogaster). The red arrows indicate the conservation of the five previously described mouse CRY1-binding amino acids (11, 12). (F) Mutating dCLK F349W/D354E increases dpCRY2’s repressive capability on dCLK:dCYC-mediated transcription (Left), but fusing the dpBMAL1 C terminus to the mutant protein has no additional effect (Right). The monarch per E box luciferase reporter (dpPerEp_Luc; 10 ng) was used in the presence of either wild-type dCLK or dCLK F349W/D354E expression plasmids (5 ng each) with (+) or without (−) exogenous dCYC or dCYC-dpBMAL1 C terminus expression plasmids (5 ng each) in the presence of dsRNA against the 5′ and 3′ UTR of endogenous dcyc (7.5 μg each). Increasing doses of dpCRY2 were provided; amounts are given in nanograms. Quantification of luciferase activity is presented as values relative to 100% for each set of conditions, and endogenous dcyc values and Western blot analysis are depicted as in B. *P < 0.05 (two-way ANOVA followed by Tukey’s post hoc test).

Fig. S5.

Absolute levels of firefly luciferase activity computed relative to renilla luciferase activity presented in Fig. 5F. The monarch per E box luciferase reporter (dpPerEp_Luc; 10 ng) was used in the presence of either wild-type dCLK or dCLK F349W/D354E expression plasmids (5 ng each) with (+) or without (−) exogenous dCYC or dCYC-dpBMAL1 C terminus expression plasmids (5 ng each) in presence of dsRNA against the 5′ and 3′ UTR of endogenous dcyc (7.5 μg each). Increasing doses of dpCRY2 were provided in the amounts indicated in nanograms. Each value is the mean ± SEM of three replicates.

Fig. 6.

The dpCLK W328 and E333 residues independently contribute to TAD-dependent and TAD-independent repression by dpCRY2. (A and B) Effects of dpCLK mutations (dpCLK W328A, dpCLK E333A, and dpCLK W328A/E333A) in the presence (A) or absence (B) of the dpBMAL1 C terminus. The monarch per E box luciferase reporter (dpPerEp_Luc; 10 ng) was used in the presence (+) of dpBMAL1 (A) or VP16–dpCYC-like (B) and dpCLK variants expression plasmids (5 ng each) with increasing doses of dpCRY2; amounts are given in nanograms. Firefly luciferase activity was computed relative to renilla luciferase activity. Each value is the mean ± SEM of three replicates. Western blots of V5-tagged dpCRY2 and Drosophila β-actin protein expression levels are shown below the graphs. *P < 0.05; **P < 0.01; ns, not significant (two-way ANOVA followed by Tukey’s post hoc test). (C) Coimmunoprecipitations (IP) of c-Myc–dpCLK and either VP16-dpBMAL1 or VP16-dpBMAL1ΔCter and of dpCYC-like–VP16 and c-Myc–dpCLK WT, c-Myc–dpCLK W328A, or c-Myc-dpCLK E333A by FLAG-dpCRY2 with anti-dpCRY2 R42 antibody from transfected S2 cells. Western blots (WB) were performed using the indicated antibodies. The double band for dpCYC-like–VP16 likely corresponds to alternatively used translation initiation sites. The top band was quantified in D. (D) Quantification of C. For each protein, the relative intensity corresponds to the intensity of IP over input signal measured using Image J in each condition, relative to the intensity of IP over input signal of dpCRY2, WT dpCLK, and WT dpBMAL1. Each value is the mean ± SEM of three independent experiments. dpCLK variants, P < 0.05; dpBMAL1 variants, P < 0.005; dpCRY2, P = 0.96 (one-way ANOVA). *P < 0.05; **P < 0.01 (Tukey’s post hoc test).

Fig. S6.

Similar to dpCLK, dpCLK W328A, E333A, and W328A/E333A do not activate transcription in absence of dpBMAL1. The monarch per E box luciferase reporter (dpPerEp_Luc; 10 ng) was used in the presence (+) of dpCLK variants (5 ng each) with (+) or without (−) dpBMAL1 expression plasmids (5 ng). Firefly luciferase activity was computed relative to renilla luciferase activity. Each value is the mean ± SEM of three replicates.