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1.
FIG. 8.

FIG. 8. From: Generation of a Novel Allelic Series of Cryptochrome Mutants via Mutagenesis Reveals Residues Involved in Protein-Protein Interaction and CRY2-Specific Repression .

CRY2G354 is immediately upstream of a region of amino acids conserved in repressive CRYs but not Photolyase. CRY2 3D homology model. This model was generated as described for Fig. 2. The N-terminal residue is shown in black, while the coiled-coil domain is shown in green. CRY2G351 is drawn in magenta, while the residues in the region adjacent to G351 that are conserved in all repressive CRYs, but not Photolyase, are shown in yellow. Under the model is a protein alignment of these residues in CRY1, CRY2, and Photolyase. Conserved residues are shaded in gray.

Ellena V. McCarthy, et al. Mol Cell Biol. 2009 October;29(20):5465-5476.
2.
FIG. 5.

FIG. 5. From: Generation of a Novel Allelic Series of Cryptochrome Mutants via Mutagenesis Reveals Residues Involved in Protein-Protein Interaction and CRY2-Specific Repression .

CRY2G354D exhibits decreased binding to CLOCK-BMAL1, PER1, and PER2, while CRY2G354D retains strong binding. (A) Coimmunoprecipitation. HEK293 cells were transfected with Flag-Clock, Bmal1-Flag3 (3XFLAG), and Cry2-GFP constructs, as indicated. CRY2 proteins were immunoprecipitated, using anti-GFP in conjunction with a protein G IP kit. Copurified proteins were detected by Western blotting (WB) using rabbit anti-GFP (αGFP) or monoclonal mouse anti-FLAG M2 (αFLAG) antibodies. The images shown are representative of three independent experiments. Underneath, the quantitation of the eluted samples from coimmunoprecipitation is shown. The ratio of CRY2 to CLOCK or CRY2 to BMAL1 in the elution was calculated. The data were normalized such that the average amount of CLOCK and BMAL1 that wild-type CRY2 pulled down was set to 1. The error bars represent the SEM. The asterisks indicate groups that were significantly different from each other (P < 0.05). (B and C) Coimmunoprecipitation of CRY2 mutants with PER proteins. In this experiment, HEK293 cells were transfected with PER1-V5 (B) or PER2-V5 (C) and CRY2-GFP constructs, as indicated. Wild-type and mutant CRY2 proteins were pulled down, as described above. Copurified proteins were detected by Western blotting, using either rabbit anti-GFP or monoclonal mouse anti-V5 antibodies. The images shown are representative of three independent experiments.

Ellena V. McCarthy, et al. Mol Cell Biol. 2009 October;29(20):5465-5476.
3.
FIG. 7.

FIG. 7. From: Generation of a Novel Allelic Series of Cryptochrome Mutants via Mutagenesis Reveals Residues Involved in Protein-Protein Interaction and CRY2-Specific Repression .

Expression of CRY2G351D causes complete loss of rhythmicity of a functional molecular clock. NIH 3T3 cultures were transfected and synchronized as described for Fig. 6, and real-time bioluminescence was recorded. (A) Detrended traces of cultures expressing 100 ng of wild-type (WT) Cry2, Cry2G351D, or Cry2G354D. (B) The rhythmicity of each culture was determined, as described for Fig. 6 and then classified as rhythmic, not rhythmic, or irregular, meaning that it was assigned a period that did not appropriately fit the trace. (C) Amplitude analysis of rhythmic cultures expressing mutant CRY2. Cultures transfected with reporter, Clock, Bmal1, and either filler DNA (black) or a low or high dose of Cry2 (wild type or mutant) were synchronized, and their light output was measured. The traces were detrended and the amplitude was determined, as described for Fig. 6. The average amplitude of cultures expressing Cry2 with different mutations is illustrated. The error bars represent the SEM. None of the cultures expressing a high dose of Cry2G351D were rhythmic, so the amplitude could not be determined. At the high dose, cultures expressing Cry2G354D showed rhythms with significantly higher amplitude than that of wild-type Cry2 (P < 0.006). (D) Period analysis of rhythmic cultures expressing high doses of CRY2. The period of the rhythms from the culture expressing a high dose of Cry2 or Cry2G354D were determined and plotted, as described for Fig. 6. NR, not rhythmic.

Ellena V. McCarthy, et al. Mol Cell Biol. 2009 October;29(20):5465-5476.
4.
FIG. 4.

FIG. 4. From: Generation of a Novel Allelic Series of Cryptochrome Mutants via Mutagenesis Reveals Residues Involved in Protein-Protein Interaction and CRY2-Specific Repression .

Identification of a repression domain unique to CRY2. (A) Mutations in CRY1 (G333D and G336D) and CRY2 (G51D and G354D) were each introduced and tested for their repression function in a luciferase assay. HEK293 cells were transfected with the Per-luciferase reporter, Clock, Bmal1, and 25 ng of either the wild-type or mutant Cry1 or Cry2, as indicated. The raw data were normalized such that the average level of activation of Clock, Bmal1, and the reporter control was equal to 100% activation. Each data point is averaged from the results of three replicates, with the error bars representing the SEM. (B) In the experiment whose results are shown, the G333D/G351D and G336D/G354D mutations were each introduced into six different constructs: wild-type CRY1 (CRY1PHR:CRY1TAIL), CRY1PHR with no tail (CRY1PHR ONLY), CRY1PHR:CRY2TAIL, wild-type CRY2 (CRY2PHR:CRY2TAIL), CRY2PHR with no tail (CRY2PHR ONLY), and CRY2PHR:CRY1TAIL. These constructs were then tested in a luciferase assay, as described for panel A, and the data were normalized such that the average repression of the wild-type CRYs was set equal to 100%. Each data point was averaged from three to nine replicates, with the error bars representing the SEM. The asterisks indicate groups that were significantly different from each other (P < 0.01).

Ellena V. McCarthy, et al. Mol Cell Biol. 2009 October;29(20):5465-5476.
5.
FIG. 3.

FIG. 3. From: Generation of a Novel Allelic Series of Cryptochrome Mutants via Mutagenesis Reveals Residues Involved in Protein-Protein Interaction and CRY2-Specific Repression .

CRY2G351D and CRY2G354D are expressed at wild-type levels and are localized to the nucleus. (A) CRY2 3D homology model highlighting two CRY2 residues mutated only in the CRY2 screen. This model was generated as described for Fig. 2. The N-terminal residue is shown in black, while the coiled-coil domain is shown in dark blue. The CRY2 mutants identified in the screen are shown in turquoise, except for the G351D and G354D mutants, drawn in magenta and light pink, respectively. Under the model is a protein alignment of residues G333 to W339 in CRY1 and G351 to W357 in CRY2. Conserved residues are represented in gray, with G351D and G354 highlighted. (B) Immunocytochemistry of CRY2 mutants. Either wild-type Cry2-GFP or mutant Cry2-GFP was transfected into COS7 cells. CRY2-GFP (wild type or mutant) is green, the nuclei are blue, and colocalization is light blue. A blind count of 200 cells for each sample was performed, and the percentage of cells in each cell compartment, nuclear (N), cytoplasmic (C), or both (N/C), was calculated. The graphs below each picture indicate the percentages of cells showing N, N/C, or C localization.

Ellena V. McCarthy, et al. Mol Cell Biol. 2009 October;29(20):5465-5476.
6.
FIG. 1.

FIG. 1. From: Generation of a Novel Allelic Series of Cryptochrome Mutants via Mutagenesis Reveals Residues Involved in Protein-Protein Interaction and CRY2-Specific Repression .

Functional screening of Cry mutant libraries reveals novel domains essential for repression of CLOCK-BMAL1-mediated transcription. Mutagenized Cry1 (yellow) (A) or Cry2 clones (red) (B) were transfected into HEK293 cells along with Clock, Bmal1, and an E-box-driven luciferase reporter plasmid in a 384-well format. Approximately 1,100 mutants were tested in duplicate. Controls, which consisted of Clock and Bmal1 plasmids only (red in panel A, yellow in panel B), reporter construct only (black), or Clock, Bmal1, and wild-type Cry (blue) were included. Luciferase assays were performed to measure transcriptional activation and then normalized to the mean of results for the wild-type Cry controls. All data shown are averages from two replicates. While most of the mutant proteins showed normal repressive activity, many showed decreased repression (brackets). In the schematics, CRY1 (C) and CRY2 (D) are drawn to scale. The PHRs of CRY1 and CRY2 (light purple and light green, respectively) and the C-terminal tails (dark purple and dark green, respectively) are represented. In CRY1, the coiled-coil domain is shown (closed arrow). The canonical NLS in CRY2 is shown (open arrow). Of the Cry mutants sequenced, seven had no changes in the coding sequence, 32 had mutations that resulted in premature stop codons (*), and 23 contained point mutations that resulted in amino acid substitutions (black arrows). Some of the mutants contained more than one mutation (double arrows).

Ellena V. McCarthy, et al. Mol Cell Biol. 2009 October;29(20):5465-5476.
7.
FIG. 6.

FIG. 6. From: Generation of a Novel Allelic Series of Cryptochrome Mutants via Mutagenesis Reveals Residues Involved in Protein-Protein Interaction and CRY2-Specific Repression .

Constitutive Cry expression does not abolish rhythmicity or alter period length but decreases rhythm amplitude. (A) Bioluminescence traces of cycling cells. NIH 3T3 cells were transfected with Per2-luciferase, Clock, and Bmal1 and then either 50 ng Cry1 or an empty vector. (B) Quantitation of culture rhythmicity. Cultures were transfected with constructs as indicated for panel A and also with various doses (1 to 100 ng) of Cry1 or Cry2. After synchronization, bioluminescence was recorded. Traces were detrended and analyzed using a chi-square periodogram (P < 0.001). Cultures were considered rhythmic if the periodogram analysis yielded one significant peak in the range of 15 to 35 h. The bars represent the averages of results from 4 to 12 culture dishes. (C) Records of rhythmic cultures from the experiment whose results are shown in panel B. The triangles represent increasing doses of Cry, with the highest dose at the bottom. (D) Quantification of rhythm amplitude. The amplitudes of the rhythms of the data in panel C were determined. While the cultures transfected with 10 ng of Cry1 exhibited some fluctuations of luciferase activity, no significant period length was detected. Amplitudes of all cultures were normalized to the average amplitude of cultures without Cry within the same experiment. Cultures transfected with all doses of Cry1 and all doses of Cry2, except 1 ng, exhibited rhythms with significantly decreased amplitude (ANOVA, P = 1.34 × 10−19; *, P < 0.0001). (E) The period of each rhythmic culture shown in panel C was determined by chi-square analysis. No significant difference in period was detected (ANOVA; P < 0.26). NR, not rhythmic.

Ellena V. McCarthy, et al. Mol Cell Biol. 2009 October;29(20):5465-5476.
8.
FIG. 2.

FIG. 2. From: Generation of a Novel Allelic Series of Cryptochrome Mutants via Mutagenesis Reveals Residues Involved in Protein-Protein Interaction and CRY2-Specific Repression .

Some CRY mutants display loss of repression dose-response profiles, while some CRY2 mutants cause increases in CLOCK-BMAL1-mediated transcription. (A) CRY1 dose-response assay. HEK293 cells were transfected with Per2-luciferase reporter, Clock, Bmal1, and 25 or 5 ng of either mutant or wild-type (WT) Cry1-Renilla Luciferase. Each data point is averaged from results of three to six replicates, with the error bars representing the standard errors of the means (SEM). The mutants exhibited three general dose-response profiles: no repression, weak repression, and attenuated repression with a significant dose response (P < 0.01). (B) Schematic of CRY1 mutants' primary structure. The domains are indicated as described in the legend of Fig. 1. Each mutant's profile is color coded (see the key for panel A). (C) Homology model of the CRY1 PHR. This model was generated using the SWISS-MODEL protein homology-modeling server (10; http://swissmodel.expasy.org/workspace/) based on the structure of A. nidulans photolyase (Protein Data Bank code 1OWL) (11) and visualized using Protein Explorer (14). The N-terminal-most residue (red) and the coiled-coil domain (green) are shown. The mutations are color coded by repression profile (see the key in panel A). (D) CRY2 dose-response assay. This experiment was performed as described for panel A, except that 150 or 20 ng of either mutant or wild-type Cry2-GFP was used. The mutants exhibited three different dose-response profiles: no repression, no repression at the low dose with activation at a high dose, and no repression at the high dose with activation at a low dose. (E) Schematic of CRY2 protein with mutant profiles indicated. (F) CRY2 3D homology model with mutant repression profiles. This model was generated as described for panel C but using the CRY2 sequence. The mutant residues are color coded as described in panel D.

Ellena V. McCarthy, et al. Mol Cell Biol. 2009 October;29(20):5465-5476.

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