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DNA damage shifts circadian clock time via Hausp-dependent Cry1 stabilization.

Papp SJ, Huber AL, Jordan SD, Kriebs A, Nguyen M, Moresco JJ, Yates JR, Lamia KA - Elife (2015)

Bottom Line: We demonstrate that genotoxic stress stimulates Cry1 phosphorylation and its deubiquitination by Herpes virus associated ubiquitin-specific protease (Hausp, a.k.a Usp7), stabilizing Cry1 and shifting circadian clock time.Indeed, the transcriptional response to genotoxic stress is enhanced in Cry1-/- and blunted in Cry2-/- cells.Furthermore, Cry2-/- cells accumulate damaged DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Physiology, Scripps Research Institute, La Jolla, United States.

ABSTRACT
The circadian transcriptional repressors cryptochrome 1 (Cry1) and 2 (Cry2) evolved from photolyases, bacterial light-activated DNA repair enzymes. In this study, we report that while they have lost DNA repair activity, Cry1/2 adapted to protect genomic integrity by responding to DNA damage through posttranslational modification and coordinating the downstream transcriptional response. We demonstrate that genotoxic stress stimulates Cry1 phosphorylation and its deubiquitination by Herpes virus associated ubiquitin-specific protease (Hausp, a.k.a Usp7), stabilizing Cry1 and shifting circadian clock time. DNA damage also increases Cry2 interaction with Fbxl3, destabilizing Cry2. Thus, genotoxic stress increases the Cry1/Cry2 ratio, suggesting distinct functions for Cry1 and Cry2 following DNA damage. Indeed, the transcriptional response to genotoxic stress is enhanced in Cry1-/- and blunted in Cry2-/- cells. Furthermore, Cry2-/- cells accumulate damaged DNA. These results suggest that Cry1 and Cry2, which evolved from DNA repair enzymes, protect genomic integrity via coordinated transcriptional regulation.

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Quantitation of in vivo Cry1 ubiquitination.Quantitation of ubiquitinated Cry1 immunoprecipitated from MEFs expressing shRNA targeting Hausp (blue) or a control sequence (black). Left, western blot from Figure 2C with boxes used for quantitation. The average signal detected in the first two lanes (background nonspecific signal from Cry1−/−;Cry2−/− cells) was subtracted from each of the other lanes to generate the data show on the right.DOI:http://dx.doi.org/10.7554/eLife.04883.009
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fig2s3: Quantitation of in vivo Cry1 ubiquitination.Quantitation of ubiquitinated Cry1 immunoprecipitated from MEFs expressing shRNA targeting Hausp (blue) or a control sequence (black). Left, western blot from Figure 2C with boxes used for quantitation. The average signal detected in the first two lanes (background nonspecific signal from Cry1−/−;Cry2−/− cells) was subtracted from each of the other lanes to generate the data show on the right.DOI:http://dx.doi.org/10.7554/eLife.04883.009

Mentions: Recombinant Hausp can deubiquitinate Cry1 in vitro (Figure 2—figure supplement 2). To examine Cry1 ubiquitination in vivo, we measured ubiquitinated Cry1 in MEFs expressing control or Hausp-targeting shRNA in the presence or absence of the proteasome inhibitor MG132 to stabilize ubiquitinated proteins. Cry1 from Hausp-depleted cells was much more highly ubiquitinated than Cry1 from control cells (Figure 2C, Figure 2—figure supplement 3) as expected if Hausp catalyzes the removal of polyubiquitin chains from Cry1 in vivo. (Note that while Cry1 is decreased in Hausp-depleted cells, we used a limiting amount of anti-Cry1 antibody for immunoprecipitation to normalize the amount of Cry1 and enable comparison between samples. Cry−/− cells were used as a control to subtract the non-specific ubiquitin signal and enable quantitative comparison of control and Hausp-depleted cells.)


DNA damage shifts circadian clock time via Hausp-dependent Cry1 stabilization.

Papp SJ, Huber AL, Jordan SD, Kriebs A, Nguyen M, Moresco JJ, Yates JR, Lamia KA - Elife (2015)

Quantitation of in vivo Cry1 ubiquitination.Quantitation of ubiquitinated Cry1 immunoprecipitated from MEFs expressing shRNA targeting Hausp (blue) or a control sequence (black). Left, western blot from Figure 2C with boxes used for quantitation. The average signal detected in the first two lanes (background nonspecific signal from Cry1−/−;Cry2−/− cells) was subtracted from each of the other lanes to generate the data show on the right.DOI:http://dx.doi.org/10.7554/eLife.04883.009
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352707&req=5

fig2s3: Quantitation of in vivo Cry1 ubiquitination.Quantitation of ubiquitinated Cry1 immunoprecipitated from MEFs expressing shRNA targeting Hausp (blue) or a control sequence (black). Left, western blot from Figure 2C with boxes used for quantitation. The average signal detected in the first two lanes (background nonspecific signal from Cry1−/−;Cry2−/− cells) was subtracted from each of the other lanes to generate the data show on the right.DOI:http://dx.doi.org/10.7554/eLife.04883.009
Mentions: Recombinant Hausp can deubiquitinate Cry1 in vitro (Figure 2—figure supplement 2). To examine Cry1 ubiquitination in vivo, we measured ubiquitinated Cry1 in MEFs expressing control or Hausp-targeting shRNA in the presence or absence of the proteasome inhibitor MG132 to stabilize ubiquitinated proteins. Cry1 from Hausp-depleted cells was much more highly ubiquitinated than Cry1 from control cells (Figure 2C, Figure 2—figure supplement 3) as expected if Hausp catalyzes the removal of polyubiquitin chains from Cry1 in vivo. (Note that while Cry1 is decreased in Hausp-depleted cells, we used a limiting amount of anti-Cry1 antibody for immunoprecipitation to normalize the amount of Cry1 and enable comparison between samples. Cry−/− cells were used as a control to subtract the non-specific ubiquitin signal and enable quantitative comparison of control and Hausp-depleted cells.)

Bottom Line: We demonstrate that genotoxic stress stimulates Cry1 phosphorylation and its deubiquitination by Herpes virus associated ubiquitin-specific protease (Hausp, a.k.a Usp7), stabilizing Cry1 and shifting circadian clock time.Indeed, the transcriptional response to genotoxic stress is enhanced in Cry1-/- and blunted in Cry2-/- cells.Furthermore, Cry2-/- cells accumulate damaged DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Physiology, Scripps Research Institute, La Jolla, United States.

ABSTRACT
The circadian transcriptional repressors cryptochrome 1 (Cry1) and 2 (Cry2) evolved from photolyases, bacterial light-activated DNA repair enzymes. In this study, we report that while they have lost DNA repair activity, Cry1/2 adapted to protect genomic integrity by responding to DNA damage through posttranslational modification and coordinating the downstream transcriptional response. We demonstrate that genotoxic stress stimulates Cry1 phosphorylation and its deubiquitination by Herpes virus associated ubiquitin-specific protease (Hausp, a.k.a Usp7), stabilizing Cry1 and shifting circadian clock time. DNA damage also increases Cry2 interaction with Fbxl3, destabilizing Cry2. Thus, genotoxic stress increases the Cry1/Cry2 ratio, suggesting distinct functions for Cry1 and Cry2 following DNA damage. Indeed, the transcriptional response to genotoxic stress is enhanced in Cry1-/- and blunted in Cry2-/- cells. Furthermore, Cry2-/- cells accumulate damaged DNA. These results suggest that Cry1 and Cry2, which evolved from DNA repair enzymes, protect genomic integrity via coordinated transcriptional regulation.

Show MeSH
Related in: MedlinePlus