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PNAS 104 (40): 15747-15752

Copyright © 2007 by the National Academy of Sciences.

Circadian control by the reduction/oxidation pathway: Catalase represses light-dependent clock gene expression in the zebrafish

Jun Hirayama*, Sehyung Cho{dagger}, and Paolo Sassone-Corsi*,{ddagger}

*Department of Pharmacology, School of Medicine, University of California, Irvine, CA 92697; and {dagger}Kyung Hee Institute of Age-Related and Brain Diseases, Kyung University, Seoul 130-701, Korea


Figure 1
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Fig. 1.. Light produces H2O2 in Z3 cells. Quantitative analysis of DCF fluorescence. Z3 cells were loaded with CM-H2DCFDA and then were exposed to light. CM-H2DCFDA fluorescence was detected by spectrofluorimetry. Values are means ± SE of three independent experiments. In each experiment, the CM-H2DCFDA fluorescence of the nontransduced cells kept in the dark was set as 1. Where indicated, the cells were transduced with zCat or luciferase genes by retroviral vector (shown by zCAT and LUC, respectively).

 

Figure 2
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Fig. 2.. H2O2 induces zCry1a and zPer2 expression in Z3 cells and this induction is mediated by MAPK pathway. (A) A time course of circadian gene induction upon H2O2 treatment of confluent Z3 cells was examined by RNA protection assay. Z3 cells permanently cultured in the dark were treated with H2O2 (100 µM). At indicated time points after the treatment, cells were harvested for RNA preparation. tRNA serves as our negative control reaction (t). Relative amounts of total RNA used for each sample are displayed (RNA). (B) Circadian gene induction by different concentrations of H2O2. Z3 cells kept in the dark and stimulated with H2O2 at the concentrations of 10–3, 10–2, 10–1, 1, 10, 50, and 100 µM. After 100 min, cells were harvested for RNA preparation. As the negative control (0 µM H2O2 concentration), we added water to the culture media. (C) Z3 cells kept in dark and stimulated with light (L), H2O2 (H) (100 µM), UV (U) (10mJ/m2, 30 seconds), or MNU (M) (2 mM). After 100 min, cells were harvested for RNA preparation. (D) Confluent Z3 cells were treated with the indicated inhibitors or with the vehicle (DMSO, 0 M). The concentrations of U0126 and SB203580 inhibitors used were 40 µM and 10 µM, respectively. After 1 h, cells were stimulated with light (L) or H2O2 (100 µM) (H), or were left in the dark (D). After 100 min, cells were harvested, and RNA was prepared. (E) Confluent Z3 cells were treated with the indicated inhibitors or with the vehicle (DMSO, 0 M). The concentrations of U0126 and SB203580 inhibitors used were 40 µM and 10 µM, respectively. After 1 h, cells were stimulated with light (L) or were left in the dark (D). After 7 h, cells were harvested, and RNA was prepared.

 

Figure 3
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Fig. 3.. H2O2 induces circadian gene oscillation in Z3 cells. Z3 cells maintained in constant darkness were stimulated with H2O2 (100 µM), and RNA was harvested at each time point indicated after the treatment. zCry1a, zPer1, zPer2, and zCat gene expression was examined by RPA, as shown in Fig. 2A.

 

Figure 4
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Fig. 4.. Oscillation of zCat and circadian clock genes under LD and DD cycles in Z3 cells. Z3 cells maintained in constant darkness were exposed to 12:12 LD cycles for 2 days and then transferred to constant dark condition for another 2 days. RNA was harvested at each time point indicated after light onset. zCry1a, zPer1, zPer2, zCat, and zAox gene expression was examined by RPA (A) or RT-PCR analysis (B), as shown in Fig. 2. In RT-PCR analysis, the value from the cells kept in dark was set as 1 for each gene, and zebrafish actin gene was used for normalization. The bar above the blots indicates light (white) and dark (black) periods.

 

Figure 5
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Fig. 5.. zCat gene expression and catalase activity in Z3 cells cycle in antiphase to zPer2 and zCry1a mRNAs. (A) Z3 cells maintained in constant darkness were exposed to light, and RNA was harvested at each time point indicated after light onset. zCry1a, zPer2, and zCat gene expression was examined by RT-PCR analysis as shown in Fig. 4B. (B) Z3 cells maintained in constant darkness were exposed to a 12:12 LD cycle. Cells were harvested at each time point indicated after light onset for catalase activity assay. The catalase activity of cells at time 0 was taken as 1. Values are means ± SE of three independent experiments. The bar above the graph indicates light (white) and dark (black) periods. (C) Catalase activity was assayed at each time point indicated, as shown in B.

 

Figure 6
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Fig. 6.. zCAT is a key negative regulator for light-dependent activation of circadian genes. (A and B) Light induction of zCry1a and zPer2 expression in non- (Ctr, black line), zCat- (zCAT, red line), and luciferase- (LUC, green line) infected Z3 cells was examined by RPA (A) or RT-PCR analysis (B), as shown in Fig. 2A and 4B, respectively. (C) Confluent Z3 cells were treated with catalase inhibitor sodium azide (350 µM) or with the vehicle (DMSO, 0 M). After 2 h, cells were stimulated with light, and RNA was harvested at each time point indicated after light onset. zCry1a, zPer2, and zAox gene expression was examined by RPA, as shown in Fig. 2D. (D) A model depicting the molecular mechanism underlying light-dependent transcription in zebrafish circadian clock.

 


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