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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 25, 2006 as doi:10.1096/fj.05-5321fje. |
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Departments of
* Cancer Biology and
Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio, USA
1Correspondence: E-mail: V.K., kondrar{at}ccf.org; M.P.A., antochm{at}ccf.org
SPECIFIC AIMS
The present study was based on our published findings that an organism’s sensitivity to genotoxic stress induced by the antitumor therapy correlates with the functional status of the CLOCK/BMAL1 transactivation complex, which presumably modulates the balance of pro-death/pro-survival genes. The present study was designed to address the underlying molecular mechanism of this phenomenon. We 1) compared the temporal expression profiles of several known direct targets of CLOCK/BMAL1 transactivation complex in the tissues of the wild-type (WT) and circadian mutant mice deficient in various components of molecular clock; and 2) characterized the role of circadian transcriptional factor BMAL1 in transcriptional regulation of circadian genes.
PRINCIPAL FINDINGS
1. The expression profiles of some transcriptional targets of the CLOCK/BMAL1 in peripheral tissues of Bmal1–/– knockout mice cannot be explained within the framework of the existing model of molecular circadian oscillator
To test the prediction that CLOCK/BMAL1-controlled genes will be down-regulated in animals deficient in circadian activators and up-regulated in animals deficient in circadian repressors, we measured the temporal expression profiles of several CLOCK/BMAL1 target genes (Per1, Per2, Cry1, Cry2, Rev-Erb 
, and Dbp) in peripheral tissues of WT, Clock/Clock, Bmal1–/–, and Cry1–/–Cry2–/– animals. In livers of WT animals, all genes tested displayed 24 h oscillations in mRNA abundance with a phase similar to that previously reported. All mutants deficient in core components of the molecular oscillator (Clock/Clock, Bmal1–/–, and Cry1–/–Cry2–/–) demonstrate disruption of the circadian profile of gene expression, though the pattern of change is different. Target genes were down-regulated (Per1, Dbp) or displayed median expression levels in livers of Clock/Clock mutant mice that are deficient in circadian transactivation function. Also consistent with our expectations, Per1 and Cry1 expression was up-regulated in the livers of Cry1–/–Cry2–/– double knockout animals lacking the CRY circadian repressor molecules. Expression patterns of the same target genes in the livers of Bmal1–/– animals did not completely fit our original prediction. Thus, while levels of Dbp, Per1, and Cry2 mRNA levels were significantly decreased, as expected, levels of Cry1 mRNA were considerably increased. Even though the Cry1 gene is a direct transcriptional target of CLOCK/BMAL1 and BMAL1 has been characterized as a transcriptional activator, we find that BMAL1 deficiency results in an elevated level of Cry1 mRNA in peripheral tissues throughout the circadian cycle, a pattern more consistent with the "lack of repression" effect characteristic of gene expression profiles in Cry1–/–Cry2–/– mice than of the "lack of activation" effect observed in Clock mutant mice and expected for Bmal1–/– mutants. Therefore, BMAL1 might possess dual functional activity, acting as a transcriptional activator for some genes and a transcriptional repressor for others, even in the same tissue.
2. The CLOCK/BMAL1 complex represses the activity of the Cry1 promoter upon interaction with CRY
To test the hypothesis that CLOCK/BMAL1 activates some promoters while repressing others, we compared regulation of the isolated Per1 (down-regulated in peripheral tissues of Bmal1–/– mice) and Cry1 (up-regulated in Bmal1–/– mice) promoters using a cell-based transient transfection approach. Both promoters were induced by CLOCK/BMAL1 coexpression (Fig. 1
A), but the fold of activation was different. While the Per1 promoter was induced by > 20-fold from its basal expression level, the Cry1 promoter, which displayed 
15-fold higher basal activity, was induced by CLOCK/BMAL1 by only 2-fold. Expression of CRY1 inhibits CLOCK/BMAL1-dependent transactivation of the Per1 promoter to a level still higher than the basal activity; on the Cry1 promoter, CRY1 expression results in promoter repression 3-fold lower than basal. As a result, both promoters demonstrate a similar change in amplitude between their activated and repressed states (6- to 10-fold, depending on the levels of CLOCK, BMAL1, and CRY1 used in transfection), but the effect is achieved via different mechanisms. In the Per1 promoter, it is based mainly on CLOCK/BMAL1-dependent transactivation; in the Cry1 it is based on CRY-mediated repression. The presence of both CLOCK and BMAL1 was crucial for the repression of Cry1 promoter because CRY1 alone, CLOCK/CRY1, or BMAL1/CRY1 were not efficient in promoter suppression (Fig. 1B
). CLOCK mutant protein demonstrated reduced transactivation function but intact transrepressor properties (Fig. 1C
). Data of the reporter assay confirmed our in vivo observation that the Per1 gene is regulated mostly by transactivation and the Cry1 mostly by transrepression.
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3. Cryptochromes do not display intrinsic transcriptional repressor activity
Our results demonstrate that suppression of the Cry1 promoter requires coexpression of all three proteins: CLOCK, BMAL1, and CRY1. The most straightforward explanation might suggest that CRYs perform their repressor function through recruitment of a transcriptional repressor or that CRYs themselves act as active repressors targeted to promoters by the CLOCK/BMAL1 complex. However, our results demonstrate that CRY1 does not possess intrinsic repressor activity. Indeed, targeting of CRY1 to the promoter through the DNA binding domain of yeast GAL4 protein did not result in suppression of the GAL4 responsive promoter, but induced promoter activation under certain conditions, suggesting that the CLOCK/BMAL1 complex acts as transcriptional activator and the CLOCK/BMAL1/CRY1 acts as an active transcriptional repressor.
4. The CLOCK/BMAL1/CRY1 complex interferes with the activity of other transcription factors.
If the CLOCK/BMAL1/CRY1 complex indeed functions as an active transcriptional repressor, then it may interfere with the ability of other transcription factors to regulate the same promoter. To check this, we analyzed the effect of the CLOCK/BMAL1/CRY1 complex on the ability of the noncircadian transcription factors N-MYC and ETS to activate the Cry1 promoter in a transient transfection system. As shown in Fig. 2
, either N-MYC or ETS expression leads to promoter activation, and CLOCK/BMAL1 cooperates with these transcription factors, resulting in even higher levels of activation. Coexpression of CRY1 alone has no effect on Cry1 transactivation induced by N-MYC and ETS, but coexpression of the CLOCK/BMAL1/CRY1 complex has a strong inhibitory effect. Thus, CLOCK/BMAL1/CRY1 complex blocks promoter activation by other, noncircadian, transcription factors, thus working as an active transcriptional repressor
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CONCLUSIONS AND SIGNIFICANCE
The dual functional role of the circadian transcriptional complex proposed here allows for introducing some important modifications to the current view of the molecular mechanism of circadian transcriptional control. The currently accepted model suggests that activation of circadian gene expression occurs through CLOCK/BMAL1-dependent recruitment of histone acetyl-transferase (HAT) to specific promoter regions with periodic disruption of CLOCK/BMAL1 interaction with HAT by CRYs. However, this mode of regulation can be effective only in situations in which target genes are controlled exclusively by circadian transcription factors and where basal promoter activity in the absence of transcriptionally active CLOCK/BMAL1 is low. In reality, the promoter regions of many clock-controlled genes contain multiple regulatory elements for other factors that may also recruit HAT and keep the promoter constitutively active. In these cases, basal promoter activity will be high and additional circadian activation will not induce significant changes in expression. Daily oscillations in gene expression levels of significant amplitude can be achieved through the proposed repressor function of CLOCK/BMAL1/CRY1 as illustrated by Cry1 promoter regulation in our study. The proposed circadian repressor model provides a feasible explanation for this phenomenon.
The circadian repressor model proposed here provides an effective mechanism for circadian control of the organism’s response to anticancer therapy. It has been reported that animals’ sensitivity to genotoxic therapies, such as chemotherapy or radiation, depends on the time of treatment. Based on our understanding of the mechanisms of circadian clock function, it has been proposed that such daily variations can be explained by CLOCK/BMAL1-dependent fluctuations in the expression level of genes that regulate cell death and proliferation. Hence, the cell’s (and consequently, the organism’s) fate upon stress at a particular time of day will depend on the balance of pro- and anti-survival factors. Different types of cellular stress lead to induction of various transcription factors (i.e., p53, NF
B, or AP1), induction by radiation, induction of NFB during the immune response, or HIF1 induction in response to low oxygen. These factors in turn activate downstream transcriptional targets important for cell death or survival. Some of these stress-induced genes may be transcriptional targets of CLOCK/BMAL1, so their induction in response to stress will occur only at those times of the day when CLOCK/BMAL1 is functioning as an activator and does not interfere with the functional activity of other transcription factors (Fig. 3
A). At times when the circadian transcriptional complex acts as an active repressor, induction of stress-induced clock-controlled genes will either be significantly attenuated or completely suppressed (Fig. 3B
). Therefore, the pattern of stress-induced gene expression will vary at different stages of the circadian cycle, which will result in daily variations in the organism’s response. It is likely that the same mechanism is involved in circadian regulation of other types of stress, such as infection or temperature shock, and in the control of response to various external stimuli (nutrients, light, toxins, etc.).
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A full understanding of the molecular details of circadian repressor function will not only allow for adjustment of existing therapeutic regimens to more favorable times, but will provide potential targets for rational pharmacological modulation of the circadian clock, which may result in improved therapeutic index.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5321fje;
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19 displays decreased transactivation but normal repressor function. Absolute luciferase signal is plotted in A to demonstrate the difference in basal Per1 and Cry1 promoter activity.
