System-level identification of transcriptional circuits underlying mammalian circadian clocks

Abstract

Mammalian circadian clocks consist of complexly integrated regulatory loops1,2,3,4,5, making it difficult to elucidate them without both the accurate measurement of system dynamics and the comprehensive identification of network circuits6. Toward a system-level understanding of this transcriptional circuitry, we identified clock-controlled elements on 16 clock and clock-controlled genes in a comprehensive surveillance of evolutionarily conserved cis elements and measurement of their transcriptional dynamics. Here we report the roles of E/E′ boxes, DBP/E4BP4 binding elements7 and RevErbA/ROR binding elements8 in nine, seven and six genes, respectively. Our results indicate that circadian transcriptional circuits are governed by two design principles: regulation of E/E′ boxes and RevErbA/ROR binding elements follows a repressor-precedes-activator pattern, resulting in delayed transcriptional activity, whereas regulation of DBP/E4BP4 binding elements follows a repressor-antiphasic-to-activator mechanism, which generates high-amplitude transcriptional activity. Our analysis further suggests that regulation of E/E′ boxes is a topological vulnerability in mammalian circadian clocks, a concept that has been functionally verified using in vitro phenotype assay systems.

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Figure 1: E/E′ boxes sufficient for circadian oscillation in phase with Per2 and antiphase to Arntl oscillation.
Figure 2: D boxes sufficient for circadian oscillation in phase with Per3 and antiphase to Arntl oscillation.
Figure 3: RREs sufficient for circadian oscillation in phase with Per2 and antiphase to Arntl oscillation.
Figure 4: Distinct roles of E/E′ boxes and RREs in noncoding regions of Cry1 and Rorc.
Figure 5: Simple design principles encoded in the complex transcription circuits underlying mammalian circadian clocks.
Figure 6: Topological and functional identification of the Achilles' heel of transcriptional circuits underlying mammalian circadian clocks.

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Acknowledgements

We thank H. Tei and Y. Sakaki for Arntl, Clock and Cry1 expression vectors; T. Kojima, T. Katakura and H. Urata for technical assistance; and D. Sipp and Y. Minami for critical reading of the manuscript. This research was done as part of a research and development project of the Industrial Science and Technology Program supported by the New Energy and Industrial Technology Development Organization and, in part, by intramural Grant-in-Aid from the CDB, Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Grant-in-Aid for Strategic Programs in R & D from the Institute of Physical and Chemical Research and Grant-in-Aid for Scientific Research from the New Energy and Industrial Technology Development Organization.

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Correspondence to Hiroki R Ueda.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Temporal expression profiles of 16 transcription factors in the suprachiasmatic nucleus and liver under constant darkness conditions. (PDF 144 kb)

Supplementary Fig. 2

Feedback and feedforward loops in the transcriptional circuits underlying mammalian circadian rhythms. (PDF 121 kb)

Supplementary Fig. 3

Positive and negative control in an in vitro circadian dynamics monitoring system. (PDF 137 kb)

Supplementary Fig. 4

Confirmation of evolutionary conserved E-box/E'-boxes on clock/clock-controlled genes. (PDF 21 kb)

Supplementary Fig. 5

Confirmation of evolutionary conserved D-boxes on clock/clock-controlled genes. (PDF 10 kb)

Supplementary Fig. 6

Confirmation of evolutionary conserved RREs on clock/clock-controlled genes. (PDF 20 kb)

Supplementary Fig. 7

Confirmation of evolutionary conserved E-box/E'-box and RREs on Cry1 and Rorc genes. (PDF 77 kb)

Supplementary Methods (PDF 108 kb)

Supplementary Note (PDF 48 kb)

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