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Antiphase Circadian Expression betweenBMAL1andperiodHomologue mRNA in the Suprachiasmatic Nucleus and Peripheral Tissues of Rats
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
253, 199 –203 (1998)
RC989779
Antiphase Circadian Expression between BMAL1 and period Homologue mRNA in the Suprachiasmatic Nucleus and Peripheral Tissues of Rats Katsutaka Oishi,* Katsuhiko Sakamoto,* Tetsuya Okada,* Takahiro Nagase,† and Norio Ishida*,1 *Ishida Group of Clock Gene, National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, MITI, Higashi 1-1, Tsukuba, Ibaraki 305-8566, Japan; and †Kazusa DNA Research Institute, Chiba, Japan
Received October 27, 1998
BMAL1 is a putative transcription factor which is involved in circadian rhythm generation in Drosophila. Northern blot analysis was performed to investigate the expression of rat BMAL1 mRNA in the suprachiasmatic nucleus (SCN) and peripheral tissues. In the SCN, circadian expression of BMAL1 mRNA which reaches its peak level at the time of dark–light transition was observed, and the expression pattern was antiphase to those of two period (per) homologues, rPer1 and rPer2. However, no circadian oscillation for rat Clock mRNA was detected. The circadian expression of BMAL1 mRNA was also observed in peripheral tissues such as brain (excluding the SCN), eye, heart, kidney, and lung. The amplitudes of BMAL1 and rPer2 mRNA expression levels were correlated between the different tissues, suggesting that the circadian expression of BMAL1 mRNA plays an important role in generating the circadian expression of per homologue genes in mammals. © 1998 Academic Press Key Words: circadian rhythm; BMAL1; period homologue; Clock; suprachiasmatic nucleus; rat.
Most organisms ranging from bacteria to human exhibit a variety of physiological and behavioral circadian rhythms controlled by endogenous oscillators. In Drosophila, two genes, period (dPer) (1) and timeless (tim) (2), are essential elements of the circadian timing system that controls circadian rhythms in eclosion and locomotor activity. The circadian changes in dPer and tim mRNA levels require their functional gene products, as these mRNA oscillations are altered in PER and TIM mutants (3, 4). In mammals, the suprachiasmatic nuclei (SCN) of the anterior hypothalamus are the site of a master clock (5). Recently, the structural homologues of the Drosophila clock gene dPer have 1 To whom correspondence should be addressed. Fax: 181-298-546095. E-mail: [email protected].
been isolated from mammals (6 –12). These mammalian per genes exhibit a robust circadian expressions not only in the SCN but in other peripheral tissues such as eye, brain (excluding the SCN), heart, lung, spleen, liver, skeletal muscle, testis, kidney, and peripheral leukocytes (7, 12–14). This resembles the situation in Drosophila, in which dPer is widely expressed throughout the animal in a circadian manner (15). Recent progress elucidating molecular components involved in the transcriptional regulation of mammalian clocks has been made (16, 17). First of all, the mouse Clock mutant, which has a phenotype affecting both the periodicity and persistence of circadian rhythms, was identified (18). Then the cloning of Clock gene revealed that the CLOCK is a member of the basic helix-loop-helix (bHLH)-PAS family, some members of which are known to function as transcription factors (19). BMAL1 was cloned as an “orphan” protein of the bHLH-PAS transcription factor family with unknown biological function (20), and two laboratories independently showed that BMAL1 (MOP3) is a potential partner heterodimerizes with mammalian CLOCK (21, 22). Gekakis et al. (21) showed that CLOCK-BMAL1 heterodimers activate transcription from E-box elements, a type of transcription factor-binding site, found adjacent to mouse per1 (mPer1) gene in mouse NIH-3T3 cells. On the other hand, the Drosophila homologues of Clock (dClock) and BMAL1 (dBMAL1) were also cloned, and these homozygous mutants (Jrk and cyc, respectively) are both arrhythmic and exhibit low and no circadian mRNA expression of the dPer and tim genes (23, 24). Darlington et al. (25) showed that Drosophila PER and its partner TIM proteins inhibited dCLOCK-dependent transcriptional activation of per promoters via the E-box in cultured cells. These observations give rise to the following negative feed back loop model (16, 17): CLOCK-BMAL1 heterodimers bind E-boxes in the promoters of per genes (dPer or mammalian Per1, Per2, and Per3) and induce their
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 12:12; lights on at zeitgeber time (ZT) 0] for at least 1 week before the day of the experiment. A white fluorescent lamp was used as a source of light during the day (150 –200 lux at the level of the cages). The experiment in the constant darkness (DD) was performed after freerunning for 3 days in DD. Cloning of rPer1 and rat Clock DNA. Total RNA from rat brain was reverse-transcribed into cDNA using oligo-dT. Fragments of rPer1 (position 143–766 of mPer1) and rat Clock (position 1196 –1438 of mouse Clock) were PCR-amplified using the following oligonucleotides: 59-TATCCAGACCTCAAAAGCCC-39 and 59-ATTCCTGGTTAGCCTGAACC-39 for rPer1, and 59-GARGARCCNAAYGARGARTT-39 and 59-CADATCCAYTGYTGNCCYTT-39 for rat Clock. These fragments were then cloned into pBluescript and sequenced to verify their identity and orientation. Northern blot analysis. Rats were decapitated, and tissues were dissected, quickly frozen, and kept in liquid nitrogen until used. Total RNA was isolated from tissues by using Isogen (a guanidine HCl/phenol procedure; Nippon Gene Co., Ltd., Tokyo, Japan) and separated on a 1% agarose/0.7 M formaldehyde gel. Each lane contained 20 mg of total RNA from each tissue or 5 mg of total SCN RNA. To ascertain the equal RNA loading in each lane, agarose gel was stained with ethidium bromide and visualized under UV light. The 32 P-labeled random primed probes were generated from rPer2 cDNA fragment (7) (bases: 1144 –1797; GenBank accession number AB016532) for rPer2, and TIC cDNA fragment (26) (bases: 231–910; GenBank Accession No. AF015953) for rat BMAL1, and the PCRamplified cDNAs for rPer1 and rat Clock. Hybridization and detection were performed as described before (27).
FIG. 1. Daily expressions of BMAL1, Clock, rPer1, and rPer2 mRNA in the SCN. Rats (n 5 3) were housed in a 12 h light: 12 h dark (LD) cycle (lights on at ZT 0). The open bars indicate the lights-on phase, and the dark bars indicate lights-off. (a) Representative Northern blots of total RNA. (b) The graph depict a comparison of the expression patterns of these four genes. The maximum value was expressed as 100% in each gene.
transcription. Then the induced PER proteins inhibit the CLOCK-BMAL1 dimerization, perhaps doing so directly via interaction of the PER PAS domain with the PAS domains of CLOCK and/or BMAL1. In this paper we report the circadian expression of rat BMAL1 mRNA in the SCN and peripheral tissues. We also evaluate the expression rhythm of rat per2 (rPer2) mRNA in the same tissues examined by BMAL1. Furthermore, the rhythmic pattern and peaktrough amplitude of these genes are compared in each tissue. MATERIALS AND METHODS Animals. Male eight week-old Wistar rats obtained from Clea Japan, Inc. (Tokyo) were housed in a 12-h light–12-h dark cycle [LD
FIG. 2. Circadian expression of BMAL1 mRNA in the SCN. Rats were held in a 12-h light:12-h dark (LD) cycle (lights on at ZT 0) before being released into constant darkness (DD). The experiment was performed after free-running for 3 days. The shaded bars indicate the subjective day [circadian time (CT) 0 –12], and the dark bars indicate subjective night (CT 12–24). (a) Representative Northern blot of total RNA. (b) The graph depict the circadian pattern of BMAL1 mRNA levels in the SCN. The maximum value was expressed as 100%.
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FIG. 3. Antiphase daily expressions of BMAL1 and rPer2 mRNA in peripheral tissues. Rats were housed in a 12-h light:12-h dark (LD) cycle (lights on at ZT 0). The open bar indicates the lights-on phase, and the dark bar indicates lights-off. (a) Representative Northern blots of total RNA prepared from the rat tissues. (b) The graph depict a comparison of the expression patterns of BMAL1 and rPer2 mRNA in each tissue. The maximum value was expressed as 100% in each gene.
RESULTS To determine whether the abundance of BMAL1 mRNA changes in the SCN, we performed Northern blot analysis using the samples collected at different times during an LD cycle. In the SCN, BMAL1 mRNA displays a robust cycling with an approximately 1.7fold peak-trough amplitude, reaching peak abundance at dawn (ZT23 to 2) and trough values around the light-dark transition at ZT12 (Fig. 1). The expression patterns of rat per homologues, rPer1 and rPer2, showed similar phases reaching peak values in the early night and strikingly antiphase to that observed for the BMAL1 mRNA (Fig. 1). The expression levels of Clock mRNA did not changed all the day (Fig. 1).
To test whether the daily cycling of BMAL1 mRNA is driven by an endogenous circadian clock, we measured its time course in DD. As shown in Fig. 2, the levels of BMAL1 mRNA continued oscillating in DD, strongly suggest that this rhythmic expression is regulated by the central clock. The expression pattern in an LD cycle was maintained in the DD condition. Whether this circadian profile is reflected at the protein level is not known. We observed the circadian expression of BMAL1 mRNA not only in the SCN but in peripheral tissues such as brain (excluding the SCN), eye, heart, kidney, and lung (Fig. 3). The expression patterns of BMAL1 mRNA in the peripheral tissues were similar to that observed in the SCN, although the peak–trough amplitude of the mRNA levels was much higher in peripheral tissues than
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in the SCN. The peak-trough amplitudes of the BMAL1 specific hybridization were 1.8-, 1.6-, 23.8-, 13.2-, and 13.7-fold in brain (excluding the SCN), eye, heart, kidney, and lung, respectively (Fig. 3), although the amplitude in the SCN was only 1.7-fold (Fig. 1). The amplitude of rPer2 expression were 2.4-, 2.9-, 7.0-, 2.6-, and 30.3-fold in brain (excluding the SCN), eye, heart, kidney, and lung, respectively (Fig. 3), while the amplitude was only 1.7-fold in the SCN (Fig. 1). DISCUSSION In this study, we showed the circadian expression of BMAL1 mRNA in the SCN. The expression reaches peak level at the time of dark-light transition (ZT23 to 2), and the expression pattern was antiphase to those of rPer1 and rPer2. Moreover, the circadian expression of BMAL1 mRNA was observed not only in the SCN but in all the other tissues examined, as those of mammalian per homologues (7, 12, 14). Interestingly, the peak-trough amplitudes of BMAL1 and rPer2 mRNA expression levels were correlated between different tissues. The amplitudes of both BMAL1 and rPer2 mRNA levels in peripheral tissues such as heart, kidney, and lung were much higher than those in the SCN. The amplitude of rPer1 mRNA expression was also higher in the peripheral tissues than in the SCN (data not shown). The data suggest that the mammalian circadian expression of BMAL1 mRNA plays an important role in generating the circadian expressions of per homologues via the binding and transactivation of CLOCK-BMAL1 heterodimers to the promotor regions of rPer1 and rPer2 genes. In Drosophila, autonomous circadian oscillators are present throughout the body (15). Furthermore, it should be noted that the treatment of cultured rat-1 fibroblasts with high concentrations of serum induces the circadian expression of rPer1, rPer2, and other various genes whose mRNA levels also oscillate in living animals (28). Multitissue expression of BMAL1 and per homologue genes leads to be hypothesized us the existence of autoregulatory feed back loops for these genes not only in the SCN but also in peripheral tissues, although the peripheral oscillators might be governed by the master clock in the SCN (7). As in Drosophila, it is likely that the mammalian clock mechanism is constituted in part by the direct or indirect repression of per genes by PER proteins (18,25). How might PER-dependent negative feedback inhibit per genes’ transcription by the CLOCK-BMAL1 heterodimer? As mentioned above, it has been suggested that PER proteins inhibit the activation, perhaps doing so directly via interaction of the PER PAS domain with the PAS domains of either CLOCK or BMAL1 (16,17), since in Drosophila, PER and TIM is sufficient to inhibit dCLOCK-BMAL1 dependent per gene activation in cultured cells (25). In this study, however, we propose another possible mechanism that
PER proteins indirectly down-regulate the BMAL1 mRNA expression, and result in inhibiting the formation of CLOCK-BMAL1 heterodimers. It should be noted that, in Drosophila, the expression levels of dBMAL1 mRNA do not oscillate at least 3 points during a day (24), while our study indicate the robust cycling of rat BMAL1 mRNA levels not only in the SCN but also in peripheral tissues. On the other hand, the bimodal cycling of Clock mRNA was shown in Drosophila (23), although our study showed the constant mRNA expression in the rat SCN. These findings suggest the functional differences of these molecules between Drosophila and mammals. Further elucidation of the molecular mechanism regulating the BMAL1 and per homologues mRNA expression will provide an understanding of the mammalian circadian clock system. ACKNOWLEDGMENTS We thank Drs. Michio Ooishi and Osamu Ohara (Kazusa DNA Research Institute) for continuous encouragement. We also thank Kayo Yokoyama and Yuka Onuma for technical assistance. This study was supported by a project grant for the Competitive Research Program from AIST, MITI, Japan.
REFERENCES 1. Ishida, N. (1995) Neurosci. Res. 23, 231–240. 2. Sehgal, A., Price, J. L., Man, B., and Young, M. W. (1994) Science 263, 1603–1606. 3. Hardin, P. E., Hall, J. C., and Rosbash, M. (1990) Nature 343, 536 –540. 4. Sehgal, A., Rothenfluh-Hilfiker, A., Hunter-Ensor, M., Chen, Y., Myers, M., and Young, M. W. (1995) Science 270, 808 – 810. 5. Silver, R., LeSauter, J., Tresco, P. A., and Lehman, M. N. (1996) Nature 382, 810 – 813. 6. Albrecht, U., Sun, Z. S., Eichele, G., and Lee, C. C. (1997) Cell 91, 1055–1064. 7. Sakamoto, K., Nagase, T., Fukui, H., Horikawa, K., Okada, T., Tanaka, H., Sato, K., Miyake, Y., Ohara, O., Kako, K., and Ishida, N. (1998) J. Biol. Chem. 273, 27039 –27042. 8. Shearman, L. P., Zylka, M. J., Weaver, D. R., Kolakowski, L. F., Jr., and Reppert, S. M. (1997) Neuron 19, 1261–1269. 9. Sun, Z. S., Albrecht, U., Zhuchenko, O., Bailey, J., Eichele, G., and Lee, C. C. (1997) Cell 90, 1003–1011. 10. Takumi, T., Taguchi, K., Miyake, S., Sakakida, Y., Takashima, N., Matsubara, C., Maebayashi, Y., Okumura, K., Takekida, S., Yamamoto, S., Yagita, K., Yan, L., Young, M. W., and Okamura, H. (1998) EMBO J. 17, 4753– 4759. 11. Tei, H., Okamura, H., Shigeyoshi, Y., Fukuhara, C., Ozawa, R., Hirose, M., and Sakaki, Y. (1997) Nature 389, 512–516. 12. Zylka, M. J., Shearman, L. P., Weaver, D. R., and Reppert, M. (1998) Neuron 20, 1103–1110. 13. Balsalobre, A., Damiola, F., and Schibler, U. (1998) Cell 93, 929 –937. 14. Oishi, K., Sakamoto, K., Okada, K., Nagase, K., and Ishida, N. (1998) Neurosci. Lett., in press. 15. Plautz, J. D., Kaneko, M., Hall, J. C., and Kay, S. A. (1997) Science 278, 1632–1635.
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16. Dunlap, J. (1998) Science 280, 1548 –1549. 17. Kako, K., and Ishida, N. (1998) Neurosci. Res., in press. 18. Vitaterna, M. H., King, D. P., Chang, A.-M., Kornhauser, J. M., Lowrey, P. L., McDonald, J. D., Dove, W. F., Pinto, L. H., Turek, F. W., and Takahashi, J. S. (1994) Science 264, 719 –725. 19. King, D. P., Zhao, Y., Sangoram, A. M., Wilsbacher, L. D., Tanaka, M., Antoch, M. P., Steeves, T. D. L., Vitaterna, M. H., Kornhauser, J. M., Lowrey, P. L., Turek, F. W., and Takahashi, J. S. (1997) Cell 89, 641– 653. 20. Ikeda, M., and Nomura, M. (1997) Biochem. Biophys. Res. Commun. 233, 258 –264. 21. Gekakis, N., Staknis, D., Nguyen, H. B., Davis, F. C., Wilsbacher, L. D., King, D. P., Takahashi, J. S., and Weitz, C. J. (1998) Science 280, 1564 –1569.
22. Hogenesch, J. B., Gu, Y.-Z., Jain, S., and Bradfield, C. A. (1998) Proc. Natl. Acad. Sci. USA 95, 5474 –5479. 23. Allada, R., White, N. E., So, W. V., Hall, J. C., and Rosbash, M. (1998) Cell 93, 791– 804. 24. Rutila, J. E., Suri, V., Le, M., So, V., Rosbash, M., and Hall, J. C. (1998) Cell 93, 805– 814. 25. Darlington, T. K., Wager-Smith, K., Ceriani, M. F., Staknis, D., Gekakis, N., Steeves, T. D. L., Weitz, C. J., Takahashi, J. S., and Kay, S. A. (1998) Science 280, 1599 –1603. 26. Wolting, C. D., and McGlade, C. J. (1998) Mamm. Genome 9, 463– 468. 27. Sakamoto, K., and Ishida, N. (1998) Neurosci. Lett. 245, 113–116. 28. Balsalobre, A., Damiola, F., and Schibler, U. (1998) Cell 93, 929 –937.
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RC989779
Antiphase Circadian Expression between BMAL1 and period Homologue mRNA in the Suprachiasmatic Nucleus and Peripheral Tissues of Rats Katsutaka Oishi,* Katsuhiko Sakamoto,* Tetsuya Okada,* Takahiro Nagase,† and Norio Ishida*,1 *Ishida Group of Clock Gene, National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, MITI, Higashi 1-1, Tsukuba, Ibaraki 305-8566, Japan; and †Kazusa DNA Research Institute, Chiba, Japan
Received October 27, 1998
BMAL1 is a putative transcription factor which is involved in circadian rhythm generation in Drosophila. Northern blot analysis was performed to investigate the expression of rat BMAL1 mRNA in the suprachiasmatic nucleus (SCN) and peripheral tissues. In the SCN, circadian expression of BMAL1 mRNA which reaches its peak level at the time of dark–light transition was observed, and the expression pattern was antiphase to those of two period (per) homologues, rPer1 and rPer2. However, no circadian oscillation for rat Clock mRNA was detected. The circadian expression of BMAL1 mRNA was also observed in peripheral tissues such as brain (excluding the SCN), eye, heart, kidney, and lung. The amplitudes of BMAL1 and rPer2 mRNA expression levels were correlated between the different tissues, suggesting that the circadian expression of BMAL1 mRNA plays an important role in generating the circadian expression of per homologue genes in mammals. © 1998 Academic Press Key Words: circadian rhythm; BMAL1; period homologue; Clock; suprachiasmatic nucleus; rat.
Most organisms ranging from bacteria to human exhibit a variety of physiological and behavioral circadian rhythms controlled by endogenous oscillators. In Drosophila, two genes, period (dPer) (1) and timeless (tim) (2), are essential elements of the circadian timing system that controls circadian rhythms in eclosion and locomotor activity. The circadian changes in dPer and tim mRNA levels require their functional gene products, as these mRNA oscillations are altered in PER and TIM mutants (3, 4). In mammals, the suprachiasmatic nuclei (SCN) of the anterior hypothalamus are the site of a master clock (5). Recently, the structural homologues of the Drosophila clock gene dPer have 1 To whom correspondence should be addressed. Fax: 181-298-546095. E-mail: [email protected].
been isolated from mammals (6 –12). These mammalian per genes exhibit a robust circadian expressions not only in the SCN but in other peripheral tissues such as eye, brain (excluding the SCN), heart, lung, spleen, liver, skeletal muscle, testis, kidney, and peripheral leukocytes (7, 12–14). This resembles the situation in Drosophila, in which dPer is widely expressed throughout the animal in a circadian manner (15). Recent progress elucidating molecular components involved in the transcriptional regulation of mammalian clocks has been made (16, 17). First of all, the mouse Clock mutant, which has a phenotype affecting both the periodicity and persistence of circadian rhythms, was identified (18). Then the cloning of Clock gene revealed that the CLOCK is a member of the basic helix-loop-helix (bHLH)-PAS family, some members of which are known to function as transcription factors (19). BMAL1 was cloned as an “orphan” protein of the bHLH-PAS transcription factor family with unknown biological function (20), and two laboratories independently showed that BMAL1 (MOP3) is a potential partner heterodimerizes with mammalian CLOCK (21, 22). Gekakis et al. (21) showed that CLOCK-BMAL1 heterodimers activate transcription from E-box elements, a type of transcription factor-binding site, found adjacent to mouse per1 (mPer1) gene in mouse NIH-3T3 cells. On the other hand, the Drosophila homologues of Clock (dClock) and BMAL1 (dBMAL1) were also cloned, and these homozygous mutants (Jrk and cyc, respectively) are both arrhythmic and exhibit low and no circadian mRNA expression of the dPer and tim genes (23, 24). Darlington et al. (25) showed that Drosophila PER and its partner TIM proteins inhibited dCLOCK-dependent transcriptional activation of per promoters via the E-box in cultured cells. These observations give rise to the following negative feed back loop model (16, 17): CLOCK-BMAL1 heterodimers bind E-boxes in the promoters of per genes (dPer or mammalian Per1, Per2, and Per3) and induce their
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 12:12; lights on at zeitgeber time (ZT) 0] for at least 1 week before the day of the experiment. A white fluorescent lamp was used as a source of light during the day (150 –200 lux at the level of the cages). The experiment in the constant darkness (DD) was performed after freerunning for 3 days in DD. Cloning of rPer1 and rat Clock DNA. Total RNA from rat brain was reverse-transcribed into cDNA using oligo-dT. Fragments of rPer1 (position 143–766 of mPer1) and rat Clock (position 1196 –1438 of mouse Clock) were PCR-amplified using the following oligonucleotides: 59-TATCCAGACCTCAAAAGCCC-39 and 59-ATTCCTGGTTAGCCTGAACC-39 for rPer1, and 59-GARGARCCNAAYGARGARTT-39 and 59-CADATCCAYTGYTGNCCYTT-39 for rat Clock. These fragments were then cloned into pBluescript and sequenced to verify their identity and orientation. Northern blot analysis. Rats were decapitated, and tissues were dissected, quickly frozen, and kept in liquid nitrogen until used. Total RNA was isolated from tissues by using Isogen (a guanidine HCl/phenol procedure; Nippon Gene Co., Ltd., Tokyo, Japan) and separated on a 1% agarose/0.7 M formaldehyde gel. Each lane contained 20 mg of total RNA from each tissue or 5 mg of total SCN RNA. To ascertain the equal RNA loading in each lane, agarose gel was stained with ethidium bromide and visualized under UV light. The 32 P-labeled random primed probes were generated from rPer2 cDNA fragment (7) (bases: 1144 –1797; GenBank accession number AB016532) for rPer2, and TIC cDNA fragment (26) (bases: 231–910; GenBank Accession No. AF015953) for rat BMAL1, and the PCRamplified cDNAs for rPer1 and rat Clock. Hybridization and detection were performed as described before (27).
FIG. 1. Daily expressions of BMAL1, Clock, rPer1, and rPer2 mRNA in the SCN. Rats (n 5 3) were housed in a 12 h light: 12 h dark (LD) cycle (lights on at ZT 0). The open bars indicate the lights-on phase, and the dark bars indicate lights-off. (a) Representative Northern blots of total RNA. (b) The graph depict a comparison of the expression patterns of these four genes. The maximum value was expressed as 100% in each gene.
transcription. Then the induced PER proteins inhibit the CLOCK-BMAL1 dimerization, perhaps doing so directly via interaction of the PER PAS domain with the PAS domains of CLOCK and/or BMAL1. In this paper we report the circadian expression of rat BMAL1 mRNA in the SCN and peripheral tissues. We also evaluate the expression rhythm of rat per2 (rPer2) mRNA in the same tissues examined by BMAL1. Furthermore, the rhythmic pattern and peaktrough amplitude of these genes are compared in each tissue. MATERIALS AND METHODS Animals. Male eight week-old Wistar rats obtained from Clea Japan, Inc. (Tokyo) were housed in a 12-h light–12-h dark cycle [LD
FIG. 2. Circadian expression of BMAL1 mRNA in the SCN. Rats were held in a 12-h light:12-h dark (LD) cycle (lights on at ZT 0) before being released into constant darkness (DD). The experiment was performed after free-running for 3 days. The shaded bars indicate the subjective day [circadian time (CT) 0 –12], and the dark bars indicate subjective night (CT 12–24). (a) Representative Northern blot of total RNA. (b) The graph depict the circadian pattern of BMAL1 mRNA levels in the SCN. The maximum value was expressed as 100%.
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FIG. 3. Antiphase daily expressions of BMAL1 and rPer2 mRNA in peripheral tissues. Rats were housed in a 12-h light:12-h dark (LD) cycle (lights on at ZT 0). The open bar indicates the lights-on phase, and the dark bar indicates lights-off. (a) Representative Northern blots of total RNA prepared from the rat tissues. (b) The graph depict a comparison of the expression patterns of BMAL1 and rPer2 mRNA in each tissue. The maximum value was expressed as 100% in each gene.
RESULTS To determine whether the abundance of BMAL1 mRNA changes in the SCN, we performed Northern blot analysis using the samples collected at different times during an LD cycle. In the SCN, BMAL1 mRNA displays a robust cycling with an approximately 1.7fold peak-trough amplitude, reaching peak abundance at dawn (ZT23 to 2) and trough values around the light-dark transition at ZT12 (Fig. 1). The expression patterns of rat per homologues, rPer1 and rPer2, showed similar phases reaching peak values in the early night and strikingly antiphase to that observed for the BMAL1 mRNA (Fig. 1). The expression levels of Clock mRNA did not changed all the day (Fig. 1).
To test whether the daily cycling of BMAL1 mRNA is driven by an endogenous circadian clock, we measured its time course in DD. As shown in Fig. 2, the levels of BMAL1 mRNA continued oscillating in DD, strongly suggest that this rhythmic expression is regulated by the central clock. The expression pattern in an LD cycle was maintained in the DD condition. Whether this circadian profile is reflected at the protein level is not known. We observed the circadian expression of BMAL1 mRNA not only in the SCN but in peripheral tissues such as brain (excluding the SCN), eye, heart, kidney, and lung (Fig. 3). The expression patterns of BMAL1 mRNA in the peripheral tissues were similar to that observed in the SCN, although the peak–trough amplitude of the mRNA levels was much higher in peripheral tissues than
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in the SCN. The peak-trough amplitudes of the BMAL1 specific hybridization were 1.8-, 1.6-, 23.8-, 13.2-, and 13.7-fold in brain (excluding the SCN), eye, heart, kidney, and lung, respectively (Fig. 3), although the amplitude in the SCN was only 1.7-fold (Fig. 1). The amplitude of rPer2 expression were 2.4-, 2.9-, 7.0-, 2.6-, and 30.3-fold in brain (excluding the SCN), eye, heart, kidney, and lung, respectively (Fig. 3), while the amplitude was only 1.7-fold in the SCN (Fig. 1). DISCUSSION In this study, we showed the circadian expression of BMAL1 mRNA in the SCN. The expression reaches peak level at the time of dark-light transition (ZT23 to 2), and the expression pattern was antiphase to those of rPer1 and rPer2. Moreover, the circadian expression of BMAL1 mRNA was observed not only in the SCN but in all the other tissues examined, as those of mammalian per homologues (7, 12, 14). Interestingly, the peak-trough amplitudes of BMAL1 and rPer2 mRNA expression levels were correlated between different tissues. The amplitudes of both BMAL1 and rPer2 mRNA levels in peripheral tissues such as heart, kidney, and lung were much higher than those in the SCN. The amplitude of rPer1 mRNA expression was also higher in the peripheral tissues than in the SCN (data not shown). The data suggest that the mammalian circadian expression of BMAL1 mRNA plays an important role in generating the circadian expressions of per homologues via the binding and transactivation of CLOCK-BMAL1 heterodimers to the promotor regions of rPer1 and rPer2 genes. In Drosophila, autonomous circadian oscillators are present throughout the body (15). Furthermore, it should be noted that the treatment of cultured rat-1 fibroblasts with high concentrations of serum induces the circadian expression of rPer1, rPer2, and other various genes whose mRNA levels also oscillate in living animals (28). Multitissue expression of BMAL1 and per homologue genes leads to be hypothesized us the existence of autoregulatory feed back loops for these genes not only in the SCN but also in peripheral tissues, although the peripheral oscillators might be governed by the master clock in the SCN (7). As in Drosophila, it is likely that the mammalian clock mechanism is constituted in part by the direct or indirect repression of per genes by PER proteins (18,25). How might PER-dependent negative feedback inhibit per genes’ transcription by the CLOCK-BMAL1 heterodimer? As mentioned above, it has been suggested that PER proteins inhibit the activation, perhaps doing so directly via interaction of the PER PAS domain with the PAS domains of either CLOCK or BMAL1 (16,17), since in Drosophila, PER and TIM is sufficient to inhibit dCLOCK-BMAL1 dependent per gene activation in cultured cells (25). In this study, however, we propose another possible mechanism that
PER proteins indirectly down-regulate the BMAL1 mRNA expression, and result in inhibiting the formation of CLOCK-BMAL1 heterodimers. It should be noted that, in Drosophila, the expression levels of dBMAL1 mRNA do not oscillate at least 3 points during a day (24), while our study indicate the robust cycling of rat BMAL1 mRNA levels not only in the SCN but also in peripheral tissues. On the other hand, the bimodal cycling of Clock mRNA was shown in Drosophila (23), although our study showed the constant mRNA expression in the rat SCN. These findings suggest the functional differences of these molecules between Drosophila and mammals. Further elucidation of the molecular mechanism regulating the BMAL1 and per homologues mRNA expression will provide an understanding of the mammalian circadian clock system. ACKNOWLEDGMENTS We thank Drs. Michio Ooishi and Osamu Ohara (Kazusa DNA Research Institute) for continuous encouragement. We also thank Kayo Yokoyama and Yuka Onuma for technical assistance. This study was supported by a project grant for the Competitive Research Program from AIST, MITI, Japan.
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