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Circadian expression of clock and screening of clock-controlled genes in peripheral lymphocytes of rat
BBRC Biochemical and Biophysical Research Communications 336 (2005) 1069–1073 www.elsevier.com/locate/ybbrc
Circadian expression of clock and screening of clock-controlled genes in peripheral lymphocytes of rat Yu-zhen Du a, Sai-jun Fan a,b, Qing-hui Meng b, Guo-qing Wang a, Jian Tong a,* a
Chronobiology Laboratory, Department of Toxicology, School of Radiation Medicine and Public Health, Soochow University, Suzhou 215007, Jiangsu, China b Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, USA Received 21 August 2005 Available online 8 September 2005
Abstract In this paper, the circadian pattern of Clock and genes mediated by the Clock was investigated in peripheral lymphocytes of rats. Circadian rhythms of Clock are found under the regimes of constant darkness (DD) and 12-h light–12-h dark (LD12:12 h), with the peak phase at CT7 and ZT21, respectively. Ten differential cDNA fragments were identified to be mediated by the Clock, including three known genes (catalase, myelin proteolipid protein, and histone acetylase), four known expressed sequence tags (ESTs), and three novel ESTs. Experiment of the RNA interference revealed that these ESTs were down-regulated by the Clock gene and three of them were identified as clock-controlled genes. Understanding of clock-mediated genes may lead to a new direction in drug design for control of circadian rhythms. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Clock gene; Clock-controlled genes; Lymphocytes
In most organisms from lower species to higher ones, physiological and behavioral functions are expressed rhythmically across days and nights. These daily rhythms, referred to as circadian, are controlled by self-sustained biological oscillators. In mammals, studies with primary neuronal cultures as well as ablation and transplantation suggest that the central component of this complex oscillatory system resides in the suprachiasmatic nuclei (SCN) of the hypothalamus [1,2], which involve transcriptional– translational feedback loops of at least eight genes, namely, Per1/2/3, Cry1/2, Clock, Bmal1, and CKIe. Of these, the Per1/2 and Cry1/2 genes function as negative regulators, while the Bmal1 and Clock gene act as positive regulators in circadian oscillation [3–7]. Clock and Bmal1 can heterodimerize and bind to the E-box sequences of other clock genes and clock-mediated genes to activate the transcrip-
*
Corresponding author. Fax: +86 512 65304830. E-mail address: [email protected] (J. Tong).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.08.228
tion. Thus, Clock and Bmal1 are believed as the most important components of the circadian oscillator. Studies have shown that the molecular clocks oscillate not only in the central nervous system, but also in peripheral organs, such as liver, kidney, and fibroblast cells [8]. Circadian fluctuations in the immune system are also observed [9–12]. The numbers of blood T and B lymphocytes in blood fluctuate about 2-fold in a circadian day. The concentrations of serum albumin and globulin, as well as the cell immunity and activation of lymphocytes in mice, vary with a 24 h period. There have been reports on rhythmic expression of clock genes Per1, Per2, and Per3 in peripheral blood mononuclear cells (PBMCs) [13]. However, no similar report was found in the literature for circadian expression of the Clock gene in peripheral lymphocytes so far. We hypothesized that a circadian expression of functional clock components in peripheral lymphocytes might be involved in regulation of the circadian immune functions. In this paper, we provided the expression profile of the Clock gene in constant darkness (DD) and light–dark
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Y. Du et al. / Biochemical and Biophysical Research Communications 336 (2005) 1069–1073
(LD12:12 h) regimes, and screened the potential genes which may be targeted by the Clock gene in peripheral lymphocytes of Sprague–Dawley rat. Materials and methods Animals and sample collection. Adult male Sprague–Dawley rats were housed in DD regime for at least 6 weeks and in LD (12:12 h) [light: 6:00– 18:00 clock, light on at zeitgeber time (ZT) 0] for at least 4 weeks before the experiment. An incandescent lamp was used as a source of light during the day (150–200 lux) at the level of the cages. Operation during the dark period was done with the help of a dim red light (0.1 lux). The rats were tested and screened in an infrared video box for the sleep–wake cycle before the sample collection in order to obtain homogeneous samples. Blood sampling was conducted by cardiac puncture at different timepoints of a day. The lymphocytes were separated by centrifugation and stored at 80 °C until use. Semi-quantitative reverse-transcription PCR. The lymphocyte samples of DD and LD (12:12 h) were collected at 6 circadian timepoints (n = 6 for each phase) of CT/ZT2, 6, 10, 14, 18, and 22 h, where the beginning of circadian time (CT0 h) in DD equals the time of light on at 6:00. Total RNA was extracted according to the protocol of the Trizol (Invitrogen). The concentration and purity of RNA samples were spectrophotometrically analyzed. The reverse-transcription reaction was carried out as described in the protocol of M-Mulv (BBI, Canada). A semiquantitative reverse-transcription PCR was performed to determine the level of Clock mRNA transcripts compared to the internally controlled gene of histone3.3 (H3.3). Primers of the Clock gene were designed by the Primer3 software. The primer sequences were as follows: Clock, 5 0 TCACCACGTTCACTCAGGACA-3 0 (forward), 5 0 -AAGGATTCCCA TGGAGC AA-3 0 (reverse); and H3.3, 5 0 -GCGTGCTAGCTGGAT GTCTT-3 0 (forward); 5 0 -CCACTGAACTTCTGATTCGC-3 0 (reverse). Rhythmicity of these genes was measured and analyzed by the Cosine software [14], and the amplitude was tested by an F test. Differential display reverse-transcriptase PCR. The lymphocyte samples were collected at ZT2 and ZT14 from the same rat with an interval of 10 days. Total RNA treated by deoxyribonuclease I (promega) was used for reverse transcription with three single base-anchored 3-end oligo(dT) primers. A total of 10 random upstream primers were chosen in combination with three downstream primers to amplify cDNA representing a subset of mRNA. To prevent isolation of ‘‘false positives,’’ all amplification experiments were performed in duplicate. Only the cDNA bands whose levels of expression were different in both duplicates were selected as interest cDNAs. Amplified cDNA fragments were separated in a 5% polyacrylamide gel with argentums (Ag) stain. The fragments of interest were excised from the gels, and then extracted and reamplified for further analysis. Reamplified fragments were cloned into TA cloning vector (pMD18-T, Takara, Japan) and prepared for later confirmation of the purported signals. Reverse Northern dot-blot. To confirm the differential expression of the identified bands, reverse Northern dot-blot analysis was performed. Briefly, cloned cDNA to be analyzed was amplified by PCR, and the products were denatured for blotting. Each sample was blotted in duplicate on two nylon membranes (Immobilon Ny+, Millipore) which were then UV cross-linked. Two single-stranded cDNA probes were prepared from 20 lg total RNA isolated at ZT2 and ZT14 by reverse transcription with 100 lCi [a-32P]dATP (10 mCi/ml, 3000 Ci/mmol, Pharmacia). Each cDNA probe was heat-denatured and then hybridized separately onto one of the two membranes. The membranes were washed for the detection of the signal intensity by a PhosphorImager (Pharmacia), and the gray of probe bound to each cDNA was quantified by ImageQuant TL software (Pharmacia). By comparing the gray scale of each homologous cDNA between ZT2 and ZT14, the expression level of cDNA changed over three times was considered as the true positive cDNA. Sequence analysis. After sequencing of the cDNA fragments, the search for homology genes was performed by the online-based BLAST program
in the GenBank Database and also by the BLAT program in the rat genome database of Medical College of Wisconsin. Quantitative real-time PCR. Because the method of reverse Northern dot-blot creates about 30% false positives [15], the differentially displayed bands were confirmed by quantitative real-time PCR. Primers of the cDNA were designed according to the guidelines of Applied Biosystems with the Primer Express 2.0 software (Applied Biosystems). The extraction of total mRNA and the reverse-transcription reaction were carried out as described before. The housekeeping gene of b-actin was used as the internal control. To avoid the interindividual variation among rats, block random design of variance analysis was used for statistical comparison of the two groups. RNA interference. The peripheral lymphocytes of rat separated from the spleen were cultured in RPMI medium 1640 (Gibco) supplemented with 10% FBS. Transfection was performed by TransIT-TKO reagent (Mirus, Madison, WI) according to the manufacturerÕs instructions. Cells were collected after the 14 h transfection, and total RNA was extracted and prepared for quantitative real-time PCR. The siRNA sequence of Clock gene is designed as CAG TGT ATC AAC TTC AAC A and synthesized by the Dharmacon. The scrambled sequence of Clock siRNA was used as a negative control (NC). Determination of circadian rhythms of the screened genes. Animals were fed and housed in LD (12:12 h). Samples of the peripheral lymphocytes were collected at timepoints of ZT2, ZT6, ZT10, ZT14, ZT18, and ZT22, with six rats for each timepoint. The transcripts of the three known genes (catalase, myelin proteolipid protein (plp), and histone acetylase) were quantified by the real-time PCR. The data were analyzed by cosine software and the amplitudes were tested by an F test.
Results Circadian alteration of clock genes First, we determined whether the expression of Clock gene altered with a circadian pattern in the peripheral lymphocytes of rat. As shown in Fig. 1, the transcripts of Clock in lymphocytes displayed a significant daily rhythm, with the peak phase at CT7 and ZT21, respectively. The amplitude of the LD group (0.062) was greater than that of the DD group (0.041). These results suggest that the Clock transcripts indeed express with circadian alteration in the peripheral lymphocytes of rat, as demonstrated with other models [3–7]. Identification of circadian genes Next, the comparison of mRNA expression patterns from the peripheral lymphocytes of rat was performed to identify up-regulated or down-regulated genes between the two timepoints, ZT2 and ZT14. Thirty-one differentially expressed cDNA bands were identified and extracted from the polyacrylamide gels to be assayed further. Among them, 10 cDNA fragments were confirmed by reverse Northern dot-blot analysis and named AP48, AP52, AP55, AP62, AP63, AP66, AP96, AP107, AP510, and AP511, respectively (Fig. 2). All the 10 cDNA fragments were sequenced and compared for the homology in the NCBI-nr database and rat genome database. Of them, AP52, AP55, and AP62 were found homologous with the same EST as the gene of XP-215032 encoding globulin b-chain. The AP510, AP63, and AP66 fragments,
Y. Du et al. / Biochemical and Biophysical Research Communications 336 (2005) 1069–1073
expression of Clock mRNA in DD LD (12:12)
1
relative level
relative level
expression of Clock mRNA in DD
0.8 0.6 0.4 0:00
6:00
12:00 18:00
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1 0.8 0.6 0.4 0:00
0:00
CT (hours)
6:00
12:00 18:00
0:00
ZT (hours)
Fig. 1. Circadian oscillation of Clock mRNA transcripts in lymphocytes in different regimes (n = 6 for each phase). The dotted line was obtained according to the parameters calculated by the cosine software. The black and white bars on the x-axis signify the times of light-off and light-on.
1
2
3
4
5
6
7
8
9
1 2
10
A
A
B
B
C
C
D
D
E
E
F
F
G
G
3 4 5 6
7 8 9
10
H
H
ZT2
ZT14
Fig. 2. Analysis of differentially displayed cDNA fragments by reverse Northern dot-blot. All the dots represent differential expressed cDNA fragments and are marked by asterisks at the left side. E1-E2: AP52; G1-G2: AP55; A3-A4: AP511; G3-G4: AP96; H3-H4: AP107; C5-C6: AP62; B7-B8: AP48; C7C8: AP510; D7-D8: AP63; G7-G8: AP66; A9-A10: negative control; H7-H8: positive control of histone 3.3; B9-B10: positive control of b-actin.
respectively, were homologous with genes of catalase, myelin proteolipid protein (plp), and histone acetylase. The AP511 was a previously identified EST, and the AP48, AP107, and AP96 were submitted to the GenBank as three novel ESTs with the registration numbers of CK807871, CK807872, and CK807873, respectively. As shown in Fig. 3, the expression levels of all 10 cDNAs were significantly higher at ZT14 than at ZT2 (p < 0.05, n = 6, a StudentÕs t test). For example, there is a 4- to 5-fold difference in amplitude for AP66, AP107, and AP511. A 20 ZT2
15
ZT14
10 5 AP48
AP510 AP511
AP63 AP66
AP96
AP107
0 -5
AP52
AP62
-10 -15 -20
Fig. 3. Relative expression levels of differentially displayed fragments at ZT2 and ZT14 (n = 6 for each phase). The y-axis indicates relative expression level compared to the internal control of b-actin.
similar result was obtained with the reverse Northern dotblot analysis (data not shown). A time-dependent alteration of these 10 cDNA transcripts with circadian rhythms is presented in Table 1. These results indicate that the expression of the 10 cDNA fragments and the Clock gene alters with circadian patterns in peripheral lymphocytes of rat. Effect of Clock-siRNA on the expression of cDNA fragments To determine the possible effect of the Clock gene on the expression of these 10 cDNA fragments, we employed a RNA interference assay to knock down the Clock expression. RNA interference is a post-transcriptional process triggered by the introduction of double-stranded RNA (dsRNA) which leads to gene silencing in a sequence-specific manner. A significant decrease of the Clock mRNA was observed in the cells transfected with the Clock-siRNA compared to those transfected with the control scrambled siRNA and the untransfected parental cells (Fig. 4). Expressions of all the cDNA fragments were depressed in the Clock-siRNA transfected cells compared with the control transfectants (P < 0.05, n = 6), as shown in Fig. 5. For example, approximately 2.5-fold decline of AP63 expression in Clock-siRNA cells was shown. The results imply that these cDNA fragments were regulated by the Clock gene.
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Table 1 Temporal changes in mRNA levels of Clock in DD and LD (12:12) in lymphocytes of rat, as determined by RT-PCR (means ± SD, n = 6 for each phase) CT2
CT6
CT10
CT14
CT18
CT22
DD
1.07 ± 0.03
1.08 ± 0.024
1.04 ± 0.08
1.04 ± 0.03
1.02 ± 0.01
0.96 ± 0.04
ZT2
ZT6
ZT10
ZT14
ZT18
ZT22
LD (12:12)
0.76 ± 0.04
0.64 ± 0.02
0.54 ± 0.02
0.72 ± 0.02
0.69 ± 0.01
0.66 ± 0.01
respectively. Additional experiments with real-time PCR showed a time-dependent alteration of these three gene transcripts with circadian rhythms (Table 2). Fig. 4. Effect of RNA interference on the level of Clock mRNA transcripts. siRNA, cells transfected with Clock-siRNA; NC, control cells transfected with scrambled siRNA; and Normal, untransfected cells.
NC
8.00
siRNA 6.00 4.00 2.00 AP48
AP510 AP511
AP63 AP66 AP96 AP107
0.00 AP52
AP62
-2.00 -4.00 -6.00 -8.00 -10.00
Fig. 5. Effect of Clock-siRNA on expression of cDNA fragments (n = 6 for each group). The y-axis indicates relative expression level compared to the internal control of b-actin.
Circadian rhythms of catalase, plp, and histone acetylase The catalase, plp, and histone acetylase genes, respectively, contain the sequences of Ap510, Ap63, and Ap66, and we thus determined whether these three genes altered with daily time course. As illustrated in Fig. 6, the expressions of the three genes appeared in a similar circadian pattern under the LD condition, with a slight difference in peak phase of about 2 h., i.e., at ZT19, ZT19, and ZT20, catalase
10 5 6:00 12:00 18:00 0:00 ZT (hours)
The circadian oscillation of the Clock mRNA revealed for the first time that the Clock gene could function in peripheral lymphocytes in a rhythmic manner. This circadian rhythm displayed not only in LD (12:12 h) but also in DD regime, indicating that it is an endogenous oscillation. On the other hand, light likely affects the oscillation intensity of Clock gene expression since the rhythm amplitude in LD (12:12) is higher than that in DD. The results may suggest that an oscillator might exist in lymphocytes and modulate daily functions of the immune cells. As shown with the DD-PCR assay, 10 differential cDNAs are mediated by the Clock gene. One of these genes, catalase, has been reported to display circadian variation in some tissues of rodents [16]. The other gene encoding histone acetylase is found in the peripheral lymphocytes of rat, although rhythmic histone acetylation has been revealed in the mammalian circadian clock before [17]. Another gene of plp is considered important for autoimmune disorder of multiple sclerosis [18,19]. The critical role of Clock in regulation of the circadian system was assumed to activate the transcription of other clock genes and the clock-controlled genes. Our results that all the examined ESTs expressed at a higher level at ZT14 than at ZT2, and the transcripts of Clock gene expressed higher at the subjective night and lower at the subjective day in LD (12:12) (Fig. 1) which coincided with those ESTs, proposed a possibility that these ESTs may be directly affected by the Clock gene. Furthermore, the experiments with RNA interference showed a notable decrease in levels of all the EST expressions upon the Clock knock-down, p/p
15
histone acetylase
20
Relative Expression Level
15
0 0:00
20 Relative Expression Level
Relative Expression Level
20
Discussion
15
10
10
5 0 0:00
6:00 12:00 18:00 0:00 ZT (hours)
5 0 0:00
6:00 12:00 18:00 0:00 ZT (hours)
Fig. 6. Circadian alteration of catalase, plp, and histone acetylase (n = 6 for each phase). The black and white bars on the x-axis signify the times of lightoff and light-on.
Y. Du et al. / Biochemical and Biophysical Research Communications 336 (2005) 1069–1073
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Table 2 Temporal changes in mRNA levels of clock-controlled genes in LD (12:12 h) in lymphocytes of rat, as determined by quantitative real-time PCR (means ± SD, n = 6 for each phase)
Catalase plp Acetylase
CT2
CT6
CT10
CT14
CT18
CT22
10.2 ± 0.67 11.0 ± 0.04 12.1 ± 0.55
7.77 ± 0.04 7.5 ± 0.69 10.8 ± 0.18
8.43 ± 0.78 8.4 ± 0.96 11.5 ± 0.91
11.4 ± 1.32 11.7 ± 1.18 13.0 ± 1.22
14.6 ± 0.88 12.4 ± 2.23 15.5 ± 0.04
16.63 ± 2.30 11.0 ± 0.08 16.5 ± 2.23
demonstrating that these candidate cDNAs or genes may act downstream of the Clock gene. For a gene to be classified as a clock-controlled gene (ccg), it should be regulated by a clock, have a circadian expression pattern, and show either a similar or an antiphase peak with the clock [5,7,20]. In fact, there are several types of ccgs according to different roles they may play and pathways being controlled directly or indirectly by the clock [21]. To those ccgs controlled by CLOCK/BMAL1 and functioning as negative components of the feedback loop, they are regulated directly by binding of CLOCK/ BMAL1 to the E-box in the enhancer region of the genes and these genes are expressed almost anti-phase to the phase of Clock and Bmal1 gene expression. On the other hand, for ccgs that function as output of the circadian signals downstream of the Clock, their peak phases need to be around that of the Clock, usually with certain lagging depending on the regulatory relationship among them. By comparison of expression pattern of the examined genes with that of the Clock gene, it was found that the circadian rhythms of all these genes and cDNA fragments displayed in a similar way, i.e., all the peaks occurred during late subjective night (Fig. 6). Based on the facts of their circadian rhythmicity, mediation by the Clock gene, and similar peak phases to the Clock gene, we conclude that catalase, plp, histone acetylase, and other cDNAs identified in this study are likely to be clock-controlled in the peripheral lymphocytes of rat. The underlying mechanism(s) has been investigating in our laboratory. In addition, the experiments are going to analyze the tissue distribution of these new genes and ESTs, and determine whether these genes specifically express in the peripheral lymphocytes in in vivo rat models. Further work will focus on determining the protein structures of these new gene/ESTs and the protein expression profile in terms of circadian rhythms. Acknowledgments This work was supported by National Natural Science Foundation of China (30170295) and National Institutes of Health, USA (ES-013199). References [1] M.R. Ralph, R.G. Foster, F.C. Davis, M. Menaker, Transplanted suprachiasmatic nucleus determines circadian period, Science 247 (1990) 975–978.
[2] D.C. Klein, R.Y. Moore, S.M. Reppert, Suprachiasmatic Nucleus: The MindÕs Clock, Oxford University Press, New York, NY, 1991. [3] J. Rutter, M. Reick, S.L. McKnight, Metabolism and the control of circadian rhythms, Annu. Rev. Biochem. 71 (2002) 307–331. [4] P.L. Lowrey, J.S. Takahashi, Genetics of the mammalian circadian system: photic entrainment, circadian pacemaker mechanisms, and posttranslational regulation, Annu. Rev. Genet. 34 (2000) 533–562. [5] J.C. Dunlap, Molecular basis for circadian clocks, Cell 96 (1999) 271– 290. [6] X. Jin, L.P. Shearman, D.R. Weaver, M.J. Zylka, G.J. De Vries, S.M. Reppert, A molecular mechanism regulating rhythmic output form the suprachiasmatic circadian clock, Cell 96 (1999) 57–68. [7] K. Oishi, H. Fukui, N. Ishida, Rhythmic expression of BMAL1 mRNA is altered in clock mutant mice: differential regulation in suprachiasmatic nucleus and peripheral tissues, Biochem. Biophys. Res. Commun. 268 (2000) 164–171. [8] S. Yamazaki, R. Numano, M. Abe, A. Hida, R. Takahashi, M. Ueda, G.D. Block, Y. Sakaki, M. Menaker, H. Tei, Resetting central and peripheral circadian oscillators in transgenic rats, Science 5466 (2000) 682–685. [9] B. Plytycz, R. Seljelid, Rhythms of immunity, Arch. Immunol. Ther. Exp. (Warsz.) 45 (1997) 157–162. [10] J. Born, T. Lange, K. Hansen, M. Molle, H.L. Fehm, Effects of sleep and circadian rhythm on human circulating immune cells, J. Immunol. 158 (1997) 4454–4464. [11] T. Wang, D.L. Delahanty, A.L. Dougall, A. Baum, Responses of natural killer cell activity to acute laboratory stressors in healthy men at different times of day, Health Psychol. 17 (1998) 428–435. [12] E. Haus, M.H. Smolensky, Biologic rhythms in the immune system, Chronobiol. Int. 16 (1999) 581–622. [13] D.B. Boivin, F.O. James, A. Wu, P.F. Cho-Park, H. Xiong, Z.S. Sun, Circadian clock genes oscillate in human peripheral blood mononuclear cells, Blood 102 (2003) 4143–4145. [14] M.H. Teicher, N.I. Barber, COSIFIT: an interactive program for simultaneous multioscillator cosinor analysis of time-series data, Comput. Biomed. Res. 23 (1990) 283–295. [15] D.W. Dilks, R.H. Ring, X.Z. Khawaja, T.J. Novak, C. Aston, Highthroughput confirmation of differential display PCR results using reverse Northern blotting, J. Neurosci. Methods 123 (2003) 47–54. [16] Z.C. Tomas, M.A. Coto, F.J. Martinez, M.J. Rodriguez Colunga, D. Tolivia, Effects of continuous light exposure on antioxidant enzymes, porphyric enzymes and cellular damage in the Harderian gland of the Syrian hamster, J. Pineal Res. 34 (2003) 60–68. [17] J.P. Etchegaray, C. Lee, P.A. Wade, S.M. Reppert, Rhythmic histone acetylation underlies transcription in the mammalian circadian clock, Nature 421 (2003) 177–182. [18] K. Bernadette, A. Hansjuerg, D.L. Fred, Characteristics of the T lymphocytes involved in experimental allergic encephalomyelitis, J. Neuroimmunol. 61 (1995) 107–116. [19] J.K. Olson, C.J. Ludovic, S.D. Miller, Innate and adaptive immune requirements for induction of autoimmune demyelinating disease by molecular mimicry, Mol. Immunol. 40 (2004) 1103–1108. [20] D.J. Kennaway, T.J. Varcoe, V.J. Mau, Rhythmic expression of clock and clock-controlled genes in the rat oviduct, Mol. Hum. Reprod. 9 (2003) 503–507. [21] N. Cermakian, D.B. Boivin, Molecular perspective of human circadian rhythm disorders, Brain Res. Rev. 42 (2003) 204–220.
Circadian expression of clock and screening of clock-controlled genes in peripheral lymphocytes of rat Yu-zhen Du a, Sai-jun Fan a,b, Qing-hui Meng b, Guo-qing Wang a, Jian Tong a,* a
Chronobiology Laboratory, Department of Toxicology, School of Radiation Medicine and Public Health, Soochow University, Suzhou 215007, Jiangsu, China b Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, USA Received 21 August 2005 Available online 8 September 2005
Abstract In this paper, the circadian pattern of Clock and genes mediated by the Clock was investigated in peripheral lymphocytes of rats. Circadian rhythms of Clock are found under the regimes of constant darkness (DD) and 12-h light–12-h dark (LD12:12 h), with the peak phase at CT7 and ZT21, respectively. Ten differential cDNA fragments were identified to be mediated by the Clock, including three known genes (catalase, myelin proteolipid protein, and histone acetylase), four known expressed sequence tags (ESTs), and three novel ESTs. Experiment of the RNA interference revealed that these ESTs were down-regulated by the Clock gene and three of them were identified as clock-controlled genes. Understanding of clock-mediated genes may lead to a new direction in drug design for control of circadian rhythms. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Clock gene; Clock-controlled genes; Lymphocytes
In most organisms from lower species to higher ones, physiological and behavioral functions are expressed rhythmically across days and nights. These daily rhythms, referred to as circadian, are controlled by self-sustained biological oscillators. In mammals, studies with primary neuronal cultures as well as ablation and transplantation suggest that the central component of this complex oscillatory system resides in the suprachiasmatic nuclei (SCN) of the hypothalamus [1,2], which involve transcriptional– translational feedback loops of at least eight genes, namely, Per1/2/3, Cry1/2, Clock, Bmal1, and CKIe. Of these, the Per1/2 and Cry1/2 genes function as negative regulators, while the Bmal1 and Clock gene act as positive regulators in circadian oscillation [3–7]. Clock and Bmal1 can heterodimerize and bind to the E-box sequences of other clock genes and clock-mediated genes to activate the transcrip-
*
Corresponding author. Fax: +86 512 65304830. E-mail address: [email protected] (J. Tong).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.08.228
tion. Thus, Clock and Bmal1 are believed as the most important components of the circadian oscillator. Studies have shown that the molecular clocks oscillate not only in the central nervous system, but also in peripheral organs, such as liver, kidney, and fibroblast cells [8]. Circadian fluctuations in the immune system are also observed [9–12]. The numbers of blood T and B lymphocytes in blood fluctuate about 2-fold in a circadian day. The concentrations of serum albumin and globulin, as well as the cell immunity and activation of lymphocytes in mice, vary with a 24 h period. There have been reports on rhythmic expression of clock genes Per1, Per2, and Per3 in peripheral blood mononuclear cells (PBMCs) [13]. However, no similar report was found in the literature for circadian expression of the Clock gene in peripheral lymphocytes so far. We hypothesized that a circadian expression of functional clock components in peripheral lymphocytes might be involved in regulation of the circadian immune functions. In this paper, we provided the expression profile of the Clock gene in constant darkness (DD) and light–dark
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Y. Du et al. / Biochemical and Biophysical Research Communications 336 (2005) 1069–1073
(LD12:12 h) regimes, and screened the potential genes which may be targeted by the Clock gene in peripheral lymphocytes of Sprague–Dawley rat. Materials and methods Animals and sample collection. Adult male Sprague–Dawley rats were housed in DD regime for at least 6 weeks and in LD (12:12 h) [light: 6:00– 18:00 clock, light on at zeitgeber time (ZT) 0] for at least 4 weeks before the experiment. An incandescent lamp was used as a source of light during the day (150–200 lux) at the level of the cages. Operation during the dark period was done with the help of a dim red light (0.1 lux). The rats were tested and screened in an infrared video box for the sleep–wake cycle before the sample collection in order to obtain homogeneous samples. Blood sampling was conducted by cardiac puncture at different timepoints of a day. The lymphocytes were separated by centrifugation and stored at 80 °C until use. Semi-quantitative reverse-transcription PCR. The lymphocyte samples of DD and LD (12:12 h) were collected at 6 circadian timepoints (n = 6 for each phase) of CT/ZT2, 6, 10, 14, 18, and 22 h, where the beginning of circadian time (CT0 h) in DD equals the time of light on at 6:00. Total RNA was extracted according to the protocol of the Trizol (Invitrogen). The concentration and purity of RNA samples were spectrophotometrically analyzed. The reverse-transcription reaction was carried out as described in the protocol of M-Mulv (BBI, Canada). A semiquantitative reverse-transcription PCR was performed to determine the level of Clock mRNA transcripts compared to the internally controlled gene of histone3.3 (H3.3). Primers of the Clock gene were designed by the Primer3 software. The primer sequences were as follows: Clock, 5 0 TCACCACGTTCACTCAGGACA-3 0 (forward), 5 0 -AAGGATTCCCA TGGAGC AA-3 0 (reverse); and H3.3, 5 0 -GCGTGCTAGCTGGAT GTCTT-3 0 (forward); 5 0 -CCACTGAACTTCTGATTCGC-3 0 (reverse). Rhythmicity of these genes was measured and analyzed by the Cosine software [14], and the amplitude was tested by an F test. Differential display reverse-transcriptase PCR. The lymphocyte samples were collected at ZT2 and ZT14 from the same rat with an interval of 10 days. Total RNA treated by deoxyribonuclease I (promega) was used for reverse transcription with three single base-anchored 3-end oligo(dT) primers. A total of 10 random upstream primers were chosen in combination with three downstream primers to amplify cDNA representing a subset of mRNA. To prevent isolation of ‘‘false positives,’’ all amplification experiments were performed in duplicate. Only the cDNA bands whose levels of expression were different in both duplicates were selected as interest cDNAs. Amplified cDNA fragments were separated in a 5% polyacrylamide gel with argentums (Ag) stain. The fragments of interest were excised from the gels, and then extracted and reamplified for further analysis. Reamplified fragments were cloned into TA cloning vector (pMD18-T, Takara, Japan) and prepared for later confirmation of the purported signals. Reverse Northern dot-blot. To confirm the differential expression of the identified bands, reverse Northern dot-blot analysis was performed. Briefly, cloned cDNA to be analyzed was amplified by PCR, and the products were denatured for blotting. Each sample was blotted in duplicate on two nylon membranes (Immobilon Ny+, Millipore) which were then UV cross-linked. Two single-stranded cDNA probes were prepared from 20 lg total RNA isolated at ZT2 and ZT14 by reverse transcription with 100 lCi [a-32P]dATP (10 mCi/ml, 3000 Ci/mmol, Pharmacia). Each cDNA probe was heat-denatured and then hybridized separately onto one of the two membranes. The membranes were washed for the detection of the signal intensity by a PhosphorImager (Pharmacia), and the gray of probe bound to each cDNA was quantified by ImageQuant TL software (Pharmacia). By comparing the gray scale of each homologous cDNA between ZT2 and ZT14, the expression level of cDNA changed over three times was considered as the true positive cDNA. Sequence analysis. After sequencing of the cDNA fragments, the search for homology genes was performed by the online-based BLAST program
in the GenBank Database and also by the BLAT program in the rat genome database of Medical College of Wisconsin. Quantitative real-time PCR. Because the method of reverse Northern dot-blot creates about 30% false positives [15], the differentially displayed bands were confirmed by quantitative real-time PCR. Primers of the cDNA were designed according to the guidelines of Applied Biosystems with the Primer Express 2.0 software (Applied Biosystems). The extraction of total mRNA and the reverse-transcription reaction were carried out as described before. The housekeeping gene of b-actin was used as the internal control. To avoid the interindividual variation among rats, block random design of variance analysis was used for statistical comparison of the two groups. RNA interference. The peripheral lymphocytes of rat separated from the spleen were cultured in RPMI medium 1640 (Gibco) supplemented with 10% FBS. Transfection was performed by TransIT-TKO reagent (Mirus, Madison, WI) according to the manufacturerÕs instructions. Cells were collected after the 14 h transfection, and total RNA was extracted and prepared for quantitative real-time PCR. The siRNA sequence of Clock gene is designed as CAG TGT ATC AAC TTC AAC A and synthesized by the Dharmacon. The scrambled sequence of Clock siRNA was used as a negative control (NC). Determination of circadian rhythms of the screened genes. Animals were fed and housed in LD (12:12 h). Samples of the peripheral lymphocytes were collected at timepoints of ZT2, ZT6, ZT10, ZT14, ZT18, and ZT22, with six rats for each timepoint. The transcripts of the three known genes (catalase, myelin proteolipid protein (plp), and histone acetylase) were quantified by the real-time PCR. The data were analyzed by cosine software and the amplitudes were tested by an F test.
Results Circadian alteration of clock genes First, we determined whether the expression of Clock gene altered with a circadian pattern in the peripheral lymphocytes of rat. As shown in Fig. 1, the transcripts of Clock in lymphocytes displayed a significant daily rhythm, with the peak phase at CT7 and ZT21, respectively. The amplitude of the LD group (0.062) was greater than that of the DD group (0.041). These results suggest that the Clock transcripts indeed express with circadian alteration in the peripheral lymphocytes of rat, as demonstrated with other models [3–7]. Identification of circadian genes Next, the comparison of mRNA expression patterns from the peripheral lymphocytes of rat was performed to identify up-regulated or down-regulated genes between the two timepoints, ZT2 and ZT14. Thirty-one differentially expressed cDNA bands were identified and extracted from the polyacrylamide gels to be assayed further. Among them, 10 cDNA fragments were confirmed by reverse Northern dot-blot analysis and named AP48, AP52, AP55, AP62, AP63, AP66, AP96, AP107, AP510, and AP511, respectively (Fig. 2). All the 10 cDNA fragments were sequenced and compared for the homology in the NCBI-nr database and rat genome database. Of them, AP52, AP55, and AP62 were found homologous with the same EST as the gene of XP-215032 encoding globulin b-chain. The AP510, AP63, and AP66 fragments,
Y. Du et al. / Biochemical and Biophysical Research Communications 336 (2005) 1069–1073
expression of Clock mRNA in DD LD (12:12)
1
relative level
relative level
expression of Clock mRNA in DD
0.8 0.6 0.4 0:00
6:00
12:00 18:00
1071
1 0.8 0.6 0.4 0:00
0:00
CT (hours)
6:00
12:00 18:00
0:00
ZT (hours)
Fig. 1. Circadian oscillation of Clock mRNA transcripts in lymphocytes in different regimes (n = 6 for each phase). The dotted line was obtained according to the parameters calculated by the cosine software. The black and white bars on the x-axis signify the times of light-off and light-on.
1
2
3
4
5
6
7
8
9
1 2
10
A
A
B
B
C
C
D
D
E
E
F
F
G
G
3 4 5 6
7 8 9
10
H
H
ZT2
ZT14
Fig. 2. Analysis of differentially displayed cDNA fragments by reverse Northern dot-blot. All the dots represent differential expressed cDNA fragments and are marked by asterisks at the left side. E1-E2: AP52; G1-G2: AP55; A3-A4: AP511; G3-G4: AP96; H3-H4: AP107; C5-C6: AP62; B7-B8: AP48; C7C8: AP510; D7-D8: AP63; G7-G8: AP66; A9-A10: negative control; H7-H8: positive control of histone 3.3; B9-B10: positive control of b-actin.
respectively, were homologous with genes of catalase, myelin proteolipid protein (plp), and histone acetylase. The AP511 was a previously identified EST, and the AP48, AP107, and AP96 were submitted to the GenBank as three novel ESTs with the registration numbers of CK807871, CK807872, and CK807873, respectively. As shown in Fig. 3, the expression levels of all 10 cDNAs were significantly higher at ZT14 than at ZT2 (p < 0.05, n = 6, a StudentÕs t test). For example, there is a 4- to 5-fold difference in amplitude for AP66, AP107, and AP511. A 20 ZT2
15
ZT14
10 5 AP48
AP510 AP511
AP63 AP66
AP96
AP107
0 -5
AP52
AP62
-10 -15 -20
Fig. 3. Relative expression levels of differentially displayed fragments at ZT2 and ZT14 (n = 6 for each phase). The y-axis indicates relative expression level compared to the internal control of b-actin.
similar result was obtained with the reverse Northern dotblot analysis (data not shown). A time-dependent alteration of these 10 cDNA transcripts with circadian rhythms is presented in Table 1. These results indicate that the expression of the 10 cDNA fragments and the Clock gene alters with circadian patterns in peripheral lymphocytes of rat. Effect of Clock-siRNA on the expression of cDNA fragments To determine the possible effect of the Clock gene on the expression of these 10 cDNA fragments, we employed a RNA interference assay to knock down the Clock expression. RNA interference is a post-transcriptional process triggered by the introduction of double-stranded RNA (dsRNA) which leads to gene silencing in a sequence-specific manner. A significant decrease of the Clock mRNA was observed in the cells transfected with the Clock-siRNA compared to those transfected with the control scrambled siRNA and the untransfected parental cells (Fig. 4). Expressions of all the cDNA fragments were depressed in the Clock-siRNA transfected cells compared with the control transfectants (P < 0.05, n = 6), as shown in Fig. 5. For example, approximately 2.5-fold decline of AP63 expression in Clock-siRNA cells was shown. The results imply that these cDNA fragments were regulated by the Clock gene.
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Table 1 Temporal changes in mRNA levels of Clock in DD and LD (12:12) in lymphocytes of rat, as determined by RT-PCR (means ± SD, n = 6 for each phase) CT2
CT6
CT10
CT14
CT18
CT22
DD
1.07 ± 0.03
1.08 ± 0.024
1.04 ± 0.08
1.04 ± 0.03
1.02 ± 0.01
0.96 ± 0.04
ZT2
ZT6
ZT10
ZT14
ZT18
ZT22
LD (12:12)
0.76 ± 0.04
0.64 ± 0.02
0.54 ± 0.02
0.72 ± 0.02
0.69 ± 0.01
0.66 ± 0.01
respectively. Additional experiments with real-time PCR showed a time-dependent alteration of these three gene transcripts with circadian rhythms (Table 2). Fig. 4. Effect of RNA interference on the level of Clock mRNA transcripts. siRNA, cells transfected with Clock-siRNA; NC, control cells transfected with scrambled siRNA; and Normal, untransfected cells.
NC
8.00
siRNA 6.00 4.00 2.00 AP48
AP510 AP511
AP63 AP66 AP96 AP107
0.00 AP52
AP62
-2.00 -4.00 -6.00 -8.00 -10.00
Fig. 5. Effect of Clock-siRNA on expression of cDNA fragments (n = 6 for each group). The y-axis indicates relative expression level compared to the internal control of b-actin.
Circadian rhythms of catalase, plp, and histone acetylase The catalase, plp, and histone acetylase genes, respectively, contain the sequences of Ap510, Ap63, and Ap66, and we thus determined whether these three genes altered with daily time course. As illustrated in Fig. 6, the expressions of the three genes appeared in a similar circadian pattern under the LD condition, with a slight difference in peak phase of about 2 h., i.e., at ZT19, ZT19, and ZT20, catalase
10 5 6:00 12:00 18:00 0:00 ZT (hours)
The circadian oscillation of the Clock mRNA revealed for the first time that the Clock gene could function in peripheral lymphocytes in a rhythmic manner. This circadian rhythm displayed not only in LD (12:12 h) but also in DD regime, indicating that it is an endogenous oscillation. On the other hand, light likely affects the oscillation intensity of Clock gene expression since the rhythm amplitude in LD (12:12) is higher than that in DD. The results may suggest that an oscillator might exist in lymphocytes and modulate daily functions of the immune cells. As shown with the DD-PCR assay, 10 differential cDNAs are mediated by the Clock gene. One of these genes, catalase, has been reported to display circadian variation in some tissues of rodents [16]. The other gene encoding histone acetylase is found in the peripheral lymphocytes of rat, although rhythmic histone acetylation has been revealed in the mammalian circadian clock before [17]. Another gene of plp is considered important for autoimmune disorder of multiple sclerosis [18,19]. The critical role of Clock in regulation of the circadian system was assumed to activate the transcription of other clock genes and the clock-controlled genes. Our results that all the examined ESTs expressed at a higher level at ZT14 than at ZT2, and the transcripts of Clock gene expressed higher at the subjective night and lower at the subjective day in LD (12:12) (Fig. 1) which coincided with those ESTs, proposed a possibility that these ESTs may be directly affected by the Clock gene. Furthermore, the experiments with RNA interference showed a notable decrease in levels of all the EST expressions upon the Clock knock-down, p/p
15
histone acetylase
20
Relative Expression Level
15
0 0:00
20 Relative Expression Level
Relative Expression Level
20
Discussion
15
10
10
5 0 0:00
6:00 12:00 18:00 0:00 ZT (hours)
5 0 0:00
6:00 12:00 18:00 0:00 ZT (hours)
Fig. 6. Circadian alteration of catalase, plp, and histone acetylase (n = 6 for each phase). The black and white bars on the x-axis signify the times of lightoff and light-on.
Y. Du et al. / Biochemical and Biophysical Research Communications 336 (2005) 1069–1073
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Table 2 Temporal changes in mRNA levels of clock-controlled genes in LD (12:12 h) in lymphocytes of rat, as determined by quantitative real-time PCR (means ± SD, n = 6 for each phase)
Catalase plp Acetylase
CT2
CT6
CT10
CT14
CT18
CT22
10.2 ± 0.67 11.0 ± 0.04 12.1 ± 0.55
7.77 ± 0.04 7.5 ± 0.69 10.8 ± 0.18
8.43 ± 0.78 8.4 ± 0.96 11.5 ± 0.91
11.4 ± 1.32 11.7 ± 1.18 13.0 ± 1.22
14.6 ± 0.88 12.4 ± 2.23 15.5 ± 0.04
16.63 ± 2.30 11.0 ± 0.08 16.5 ± 2.23
demonstrating that these candidate cDNAs or genes may act downstream of the Clock gene. For a gene to be classified as a clock-controlled gene (ccg), it should be regulated by a clock, have a circadian expression pattern, and show either a similar or an antiphase peak with the clock [5,7,20]. In fact, there are several types of ccgs according to different roles they may play and pathways being controlled directly or indirectly by the clock [21]. To those ccgs controlled by CLOCK/BMAL1 and functioning as negative components of the feedback loop, they are regulated directly by binding of CLOCK/ BMAL1 to the E-box in the enhancer region of the genes and these genes are expressed almost anti-phase to the phase of Clock and Bmal1 gene expression. On the other hand, for ccgs that function as output of the circadian signals downstream of the Clock, their peak phases need to be around that of the Clock, usually with certain lagging depending on the regulatory relationship among them. By comparison of expression pattern of the examined genes with that of the Clock gene, it was found that the circadian rhythms of all these genes and cDNA fragments displayed in a similar way, i.e., all the peaks occurred during late subjective night (Fig. 6). Based on the facts of their circadian rhythmicity, mediation by the Clock gene, and similar peak phases to the Clock gene, we conclude that catalase, plp, histone acetylase, and other cDNAs identified in this study are likely to be clock-controlled in the peripheral lymphocytes of rat. The underlying mechanism(s) has been investigating in our laboratory. In addition, the experiments are going to analyze the tissue distribution of these new genes and ESTs, and determine whether these genes specifically express in the peripheral lymphocytes in in vivo rat models. Further work will focus on determining the protein structures of these new gene/ESTs and the protein expression profile in terms of circadian rhythms. Acknowledgments This work was supported by National Natural Science Foundation of China (30170295) and National Institutes of Health, USA (ES-013199). References [1] M.R. Ralph, R.G. Foster, F.C. Davis, M. Menaker, Transplanted suprachiasmatic nucleus determines circadian period, Science 247 (1990) 975–978.
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