Role of circadian gene Clock during differentiation of mouse pluripotent stem cells

Abstract

Biological rhythms controlled by the circadian clock are absent in embryonic stem cells (ESCs). However, they start to develop during the differentiation of pluripotent ESCs to downstream cells. Conversely, biological rhythms in adult somatic cells disappear when they are reprogrammed into induced pluripotent stem cells (iPSCs). These studies indicated that the development of biological rhythms in ESCs might be closely associated with the maintenance and differentiation of ESCs. The core circadian gene Clock is essential for regulation of biological rhythms. Its role in the development of biological rhythms of ESCs is totally unknown. Here, we used CRISPR/CAS9-mediated genetic editing techniques, to completely knock out the Clock expression in mouse ESCs. By AP, teratoma formation, quantitative real-time PCR and Immunofluorescent staining, we did not find any difference between Clock knockout mESCs and wild type mESCs in morphology and pluripotent capability under the pluripotent state. In brief, these data indicated Clock did not influence the maintaining of pluripotent state. However, they exhibited decreased proliferation and increased apoptosis. Furthermore, the biological rhythms failed to develop in Clock knockout mESCs after spontaneous differentiation, which indicated that there was no compensational factor in most peripheral tissues as described in mice models before (DeBruyne et al., 2007b). After spontaneous differentiation, loss of CLOCK protein due to Clock gene silencing induced spontaneous differentiation of mESCs, indicating an exit from the pluripotent state, or its differentiating ability. Our findings indicate that the core circadian gene Clock may be essential during normal mESCs differentiation by regulating mESCs proliferation, apoptosis and activity.

Key words： Circadian gene Clock; mouse embryonic stem cells; gene knockout; pluripotency; cell proliferation; cell apoptosis; cell differentiation

References

1 Balsalobre A, Damiola F, Schibler U (1998) A serum shock induces circadian gene expression in mammalian tissue culture cells. Cell 93:929–937

2 Borgs L, Beukelaers P, Vandenbosch R, Nguyen L, Moonen G, Maquet P, Albrecht U, Belachew S, Malgrange B (2009) Period 2 regulates neural stem/progenitor cell proliferation in the adult hippocampus. BMC Neurosci 10:30

3 Cardinal-Aucoin M, Steel CG (2016) Circadian control of prothoracicotropic hormone release in an adult insect and the induction of its rhythmicity by light cues. Comp Biochem Physiol A Mol Integr Physiol 201:46–52

4 Casanova-Acebes M, Pitaval C, Weiss LA, Nombela-Arrieta C, Chevre R, Noelia AG, Kunisaki Y, Zhang D, van Rooijen N, Silberstein LE (2013) Rhythmic modulation of the hematopoietic niche through neutrophil clearance. Cell 153:1025–1035

5 Dashti HS, Aslibekyan S, Scheer FA, Smith CE, Lamon-Fava S, Jacques P, Lai CQ, Tucker KL, Arnett DK, Ordovas JM (2016) Clock genes explain a large proportion of phenotypic variance in systolic blood pressure and this control is not modified by environmental temperature. Am J Hypertens 29:132–140

6 Debruyne JP (2008) Oscillating perceptions: the ups and downs of the CLOCK protein in the mouse circadian system. J Genet 87:437–446

7 DeBruyne JP, Weaver DR, Reppert SM (2007a) CLOCK and NPAS2 have overlapping roles in the suprachiasmatic circadian clock. Nat Neurosci 10:543–545

8 DeBruyne JP, Weaver DR, Reppert SM (2007b) Peripheral circadian oscillators require CLOCK. Curr Biol 17:R538–539

9 Dubrovsky YV, Samsa WE, Kondratov RV (2010) Deficiency of circadian protein CLOCK reduces lifespan and increases agerelated cataract development in mice. Aging (AlbanyNY) 2:936–944

10 Dunlap JC (1999) Molecular bases for circadian clocks. Cell 96:271–290

11 Fu L, Pelicano H, Liu J, Huang P, Lee C (2002) The circadian gene Period2 plays an important role in tumor suppression and DNA damage response in vivo. Cell 111:41–50

12 Gachon F, Nagoshi E, Brown SA, Ripperger J, Schibler U (2004) The mammalian circadian timing system: from gene expression to physiology. Chromosoma 113:103–112

13 Gaddameedhi S, Selby CP, Kemp MG, Ye R, Sancar A (2015) The circadian clock controls sunburn apoptosis and erythema in mouse skin. J Invest Dermatol 135:1119–1127

14 Hu K, Van Someren EJ, Shea SA, Scheer FA (2009) Reduction of scale invariance of activity fluctuations with aging and Alzheimer’s disease: Involvement of the circadian pacemaker. Proc Natl Acad Sci USA 106:2490–2494

15 Janich P, Toufighi K, Solanas G, Luis NM, Minkwitz S, Serrano L, Lehner B, Benitah SA (2013) Human epidermal stem cell function is regulated by circadian oscillations. Cell Stem Cell 13:745–753

16 Johnson MH, Lim A, Fernando D, Day ML (2002) Circadian clockwork genes are expressed in the reproductive tract and conceptus of the early pregnant mouse. Reprod Biomed Online 4:140–145

17 Johnston JD, Ordovas JM (2016) Circadian rhythms, metabolism, and chrononutrition in rodents and humans. Adv Nutr 7:399–406

18 Karpowicz P, Zhang Y, Hogenesch JB, Emery P, Perrimon N (2013) The circadian clock gates the intestinal stem cell regenerative state. Cell Rep 3:996–1004

19 King DP, Zhao Y, Sangoram AM, Wilsbacher LD, Tanaka M, Antoch MP, Steeves TD, Vitaterna MH, Kornhauser JM, Lowrey PL (1997) Positional cloning of the mouse circadian clock gene. Cell 89:641–653

20 Koike N, Yoo SH, Huang HC, Kumar V, Lee C, Kim TK, Takahashi JS (2012) Transcriptional architecture and chromatin landscape of the core circadian clock in mammals. Science 338:349–354

21 Kovanen L, Saarikoski ST, Aromaa A, Lonnqvist J, Partonen T (2010) ARNTL (BMAL1) and NPAS2 gene variants contribute to fertility and seasonality. PLoS One 5:e10007

22 Li R, Cheng S, Wang Z (2015) Circadian clock gene plays a key role on ovarian cycle and spontaneous abortion. Cell Physiol Biochem 37:911–920

23 Liang X, Cheng S, Jiang X, He X, Wang Y, Jiang Z, Hou W, Li S, Liu Y, Wang Z (2013) The noncircadian function of the circadian Clock gene in the regulation of male fertility. J Biol Rhythms 28:208–217

24 Lopez-Minguez J, Gomez-Abellan P, Garaulet M (2016) Circadian rhythms, food timing and obesity. Proc Nutr Soc.

25 Lowrey PL, Takahashi JS (2004) Mammalian circadian biology: elucidating genome-wide levels of temporal organization. Annu Rev Genomics Hum Genet 5:407–441

26 McAlpine CS, Swirski FK (2016) Circadian influence on metabolism and inflammation in atherosclerosis. Circ Res 119:131–141

27 Mizuno S, Dinh TT, Kato K, Mizuno-Iijima S, Tanimoto Y, Daitoku Y, Hoshino Y, Ikawa M, Takahashi S, Sugiyama F (2014) Simple generation of albino C57BL/6J mice with G291T mutation in the tyrosinase gene by the CRISPR/Cas9 system. Mamm Genome 25:327–334

28 Mukherjee S, Coque L, Cao JL, Kumar J, Chakravarty S, Asaithamby A, Graham A, Gordon E, Enwright JF III, DiLeone RJ (2010) Knockdown of Clock in the ventral tegmental area through RNA interference results in a mixed state of mania and depression-like behavior. Biol Psychiatry 68:503–511

29 Nelakanti RV, Kooreman NG, Wu JC (2015) Teratoma formation: a tool for monitoring pluripotency in stem cell research. Curr Protoc Stem Cell Biol 32(4a.8):1–17

30 Nitschke S, Cortleven A, Iven T, Feussner I, Havaux M, Riefler M, Schmulling T (2016) Circadian stress regimes affect the circadian clock and cause jasmonic acid-dependent cell death in cytokinindeficient arabidopsis plants. Plant Cell.

31 Peyric E, Moore HA, Whitmore D (2013) Circadian clock regulation of the cell cycle in the zebrafish intestine. PLoS One 8:e73209

32 Tobback J, Vuerinckx K, Boerjan B, Huybrechts R (2012) RNA interference mortality points to noncircadian functions for the clock gene in the desert locust Schistocerca gregaria. Insect Mol Biol 21:369–381

33 Tracey CJ, Pan X, Catterson JH, Harmar AJ, Hussain MM, Hartley PS (2012) Diurnal expression of the thrombopoietin gene is regulated by CLOCK. J Thromb Haemost 10:662–669

34 Witting W, Kwa IH, Eikelenboom P, Mirmiran M, Swaab DF (1990) Alterations in the circadian rest-activity rhythm in aging and Alzheimer’s disease. Biol Psychiatry 27:563–572

35 Wood PA, Yang X, Taber A, Oh EY, Ansell C, Ayers SE, Al-Assaad Z, Carnevale K, Berger FG, Pena MM (2008) Period 2 mutation accelerates ApcMin/+ tumorigenesis. Mol Cancer Res 6:1786–1793

36 Wyse CA, Coogan AN (2010) Impact of aging on diurnal expression patterns of CLOCK and BMAL1 in the mouse brain. Brain Res 1337:21–31

37 Xiang S, Mao L, Duplessis T, Yuan L, Dauchy R, Dauchy E, Blask DE, Frasch T, Hill SM (2012) Oscillation of clock and clock controlled genes induced by serum shock in human breast epithelial and breast cancer cells: regulation by melatonin. Breast Cancer (Auckl) 6:137–150

38 Xue T,Song C, Wang Q, Wang Y, Chen G (2016) Investigations of the CLOCK and BMAL1 proteins binding to DNA: a molecular dynamics simulation study. PLoS One 11:e0155105

39 Yagita K, Horie K, Koinuma S, Nakamura W, Yamanaka I, Urasaki A, Shigeyoshi Y, Kawakami K, Shimada S, Takeda J (2010) Development of the circadian oscillator during differentiation of mouse embryonic stem cells in vitro. Proc Natl Acad Sci USA 107:3846–3851

40 Yang G, Chen L, Grant GR, Paschos G, Song WL, Musiek ES, Lee V, McLoughlin SC, Grosser T, Cotsarelis G (2016) Timing of expression of the core clock gene Bmal1 influences its effects on aging and survival. Sci Transl Med 8:324316

41 Yee JK (2016) Off-target effects of engineered nucleases. FEBS J.