The bHLH-PAS transcription factors clock circadian regulator (CLOCK) and brain and muscle ARNT-like protein 1 (BMAL1) play essential roles in the generation of circadian gene expression rhythms through the activation of E-box-mediated transcription. Importantly, circadian transcriptional rhythms mediated by CLOCK/BMAL1 are observed in peripheral tissues as well as in the suprachiasmatic nucleus and contribute to tissue-specific functions. These findings suggest that CLOCK/BMAL1 have roles in many biological phenomena by interacting with various cellular regulators. In the present study, to understand the mechanisms underlying the multiple functional roles of CLOCK, we tried to identify new proteins that interact with CLOCK using a yeast two-hybrid system. We identified neuroendocrine-specific protein (NSP)-C, which is highly expressed in the brain, as a positive regulator of CLOCK/BMAL1-mediated transcription. We found that NSP-C interacted with CLOCK in mammalian cells. Co-expression of NSP-C with CLOCK/BMAL1 enhanced the transcriptional activation by CLOCK/BMAL1. Furthermore, knockdown of endogenous NSP-C by small interfering RNA (siRNA) suppressed E-box-mediated transcription, while this reduction of transcription was rescued by the expression of NSP-C protected from the action of siRNA. These observations suggest that NSP-C contributes to the upregulation of CLOCK/BMAL1-mediated transcription.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
Albrecht U, Sun ZS, Eichele G, Lee CC (1997) A differential response of two putative mammalian circadian regulators, mper1 and mper2, to light. Cell 91:1055–1064. https://doi.org/10.1016/S0092-8674(00)80495-X
Balsalobre A, Damiola F, Schibler U (1998) A serum shock induces circadian gene expression in mammalian tissue culture cells. Cell 93:929–937. https://doi.org/10.1016/S0092-8674(00)81199-X
Bass J, Takahashi JS (2010) Circadian integration of metabolism and energetics. Science 330:1349–1354. https://doi.org/10.1126/science.1195027
Bunger MK, Wilsbacher LD, Moran SM, Clendenin C, Radcliffe LA, Hogenesch JB, Simon MC, Takahashi JS, Bradfield CA (2000) Mop3 is an essential component of the master circadian pacemaker in mammals. Cell 103:1009–1017. https://doi.org/10.1016/S0092-8674(00)00205-1
Cermakian N, Sassone-Corsi P (2000) Multilevel regulation of the circadian clock. Nat Rev Mol Cell Biol 1:59–67. https://doi.org/10.1038/35036078
Cho H, Zhao X, Hatori M, Yu RT, Barish GD, Lam MT, Chong LW, DiTacchio L, Atkins AR, Glass CK, Liddle C, Auwerx J, Downes M, Panda S, Evans RM (2012) Regulation of circadian behaviour and metabolism by REV-ERB-α and REV-ERB-β. Nature 485:123–127. https://doi.org/10.1038/nature11048
Christiansen SL, Bouzinova EV, Fahrenkrug J, Wiborg O (2016) Altered expression pattern of clock genes in a rat model of depression. Int J Neuropsychopharmacol 19:1–13. https://doi.org/10.1093/ijnp/pyw061
Dibner C, Schibler U, Albrecht U (2010) The mammalian circadian timing system: organization and coordination of central and peripheral clocks. Annu Rev Physiol 72:517–549. https://doi.org/10.1146/annurev-physiol-021909-135821
Dunlap JC (1999) Molecular bases for circadian clocks. Cell 96:271–290. https://doi.org/10.1016/S0092-8674(00)80566-8
Fuller PM, Lu J, Saper CB (2008) Differential rescue of light- and food-entrainable circadian rhythms. Science 320:1074–1077. https://doi.org/10.1126/science.1153277
Gekakis N, Staknis D, Nguyen HB, Davis FC, Wilsbacher LD, King DP, Takahashi JS, Weitz CJ (1998) Role of the CLOCK protein in the mammalian circadian mechanism. Science 280:1564–1569. https://doi.org/10.1126/science.280.5369.1564
Goldberg JL, Barres BA (2000) Neurobiology: nogo in nerve regeneration. Nature 403:369–370. https://doi.org/10.1038/35000309
GrandPré T, Nakamura F, Vartanian T, Strittmatter SM (2000) Identification of the Nogo inhibitor of axon regeneration as a Reticulon protein. Nature 403:439–444. https://doi.org/10.1038/35000226
Hens J, Nuydens R, Geerts H, Senden NH, Van de Ven WJ, Roebroek AJ, van de Velde HJ, Ramaekers FC, Broers JL (1998) Neuronal differentiation is accompanied by NSP-C expression. Cell Tissue Res 292:229–237
Hirayama J, Sahar S, Grimaldi B, Tamaru T, Takamatsu K, Nakahata Y, Sassone-Corsi P (2007) CLOCK-mediated acetylation of BMAL1 controls circadian function. Nature 450:1086–1090. https://doi.org/10.1038/nature06394
Hogenesch JB, Gu YZ, Jain S, Bradfield CA (1998) The basic-helix-loop-helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors. Proc Natl Acad Sci USA 95:5474–5479. https://doi.org/10.1073/pnas.95.10.5474
Hosoda H, Asano H, Ito M, Kato H, Iwamoto T, Suzuki A, Masushige S, Kida S (2009) CBP/p300 is a cell type-specific modulator of CLOCK/BMAL1-mediated transcription. Mol Brain 2:34. https://doi.org/10.1186/1756-6606-2-34
Hosoda H, Miyao T, Uchida S, Sakai S, Kida S (2011) Development of a tightly-regulated tetracycline-dependent transcriptional activator and repressor co-expression system for the strong induction of transgene expression. Cytotechnology 63:211–216. https://doi.org/10.1007/s10616-011-9335-z
Ikeda M, Ikeda M (2014) BMAL1 is an essential regulator for circadian cytosolic Ca2+ rhythms in suprachiasmatic nucleus neurons. J Neurosci 34:12029–12038. https://doi.org/10.1523/JNEUROSCI.5158-13.2014
Iwamoto T, Mamiya N, Masushige S, Kida S (2005) PLCgamma2 activates CREB-dependent transcription in PC12 cells through phosphorylation of CREB at serine 133. Cytotechnology 47:107–116. https://doi.org/10.1007/s10616-005-3763-6
Kim SH, Yoo BC, Broers JL, Cairns N, Lubec G (2000) Neuroendocrine-specific protein C, a marker of neuronal differentiation, is reduced in brain of patients with Down syndrome and Alzheimer’s disease. Biochem Biophys Res Commun 276:329–334. https://doi.org/10.1006/bbrc.2000.3464
King DP, Takahashi JS (2000) Molecular genetics of circadian rhythms in mammals. Annu Rev Neurosci 23:713–742. https://doi.org/10.1146/annurev.neuro.23.1.713
King DP, Zhao Y, Sangoram AM, Wilsbacher LD, Tanaka M, Antoch MP, Steeves TD, Vitaterna MH, Kornhauser JM, Lowrey PL, Turek FW, Takahashi JS (1997) Positional cloning of the mouse circadian clock gene. Cell 89:641–653. https://doi.org/10.1016/S0092-8674(00)80245-7
Kwon I, Lee J, Chang SH, Jung NC, Lee BJ, Son GH, Kim K, Lee KH (2006) BMAL1 shuttling controls transactivation and degradation of the CLOCK/BMAL1 heterodimer. Mol Cell Biol 26:7318–7330. https://doi.org/10.1128/MCB.00337-06
Liu C, Li S, Liu T, Borjigin J, Lin JD (2007) Transcriptional coactivator PGC-1 integrates the mammalian clock and energy metabolism. Nature 447:477–481. https://doi.org/10.1038/nature09253
Luciano AK, Zhou W, Santana JM, Kyriakides C, Velazquez H, Sessa WC (2018) CLOCK phosphorylation by AKT regulates its nuclear accumulation and circadian gene expression in peripheral tissues. J Biol Chem 293:9126–9136. https://doi.org/10.1074/jbc.RA117.000773
Marcheva B, Ramsey KM, Buhr ED, Kobayashi Y, Su H, Ko CH, Ivanova G, Omura C, Mo S, Vitaterna MH, Lopez JP, Philipson LH, Bradfield CA, Crosby SD, JeBailey L, Wang X, Takahashi JS, Bass J (2010) Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes. Nature 466:627–631. https://doi.org/10.1038/nature09253
McDearmon EL, Patel KN, Ko CH, Walisser JA, Schook AC, Chong JL, Wilsbacher LD, Song EJ, Hong HK, Bradfield CA, Takahashi JS (2006) Dissecting the functions of the mammalian clock protein BMAL1 by tissue-specific rescue in mice. Science 314:1304–1308. https://doi.org/10.1126/science.1132430
Mishra I, Singh D, Kumar V (2016) Daily expression of genes coding for neurotransmitters in central and peripheral tissues of redheaded bunting: implication for circadian regulation of physiology in songbirds. Chronobiol Int 33:280–292. https://doi.org/10.3109/07420528.2016.1139587
Panda S, Hogenesch JB, Kay SA (2002) Circadian rhythms from flies to human. Nature 417:329–335. https://doi.org/10.1038/417329a
Reppert SM, Weaver DR (2002) Coordination of circadian timing in mammals. Nature 418:935–941. https://doi.org/10.1038/nature00965
Sahar S, Zocchi L, Kinoshita C, Borrelli E, Sassone-Corsi P (2010) Regulation of BMAL1 protein stability and circadian function by GSK3beta-mediated phosphorylation. PLoS ONE 5:e8561. https://doi.org/10.1371/journal.pone.0008561
Shearman LP, Zylka MJ, Weaver DR, Kolakowski LF Jr, Reppert SM (1997) Two period homologs: circadian expression and photic regulation in the suprachiasmatic nuclei. Neuron 19:1261–1269. https://doi.org/10.1016/S0896-6273(00)80417-1
Shimizu K, Kobayashi Y, Nakatsuji E, Yamazaki M, Shimba S, Sakimura K, Fukada Y (2016) SCOP/PHLPP1β mediates circadian regulation of long-term recognition memory. Nat Commun 7:12926. https://doi.org/10.1038/ncomms12926
Spengler ML, Kuropatwinski KK, Schumer M, Antoch MP (2009) A serine cluster mediates BMAL1-dependent CLOCK phosphorylation and degradation. Cell Cycle 8:4138–4146. https://doi.org/10.4161/cc.8.24.10273
Steiner P, Kulangara K, Sarria JC, Glauser L, Regazzi R, Hirling H (2004) Reticulon 1-C/neuroendocrine-specific protein-C interacts with SNARE proteins. J Neurochem 89:569–580. https://doi.org/10.1111/j.1471-4159.2004.02345.x
Tagami S, Eguchi Y, Kinoshita M, Takeda M, Tsujimoto Y (2000) A novel protein, RTN-XS, interacts with both Bcl-XL and Bcl-2 on endoplasmic reticulum and reduces their anti-apoptotic activity. Oncogene 19:5736–5746. https://doi.org/10.1038/sj.onc.1203948
Takahashi JS (2015) Molecular components of the circadian clock in mammals. Diabetes Obes Metab 17:6–11. https://doi.org/10.1111/dom.12514
Tamaru T, Hirayama J, Isojima Y, Nagai K, Norioka S, Takamatsu K, Sassone-Corsi P (2009) CK2α phosphorylates BMAL1 to regulate the mammalian clock. Nat Struct Mol Biol 16:446–448. https://doi.org/10.1038/nsmb.1578
Trott AJ, Menet JS (2018) Regulation of circadian clock transcriptional output by CLOCK:BMAL1. PLoS Genet 14:e1007156. https://doi.org/10.1371/journal.pgen.1007156
Verwey M, Dhir S, Amir S (2016) Circadian influences on dopamine circuits of the brain: regulation of striatal rhythms of clock gene expression and implications for psychopathology and disease. F1000Res. https://doi.org/10.12688/f1000research.9180.1
We strongly acknowledge Akemi KAMETA and Hidenori ASANO (Department of Agricultural Chemistry, Faculty of Applied Bioscience, Tokyo University of Agriculture) for their great technical supports. SK was supported by Grant-in-Aids for Scientific Research (A) [KAKENHI; 15H02488, 18H03944], Scientific Research (B) [KAKENHI; 23300120, 20380078], and Challenging Exploratory Research [KAKENHI; 24650172, 26640014, 17K19464], Grant-in-Aids for Scientific Research on Priority Areas – Molecular Brain Science- [KAKENHI; 18022038, 22022039], Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area) [KAKENHI; 24116008, 24116001, 23115716, 17H06084, 17H05961, 17H05581, 18H05428, 18H05434], Core Research for Evolutional Science and Technology (CREST), Japan, The Science Research Promotion Fund, The Promotion and Mutual Aid Corporation for Private Schools of Japan, The Sumitomo Foundation, The Naito Foundation, The Uehara Memorial Foundation and the Takeda Science Foundation, Japan.
About this article
Cite this article
Hosoda, H., Kida, S. NSP-C contributes to the upregulation of CLOCK/BMAL1-mediated transcription. Cytotechnology 71, 453–460 (2019). https://doi.org/10.1007/s10616-018-0266-9
- Transcription activation
- Yeast-two hybrid