NSP-C contributes to the upregulation of CLOCK/BMAL1-mediated transcription
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.
KeywordsCLOCK NSP-C Interaction Transcription activation Yeast-two hybrid
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.
SK is responsible for the hypothesis development and overall design of the research and experiment, and supervised the experimental analyses and wrote the paper. HH performed and analyzed experiments and wrote the paper.
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