Hosoda Hiroshi, Kida Satoshi
Department of Molecular Microbiology, Faculty of Life Science, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo, 156-8502, Japan.
Department of Bioscience, Faculty of Life Science, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo, 156-8502, Japan.
Cytotechnology. 2019 Feb;71(1):453-460. doi: 10.1007/s10616-018-0266-9. Epub 2019 Jan 1.
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.
bHLH-PAS转录因子生物钟昼夜调节因子（CLOCK）和脑与肌肉芳香烃受体核转运蛋白样蛋白1（BMAL1）通过激活E盒介导的转录在昼夜节律基因表达节律的产生中发挥重要作用。重要的是，由CLOCK/BMAL1介导的昼夜转录节律在外周组织以及视交叉上核中均有观察到，并有助于组织特异性功能。这些发现表明，CLOCK/BMAL1通过与各种细胞调节因子相互作用，在许多生物学现象中发挥作用。在本研究中，为了了解CLOCK多种功能作用的潜在机制，我们尝试使用酵母双杂交系统鉴定与CLOCK相互作用的新蛋白。我们鉴定出在脑中高度表达的神经内分泌特异性蛋白（NSP）-C，它是CLOCK/BMAL1介导转录的正向调节因子。我们发现NSP-C在哺乳动物细胞中与CLOCK相互作用。NSP-C与CLOCK/BMAL1共表达增强了CLOCK/BMAL1的转录激活作用。此外，用小干扰RNA（siRNA）敲低内源性NSP-C可抑制E盒介导的转录，而这种转录减少可通过表达不受siRNA作用影响的NSP-C得到挽救。这些观察结果表明，NSP-C有助于上调CLOCK/BMAL1介导的转录。
