Hirata Yuki, Toyono Takashi, Kokabu Shoichiro, Obikane Yui, Kataoka Shinji, Nakatomi Mitsushiro, Masaki Chihiro, Hosokawa Ryuji, Seta Yuji
Division of Oral Reconstruction and Rehabilitation, Department of Oral Functions, Kyushu Dental University.
Division of Anatomy, Department of Health Promotion, Kyushu Dental University.
Biomed Res. 2019;40(2):67-78. doi: 10.2220/biomedres.40.67.
T1R1 and T1R3 are receptors expressed in taste buds that detect L-amino acids. These receptors are also expressed throughout diverse organ systems, such as the digestive system and muscle tissue, and are thought to function as amino acid sensors. The mechanism of transcriptional regulation of the mouse T1R1 gene (Tas1r1) has not been determined; therefore, in this study, we examined the function of Tas1r1 promoter in the mouse myoblast cell line, C2C12. Luciferase reporter assays showed that a 148-bp region upstream of the ATG start codon of Tas1r1 had a promoter activity. The GT box in the Tas1r1 promoter was conserved in the dog, human, mouse, and pig. Site-directed mutagenesis of this GT box significantly reduced the promoter activation. The GT box in promoters is a recurring motif for Sp/KLF family members. RNAi-mediated depletion of Sp4 and Klf5 decreased Tas1r1 expression, while overexpression of Klf5, but not Sp4, significantly increased Tas1r1 expression. The ENCODE data of chromatin immunoprecipitation and sequencing (ChIP-seq) showed that Klf5 bound to the GT box during the myogenic differentiation. Furthermore, the Klf5 knockout cell lines led to a considerable decrease in the levels of Tas1r1 expression. Collectively, these results showed that Klf5 binds to the GT box in the Tas1r1 promoter and regulates Tas1r1 expression in C2C12 cells.
T1R1和T1R3是味蕾中表达的受体，可检测L-氨基酸。这些受体也在多种器官系统中表达，如消化系统和肌肉组织，被认为起着氨基酸传感器的作用。小鼠T1R1基因（Tas1r1）的转录调控机制尚未确定；因此，在本研究中，我们检测了Tas1r1启动子在小鼠成肌细胞系C2C12中的功能。荧光素酶报告基因检测表明，Tas1r1的ATG起始密码子上游148 bp区域具有启动子活性。Tas1r1启动子中的GT框在狗、人、小鼠和猪中是保守的。对该GT框进行定点诱变显著降低了启动子活性。启动子中的GT框是Sp/KLF家族成员的重复基序。RNAi介导的Sp4和Klf5缺失降低了Tas1r1的表达，而Klf5的过表达（而非Sp4的过表达）显著增加了Tas1r1的表达。染色质免疫沉淀和测序（ChIP-seq）的ENCODE数据表明，在成肌分化过程中，Klf5与GT框结合。此外，Klf5基因敲除细胞系导致Tas1r1表达水平显著降低。总的来说，这些结果表明，Klf5与Tas1r1启动子中的GT框结合，并调节C2C12细胞中Tas1r1的表达。
