Myogenic differentiation 1 and transcription factor 12 activate the gene expression of mouse taste receptor type 1 member 1.

OBJECTIVES: Myogenic differentiation 1 (Myod1) is involved in the expression of taste receptor type 1 member 1 (Tas1r1) during myogenic differentiation. Further, the target genes of Myod1 participate in transcriptional control, muscle development, and synaptic function. We examined, for the first time, the function of Myod1 in the transcriptional regulation of Tas1r1. METHODS: ENCODE chromatin immunoprecipitation and sequencing (ChIP-seq) data of myogenically differentiated C2C12 cells were analyzed to identify the Myod1 and transcription factor 12 (Tcf12) binding sites in the Tas1r1 promoter region. Luciferase reporter assays, DNA affinity precipitation assays, and co-immunoprecipitation assays were also performed to identify the functions of Myod1, Tcf12, and Krüppel-like factor 5 (Klf5). RESULTS: Based on ENCODE ChIP-seq, Myod1 bound to the Tas1r1 promoter region containing E-boxes 1-3. Luciferase reporter assays revealed that site-directed E-box1 mutations significantly reduced promoter activation induced by Myod1 overexpression. According to the DNA affinity precipitation assay and co-immunoprecipitation assay, Myod1 formed a heterodimer with Tcf12 and bound to E-box1. Further, Klf5 bound to the GT box near E-box1, activating Tas1r1 expression. CONCLUSIONS: During myogenic differentiation, the Myod1/Tcf12 heterodimer, in collaboration with Klf5, binds to E-box1 and activates Tas1r1 expression.

Krüppel-like factor 5 (Klf5) regulates expression of mouse T1R1 amino acid receptor gene (Tas1r1) in C2C12 myoblast cells.

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.