Fig. 5

In silico predictions and dual-luciferase reporter gene assays identified CEBPB as a key transcription factor (TF) binding to the upstream region of CPEB1. a–c Dual-luciferase reporter assays revealed that CEBPB binds at the CPEB1 upstream region in HCT116 cells and functions as a key TF. CEBPB, cells transfected with the pcDNA3.1-CEBPB vector; CPEB1-WT, cells transfected with the pGL3-CPEB1 vector that contains the wild-type CPEB1 promoter region; CPEB1-Mut, cells transfected with the pGL3-CPEB1 vector that contains a mutated version of the CPEB1 promoter region; GATA2, cells transfected with a pcDNA3.1-GATA2 vector; TP53, cells transfected with a pcDNA3.1-TP53 vector; **P < 0.01; ***P < 0.001. d Dual-luciferase reporter assays revealed that hypermethylation of the CEBPB-binding region resulted in diminished activation of CPEB1 by CEBPB. CPEB1-WT and CPEB1-Mut represent the wild-type and mutated forms of the CPEB1 promoter, respectively, inserted into the (CpG-free) pCpGL vector; M.Sssl, CpG methyltransferase, an enzyme that can methylate the CpGs in sequences inserted into the pCpGL vector; **P < 0.01; ***P < 0.001, ^#P > 0.05. e The flowchart documenting the steps involved in the ChIP-PCR assay. The upstream region of CPEB1 (approximately 2020 bp) was divided into PR1, PR2, and PR3. PR2, which was the predicted core TF-binding region (− 993 to − 779) was further divided into PCR1, PCR2, and PCR3 as shown; TSS, transcription start site. f ChIP-PCR assay validated CEBPB binding to the CPEB1 core TF-binding region (− 993 to − 779) in HCT116 cells. CPEB1 expression levels were determined using the DNA pulled-down in the ChIP assay with various antibodies. Relative input (%) = CPEB1 expression in the DNA pulled-down with anti-CEBPB or anti-IgG (control)/Input. ***P < 0.001