Fig. 3

C/EBPβ auto-regulates its enhancer activity. a qChIP-PCR showed enrichment of C/EBPβ, BRD4, H3K27ac, and RNPII at C/EBPβ enhancer after 5-aza-dC treatment. b Western blot analysis of C/EBPβ level in liver cells upon siRNA-mediated knockdown. β-actin was used as loading control. c, d qRT-PCR analysis of C/EBPβ eRNA upon (c) C/EBPβ knockdown and (d) treatment with BRD4 inhibitor JQ1. C/EBPβ eRNA/mRNA levels were calculated by the 2^−ΔΔCt method using 18s rRNA as internal control, and are presented as fold-changes against the average values of the respective control groups (siCtrl/DMSO). e Schematic diagrams of C/EBPβ promoter-, C/EBPβ promoter-C/EBPβ enhancer-, and C/EBPβ promoter-C/EBPβ enhancer mutant-luciferase reporter constructs. The C/EBPβ promoter (−285 to + 808-bp relative to TSS) was cloned to the PGL3 vector, in the absence or presence of the C/EBPβ enhancer with the WT or deleted C/EBP consensus sequence. f The relative luciferase activity in liver cells upon transfection with different reporter constructs. g The relative luciferase activity of the C/EBPβ enhancer construct upon siRNA-mediated knockdown of C/EBPβ. Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 as calculated by unpaired two-tailed Student’s t-test (a, c, d, f, g)