Figure 2. HBx up-regulates the activity of mEZH2 promoter and mEZH2 expression through E2F1 transcription factor. A, HBx up-regulates the activity of mEZH2 promoter through −486/−214 region. The truncated mEZH2 promoter vector pGL3-pmEZH2 (−486/+22) or pGL3-pmEZH2 (−214/+22) was co-transfected with HBx expression vector pcDNA4.0-EGFP-HBx, and the luciferase activity was detected 36 h after tranfection. The mEZH2 promoter vector pGL3-pmEZH2 (−214/−50), of which candidate transcriptional start site deleted, was used as the negative control. B, mutation of E2F1-binding site leads to noticeable decline of HBx activation to mEZH2 promoter. The mutated mEZH2 promoter vector pGL3-pmEZH2 (−486/+22M) or pGL3-pmEZH2 (−486/+22) was co-transfected with HBx expression vector pcDNA4.0-EGFP-HBx, the luciferase activity was detected 36 h after tranfection. The mEZH2 promoter vector pGL3-pmEZH2 (−214/−50), in which the candidate transcription start site was deleted, and the pGL3-pmEZH2 (−214/+22), in which the candidate E2F1-binding site was deleted, were used as the negative and positive controls, respectively. C, HBx up-regulates the activity of mEZH2 promoter through E2F1 transcription factor. The mEZH2 promoter pGL3-pmEZH2 (−486/+22) was co-transfected with E2F1 shRNA expression vector pRS-puro-mE2F1 and HBx expression vector pcDNA4.0-EGFP-HBx, and the luciferase activity was detected 36 h after tranfection. The mutated mEZH2 promoter vector pGL3-pmEZH2 (−486/+22M) co-transfected with pRS-puro-mE2F1 and pcDNA4.0-EGFP-HBx was used as negative control. D, HBx up-regulates the expression level of mEZH2 through E2F1 transcription factor. The E2F1 shRNA expression vector pRS-puro-mE2F1 was co-transfected with HBx expression vector pcDNA4.0-EGFP-HBx (HBx) or empty vector pcDNA4.0-EGFP (Vec), and the protein expression level was analyzed by Western blotting 48 h after tranfection. The control shRNA expression vector pRS-puro-control co-transfected with pcDNA4.0-EGFP-HBx (HBx) or pcDNA4.0-EGFP (Vec) was used as negative control.