TWIST directly inhibits E2A transcription by binding to E-box motif in E2A promoter. (A) Genomic organization of the region flanking the promoter region of human E2A (top panel) and the schematic representation of the pGL3-E2A reporter construct. Transcription start site, TSS. Reporter assays showing, in immortalized MSCs (B) or 293T (C), TWIST represses the E2A promoter in a dose dependent manner (n = 3). β-galactosidase was used as a control of transfection efficiency. (D) ChIP analysis of immortalized MSCs after transfection of pFLAG-TWIST. The chromatin was incubated either without antibodies, with an anti-TWIST antibody or with an isotype IgG antibody. Fragments of the E2-(147bp) and E5-(115bp) box in the E2A promoter were amplified by PCR (left panel) and were also quantified with quantitative RT-PCR (right panel). Input, 2% of total input lysate. Results are shown as the mean ± SD values (black bar for E2 box; white bar for E5 box). (E) Mutational analysis of E2-box and E5-box sites in the E2A promoter in 293T cells. Reporter constructs containing wild-type E2A (WT), E2-box (E2M) or E5-box (E5M) mutations, or double mutations (E2E5M) were generated and used to analyze the importance of these sites in mediating repression by TWIST (n = 3). (F) Truncation of the bHLH domain (tbTWIST) inhibits TWIST repression of E2A promoter (n = 3). Each ratio was normalized to the control (pGL3 basic vector), and significance was determined by Student t test. *P < .05, **P < .01 vs control.