Figure 3. TCF3/E2A affects p21 mRNA stability.

A–E. TCF3/E2A binds to the p21 proximal promoters and its depletion does not affect p53 binding or RNAPII transactivation. ChIP analysis of the p21 locus with antibodies against TCF3/E2A (A), p53 (B), total RNAPII (C), Serine 5-phosphorylated RNAPII (S5P, D) and Serine 2-phoshoryalted RNAPII (E), in HCT116 cells expressing control shRNA of shRNA against TCF3/E2A, treated with DMSO or 10 μM Nutlin-3 (24 hours). The p21 gene map is shown at the bottom, with black boxes representing exons and the transcribed region highlighted by a gray box. Numbers at the bottom indicate the position of PCR amplicons used to analyze ChIP-enriched DNA relative to the transcription start site. n indicates the number of ChIPs performed with each antibody. F. TCF3/E2A depletion decreases p21 mRNA half life. mRNA half life assays were performed for the p21 and 14-3-3s (SFN) mRNAs by collecting RNA samples at the indicated times after Actinomycin D treatment in HCT116 cells expressing control or TCF3/E2A shRNAs, treated with DMSO or 10 μM Nutlin-3 (24 hours). Asterisks indicate significant different (p<0.05) between shControl and shTCR3/E2A samples treated with DMSO (blue) or Nutlin-3 (red) within a given time point. Data are expressed as averages of three experiments −/+ s.d.