Tandem E2F Binding Sites in the Promoter of the p107 Cell Cycle Regulator Control p107 Expression and Its Cellular Functions
Figure 5
E2F binding sites mediate activation of the p107 promoter in cycling cells.
(A) RT-qPCR analysis of p107 mRNA relative to TBP in asynchronously cycling primary wild-type and p107E2F-1*2*/1*2* MEFs. (n = 12) (B) Immunoblot analysis (left panel) of p107 expression in wild-type and p107E2F-1*2*/1*2* MEFs as in A. Tubulin expression is shown as a loading control. p107 protein quantification (right panel) is shown relative to Tubulin levels. (n = 3) (C) Representative example of Hoescht33342 staining of asynchronously cycling MEFs showing G1 and S phase populations; wild-type and mutant cells have similar profiles (data not shown). (D) RT-qPCR analysis of immortalized WT, p107E2F-1*/1* and p107E2F-1*2*/1*2* MEFs. For each genotype, G0 samples were collected after at least three days of serum starvation. Asynchronous cells were stained with Hoechst33342 and sorted by their DNA content into G1 and S-phase samples. (n≥2) (E) and (F) RT-qPCR analysis of primary wild-type and p107E2F-1*2*/1*2* MEFs that have been synchronized in G0 by serum starvation. DMEM supplemented with 20% serum was added at time 0, and extracts were collected at 10 hrs, 16 hrs, 22 hrs, and 28 hrs post-stimulation. (E) p107 mRNA and (F) Cdc6 mRNA. n≥8 for both genotypes at all time points. (G) Percentage of cells in S-phase in primary MEFs collected during cell-cycle re-entry as in E. and F. Percentages were calculated by BrdU/PI analysis (n = 3). (H) Immunoblot analysis of p107 protein expression in primary wild-type and p107E2F-1*2*/1*2* MEF extracts collected at 0 hr, 8 hrs, 12 hrs, 16 hrs, 20 hrs, and 24 hrs post-stimulation with 20% serum. MCM6 expression is shown as a positive control for cell cycle re-entry, and Tubulin levels are shown as a loading control. Note that the second, slowly migrating form of p107 at later time points probably reflects p107 phosphorylation during S phase.