Figure 2.

Arf promoter regulation, p19^Arf and p21^Cip1 expression and asynchronous proliferation properties of E2f3a- and E2f3b-deficient MEFs. (a) Chromatin immunoprecipitation (ChIP) was performed using asynchronously proliferating wildtype and E2f3a^-/- littermate MEFs, or (b) wildtype and E2f3b^-/- littermate MEFs. Sonicated cross-linked chromatin was immunoprecipitated with antibodies to E2f3, E2f1, E2f4, or control IgG. The purified DNA was analyzed by PCR with primers specific for the p107 or Arf promoters, or a control sequence lacking E2f binding sites (1kb upstream of a control promoter). Input, 0.5% of chromatin in IP reaction was analyzed by PCR. These analyses show that in the absence of E2f3b, E2f3a is detected bound to the Arf promoter. (c) The majority of E2f3a mutant MEFs show little or no increase in p19^Arf and p21^Cip1 levels relative to wildtype controls, as illustrated by western blot analysis of two representative sets of serum arrested MEFs. Gapdh is shown as a loading control. (d) No increase in p19^Arf or p21^Cip1 levels are observed in E2f3b mutant MEFs relative to wildtype littermate controls. (e) E2f3a^-/-, E2f3a^+/- and E2f3a^+/+ MEFs or (f) E2f3b^-/-, E2f3b^+/-, and E2f3b^+/+ MEFs were assayed for asynchronous proliferation. Cells were plated in duplicate at 6×10⁴/3cm dish and their growth monitored by daily counting for six days. No significant growth defect was observed in the isoform specific mutant cells.