Figure 3. p107 repression in quiescent MEFs is mediated by the two E2F binding sites.

(A) mESCs targeted by the neomycin resistance cassette but retaining a wild-type p107 promoter and mESCs targeted by homozygous mutations into the distal (1*/1*) or both E2F sites (1*2*/1*2*) were injected to generate chimeric embryos. Wild-type, p107^E2F-1*/1* and p107^{E2F-1*2*/1*2*} MEFs derived from chimeric embryos were selected for Neomycin resistance to generate pure populations. (B) RT-qPCR analysis of p107 expression in quiescent wild-type and p107^{E2F-1*2*/1*2*} MEFs. (n≥9) (C) Immunoblot analysis of p107 in the same conditions. Tubulin expression is shown as a loading control. (D) Quantitative ChIP analysis of E2F4, p107, and p130 binding on the p107 promoter in quiescent immortalized wild-type, p107^E2F-1*/1*, and p107^{E2F-1*2*/1*2*} MEFs. The B-Myb promoter is shown as a control. (n = 3) (E) Quantitative ChIP analysis of Rb binding to the p107 and Mcm3 promoters in cycling immortalized wild-type and p107^{E2F-1*2*/1*2*} MEFs. Mouse IgG antibodies serve as a negative control. (n≥3) For (D,E), fold enrichment is calculated over actin and the y-axis is plotted on a log2 scale.