Macrophage Migration Inhibitory Factor MIF Interferes with the Rb-E2F Pathway  Oleksi Petrenko, Ute M. Moll  Molecular Cell  Volume 17, Issue 2, Pages 225-236 (January 2005) DOI: 10.1016/j.molcel.2004.11.052

Figure 1 MIF Affects Cellular Proliferation through E2F-Dependent Mechanisms (A) Growth characterization of MIF/p53 double-knockout (DKO) fibroblasts. For each curve, cells from eight embryos were cultured on the 3T3 protocol. The data are presented as fold cell accumulation after each successive passage. Inset, MIF expression in primary fibroblasts of the indicated genotypes. Erk is a loading control. Error bars represent the standard deviation. (B) Total cell accumulation of p53−/− and DKO MEFs maintained on the 3T3 protocol. Circles depict the average of eight embryos each. (C) Growth curves of p53−/− and DKO MEFs expressing ectopic CDK4 alone or in combination with E2F1. Error bars represent the standard deviation. (D) Growth curves of p53−/− and DKO MEFs expressing ectopic E2F1 or an E2F1 mutant lacking the C-terminal domain (DbE2F1). Error bars represent the standard deviation. Molecular Cell 2005 17, 225-236DOI: (10.1016/j.molcel.2004.11.052)

Figure 2 MIF Deficiency Impairs Tumor Formation In Vivo (A) Growth curves of p53−/− and DKO MEFs transduced with oncogenic H-RasV12. Equal Ras expression was verified by immunoblot analysis. Odd lanes correspond to p53−/− MEFs, whereas even lanes correspond to DKO cells. Error bars represent the standard deviation. (B) Tumor development in nude mice by H-RasV12-expressing (top) and K-RasV12-expressing (bottom) p53−/− and DKO MEFs. Error bars represent the standard deviation. (C) Tumors developed by H-RasV12- and K-RasV12-expressing p53−/− and DKO MEFs were excised 24 days after injection and weighed. Student's t test was used to determine statistical significance. Error bars represent the standard deviation. Molecular Cell 2005 17, 225-236DOI: (10.1016/j.molcel.2004.11.052)

Figure 3 MIF-Deficient Cells Exhibit an Aberrant E2F4-Dependent Response (A) Focus formation assays of p53−/− and DKO MEFs expressing H-RasV12 plus wtE2F1 or E2F1 mutants Y411C and DbE2F1. Error bars represent the standard deviation. (B) Focus formation assays of p53−/− and DKO MEFs expressing H-RasV12 plus wtE2F4 or E2F4 mutants T391, E2F4 Δ332, and DbE2F4. Error bars represent the standard deviation. (C and D) CDK4 overexpression indirectly upregulates endogenous CDK2 activity. p53−/− and DKO MEFs were transduced with H-RasV12 alone or H-RasV12 plus CDK4 and (C) immunoprecipitated with the indicated antibodies. CDK4 and CDK2 kinase activities were determined with γ-32P-ATP and GST-Rb protein as substrate. (D) Accumulation of hyperphosphorylated endogenous Rb in CDK4-overexpressing cells was revealed by immunoblot analysis. (E) Focus formation assays of p53−/− and DKO MEFs expressing H-RasV12 plus CDK4 and either E2F1, E2F4, DbE2F1, or DbE2F4. Error bars represent the standard deviation. Molecular Cell 2005 17, 225-236DOI: (10.1016/j.molcel.2004.11.052)

Figure 4 The C-Terminal E2F1 Domain Rescues the Transformation Defect of MIF-Deficient Fibroblasts (A) Immunoblot analysis of p53−/− and DKO MEFs expressing H-RasV12 plus E2F4C1 or DbE2F4. (B) Focus formation assays of H-RasV12-transformed p53−/− and DKO MEFs expressing E2F1, E2F4, E2F4C1, or DbE2F4. Error bars represent the standard deviation. Molecular Cell 2005 17, 225-236DOI: (10.1016/j.molcel.2004.11.052)

Figure 5 MIF Deficiency Alters the DNA Binding Properties of E2Fs and Affects the Composition of Rb/E2F Complexes (A) ChIP analysis of cdc6, p107, and cdc2 promoters for occupancy by endogenous E2F1, E2F3, and E2F4 before and after expression of H-RasV12. Top of each pair is in the absence of Ras, bottom is in the presence of Ras. Neo-specific primers were used to control for equal DNA input. (B) ChIP analysis of promoter occupancy by endogenous Rb family proteins before and after expression of H-RasV12. (C and D) Lysates from vector- and H-RasV12-transduced p53−/− and DKO MEFs were immunoprecipitated with an E2F1-specific Ab (C) or E2F4-specific Abs (D) followed by immunoblotting with the indicated antibodies. Molecular Cell 2005 17, 225-236DOI: (10.1016/j.molcel.2004.11.052)

Figure 6 Loss of MIF Represses DNA Replication Initiation Sites (A) Hygromycin-resistant colony formation by vector- or H-RasV12-transduced p53−/− and DKO MEFs after transfection with oriP reporter pREPΔ. (A–D) Trichostatin A (TSA) was added at 10−9 M where indicated. Error bars represent the standard deviation. (B) ChIP analysis of vector- or H-RasV12-transduced p53−/− and DKO MEFs stably transfected with pREPΔ. Immunoprecipitations with the indicated antibodies followed by PRC amplification with the oriP primers. Neo-specific primers were used to control for equal DNA input. (C and D) Colony formation by p53−/− and DKO MEFs transfected with pREPΔ. Prior to transfection, cells were transduced with the indicated combination of genes. Error bars represent the standard deviation. (E) ChIP analysis of p53−/− and DKO MEFs transduced with H-RasV12 or H-RasV12 plus E2F1 or E2F4. Immunoprecipitations with the indicated antibodies followed by PRC amplification with Ori primers. Control for equal DNA input as in (B). Molecular Cell 2005 17, 225-236DOI: (10.1016/j.molcel.2004.11.052)

Figure 7 The N-Terminal E1A Domain Rescues the Transformation Defect of MIF-Deficient Fibroblasts (A) Focus formation assay of p53−/− and DKO MEFs expressing H-RasV12 plus wtE1A, ΔN E1A, or ΔCR2 E1A. Error bars represent the standard deviation. (B) Focus formation assays of H-RasV12-transformed p53−/− and DKO MEFs expressing vector, N-Myc, N-MycΔ (deletion of the MBII region), or N-MycΔ proteins fused to E1A10–E1A33 or E1A10–E1A39. Immunoblot analysis of p53−/− (odd lanes) and DKO (even lanes) MEFs confirms equal expression of transduced genes in (A) and (B). Error bars represent the standard deviation. (C) ChIP analysis of p53−/− and DKO MEFs transduced with H-RasV12 plus E1A, N-Myc, or N-MycD fused to E1A10–E1A39. Immunoprecipitations were performed with the indicated antibodies followed by PRC amplification with Ori primers. (D) ChIP analysis of p53−/− and DKO MEFs transduced with H-RasV12 plus vector or CDK4. Immunoprecipitations were performed with the indicated antibodies followed by PRC amplification with Ori primers. Neo-specific primers (bottom) were used to control for equal DNA input. Molecular Cell 2005 17, 225-236DOI: (10.1016/j.molcel.2004.11.052)