FIGURE 6. Mdm4 cooperates with Polycomb Repressor Complex members and regulates H2A ubiquitination.

(A) Co-immunoprecipitation of Mdm4 and RNF4. Expression plasmids for Flag-tagged Mdm4 and/or non-tagged RNF2, or an empty control plasmid, were transfected into H1299 cells. Cell lysates (input) and the immunoprecipitated (IP) material were analyzed by immunoblotting (IB) with antibodies detecting the Flag-tag and RNF2, respectively. Immunoprecipitation was conducted using antibodies against RNF2 (rabbit antibody), and pre-immune IgG (rabbit antibody) was used as a negative control.

* indicates a background band, presumably dimerized IgH; ** indicates monomeric IgH.

(B) Co-immunoprecipitation of Mdm4 and EZH2. H1299 cells were transfected with expression plasmids for Flag-tagged Mdm4, non-tagged EZH2, and/or a control plasmid, for 30 h. Cell lysates were immunoprecipitated using Mdm4 (mouse antibody) and βGal (mouse antibody, negative control) antibodies and subjected to immunoblot analysis with antibodies detecting the Flag-tag and EZH2. An additional Co-IP upon ectopic expression of Mdm4 and/or Mdm2 and EZH2 is shown in Suppl. Fig. 6 A.

(C) DNA replication upon Mdm4 depletion and overexpression of RNF2. Representative images of immunostained tracks of CldU (red) and IdU (green) of H1299 cells upon transfection with Mdm4 siRNA and overexpression of RNF2.

(D) Boxplot analysis of fork progression in the IdU label (green) with 10–90 percentile whiskers of the experiment described in (C). Two biological replicates are shown in Suppl. Fig. 6 C–D.

(E) Immunoblot analysis of cells depleted of Mdm4, RNF2 or both. Samples stained for RNF2 as well as H2AK119ub1 and HSC70 and total H2A. A biological replicate is shown in Suppl. Fig. 6 E.

(F) Chromatin immunoprecipitation (ChIP) analysis of H2AK119ub1 on PRC1 target gene promotors and C-FOS as negative control after H1299 cells were transfected with siRNA against Mdm4 or Mdm2. (Enrichment normalized to input and total H2A shown as mean + SEM from three biological replicates).

(G) Overexpressed H2A rescuing DNA replication in Mdm4-depleted cells. Representative images of tracks of newly synthesized DNA that were visualized using immunofluorescence.

(H) Fork progression after overexpression of wild-type H2A (pcDNA3.1-Flag-H2A) or a mutant lacking the lysine residue that is known to be ubiquitinated by Mdm2 and RNF2 (pcDNA3.1-Flag-H2A-K118–119R), upon Mdm4 depletion. H2A but not the mutant rescued DNA replication. Two biological replicates are shown in Suppl. Fig. 6 F–G.

(I) Overexpressed H2A rescuing DNA replication in MEFs lacking p53 and the respective Mdm proteins. Representative images of immunostained tracks of CldU (red) and IdU (green) of a fiber assay with mouse embryonic fibroblasts (MEFs).

(J) MEFs with targeted deletions of p53 alone, p53 in combination with Mdm4 or Mdm2, as well as a triple knockout were transfected with a plasmid carrying wildtype H2A or an empty plasmid as a control. Fork progression analysis of these cells is displayed in a boxplot with 10–90 percentile whiskers. A biological replicate is shown in Suppl. Fig. 6 I.