Fig. 4

The interaction between Trim28 and H3.3 is not mediated by DAXX. A RT-qPCR for DAXX expression on WT and DAXX KO cells, normalized to UBC control gene. Data are the mean ± s.e.m. (n = 4) P value was calculated using Mann–Whitney U unpaired nonparametric test, *P < 0.05. B immunoblotting WT and DAXX KO cells using anti-DAXX and H3 antibody as a control. C Percentage of GFP expressing cells following DAXX depletion. Cells infected with PBSpro or D PBSgln virus. Data are the mean ± s.e.m. (n = 3) P value was calculated using Kruskal–Wallis nonparametric test with Dunns' multiple comparison, **P < 0.01 E Immunoprecipitation (IP) of nuclear extracts from WT or DAXX KO ESCs carrying the H3.3-HA-tag, followed by immunoblotting with the indicated Antibodies. F Suggested model describing three phases of silencing onset: (1) H3.3 hyper dynamic nucleosome deposition is required for proper (2) Trim28 recruitment to the proviral DNA, which then (3) facilitates heterochromatinization of the region. At this point, DAXX is not required for H3.3 accumulation. Created with BioRender.com