Figure 6. Regulation of H3K79me2 marks by Cdk1 During ES Cell Differentiation.
(A) Cdk1 or Cdk2 were immunoprecipitated from in vitro cultured 2 different ES cell lines, or mouse embryonic fibroblasts (MEFs) derived from 3 different embryos, or 2 independent lines of induced pluripotent stem cells (iPSC; line iPSC-1 was derived from MEF-3 cells, by reprogramming), and used for in vitro kinase reactions with histone H1 as a substrate. For control, immunoprecipitates from ES-J1 with normal IgG (IgG) were used.
(B) Wild-type ES cells and MEFs were transduced with a lentivirus encoding Flag-tagged wild-type Dot1l. Undifferentiated ES cells were maintained by culturing in a stem cell medium containing serum and LIF (SL), or were induced to differentiate with retinoic acid (RA) or Wnt3a and activin A (Wnt/Act). The localization of Dot1l was assessed by immunostaining with an anti-Flag antibody. DAPI was used to visualize cell nuclei. Scale bar, 20 μm.
(C) Densitometric quantification of H3K79me2 marks in 3 ES cell lines and MEFs derived from 3 independent mouse embryos, normalized against total histone H3 levels. Below, immunoblotting of chromatin fractions for H3K79me2 and total histone H3.
(D) Similar analysis as in C, using in vitro cultured MEFs derived from 3 different mice and 3 iPSC lines (iPSC-1 cells were derived from MEF-3 cells, by reprogramming).
(E) Cdk1AS/AS ES cells were treated with the increasing concentrations of 3-MB-PP1. Total lysates were probed with antibodies against H3K79me2 or histone H3. Shown is densitometric quantification of H3K79me2 marks, normalized against total histone H3 levels (mean values ±SD from 2 independent experiments). *, p<0.05 (one-way Anova with Tukey’s multiple comparison test). Identical results were obtained when chromatin fraction was analyzed this way.
(F) Wild-type ES cells were cultured in a stem cell medium (SL), or switched to differentiation medium containing retinoic acid (RA) or Wnt3a and activin (Wnt/Act) for 4 or 7 days. Cdk1 or Cdk2 were immunoprecipitated and subjected to in vitro kinase assays using histone H1 as a substrate. For control, immunoprecipitates from pluripotent ES cells with normal IgG (IgG) were used
(G) Densitometric quantification of H3K79me2 marks in chromatin fractions of Cdk1AS/AS ES cells cultured in stem cell medium (SL), or switched to differentiation medium containing Wnt3a and activin (Wnt/Act) for 4 and 7 days.
Mean values ±SD from 2 replicates. **, p<0.05 (paired t-test).