Figure 3: H3K9 methylation affects H1 dynamics and differentiation.

(a,b) H1e–GFP FRAP analysis in euchromatin (a) and heterochromatin (b) of WT ESCs, undifferentiated (LIF, grey) or differentiated with retinoic acid for 4 days (RA, black). Changes are significant (P<0.01, 2-tailed t-test). (c,d) H1e–GFP FRAP analysis in euchromatin (c) and heterochromatin (d) of G9a^−/− ESCs, undifferentiated (grey) or differentiated with RA for 4 days (black). No restriction of H1 dynamics is observed following differentiation. (e,f) H1e–GFP FRAP analysis in euchromatin (e) and heterochromatin (f) of G9a^−/− ESCs in which WT G9a was added-back (G9a-Null-add-WT), undifferentiated (grey) or differentiated with RA for 4 days (black). Changes are significant (P<0.05, 2-tailed t-test). (g,h) H1e–GFP FRAP analysis in euchromatin (g) and heterochromatin (h) of G9a^−/− ESCs in which a mutant form of G9a lacking catalytic activity was added-back (G9a-Null-add-Mut), undifferentiated (grey) or differentiated with RA for 4 days (black). No restriction of chromatin protein dynamics is observed following differentiation. (i,j) H1e–GFP FRAP analysis in G9a^−/− ESCs, undifferentiated (i) and differentiated for 4 days with RA (j), treated (black) or untreated (grey) with 0.5 mM VPA for 24 h. In both cases, changes are significant (P<0.005, 2-tailed t-test). (k) Neuronal differentiation of G9a^−/−, G9a-addback and Suv39h1/2^−/− ESCs. All cells reach the NPC stage at day 7 (top) but the G9a^−/− cells die between day 7 and day 10 while the G9a-addback and the Suv39h1/2^−/− cells continue to differentiate normally. Scale bar, 100 μm. (l) Teratoma analysis in SCID mice injected with WT (top) or G9a^−/− (bottom) ESCs. WT ESCs displayed normal differentiation with apparent endoderm, mesoderm and ectoderm. However, teratomas made from G9a^−/− ESCs were significantly smaller (inset) and did not show neuroectodermal differentiation. Scale bar, 0.5 mm; inset scale bar, 5 mm. Values are from at least 2 independent experiments, at least 20 cells each.