Fig. 5. Histone methylation patterns are disturbed in the absence of Ezh2. (A) Histone H3-me₃-K9 modification is present mainly in pericentric heterochromatic regions and can be detected as several foci in all cells of controls (left). The number and intensity of these foci is highly reduced in most cells of PG^-/- embryos (far-right). (B) Histone H3-me₂-K27 accumulates to one region in cells of the TE and has a stronger overall staining in cells of the presumptive ICM (left). By contrast, this accumulation is not detectable in PG^-/- embryos (far right). (C) Histone H3-me₃-K27 also accumulates in TE cells of control embryos, which is abolished in PG^-/- blastocysts (not shown). Eed accumulation (in green) co-localises with H3-me₃-K27 (in red) in TE cells of controls, presumably at the X_i, whereas cells of the ICM show a bright staining throughout the nucleus. DNA is counterstained with Toto3 (blue). The insets show a TE cell at a higher magnification with a clear co-localisation of Eed and H3-me₃-K27. (D) Fluorescence in-situ hybridisation (FISH) using a mouse X chromosome specific paint (red) combined with immunofluorescence shows that H3-me₃-K27 (green) is associated with one X chromosome in a TE cell at the blastocyst stage. DNA is counterstained with Toto3 (blue). Metaphase chromosomes stained for H3-me₃-K9 and H3-me₁-K27 stained very brightly in control embryos and in PG^-/-, owing to a higher accessibility of antibodies to metaphase chromosomes in early embryos. (E) Eed (green) accumulation at the X_i does not co-localise with H3-me₃-K9 (red) in TE cells of controls. (F) Metaphase chromosomes in control embryos stain brightly for H3-me₃-K9 in the pericentric heterochromatin (left, yellow staining). At later stages of development (9.5dpc), when Ezh2/Eed do not co-localise to the X_i any more (not shown), H3-me₃-K9 specific antibodies stain one entire chromosome (white arrow head) in addition to pericentric heterochromatin in female embryos (middle). By contrast, me₂- (and me₃-) K27 is highly associated with one chromosome in control embryos at the blastocyst stage (right, white arrowheads), as well as in 50% of wild-type embryos at the blastocyst stage (not shown). D-F show TE cells in interphase (D,E) or metaphase (F) of normal fertilised embryos.