Fig. 1

Subnuclear distribution of facultative and constitutive heterochromatin marks in female Microtus cabrerae fibroblasts. a Metaphase chromosomes from female Microtus cabrerae cell line were analyzed with a painting probe from the X chromosome. Arrow points to the heterochromatic block, which occupies the whole short arm of the X chromosome. b Prominent chromatin marks were analyzed by transient transfections and immunostaining. MacroH2A1 and H3K27me3 signals were simultaneously visualized by transient transfection with GFP-macroH2A1 and immunostaining using anti-H3K27me3 antibody (upper row). H3K9me3 and H3K27me3 marks, typical for constitutive and facultative heterochromatin, were simultaneously analyzed by double immunostaining (middle row). HP1 beta and H3K9me3 signals were simultaneously visualized by transient transfection with DsRed-HP1 beta and immunostaining against H3K9me3 (lower row). DNA was counterstained with DAPI. Protein and merge signals of all channels are shown. Single optical sections acquired on a spinning disk confocal microscope are shown. Scale bar 5 µm. c Immunofluorescence detection of histone H3 posttranslational modifications on metaphase chromosomes. Upper row female Microtus cabrerae cells (tetraploid) show two pairs of either H3K27me3 or H3K9me3 decorated giant sex chromosomes. The line plots along the enlarged metaphase sex chromosomes depict the fluorescence intensity distribution of the H3K27me3-enriched sex chromosome (X*) and the H3K9me3-enriched sex chromosome (X). In contrast, in the male Microtus cabrerae cell line (nearly diploid) only one giant X chromosome is found that is enriched in H3K9me3, especially on the long arm. Scale bar 5 µm. d Overview of subnuclear distribution of facultative and constitutive heterochromatin marks in female Microtus cabrerae cell line. Euchromatin is enriched for euchromatic marks such as H3K9ac and H4K8ac, but depleted for heterochromatin marks such as H3K27me3 and H3K9me3. Facultative heterochromatin is enriched for H3K27 trimethylation and macroH2A1. The constitutive heterochromatin is characterized by marks such as H3K9me3 and HP1 beta accumulation