MOESM1 of DNA replication dynamics of vole genome and its epigenetic regulation

Additional file 1. Subnuclear distribution of facultative and constitutive heterochromatin marks in female Microtus cabrerae fibroblasts and transcription at X chromosomes. (A) Prominent chromatin marks were analyzed by immunostaining: H3K4me3, H3K9ac, H4K8ac (euchromatin), HP1 alpha (constitutive heterochromatin), MBD (DNA methylation) and 5-methyl-cytosine (5mC). Since 5mC staining was barely detectable in Microtus cabrerae cells, 5mC staining is also shown in MEF W8 cells where prominent staining at pericentromeric heterochromatin blocks is visible. DAPI-stained DNA (blue), immunostaining (red) and merge of both channels are depicted. A ROI around one heterochromatic block was selected, and a line intensity plot analysis was performed. Red arrows point to characteristic hypoacetylation of heterochromatic blocks. Scale bar 5 µm. (B) Transcriptional activity of heterochromatic blocks was assessed by the colocalization of nascent transcripts (10 minutes pulse of 1 mM EU) with the H3K9me3 and H3K27me3 enriched heterochromatin blocks. Quantification of EU signal within the two respective volumes after 3D segmentation showed significant higher levels in H3K9me3 blocks (***P < 0.001, paired t-test).