Fig. 2

DNA replication dynamics and epigenetic constitution of facultative and constitutive heterochromatin in female Microtus cabrerae fibroblasts. a Schematic representation of the experimental strategy: Asynchronously growing cultures of female Microtus cabrerae cells were transfected with CFP-PCNA plasmid and pulse labeled with 10 μM EdU for 20 min. This nucleotide pulse was followed by a 1-h chase before fixation with 4% paraformaldehyde and methanol. EdU was detected with the ClickIT system with Alexa488 followed by immunostaining against H3K27me3 (facultative heterochromatin), H3K9me3 (constitutive heterochromatin) and PCNA to visualize ongoing replication at the time of fixation. This strategy allowed us to determine two time points of S-phase in every cell, which are 1 h apart determined by the chase period. The combination with the two histone mark antibody staining permits then the identification of the replication order of the chromatin marked with one versus the other histone modification. The data indicated that the H3K27me3-enriched heterochromatic block is replicated prior to the H3K9me3-enriched heterochromatic block. Maximum intensity projections of confocal images are shown. Scale bar 5 μm. b Live images from female Microtus cabrerae cells triple transfected with CFP-PCNA, GFP-macroH2A1 and DsRedHP1 beta. Maximum intensity projection of z-stacks acquired on a spinning disk confocal microscope at 20-min time intervals. Yellow arrows denote ongoing replication of the heterochromatic blocks of the chromosomes. Exemplary images depict three distinct PCNA patterns, which can be assigned to three different types of chromatin. In early S-phase, a multitude of small replication foci was distributed throughout the whole nucleus, excluding the nucleolus, when euchromatin was replicated. In mid-S-phase, the replication foci became more organized and a perinuclear pattern arises, with foci at the nucle(ol)ar periphery. A first big blob was identified colocalizing with the heterochromatic block of the X chromosome, which is enriched for macroH2A1, whereas in late S-phase the replication foci were consolidated together in large blobs of replication foci colocalizing with the heterochromatic block of the X chromosome, which is enriched in HP1 beta. Both heterochromatic blocks were replicated later than the euchromatin. The orientation of the cell at the end of the time lapse is different due to cell movement over the hours of imaging. Scale bar 5 μm. For full time-lapse, see Additional file 2 Movie 1. c Summary of the replication timing of the H3K9me3- (X) and the H3K27me3 (X*)-enriched heterochromatic blocks of the X chromosomes