Fig. 2: Perturbed heterochromatin organisation in MEF cells expressing MSR-dCas9-effector components.

a Immuno-DNA FISH analysis for MSR sequences of inducible MSR-dCas9-Control, MSR-dCas9-Repressor and MSR-dCas9-Activator MEF cells. In addition to the DNA-FISH signal, 3D masks were created to quantify changes in sphericity and volume of MSR-positive domains (right panels). MSR-positive domains analysed: MSR-dCas9-Control n = 509 (–dox), n = 469 (+dox); MSR-dCas9-Repressor n = 404 (–dox), n = 371 ( + dox); MSR-dCas9-Activator n = 344 (–dox), n = 254 ( + dox). Box plots shown are centred around the median value with an interquartile range (IQR) defined by the 1st and 3rd quartiles and whiskers extending to 1.5xIQR. Outliers are plotted as individual points. Asterisks indicate statistically significant differences (***, p ≤ 0.001, ****, p ≤ 0.0001, two-sided unpaired Mann–Whitney test). Scale bar is 5 μm. b Double immunofluorescence in inducible MSR-dCas9-Control, MSR-dCas9-Repressor and MSR-dCas9-Activator MEF cells for localisation of dCas9-effector components and HP1α. Nuclei were counterstained with DAPI. Linescans were used to visualise the colocalisation of DAPI (blue), Cas9 (orange), and HP1α (grey) signals as shown in the panels on the right. For each sample n ≥ 30 cells were analysed. Scale bar is 5 μm. c Double immunofluorescence in inducible MSR-dCas9-Control, MSR-dCas9-Repressor and MSR-dCas9-Activator MEF cells for localisation of MSR-dCas9-effector components and initiating RNAPII (Ser5phos) (left panel) or for MSR-dCas9-effector components and H3 panAc (right panel). Nuclei were counterstained with DAPI. For each sample n ≥ 30 cells were analysed. Scale bar is 5 μm.