Fig. 5: RNA transcription and hyperacetylated chromatin drive heterochromatin disruption.

a Western blot analysis for expression of MSR-dCas9-Activator in MEF cells (see flow diagram for induction and inhibition conditions). GAPDH expression is shown as a loading control. b RT-qPCR analysis for MSR transcripts in MEF cells treated as in (a). Values were normalised to 5S RNA and are relative to MSR-dCas9-Control (–dox, –DRB) (mean ± SD) (****, p ≤ 0.0001, one-way ANOVA, Tukey’s test). n = 3 independent experiments. c Confocal imaging of DAPI-dense regions in MSR-dCas9-Activator MEF cells that were treated as in (a). Scale bar is 5 μm. The percentages reflect the fraction of cells with either undispersed (white) or dispersed (yellow) DAPI-dense regions. For each condition n ≥ 200 cells were analysed from n = 3 independent experiments. Quantification of the imaging data is shown on the right (mean ± SD). d Western blot analysis for expression of MSR-dCas9-Activator and pan-acetylated H3 in MEF cells (see flow diagram for induction and inhibition conditions). GAPDH and H3 expression are shown as loading controls. e RT-qPCR analysis for MSR transcripts in MEF cells treated as in (d). Values were normalised to Hprt and are relative to the MSR-dCas9-Control (-dox,-A-485) (mean ± SD). n = 3 independent experiments. f Immunofluorescence of MSR-dCas9-Activator MEF cells treated as in (d). Cells were immunolabelled for Cas9 and pan-acetylated H3 and counterstained with DAPI. Scale bar is 5 μm. The percentages reflect the fraction of cells with either dispersed (yellow) or undispersed (white) DAPI-dense regions. For each condition n ≥ 135 cells were analysed from three independent experiments. Quantification is shown on the right (mean ± SD). g Confocal imaging of DAPI-dense regions after combined inhibition of RNAPII and HAT enzymes in induced MSR-dCas9-Activator MEF cells (see flow diagram). Scale bar is 5 μm. The percentages reflect the fraction of cells with either dispersed (yellow) or undispersed (white) DAPI-dense regions. The total number of cells analysed per condition are as follows: (–DRB,–A-485) n = 159, (–DRB,1 µM A-485) n = 197, (–DRB,5 µM A-485) n = 172, (–DRB,10 µM A-485) n = 205, ( + DRB,–A-485) n = 157, ( + DRB,1 µM A-485) n = 186, (–DRB,5 µM A-485) n = 172, (–DRB,10 µM A-485) n = 220. n = 3 independent experiments. Quantification of the imaging data is displayed on the right (mean ± SD) (**, p ≤ 0.0044, two-way ANOVA, Šídák’s test).