Fig. 3: Upregulated MSR repeat transcripts remain associated with MSR-targeted dCas9-Activator domains and form RNA:DNA hybrids.

a Immuno-RNA FISH in inducible MSR-dCas9-Control, MSR-dCas9-Repressor and MSR-dCas9-Activator MEF cells for localisation of MSR-dCas9-effector components and forward (purine-rich) MSR repeat transcripts (left panel) or for MSR-dCas9-effector components and reverse (pyrimidine-rich) MSR repeat transcripts (right panel). Nuclei were counterstained with DAPI. In parallel, cells were incubated with RNase A ( + RNase A) prior to MSR probe hybridisation. For each sample n ≥ 60 cells were analysed. Scale bar is 5 μm. b RNA:DNA hybrid dot blot with the S9.6 antibody on nucleic acid samples from MSR-dCas9-Control, MSR-dCas9-Repressor and MSR-dCas9-Activator MEF cells uninduced or induced with doxycycline (dox). Samples were also treated with or without RNase H to determine RNase H sensitivity of the immunoprecipitated nucleic acids. The membrane was then stained with SYBR Gold to quantify total nucleic acids. As a control for RNase H activity, a cDNA sample was included. The histogram on the right is the quantification of the S9.6 signal from the dot blot normalised to the SYBR Gold signal (mean ± SD). n = 3 independent experiments. Asterisks indicate statistically significant differences (**, p ≤ 0.0019, two-way ANOVA, Šídák’s test). c RNA:DNA hybrid immunoprecipitation (RDIP) with the S9.6 antibody on genomic DNA from MSR-dCas9-Control, MSR-dCas9-Repressor and MSR-dCas9-Activator MEF cells uninduced or induced with doxycycline (dox). Samples were treated with or without RNase H to determine RNase H sensitivity of the immunoprecipitated nucleic acids. RNA: DNA hybrids were quantified by qPCR using primers against MSR, minor satellite repeats and LINE-1 (L1Md_A) repeats. For each histogram, values were normalised relative to MSR-dCas9-Control (–dox, –RNase H) (mean ± SD). n = 3 independent experiments. Asterisks indicate statistically significant differences (*, p ≤ 0.0102, ***, p ≤ 0.001, ****, p ≤ 0.0001, two-way ANOVA, Šídák’s test).