Forced expression of MSR repeat transcripts above a threshold limit breaks heterochromatin organization

Mouse heterochromatin is characterised by transcriptionally competent major satellite repeat (MSR) sequences and it has been proposed that MSR RNA contributes to the integrity of heterochromatin. We established an inducible dCas9-effector system in mouse embryonic fibroblasts, where we can modulate MSR transcription through the targeting of a dCas9-Repressor or a dCas9-Activator. With this system, we can define a threshold limit of >300-fold deregulation of MSR transcript levels, above which the structural organisation of heterochromatin becomes disrupted. MEF cells expressing MSR RNA above this threshold limit are not viable and the defects in heterochromatin organisation and chromosome segregation cannot be reverted. This study highlights the importance of restricting MSR RNA output to maintain heterochromatin integrity and relates MSR transcript levels to either physiological or pathological conditions. It also reveals that the structural organisation of heterochromatin is governed by the transcriptional chromatin state and associated MSR RNA of the MSR repeats. Overall design: To investigate the effect of modulating MSR repeat transcription on repeat dysregulation genomewide. MEF cell lines were created where chromatin effectors can be inducibly expressed. These chromatin effectors are targeted to MSR repeats utilizing the CRISPR/dCas9 system: MSR-dCas9-Control, MSR- dCas9-Repressor, MSR-dCas9-Activator.