Forced expression of MSR repeat transcripts breaks heterochromatin organization

Mouse heterochromatin is characterized by transcriptionally competent major satellite repeat (MSR) sequences and it has been proposed that MSR repeat RNA contributes to the integrity of heterochromatin. We established an inducible dCas9-effector system in mouse embryonic fibroblasts, where we can target dCas9-VPR (transcriptional activator) and dCas9-KM (transcriptional repressor) to MSR repeat sequences. We show that induction of dCas9-VPR forces significant MSR RNA output, while induction of dCas9-KM silences MSR transcription. Both approaches perturb heterochromatin organization, where the dCas9-VPR leads to an immediate dispersion of heterochromatin and dCas9-KM induction results in a delayed aggregation of heterochromatin. MEF cells with the forced expression of MSR RNA are not viable and the defects in heterochromatin organization cannot be reverted. This study highlights the importance of restricting MSR RNA output to maintain heterochromatin integrity and also reveals that the structural organization of heterochromatin is governed by the transcriptional state of the underlying MSR repeats. 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: dCas9-control, dCas9-KM (transcriptional repressor), dCas9-VPR (transcriptional activator).

小鼠异染色质以具备转录活性的主要卫星重复序列（major satellite repeat, MSR）为特征，已有研究提出MSR重复RNA对维持异染色质完整性具有重要作用。本研究在小鼠胚胎成纤维细胞（mouse embryonic fibroblasts, MEF）中构建了诱导型dCas9效应器系统，可将dCas9-VPR（转录激活因子）与dCas9-KM（转录抑制因子）靶向结合至MSR重复序列。实验结果显示，诱导dCas9-VPR表达可显著提升MSR RNA的转录水平，而诱导dCas9-KM表达则会沉默MSR的转录过程。两种操作均会干扰异染色质的组织架构：dCas9-VPR诱导会快速引发异染色质分散，而dCas9-KM诱导则会延迟异染色质聚集。过表达MSR RNA的MEF细胞无法存活，且异染色质组织的缺陷无法被逆转。本研究凸显了限制MSR RNA转录水平对维持异染色质完整性的关键意义，同时揭示了异染色质的结构组织受其所在区域MSR重复序列的转录状态调控。为探究调控MSR重复序列转录对全基因组范围内重复序列失调的影响，本研究构建了可诱导表达染色质效应因子的MEF细胞系。这些染色质效应因子可通过CRISPR/dCas9系统靶向结合至MSR重复序列，包括dCas9-对照（dCas9-control）、dCas9-KM（转录抑制因子）与dCas9-VPR（转录激活因子）。