Spreading of Chromatin-Mediated Gene Silencing and Reactivation in Mammalian Cells

Chromatin regulators can modify the epigenome through post-translational modifications of histones. Certain modifications have been shown to spread along the chromatin fiber, establishing heterochromatin over large genomic distances. One such modification is histone methylation resulting from recruitment of the Kruppel associated box (KRAB) repressor.However, CRISPRi technology, a commonly used epigenetic tool for targeted silencing with KRAB, is reported to be highly specific to the single gene targeted. Therefore, it is unclear what rules are controlling the spreading of silencing modifications and their epigenetic memory. To determine these rules, we engineered a two-gene reporter system that allows us to target one of the genes with a chromatin regulator and measure the silencing and reactivation dynamics of both genes. This synthetic inducible silencing system, consisting of two fluorescent reporter genes separated by DNA spacers varying in length between from 0 bp (no spacer) to 5000 bp, allowed us to measure the physical extent of spreading. We induce recruitment of KRAB near the upstream gene with addition of doxycycline, and measure spreading of gene silencing and reactivation dynamics between these genes with flow cytometry and time-lapse microscopy. We observed spreading of silencing between the two genes in response to KRAB recruitment for all lengths of spacers we tested: zero, 250 bp, and 5000 bp. Interestingly, both silencing and reactivation of the two fluorescent reporters are highly correlated in the zero and 250 bp spacer cell lines, taking place at the same time in a given cell. When the genes are separated by 5000 bp, the silencing and reactivation of the two genes become less correlated. These results provide additional evidence for spreading of silencing and activation in mammalian cells for distances up to 5000 bp and suggest that epigenetic editing in mammalian cells allows simultaneous control of genes in close proximity.