Engineering Epigenetic Memory Requires Both DNA and Histone Methylation

Distinct epigenomic profiles of histone marks have been associated with gene expression, which is tightly regulated during development and important for cell identity. Acquired epigenetic alterations can lead to diseases, such as cancers, cardiovascular disease and mental disorders. It should now be possible to treat such disorders by altering the epigenetic information at specific loci. However, to avoid the need for lifelong expression of an exogenous modifier protein, we need to establish a persistent epigenetic state, as nature does. That way the activity of our modifier could be transient, but its effect on gene expression would be long-lasting. Towards this goal, we have created a dCas9-based toolbox to precisely target and correct epigenetic alterations. We investigated the activity of a broad collection of genomically targeted epigenetic regulators that could write epigenetic marks associated with a repressed chromatin state (G9A, SUV39H1, KRAB, DNMT3A, Ezh2 and FOG1). Individual dCas9 fusions were only able to transiently repress the target gene. Long-term gene silencing required the action of both DNA methylatransferase and histone methyltransferases. Epigenetic switching at the target site was confirmed and the impact of combinatorial epigenome editing on the global epigenetic landscape was evaluated. We then investigated whether combinatorial epigenetic treatment could have a long-term effect for correction of Angelman Syndrome in the mouse brain. Angelman Syndrome is a rare neurologic disorder caused by the lack of maternally expressed UBE3A protein in the brain. A long non-coding RNA (UBE3A-ATS) prevents expression of the paternal UBE3A allele. Repeated delivery of a zinc finger (ZF)-KRAB fusion targeting Ube3a-ATS allowed us to upregulate paternal Ube3a expression in the brain of a mouse model. Towards the goal of a single treatment to upregulate Ube3a, we demonstrate for the first time that combinatorial epi-ZF fusions establish longer-lasting upregulation of Ube3a.