Targeted DNA methylation of neurodegenerative disease genes via homology directed repair

Abstract

DNA methyltransferases (DNMTs) are thought to be involved in the cellular response to DNA damage, thus linking DNA repair mechanisms with DNA methylation. In this study we present Homology Assisted Repair Dependent Epigenetic eNgineering (HARDEN), a novel method of targeted DNA methylation that utilizes endogenous DNA double strand break repair pathways. This method allows for stable targeted DNA methylation through the process of homology directed repair (HDR) via an in vitro methylated exogenous repair template. We demonstrate that HARDEN can be applied to the neurodegenerative disease genes C9orf72 and APP, and methylation can be induced via HDR with both single and double stranded methylated repair templates. HARDEN allows for higher targeted DNA methylation levels than a dCas9-DNMT3a fusion protein construct at C9orf72, and genome-wide methylation analysis reveals no significant off-target methylation changes when inducing methylation via HARDEN, whereas the dCas9-DNMT3a fusion construct causes global off-target methylation. HARDEN is applied to generate a patient derived iPSC model of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) that recapitulates DNA methylation patterns seen in patients, demonstrating that DNA methylation of the 5′ regulatory region directly reduces C9orf72 expression and increases histone H3K9 tri-methylation levels.

Large-scale manipulation of promoter DNA methylation reveals context-specific transcriptional responses and stability

“…Even in downregulated promoter-DMRs, we do not observe a major reconfiguration of nucleosome positioning around the TSS, which clearly represents a roadblock to methylation induction strategies relying on fusion DNMTs with either CRISPR or ZF effectors. These challenges are more likely to be bypassed through epigenome editing approaches relying on in vitro methylation coupled to homologous recombination [ 78 , 79 ], or simultaneous recruitment of other factors that alter nucleosome positioning. Additionally, we show that methylation of the primary promoter of many genes leads to activation of intragenic secondary promoters, which needs to be accounted for when designing promoter-silencing approaches.…”

Staying true to yourself: mechanisms of DNA methylation maintenance in mammals

“…A situation we have not discussed is that of DNA repair. When a DNA break is repaired by homologous recombination, a long tract of hemi-methylated DNA is generated, and it must be converted back to fully methylated DNA ( 165 ). It is unknown if the same events govern replication-coupled DNA methylation maintenance, and repair-coupled maintenance.…”

Roles of physical exercise in neurodegeneration: reversal of epigenetic clock

“…Hexanucleotide repeat expansion of C9orf72 causes downstream molecular aberrations and leads to overt cellular toxicity [ 167 ]. C9orf72 promoter hypermethylation induces transcriptional silencing of C9orf72, which could maintain motor neuronal survival and serve as an endogenous protective regulator of neuropathology [ 167 , 168 ]. In conclusion, most neurodegenerative diseases are related to epigenetic changes (i.e., hypomethylation and hypermethylation) in pathogenesis.…”