Juan Ouyang, Kaan Okay, Katrín Möller, Kasper Daniel Hansen and Hans Tómas Björnsson
Introduction: Mutations in epigenetic machinery (EM) components often lead to intellectual disability (ID) and growth retardation. Mendelian disorders of Epigenetic Machinery (MDEM) occur because of mutations in these EM factors. Many of these disorders have specific DNA methylation (DNAm) signatures in peripheral blood cells. This project aims to uncover DNAm signatures for 60 different EM genes in neuronal progenitors (NPCs) as a proxy for disrupted chromatin status at critical regulatory sites that mediate phenotypic overlap among MDEM.
Methods: 37 EM genes were individually knocked out (KO) with CRISPR-Cas9 in NPCs and DNA epi-signatures were extracted using Oxford Nanopore Technology (ONT) sequencing data.
Results: We have identified hundreds of differentially methylated CpG regions (DMRs) by comparing individual EM gene knockouts and controls. Interestingly, some shared DMRs (41) were identified by overlapping multiple different EM KOs, and a subset of these shared DMRs appear to occur at functionally important regions as they occur at regulatory regions and the observed DNAm changes are associated with changes in gene expression. In addition, a subset of our knockouts appears to lead to abnormalities in normal differentiation rate (Kmt2a, Dnmt1 and Chd3) which we have subsequently validated by Western blot.
Conclusions: We have developed a high-throughput strategy to identify shared DNAm abnormalities in EM-deficient cells using CRISPR-Cas9 and ONT-sequencing. We find an enrichment of regulatory material among shared sites. Interestingly, our results have also led to the discovery of several genes for which loss alters differentiation rate during neurodevelopment.
