Assessment and site-specific manipulation of DNA (hydroxy-)methylation during mouse corticogenesis

Purpose: In this study we investigated the molecular role of cytosine modification in the developing cortex of mice. To this aim we isolated thee linage related cell populations of the developing cortex and mapped both, 5mC as well as 5hmC on a genome-wide scale. Furthermore we established a system to site-specifcally demethylate DNA by using a dCas9-Tet1 fusion protein. Methods: Neuronal stem cells (Btg2-/Tubb3-), neurogenic progenitors (Btg2+/Tubb3-) and neurons (Tubb3+) were isolated from E14.5 Btg2-RFP/Tubb3-GFP double heterozygous mouse embryos (Aprea et al. 2013) and were used for a comparative immunoprecipitation of 5mC and 5hmC (Tan et al. 2013). Results: We were able to identify several genomic regions that change dynamically (hydroxy-)methylation patterns in specific cell type. Specifically for regions losing 5mC and gaining 5hmC we found a consistent enrichment in neurogenesis related GOterms, genomic features, transcription factor binding motifs and different histone marks. Furthermore we found that loss in 5mC and gain in 5hmC correlate with each other and lead to the upregulation of nearby genes. Site-specific demethylation of the identified differentially (hydroxy-)methylated regions in vivo, led to a premature upregulation of the nearby genes which resulted in an altered development of the brain. Genome-wide maps of 5-methylcytosine as well as 5-hydroxymethylcytosine in three lineage-related cell populations of the developing cortex of mice (E14.5).