Whole-genome 5-hydroxymethylation profiling of human neurons for regulatory and biological insight.

While 5-methylcytosine (5mC) DNA methylation is a well-known player in genome stability and transcriptional regulation, the role of 5-hydroxymethylcytosine (5hmC), particularly prevalent in neurons, remains largely unknown. Here, we used long-read Oxford Nanopore Technology (ONT) to profile whole-genome, native 5mC and 5hmC levels in sorted neuronal nuclei samples from human post-mortem brain tissue. Genomic annotation revealed high 5hmC levels in actively transcribed genes and enhancers, which were enriched in neuron-related pathways, with functional variety when stratifying across chromatin states. Furthermore, 5hmC and 5mC modifications were enriched at transcription factor binding sites, affecting specific downstream regulatory networks. Altogether, our study demonstrates the potential of 5hmC and 5mC DNA modifications to provide biological insight into the regulatory landscape and function of human neurons.