Epigenetic regulation is fundamental to gene expression control and cellular differentiation, with chromatin accessibility, DNA methylation, and hydroxymethylation serving as key transcriptional modulators. These regulatory layers function in a highly dynamic and interdependent manner, shaping the epigenomic landscape that underlies cell fate decisions. However, current approaches often require separate assays to investigate each of these regulators, increasing experimental and analytical costs while limiting insights into their interrelation. To address this, we developed Mx-TOP [1], a novel multiomic method that enables simultaneous profiling of chromatin accessibility, general DNA modification, and hydroxymethylation from a single sequencing library.
Mx-TOP is based on a chemo-enzymatic covalent tagging strategy to selectively label unmodified CG sites, hydroxymethylated cytosines (5hmC), and GC sites within chromatin, preserving base-resolution information through tag-selective TOP-seq [2] sequencing. This integrated approach enhances epigenetic profiling resolution while reducing experimental complexity. To identify open chromatin regions, we applied two peak-calling strategies: seed-and-extend and hidden Markov models, enabling robust detection of accessible genomic loci. Both strategies delineate chromatin accessibility patterns and their interplay with DNA modifications, offering deeper insights into transcriptional regulation.
We validated Mx-TOP through extensive benchmarking, demonstrating its high sensitivity in capturing chromatin accessibility and DNA modification dynamics in mouse embryonic stem cells. We then applied it to study chromatin remodeling and DNA demethylation during in vitro neuronal differentiation, a process characterized by extensive epigenetic reprogramming. Our results revealed decoupling between promoter accessibility and transcription, with gene body 5hmC emerging as a key gene expression modulator. Specifically, higher 5hmC levels within gene bodies correlated with transcriptional activity, even when promoter accessibility remained unchanged or decreased. Additionally, we identified developmentally regulated open chromatin loci resistant to global 5hmC erasure during epigenome reprogramming, suggesting that 5hmC in open chromatin plays a critical role in differentiation.
These findings provide new insights into gene body 5hmC’s functional role in transcriptional regulation and chromatin remodeling, particularly in neuronal lineage commitment. By offering a high-resolution, multi-dimensional view of epigenetic landscapes, Mx-TOP serves as a powerful tool for studying complex gene regulatory networks. Its ability to integrate multiple epigenetic layers within a single framework holds great potential for developmental biology, neuroepigenetics, and disease-related epigenetic reprogramming.