Mapping Protein Occupancy on DNA with an Unnatural Cytosine Modification in Bio-orthogonal Contexts

The epigenome provides a dynamic layer of gene regulatory control above the static genetic sequence. Epigenetic DNA base modifications are a key regulator of gene expression, which in mammalian genomes predominantly occur in CpG contexts and are disproportionately distributed in regulatory regions like promoters. Chromatin-associated proteins and transcription factors work in tandem with these modifications to further control gene regulation. Given the interplay of these factors, mapping DNA base modifications concurrently with protein-DNA occupancy is therefore critical to interpreting the epigenome. Current multi-modal mapping methods employ DNA methyltransferases that mark accessible protein-unbound DNA in non-CpG contexts. However, the overlap of this exogenous DNA methylation with naturally occurring modifications can confound readouts and significantly limit compatibility with methods to simultaneously read epigenetic states. To circumvent these limitations, we explored the possibility of introducing an unnatural DNA base modification as an alternative label for protein occupancy. Here, we report our efforts to rationally engineer non-CpG-specific DNA carboxymethyltransferases, characterize their neomorphic activity, and assess DNA carboxymethylation as a reporter of protein occupancy on DNA. We find that DNA carboxymethylation of cytosines in GpC contexts shows broad compatibility with the most widely used epigenetic detection methods and reliably reports on protein occupancy state. Our results demonstrate that unnatural DNA modifications in bio-orthogonal sequence contexts, coupled with either chemical or enzymatic deamination, can potentiate new approaches to multimodal epigenetic profiling.