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bioRxiv · 10.1101/2025.11.24.690163

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

Zhu, R.·Loo, C. E.·Hurley, C. M.·Parker, J. B.·Kohli, R. M.
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Abstract

The epigenome provides a dynamic layer of gene regulatory control above the static genetic sequence. DNA base modifications are key epigenetic regulators, predominantly found within CpG contexts in mammalian genomes. Working in tandem with these DNA modifications, chromatin-associated proteins and transcription factors further control gene expression. Given the interplay of these factors, concurrent mapping of DNA base modifications with protein-DNA occupancy can greatly aid in interpreting the epigenome. Existing multi-modal mapping methods include the use of DNA methyltransferases to mark accessible, protein-unbound DNA in non-CpG contexts. However, such approaches can either confound readouts with native DNA modifications or constrain users to third-generation sequencing approaches. To circumvent these limitations, we explored the possibility of introducing an unnatural DNA base modification, 5-carboxymethylcytosine, as an alternative label for protein occupancy. Here, we report our efforts to rationally engineer non-CpG-specific DNA methyltransferases to take on neomorphic DNA carboxymethyltransferase (CxMTase) activities. We find that DNA carboxymethylation of cytosines in GpC contexts shows broad compatibility with the most widely used epigenetic detection methods and can be used to reliably report on protein occupancy states. Using this approach, we reveal the single-molecule binding patterns of LexA, a master repressor in the bacterial DNA damage (SOS) response, at its self-regulated and endogenously methylated promoter. We thus show that unnatural DNA modifications in bio-orthogonal contexts can uncover biological insights and potentiate new approaches to multimodal epigenetic profiling. TOC GRAPHIC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/690163v3_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1817a46org.highwire.dtl.DTLVardef@182ca09org.highwire.dtl.DTLVardef@19e3572org.highwire.dtl.DTLVardef@1c6e1a2_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Zhu, R., Loo, C. E., Hurley, C. M., Parker, J. B., Kohli, R. M.. 2025-11-25. Mapping Protein Occupancy on DNA with an Unnatural Cytosine Modification in Bio-orthogonal Contexts. https://doi.org/10.1101/2025.11.24.690163

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