Abstract
Epigenetic modifications govern chromatin dynamics and cell state. However, current methods cannot simultaneously resolve the presence of multiple DNA modifications at co-occurring chromatin-associated features. It is thus not clear how these features are physically coupled and how their combinations regulate genome function. To address this key question, we report 6-base-CUT&Tag, a method for simultaneous 6-base DNA sequencing at target chromatin features. Using 6-base-CUT&Tag to profile 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) at co-occurring histone modifications in mouse embryonic stem cells (mESCs), we identify feature-dependent 5mC/5hmC signatures previously unresolvable with untargeted or bisulfite-based workflows. We show that DNA methylation and hydroxymethylation are specifically coupled with the H3K4me1 mark in mESC enhancers and that H3K4me1-derived signatures robustly distinguish different enhancer functional states.
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Data availability
Sequencing data generated in this study is available through NCBI Gene Expression Omnibus under accession code GSE296587, with no access restrictions. Publicly available mouse ESC CUT&Tag-BS data were obtained from GEO under accession code GSE179266 and ChIP-seq datasets were obtained from GEO under accession code GSE89211. Source data are provided with this paper.
Code availability
Code is available on GitHub at https://github.com/sblab-informatics/6B-CUTnTag, with no access restrictions.
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Acknowledgements
We thank staff from the Research Instrumentation and Cell Services and the Genomics core facilities at the Cancer Research UK Cambridge Institute for research support. We thank previous and former members of the Balasubramanian group for helpful discussions on the development and application of 6B-C&T, especially Alice Dubois-Veltman, Zutao Yu, Sean M Flynn, and Larry Melidis. The Balasubramanian laboratory is supported by CRUK core funding (SEBINT-2024/100003 to S.B.), CRUK programme funding (C9681/A29214 to S.B.), and University of Cambridge Herchel Smith funds (S.B.). S.B. is a Wellcome Trust Investigator (209441/Z/17/Z).
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R.C.A.T., D.T., and S.B. conceived the study. R.C.A.T. led the study, developed and optimised the 6B-C&T workflow, with wet-lab and computational input from S.D., J.M., M.T., P.G., A.C-U., and W.G. S.D. performed bioinformatic analyses with input from J.M. X.H. performed the machine learning analysis. All authors contributed to interpreting the results and provided critical feedback. R.C.A.T. created the figures with input from S.D., X.H. and J.M. R.C.A.T., D.T., and S.B. wrote the manuscript with contributions from all authors.
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S.B. is a founder and shareholder of biomodal Ltd, GenomeTx and RNAvate Ltd and a science partner and paid adviser to Ahren Innovation Capital LLC. J.M., M.T., P.G., A.C-U., and W.G. are employees of biomodal Ltd. The remaining authors declare no competing interests.
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Araujo Tavares, R.d.C., Dhir, S., He, X. et al. Sequencing DNA methylation and hydroxymethylation at co-occurring chromatin features. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69429-6
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DOI: https://doi.org/10.1038/s41467-026-69429-6
