Simultaneous global labeling (SiGL) of 5-methylcytosine and 5-hydroxymethylcytosine by DNA alkylation with a synthetic cofactor and engineered methyltransferase

Avraham, S.; schuetz, L.; Kaever, L.; Dankers, A.; Margalit, S.; Michaeli, Y.; Zirkin, S.; Torchinsky, D.; Gilat, N.; Bahr, O.; Nifker, G.; Koren-Michowitz, M.; Weinhold, E.; Ebenstein, Y.

doi:10.1101/2022.10.31.513813

bioRxiv · 10.1101/2022.10.31.513813

Simultaneous global labeling (SiGL) of 5-methylcytosine and 5-hydroxymethylcytosine by DNA alkylation with a synthetic cofactor and engineered methyltransferase

Avraham, S.·schuetz, L.·Kaever, L.·Dankers, A.·Margalit, S.·Michaeli, Y.·Zirkin, S.·Torchinsky, D.·Gilat, N.·Bahr, O.·Nifker, G.·Koren-Michowitz, M.·Weinhold, E.·Ebenstein, Y.

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Abstract

5-methylcytosine and 5-hydroxymethylcytosine are epigenetic modifications involved in gene regulation and cancer. Here, we describe a new, simple, and high-throughput platform for multi-colour epigenetic analysis. The novelty of our approach is the ability to multiplex methylation and de-methylation signals in the same assay. We utilize an engineered methyltransferase enzyme that recognizes and labels all unmodified CpG sites with a fluorescent cofactor. In combination with the already established labelling of the de-methylation mark 5-hydroxymethylcytosine via enzymatic glycosylation, we obtained a robust platform for simultaneous epigenetic analysis of these marks. We assessed the global epigenetic levels in multiple samples of colorectal cancer and observed a reduction in 5-hydroxymethylcytosine levels, but no change in DNA methylation levels between sick and healthy individuals. We also measured epigenetic modifications in chronic lymphocytic leukaemia and observed a decrease in both modification levels. Our results indicate that this assay may be used for the epigenetic characterization of clinical samples for research and patient management.

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Avraham, S., schuetz, L., Kaever, L., Dankers, A., Margalit, S., Michaeli, Y., Zirkin, S., Torchinsky, D., Gilat, N., Bahr, O., Nifker, G., Koren-Michowitz, M., Weinhold, E., Ebenstein, Y.. 2022-10-31. Simultaneous global labeling (SiGL) of 5-methylcytosine and 5-hydroxymethylcytosine by DNA alkylation with a synthetic cofactor and engineered methyltransferase. https://doi.org/10.1101/2022.10.31.513813

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