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

Author:

Avraham Sigal,Schütz Leonie,Käver Larissa,Dankers Andreas,Margalit Sapir,Michaeli Yael,Zirkin Shahar,Torchinsky Dmitry,Gilat Noa,Bahr Omer,Nifker Gil,Koren-Michowitz Maya,Weinhold Elmar,Ebenstein Yuval

Abstract

Abstract5-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.

Publisher

Cold Spring Harbor Laboratory

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