Base-resolution profiling of active DNA demethylation using MAB-seq and caMAB-seq

Area of Science:

• Epigenetics
• Molecular Biology
• Genomics

Background:

• Understanding ten-eleven translocation (TET) dioxygenase function in DNA demethylation requires precise mapping of oxidized 5-methylcytosine (5mC) bases.
• Standard bisulfite sequencing (BS-seq) struggles to distinguish unmodified cytosine (C) from 5fC and 5caC, hindering the study of active demethylation.
• Existing subtraction-based mapping methods for 5fC and 5caC are less efficient and robust.

Purpose of the Study:

• To develop novel, high-resolution methods for quantitatively mapping oxidized 5mC bases, specifically 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC).
• To overcome the limitations of standard BS-seq in distinguishing these key epigenetic marks.
• To provide more efficient and statistically robust tools for studying DNA demethylation.

Main Methods:

• Methylase-assisted bisulfite sequencing (MAB-seq) was developed, enzymatically converting unmodified C to 5mC for direct mapping of 5fC and 5caC.
• caMAB-seq was created by combining MAB-seq with chemical reduction of 5fC to 5-hydroxymethylcytosine (5hmC) for direct 5caC mapping.
• MAB-seq and caMAB-seq were adapted for whole-genome (WG-MAB-seq), ChIP-enriched (ChIP-MAB-seq), and reduced-representation (RR-MAB-seq) analyses.

Main Results:

• MAB-seq and caMAB-seq enable base-resolution mapping of 5fC and 5caC.
• These methods require less sequencing effort compared to subtraction-based approaches.
• Robust statistical calling of 5fC and/or 5caC is achieved, facilitating the study of active DNA demethylation.

Conclusions:

• MAB-seq and caMAB-seq represent significant advancements in the quantitative mapping of oxidized 5mC bases.
• These methods provide efficient, robust, and adaptable tools for epigenomic research, particularly in studying DNA demethylation.
• The protocols are relatively rapid, completable within 6-8 days, accelerating research timelines.