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

Area of Science:

• Molecular Biology
• Epigenetics
• Genomics

Background:

• The epigenome dynamically regulates gene expression through DNA modifications and protein-DNA interactions.
• Current methods for mapping DNA modifications and protein occupancy concurrently face limitations due to overlapping signals and method incompatibility.
• Accurate interpretation of the epigenome requires simultaneous profiling of DNA base modifications and protein-DNA occupancy.

Purpose of the Study:

• To engineer non-CpG-specific DNA carboxymethyltransferases for labeling protein occupancy.
• To assess DNA carboxymethylation as a reporter for protein occupancy in GpC contexts.
• To develop new approaches for multimodal epigenetic profiling.

Main Methods:

• Rational engineering of DNA carboxymethyltransferases with neomorphic activity.
• Characterization of the enzymatic activity and specificity of engineered enzymes.
• Assessment of DNA carboxymethylation compatibility with standard epigenetic detection techniques.

Main Results:

• Successfully engineered non-CpG-specific DNA carboxymethyltransferases.
• Demonstrated that DNA carboxymethylation in GpC contexts reliably reports protein occupancy.
• Showcased broad compatibility of DNA carboxymethylation with established epigenetic profiling methods.

Conclusions:

• Unnatural DNA modifications in bio-orthogonal contexts offer a viable alternative for labeling protein occupancy.
• DNA carboxymethylation provides a compatible readout for multimodal epigenetic profiling.
• This work enables new strategies for comprehensive epigenome analysis.