Pairwise Proximity-Differentiated Visualization of Single-Cell DNA Epigenetic Marks

Area of Science:

• Epigenetics and Molecular Biology
• Single-cell analysis
• Biomolecular imaging

Background:

• Understanding the spatial organization of DNA epigenetic marks is crucial for deciphering cellular functions.
• Current methods for visualizing multiple epigenetic marks like 5-formylcytosine (5fC) and 5-hydroxymethylcytosine (5hmC) at the single-cell level are limited.
• The spatial proximity of these marks is poorly understood but potentially key to chromatin regulation.

Purpose of the Study:

• To develop a novel technique for visualizing the pairwise proximity of 5fC and 5hmC in single cells.
• To enable differentiated imaging of individual epigenetic marks and their spatial relationships.
• To provide multi-level subcellular information for understanding chromatin modification mechanisms.

Main Methods:

• Sequential labeling and crosslinking of 5fC and 5hmC with DNA primer probes using click chemistry.
• A pairwise proximity-differentiated mechanism to encode proximal and residual sites with unique circularized barcodes.
• Simultaneous amplification of barcodes for multiplexed single-molecule imaging.

Main Results:

• Successful differentiated visualization of 5fC and 5hmC spatial positioning within single cells.
• Demonstration of the pairwise proximity of 5fC and 5hmC sites.
• Generation of multi-level subcellular information regarding chromatin modifications.

Conclusions:

• The developed method allows for unprecedented visualization of epigenetic mark proximity in single cells.
• This spatial information can elucidate the regulatory functions and mechanisms of chromatin modifications.
• Insights into potential crosstalk or interactions between reader proteins of 5fC and 5hmC can be gained.