Identifying Genomic DNA Sequences Near the Nuclear Lamina Using Proximity Biotinylation with Ascorbate Peroxidase.

Area of Science:

• Biochemistry
• Molecular Biology
• Genomics

Background:

• Engineered ascorbate peroxidase (APEX2) facilitates proximity-dependent biotinylation of biomolecules.
• Targeting APEX2 to specific subcellular locations, such as the nuclear lamina via Lamin B1 fusion, enables localized labeling.
• Previous methods for identifying DNA proximity to nuclear structures were limited.

Purpose of the Study:

• To develop and detail a protocol for identifying DNA proximal to the nuclear lamina using APEX2-Lamin B1 fusion.
• To characterize lamina-associated domains (LADs) through proximity-based biotinylation.
• To establish a robust method applicable to both living and formaldehyde-fixed cells.

Main Methods:

• Construction of an APEX2-Lamin B1 fusion protein.
• Proximity ligation reaction to biotinylate DNA and proteins near the nuclear lamina.
• Streptavidin bead precipitation of biotinylated molecules for downstream analysis.
• Application of the protocol in both live and formaldehyde-fixed cells.

Main Results:

• The APEX2 proximity ligation reaction proved robust and effective in various cellular states.
• The APEX-Lamin B1 fusion successfully identified DNA in proximity to the nuclear lamina.
• The protocol enabled the characterization of lamina-associated domains (LADs).

Conclusions:

• The APEX2-Lamin B1 fusion protein provides a powerful tool for investigating DNA organization at the nuclear periphery.
• This protocol offers a versatile method for mapping DNA-protein and DNA-DNA interactions within specific subcellular compartments.
• The technique is applicable for identifying genomic regions associated with nuclear structures like LADs.