DNA fragility at topologically associated domain boundaries is promoted by alternative DNA secondary structure and...

Area of Science:

• Genomics
• Molecular Biology
• Biochemistry

Background:

• CCCTC-binding factor (CTCF) binding sites are implicated in genome instability.
• The interplay of factors contributing to CTCF site fragility remains incompletely understood.
• Previous studies have not holistically investigated mechanisms of DNA fragility at CTCF sites.

Purpose of the Study:

• To elucidate the mechanisms underlying DNA double-strand break (DSB) enrichment at CCCTC-binding factor (CTCF) binding sites.
• To investigate the role of alternative DNA secondary structures and topoisomerase II (TOP2) in CTCF site fragility.
• To determine the impact of CTCF loss on genome instability, particularly at topologically associated domain (TAD) boundaries.

Main Methods:

• Genome-wide analysis of DNA double-strand breaks (DSBs) in relation to CTCF binding sites across five human cell types.
• Computational identification of energetically favorable alternative DNA secondary structures at CTCF binding sites.
• Assessment of topoisomerase II (TOP2) cleavage complex localization.
• Experimental manipulation via CTCF knockdown to evaluate its effect on DSBs at CTCF sites and TAD boundaries.
• Analysis of G-quadruplex enrichment at CTCF sites affected by knockdown.

Main Results:

• DSBs are significantly enriched at strong CTCF binding sites, but not weak ones.
• Alternative DNA secondary structures are prevalent at strong CTCF binding sites and correlate with TOP2 cleavage complex sites.
• CTCF knockdown leads to increased DSBs at strong CTCF binding sites and at TAD boundary sites.
• CTCF sites at TAD boundaries that lose CTCF upon knockdown show higher DSB levels compared to gained sites.
• Lost CTCF sites at TAD boundaries are enriched for G-quadruplexes, suggesting a role in insulation.

Conclusions:

• Alternative DNA secondary structures recruit TOP2 to CTCF binding sites, contributing to DNA fragility.
• CTCF plays a crucial role in maintaining genome stability at its binding sites and TAD boundaries.
• G-quadruplex structures may function as boundary insulators in the absence of CTCF, but also contribute to DSBs.
• This study provides a mechanistic model for DNA fragility at CTCF binding sites involving DNA structure and TOP2 activity.