DNA strand asymmetry generated by CpG hemimethylation has opposing effects on CTCF binding

Area of Science:

• Epigenetics
• Molecular Biology
• Genomics

Background:

• CpG methylation is crucial for mammalian development and differentiation.
• Hemimethylation was previously considered a transient DNA synthesis state.
• Heritable hemimethylation at CCCTC-binding factor (CTCF) sites suggests a potential biological role.

Purpose of the Study:

• To investigate the impact of DNA strand-specific methylation on CTCF binding.
• To elucidate the mechanism by which CTCF senses hemimethylation.
• To explore the functional implications of hemimethylation in CTCF-mediated chromatin interactions.

Main Methods:

• In vitro assays measuring CTCF binding to methylated and unmethylated DNA motifs.
• Site-directed mutagenesis to identify key residues in CTCF involved in methylation sensing.
• Analysis of strand-specific methylation effects on CTCF binding affinity and kinetics.

Main Results:

• CpG methylation on the CTCF motif strand inhibits binding (up to 7-fold).
• CpG methylation on the opposite strand stimulates binding (up to 4-fold).
• Hemimethylation can alter CTCF binding by up to 28-fold, depending on strand and motif. V454 and S364 in CTCF zinc fingers are critical for sensing opposite-strand methylation. Hydroxymethylation shows similar, but distinct, effects.

Conclusions:

• Strand-specific DNA methylation states significantly modulate CTCF binding affinity.
• CTCF utilizes specific residues to sense methylation on the opposite DNA strand.
• This strand-specific regulation by hemimethylation offers a mechanism for dynamic CTCF-mediated chromatin interactions.