Two CTCF motifs impede cohesin-mediated DNA loop extrusion

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Human cohesin mediates DNA loop extrusion (LE), a fundamental process in genome organization.
• Cohesin stalling at the CCCTC-binding factor (CTCF) is crucial for genome positioning but the underlying mechanism remains elusive.
• The N-terminal region (NTR) of CTCF is implicated in stalling cohesin's DNA loop extrusion activity.

Purpose of the Study:

• To elucidate the molecular mechanism by which CTCF's N-terminal region (NTR) stalls cohesin-mediated DNA loop extrusion (LE).
• To identify specific amino acid motifs within the CTCF NTR responsible for hindering cohesin's extrusion activity.

Main Methods:

• Employed single-molecule assays to directly monitor DNA loop extrusion (LE) dynamics.
• Utilized fragments of the CTCF N-terminal region (NTR) containing specific amino acid motifs (YDF, KTYQR) to probe interactions with cohesin.
• Assessed the impact of these motifs on cohesin's LE step cycles and directionality.

Main Results:

• Identified two critical amino acid motifs in CTCF's NTR: YDF and KTYQR, which impede cohesin's DNA loop extrusion (LE).
• The KTYQR motif completely abolished cohesin's LE activity.
• The YDF motif hindered the completion of LE step cycles and induced unidirectional extrusion by enhancing STAG1-DNA binding.

Conclusions:

• Two distinct motifs within CTCF's NTR, YDF and KTYQR, stall cohesin-mediated DNA loop extrusion (LE) through different yet synergistic mechanisms.
• These findings reveal how CTCF utilizes specific motifs to modulate cohesin's activity, thereby shaping and regulating genome architecture.
• Highlights the complex interplay between CTCF and cohesin in establishing higher-order chromatin structures.