A genome-wide map of CTCF multivalency redefines the CTCF code.

Area of Science:

• Genomics
• Molecular Biology
• Epigenetics

Background:

• The "CTCF code" hypothesis suggests CTCF's diverse functions arise from combinatorial use of its 11 zinc fingers (ZFs) to recognize various DNA sequences.
• Previous evidence relied on limited in vitro binding studies, necessitating in vivo validation.

Purpose of the Study:

• To investigate the in vivo binding requirements and multivalency of CTCF across thousands of genomic sites.
• To elucidate how CTCF's zinc fingers contribute to sequence recognition and chromatin association.

Main Methods:

• Defined CTCF zinc finger binding requirements at approximately 50,000 genomic sites in primary lymphocytes.
• Analyzed DNA sequence motifs and their association with CTCF occupancy and stability.

Main Results:

• CTCF recognizes DNA sequence diversity through specific zinc finger (ZF) clustering.
• ZFs 4-7 anchor CTCF to the core motif at most targets; ZFs 1-2 and 8-11 recognize flanking sequences, stabilizing binding.
• A second upstream motif recognized by ZFs 9-11 was identified at 15% of sites.
• A downstream motif was found to destabilize CTCF occupancy.

Conclusions:

• CTCF utilizes a combinatorial clustering of its 11 zinc fingers to associate with a wide array of DNA modules.
• Individual ZFs contribute to binding affinity and chromatin residence time.
• The study reveals novel DNA motifs influencing CTCF binding dynamics, including a destabilizing element.