DNA recognition patterns of the multi-zinc-finger protein CTCF: a mutagenesis study

CCCTC-binding factor (CTCF) is a zinc-finger protein, serving an important part in the genome architecture as well as some biochemical processes. Over 70,000 CTCF binding DNA sites have been detected genome-wide, and most anchors of chromatin loops are demarcated with the CTCF binding. Various protein or RNA molecules interact with DNA-bound CTCF to conduct different biological functions, and potentially the interfaces between CTCF and its cofactors can be targets for drug development. Here we identify the effective region of CTCF in DNA recognition, which defines the exposed CTCF surface feature for the interaction of cofactors. While the zinc-finger region contributes the most in DNA association, its binding affinity varies based on different DNA sequences. To investigate the effectiveness of individual zinc-fingers, the key residues are mutated to inactivate the DNA binding ability, while the finger configuration and the spacing between fingers are preserved. The strategy is proved to be successful, while clear differences are observed in the DNA binding affinities among the 11 finger mutants and the result is consistent to previous studies in general. With the help of inactivated finger mutants, we identify the ineffective fingers and the dominant effective fingers, which form distinctive patterns on different DNA targets. KEY WORDS: CTCF, Zinc-finger, Structure integrity, Mutagenesis, DNA recognition patterns