Investigation of the interaction between CTCF and DNA using AFM imaging

CCCTC binding factor (CTCF) is a highly conserved zinc finger protein found in all eukaryotes. Originally it was studied as a traditional transcription factor. Later on, the CTCF binding sites distribution was found to be correlated with gene density, and CTCF was hypothesized to be a global insulator. Recently, the spatial structure of chromatin was shown to play an important role in transcription regulation. CTCF is now considered as a "weaver "of genome because it is able to mediate functional long range DNA contacts at multiple gene loci. Our goal is to understand how CTCF induces such DNA conformation. In our experiment, DNA molecules with~1000 base pairs long are examined. Atomic force microscopy (AFM) is applied to scan and acquire the CTCF-DNA complexes formed in vitro. Our analysis demonstrates that the CTCF-DNA complexes are likely to adopt a loop conformation with a contour length of~160nm. Similar loop conformations are observed when we eliminated the CTCF specific binding site. But when a part of the zinc-finger motifs of CTCF is deleted from the sequence, the loop conformations are no longer observed. We attempt to interpret the results using free energy approach. We quantify the elastic energy of the DNA and the binding energy. We also apply three-dimensional Gaussian model to estimate the entropy contribution to the free energy of the DNA-CTCF system. Based on our analysis we propose that, CTCF can simultaneously bind at multiple sites to the DNA using all11 zinc fingers, and that the loop conformation in the presence of binding is formed as a result of a competition between the entropic contribution favoring small loops and elastic contribution favoring large loops, thereby minimizing the free energy.