The CCCTC-binding factor (CTCF) organises the genome in 3D through DNA loops and in 1D by setting boundaries isolating different chromatin states, but these processes are not well understood. Here we investigate chromatin boundaries in mouse embryonic stem cells, defined by the regions with decreased Nucleosome Repeat Length (NRL) for ∼20 nucleosomes near CTCF sites, affecting up to 10% of the genome. We found that the nucleosome-depleted region (NDR) near CTCF is asymmetrically located >40 nucleotides 5′-upstream from the centre of CTCF motif. The strength of CTCF binding to DNA and the presence of cohesin is correlated with the decrease of NRL near CTCF, and anti-correlated with the level of asymmetry of the nucleosome array. Individual chromatin remodellers have different contributions, with Snf2h having the strongest effect on the NRL decrease near CTCF and Chd4 playing a major role in the symmetry breaking. Upon differentiation, a subset of preserved, common CTCF sites maintains asymmetric nucleosome pattern and small NRL. The sites which lost CTCF upon differentiation are characterized by nucleosome rearrangement 3′-downstream, with unchanged NDR 5′-upstream of CTCF motifs. Boundaries of topologically associated chromatin domains frequently contain several inward-oriented CTCF motifs whose effects, described above, add up synergistically.

The CCCTC-binding factor (CTCF) organises the genome in 3D through DNA loops and in 1D by setting boundaries isolating different chromatin states, but these processes are not well understood. Here we focus on the relationship between CTCF binding and the decrease of the Nucleosome Repeat Length (NRL) for ~20 adjacent nucleosomes, affecting up to 10% of the mouse genome. We found that the chromatin boundary near CTCF is created by the nucleosome-depleted region (NDR) asymmetrically located >40 nucleotides 5'-upstream from the centre of CTCF motif. The strength of CTCF binding to DNA is correlated with the decrease of NRL near CTCF and anti-correlated with the level of asymmetry of the nucleosome array. Individual chromatin remodellers have different contributions, with Snf2h having the strongest effect on the NRL decrease near CTCF and Chd4 playing a major role in the symmetry breaking. Upon differentiation of embryonic stem cells to neural progenitor cells and embryonic fibroblasts, a subset of common CTCF sites preserved in all three cell types maintains a relatively small local NRL despite genome-wide NRL increase. The sites which lost CTCF upon differentiation are characterised by nucleosome rearrangement 3'-downstream, but the boundary defined by the NDR 5'-upstream of CTCF motif remains.