CTCF-dependent chromatin boundaries formed by asymmetric nucleosome arrays with decreased linker length.

Christopher T Clarkson,Ralph Samarista,Vladimir B Teif,Emma A Deeks,Hulkar Mamayusupova,Victor B Zhurkin

doi:10.1093/nar/gkz908

Christopher T Clarkson, Ralph Samarista + Show 4 more

Open Access

https://doi.org/10.1093/nar/gkz908

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Abstract

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.

Highlights

Nucleosomes are positioned along the genome in a non-random way [1,2,3], which is critical for determining the DNA accessibility and genome organisation [4]
To perform bulk calculations of Nucleosome Repeat Length (NRL) for many genomic subsets of interest we developed software NRLcalc, which loads the phasograms computed in NucTools [43] and performs linear fitting to calculate the NRL
Using coordinates of ChIP-seq peaks of RNA Pol III determined previously in embryonic stem cells (ESC) [53], we found that 33% of co-localisations of TFIIIC and Pol III and 17% of co-localisations of SINE repeats and Pol III overlapped with our CTCF motifs

Summary

Introduction

Nucleosomes are positioned along the genome in a non-random way [1,2,3], which is critical for determining the DNA accessibility and genome organisation [4]. A important nucleosome positioning signal is provided by CTCF, an architectural protein that maintains 3D genome architecture [5,6,7] and can organise up to 20 nucleosomes in its vicinity [8] (Figure 1A). CTCF has ~100,000 potential binding sites in the mouse genome. There are ~30,000-60,000 CTCF sites bound in a given cell type, which translates to about 1 million of affected nucleosomes (up to 10% of the mouse genome) [9,10,11,12]. CTCF is able to act as an insulator between genomic regions with different chromatin states, but how exactly this is achieved is not known. We explore molecular mechanisms of the insulator boundary formation by CTCF through rearrangement of surrounding nucleosome arrays

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