Epigenetic rewriting at centromeric DNA repeats leads to increased chromatin accessibility and chromosomal instability.

BACKGROUND: Centromeric regions of human chromosomes contain large numbers of tandemly repeated α-satellite sequences. These sequences are covered with constitutive heterochromatin which is enriched in trimethylation of histone H3 on lysine 9 (H3K9me3). Although well studied using artificial chromosomes and global perturbations, the contribution of this epigenetic mark to chromatin structure and genome stability remains poorly known in a more natural context. RESULTS: Using transcriptional activator-like effectors (TALEs) fused to a histone lysine demethylase (KDM4B), we were able to reduce the level of H3K9me3 on the α-satellites repeats of human chromosome 7. We show that the removal of H3K9me3 affects chromatin structure by increasing the accessibility of DNA repeats to the TALE protein. Tethering TALE-demethylase to centromeric repeats impairs the recruitment of HP1α and proteins of Chromosomal Passenger Complex (CPC) on this specific centromere without affecting CENP-A loading. Finally, the epigenetic re-writing by the TALE-KDM4B affects specifically the stability of chromosome 7 upon mitosis, highlighting the importance of H3K9me3 in centromere integrity and chromosome stability, mediated by the recruitment of HP1α and the CPC. CONCLUSION: Our cellular model allows to demonstrate the direct role of pericentromeric H3K9me3 epigenetic mark on centromere integrity and function in a natural context and opens interesting possibilities for further studies regarding the role of the H3K9me3 mark.

Molecular basis of CTCF binding polarity in genome folding.

Current models propose that boundaries of mammalian topologically associating domains (TADs) arise from the ability of the CTCF protein to stop extrusion of chromatin loops by cohesin. While the orientation of CTCF motifs determines which pairs of CTCF sites preferentially stabilize loops, the molecular basis of this polarity remains unclear. By combining ChIP-seq and single molecule live imaging we report that CTCF positions cohesin, but does not control its overall binding dynamics on chromatin. Using an inducible complementation system, we find that CTCF mutants lacking the N-terminus cannot insulate TADs properly. Cohesin remains at CTCF sites in this mutant, albeit with reduced enrichment. Given the orientation of CTCF motifs presents the N-terminus towards cohesin as it translocates from the interior of TADs, these observations explain how the orientation of CTCF binding sites translates into genome folding patterns.

Single molecule study of non-specific binding kinetics of LacI in mammalian cells.

Many key cellular processes are controlled by the association of DNA-binding proteins (DBPs) to specific sites. The kinetics of the search process leading to the binding of DBPs to their target locus are largely determined by transient interactions with non-cognate DNA. Using single-molecule microscopy, we studied the dynamics and non-specific binding to DNA of the Lac repressor (LacI) in the environment of mammalian nuclei. We measured the distribution of the LacI-DNA binding times at non-cognate sites and determined the mean residence time to be τ(1D) = 182 ms. This non-specific interaction time, measured in the context of an exogenous system such as that of human U2OS cells, is remarkably different compared to that reported for the LacI in its native environment in E. coli (<5 ms). Such a striking difference (more than 30 fold) suggests that the genome, its organization, and the nuclear environment of mammalian cells play important roles on the dynamics of DBPs and their non-specific DNA interactions. Furthermore, we found that the distribution of off-target binding times follows a power law, similar to what was reported for TetR in U2OS cells. We argue that a possible molecular origin of such a power law distribution of residence times is the large variability of non-cognate sequences found in the mammalian nucleus by the diffusing DBPs.