Comprehensive study of 3D chromatin structure

Abstract

The connection between DNA folding and chromatin conformation has been much studied but the relation between the modulation of chromatin conformation and biological processes such as DNA damage, cell differentiation and DNA replication, still remains unclear. To better understand these mechanisms, an interdisciplinary study combining several technologies is required to provide an integrative view of what happens from the level of nucleosome interactions up to whole genome organisation.

In this thesis, we aimed to set up chromatin conformation methodologies such as Hi-C and Micro-C to analyse the effect of DNA damage and cell differentiation on 3D chromatin structure.

Chromatin structure is affected by DNA damage caused by UV irradiation or oxidative stress. In our study, the effects of UV irradiation and oxidative stress on 3D genome structure were analysed from nucleosome positioning to chromatin conformation. Molecular Dynamics simulation and Coarse grained modelling provided a 3D model of the whole genome of Saccharomyces cerevisiae in normal and stressed conditions.

The conditions of UV irradiation that was applied created a decrease of chromatin interactions increasing chromatin flexibility. In addition, oxidative stress induced chromatin changes with an increase of inter-chromosomal interactions and a reduction of intra-chromosomal interactions. These effects were associated with a decrease in the number of chromatin interaction domains (CID) as well as the insulation score. The reduction of CID is correlated with a reduction of cohesin loading factor complex (SCC2/SCC4) bound to the chromatin upon oxidative stress. In addition, nucleosome contacts were globally reduced, especially in upregulated genes and DNA damaged regions. Concerning nucleosome positioning, a general increase of the fuzziness score was observed both in upregulated and downregulated genes, and in regions of DNA damage in response to oxidative stress.

Overall, our results showed a decrease in the number of nucleosomes and a tendency of the chromatin to be more open at short-range distances while, at long-range distances, the genome seems to be more compact in response to oxidative stress. One hypothesis could be that chromatin decondensation can contribute to facilitate DNA repair. MD simulations were used to build two models: one at the scale of the chromatin fibre and the other one for the whole genome.

Finally, high resolution microscopy combined with Capture-MNase-Seq, Capture-Hi-C and coarse grain models were used to study the conformational changes occurring during cell reprograming from fibroblast to hiPSC. A specific region containing NANOG and STELLA loci as well as GADPH and IFFO1 as control genes was selected. Capture- MNase-Seq and Capture-Hi-C revealed that nucleosomes tend to be fuzzier in the pluripotent hiPSC cells and that the TADs were reorganized during cell differentiation.