Comprehensive mapping of the 3D epigenome by high-content super-resolution image analysis

A full understanding of the relationship between the density and packing arrangements of chromatin in interphase mammalian nuclei and epigenetic function at the nanometer size scale remains an elusive goal in the field of chromatin biology. Over the last years great technical leaps have been made through sequencing-based methods to address this question. Advancements have also allowed research to bypass long-held optical limits to resolve chromatin at a scale where its 3D topology can start to be analysed. The work in this thesis represents attempts at high-throughput data mining of 3D SIM datasets, encoding rich nanometer-scale spatial information from immunofluorescence detection of histone modifications and key epigenetic markers relative to a chromatin landscape. To do so, an automated and high-throughput image analysis workflow (ChaiN , for ChaiN analysis of the in situ Nucleome) was developed. Novel metrics for the quantitation and correlation of chromatin and the 3D epigenome reveal a chromatin network of filaments at the size scale proposed from sequencing approaches, and with segregated regions of genomic activities as a function of chromatin accessibility (its density and depth). Furthermore, ChaiN has allowed characterisation of the local and global rearrangements of chromatin when subjected to replication pressures or exogenous perturbation showing for the first time how individual genomic markers are affected at different locations throughout the chromatin network. The model hypothesised from these results tries to reconcile previous data obtained from Hi-C population ensemble studies and in silico modelling, to single cell observations at super-resolution.