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Abstract

Whole genome doubling (WGD) events are drivers of genetic innovation across vertebrate evolution. While generally detrimental to mammalian organisms, WGDs are crucial in the development of various plants and fungi, as well as for the terminal differentiation of some mammalian cell types. WGD is also a recurrent genetic abnormality supporting cancer growth through its ability to promote chromosomal instability and aneuploidy. However, the chromatin organisation of WGD cells and its impact on tumour development and evolution have yet to be understood. In the interphase nucleus, the DNA is organised in a multi-layered 3D structure which includes chromosomal territories, chromatin compartments, chromatin domains, and loops, all of which are associated with chromatin activity. A myriad of genetic and structural abnormalities can alter this organisation, leading to aberrant phenotypes which sustain tumorigenesis. In this thesis, I present my work focused on how the chromatin structure responds to WGD events. More specifically, the study investigated how the 3D chromatin is organised in cells that underwent WGD and whether these changes contributed to tumorigenesis. We showed that in cells with a defective p53-dependent tumour suppressor pathway, WGD leads to loss of chromatin segregation (LCS), a phenomenon defined by increased contacts between otherwise well segregated chromatin structures. We detected LCS at the level of chromosomal territories, chromatin compartments, and topologically associating domains. LCS was prompted by the cells' inability to upscale production of the architectural protein CTCF and the histone mark H3K9me3, both involved in chromatin structure maintenance. Activation of a p53-dependent tetraploid checkpoint or temporarily preventing the cell cycle progression restored protein production and resolved LCS in post-WGD cells. Importantly, using single cell chromatin conformation capture, we confirmed that LCS is displayed by single cells and it is not a feature emerging from increased heterogeneity of chromatin conformations after WGD. Additionally, while WGD is linked to chromosomal instability, the latter cannot induce LCS by itself. Longitudinal analyses demonstrated that post-WGD cells are able to form soft-agar colonies in vitro, as well as tumours in vivo. In both cases, the tumorigenic cells presented aneuploid karyotypes, as well as repositioning of chromatin regions to different compartments compared to non-WGD cells. These compartment repositioning events were independent of chromosomal alterations, but were displaying epigenetic and transcriptional reprogramming affecting oncogenic loci. Remarkably, across experiments, the compartment repositioning events could be traced back to concordant changes already present in cells immediately after WGD, preceding the acquisition of chromosomal alterations, suggesting that regions of chromatin can be primed for repositioning upon tetraploidisation. In conclusion, our study reveals that the oncogenic abilities of WGD are fuelled not only by induction of chromosomal instability as previously demonstrated, but also by altering chromatin organisation through loss of chromatin segregation and compartment repositioning.

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