Peripolli, Silvia; (2021) Oncogenic c-Myc induces DNA replication stress by increasing cohesins chromatin occupancy. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Oncogene-induced Replication Stress (RS) has a major role in driving genomic instability and in promoting both cancer initiation and evolution. Whilst several mechanisms of oncogene-induced RS have been proposed, the exact mechanism still remains unclear. In this thesis, I investigate how the oncogene c-Myc generates RS using a c-Myc inducible cell line, and I propose a new mechanism for oncogene-induced RS. Reported mechanisms of c-Myc-induced RS are based on deregulation of DNA replication initiation events. My data suggests that these mechanisms, involving DNA over- and under-replication, and collisions between the replication and transcription machineries do not have a prominent role in generating RS in the first S-phase upon c-Myc activation. My findings support a model where increased binding of the cohesin complex to DNA in the G1-phase of the cell cycle generates RS in the following S-phase. In fact, preventing an increase in the levels of chromatin-bound cohesin by either depleting cohesin subunits or regulators partially prevents the onset of RS and DNA damage in c-Myc-activated cells. c-Myc activation leads to increased expression of the cohesin loader Mau2 and Establishment of Sister Chromatid Cohesion N-Acetyltransferase 1 (ESCO1), which respectively increase cohesin loading on the DNA and cohesin stabilisation, i.e. stable interaction of cohesins with DNA. I established by Chromatin immunoprecipitation (ChIP)-seq analysis that the cohesin complex binds preferentially to CCCTC-binding factor (CTCF) sites upon c-Myc activation. The accumulation of cohesins at these sites seems to be key in the process of c-Myc-induced RS, since depleting CTCF in c-Myc-activated cells prevents RS. Finally, I investigate whether the biological relevance of this novel mechanism of c-Myc- induced RS in cancer cell lines that display various levels of c-Myc overexpression. My data shows that depleting the loader Mau2 and the cohesin subunit Rad21 partially rescues RS in cancer cells with high levels of c-Myc overexpression, similarly to our c-Myc-inducible cell line. Since c-Myc activation is a crucial event in many human cancers, identifying the mechan- isms through which it promotes RS and genomic instability provides critical insights into cancer biology and therapy.

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