Epigenetic control of DNA replication dynamics in mammals

One of the most critically important processes in any living organism, essential for development and reproduction, is that of the accurate replication of its genome before each cell division. The process of DNA replication can take place millions of times in a single organism and any mistake, if left unrepaired, is potentially transmitted into the next generation. Errors during replication can result in genetic mutations or karyotype aberrations, both of which can lead to disease or death. The duplication of the genome happens in a well-conserved spatio-temporal manner, a phenomenon implicated in development and disease. This fact indicates that DNA replication needs to be tightly regulated. Further, its precise coordination suggests that distinct genomic regions undergo replication at specific times during S-phase. On the other hand, the regulation of replication is a flexible process throughout development and is, therefore, proposed to be controlled epigenetically. However, the complexity of the mammalian nucleus has hampered the elucidation of how chromatin structure can regulate replication timing. In fact, our understanding of the regulation of replication timing in mammals is restricted to only a few studies with, in part, seemingly contradicting results. In the context of the present thesis, I set out to study the epigenetic mechanisms that control DNA replication dynamics in mammalian cells. To this end, I took advantage of the most prominent example of facultative heterochromatin, the epigenetically silenced X chromosome (Xi) of female mammalian cells, as well as of the mouse chromocenters, formed by clusters of constitutive heterochromatin. To study their particular replication dynamics and the epigenetic mechanisms controlling them, I used a set of genetic (conditional) knockouts, chemical inhibitory treatments and differentiation assays. The latter allowed me to control whole-chromosome inactivation and the subsequent establishment of the corresponding replication pattern, as well as to distinguish the contribution of different epigenetic markers in this process. I visualized the epigenetic changes and their effects on the replication program in situ by immunostainings, also in combination with fluorescence in situ hybridization (FISH), confocal and super resolution light microscopy, as well as in vivo by time-lapse microscopy over peri-ods of up to two days. This approach prompted the development of several tools for live-cell analysis. Using established and new tools, I comprehensively assessed the Xi replication dynamics and the effects of modulating different epigenetic modifications of heterochromatin, their cros-stalk and the subsequent effects on DNA replication timing and was able to show that histone hypoacetylation, a common mark of the Xi and chromocenters, is responsible for the delayed initiation in replication of both heterochromatic regions. Consequently, I propose that histone hyperacetylation, probably due to its opening effect on chromatin structure, renders some genomic regions prone to be bound by initiation factors earlier and / or more abundantly. This preferential binding, e.g. by replication initiation factors, would thus lead to earlier and concomitantly more efficient replication origin firing. Moreover, I discuss the causal relation between transcriptional inactivity and synchronous replication dynamics, a common feature of developmentally opposite systems, such as the mammalian Xi and the embryos of flies and frogs