title: Cytosine Base Modifications, DNA Metabolism and Development
Creators: name: Rausch, Cathia
Publisher: name: UNSPECIFIED
Year: 2021

description: While the basic composition of the DNA double helix is known for decades, there is increasing evidence for the stable presence of modified DNA bases in the genome. It is also well known that the majority of our genetic material consists of non-coding and repeat DNA which have important functions in genome regulation, maintenance and organization. Importantly, to avert genome instability through the process of (epi)genome duplication, the underlying processes are tightly regulated in space and time. Whereas many studies correlate replication timing and chromatin landscapes of non-repeat DNA, repetitive regions are difficult to map and, thus, are commonly excluded in genome-wide assays. To overcome this drawback, we developed a protocol to simultaneously visualize DNA replication (or DNA repair) and specific DNA repeat elements in individual and structurally intact 3-dimensional cell nuclei (Repli-FISH). Making use of this single cell microscopy approach in human somatic cells, we find that the euchromatic Alu element (short interspersed nuclear element (SINE)) is replicated early during the synthesis (S-) phase, while the pericentromeric satellite III is exclusively replicated in late S-phase. Long interspersed nuclear element 1 (LINE-1) replication, though, is performed throughout S-phase. Together with available population genomics analyses, we demonstrate that the majority of LINE-1 elements is duplicated according to their distinct histone modifications. In contrast, pericentromeric heterochromatin (chromocenters) in mouse embryonic stem (mES) cells is replicated during early/mid S-phase (stage II). Similarly, minor satellite and p-arm telomeres are also duplicated during this timeframe, as a consequence of a domino-like replication propagation. We find that this shifted replication timing depends on an increased level of histone acetylation and a more open chromocenter structure. Likewise, we unveil a synchronous replication of the Y chromosome at the end of S-phase, independently of the pluripotency state of the cell. We additionally investigated the properties of the mES cell replicon, i.e. the DNA replicated from one origin, and the number of active replication sites. By combining genome-wide origin mapping, single-molecule analysis and super-resolution microscopy (3D-SIM), we demonstrate that each replication focus visualized by 3D-SIM corresponds to an individual replicon. Moreover, up to one quarter of the foci represent unidirectional forks. Furthermore, mES cell replicons are smaller than in somatic cells, resulting from the activation of twice as many origins spaced at half the distance, highlighting fundamental developmental differences in genome replication organization in pluripotent cells. In the light of the dramatic effects of histone acetylation level changes on DNA replication, we investigated the influence of cytosine modifications on nuclear processes. We, therefore, established cellular systems to modulate the extent of cytosine variants. Those were based on changing the expression levels of modification writer enzymes, as well as on transfection of modified nucleotides into cells. We find highest DNA duplex stabilities for methylated DNA (5mC) in vitro and in vivo. This effect is reverted by all oxidized cytosine variants. Accordingly, we find enhanced transcription, replication and DNA unwinding rates in the presence of the destabilizing 5-hydroxymethylcytosine (5hmC) and the absence of the stabilizing 5mC. We show that mES cells deficient of 5mC pass more rapidly through S-phase stage II and, consistently, have faster replication forks. These observations are not the result of altered chromatin condensation, structure, accessibility or histone marks and we conclude that the absence of 5mC enhances DNA unwinding and consequently DNA replication. Thereby, modified cytosines may constitute a mechanism for local fine-tuning of DNA processes. In summary, our data contribute to a better understanding of different levels of epigenetic regulation involved in nuclear metabolic processes.