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High-resolution analysis of the spatio-temporal control of the DNA replication activation

Subject Area Cell Biology
Term from 2013 to 2024
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 232488461
 
Final Report Year 2024

Final Report Abstract

The interphase nucleus contains the genetic information and acts as the host of a plethora of vital functions, such as genome duplication before cell division, differential gene transcription, and processing, in addition to the assembly of the protein synthesis machinery, the ribosomes. Among all the DNA-dependent processes, chromatin duplication is the most global one, as the full genome is duplicated by replisomes from thousands of sites called replication origins, and the newly synthesized genome is then rewrapped with histones and other proteins and modifications maintaining the epigenetic information. This process is spatiotemporally regulated and depends on chromatin structure and epigenetic modifications. While the open chromatin (euchromatin) replicates early in S-phase, the compacted chromatin, such as facultative or constitutive heterochromatin, are known to replicate in mid and late in S-phase. The underlying regulation remained elusive. Using preexisting and data acquired during this project in combination with computational tools, we developed a model for genome replication called the “domino” model of genome replication progression. We further validated the model using synthetic tools by repositioning the late replicating constitutive heterochromatin close to the facultative heterochromatin and revealing the dynamic regulation of the chromatin structure and replication dynamics. Preexisting studies regarding genome replication progression from the perspective of development were mostly restricted to specific loci. Using mouse and human cell lines at different developmental stages, we used a holistic approach to dissect the global genome replication progression with a focus on less explored DNA repeat elements. The latter make up more than 50% of the mammalian genome. We found key differences in their replication timing in cells at different developmental stages, which was regulated by changes in the epigenetic state of chromatin. We also addressed what DNA modifications do to the DNA duplex itself, and to all DNA-dependent processes. We revealed that such modifications change the stability of the DNA duplex and directly affect the efficacy of DNA-dependent processes with DNA methylation increasing the DNA duplex stability and its oxidation resulting in the opposite. In addition to epigenetics, proteins such as CCCTC-binding factor (CTCF) and cohesins are known to regulate genome architecture. CTCF, initially discovered for its role in enhancerpromoter insulation in association with other essential proteins, organizes the DNA/protein complex in hierarchical chromatin structure. We characterized the less-studied role of CTCF in cell survival and genome maintenance upon stress. The cohesin protein complex is known for its role in chromatin loop extrusion and entrapment of sister chromatids after replication. We investigated its role in genome replication and architecture and maintaining the function integrity of chromosomes.

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