Hochauflösende Analyse der räumlich-zeitlichen Kontrolle des DNA Replikationsaktivierungsmechanismus
Zusammenfassung der Projektergebnisse
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.
Projektbezogene Publikationen (Auswahl)
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Spatiotemporal Visualization of DNA Replication Dynamics. Methods in Molecular Biology (2013), 213-225. Humana Press.
Reinhart, Marius; Casas-Delucchi, Corella S. & Cardoso, M. Cristina
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Separation of replication and transcription domains in nucleoli. Journal of Structural Biology, 188(3), 259-266.
Smirnov, E.; Borkovec, J.; Kováčik, L.; Svidenská, S.; Schröfel, A.; Skalníková, M.; Švindrych, Z.; Křížek, P.; Ovesný, M.; Hagen, G.M.; Juda, P.; Michalová, K.; Cardoso, M.C.; Cmarko, D. & Raška, I.
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High-Resolution Analysis of Mammalian DNA Replication Units. Methods in Molecular Biology (2015), 43-65. Springer New York.
Chagin, Vadim O.; Reinhart, Marius & Cardoso, M. Cristina
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3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression. Nature Communications, 7(1).
Löb, D.; Lengert, N.; Chagin, V. O.; Reinhart, M.; Casas-Delucchi, C. S.; Cardoso, M. C. & Drossel, B.
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4D Visualization of replication foci in mammalian cells corresponding to individual replicons. Nature Communications, 7(1).
Chagin, V. O.; Casas-Delucchi, C. S.; Reinhart, M.; Schermelleh, L.; Markaki, Y.; Maiser, A.; Bolius, J. J.; Bensimon, A.; Fillies, M.; Domaing, P.; Rozanov, Y. M.; Leonhardt, H. & Cardoso, M. C.
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Identification of the elementary structural units of the DNA damage response. Nature Communications, 8(1).
Natale, Francesco; Rapp, Alexander; Yu, Wei; Maiser, Andreas; Harz, Hartmann; Scholl, Annina; Grulich, Stephan; Anton, Tobias; Hörl, David; Chen, Wei; Durante, Marco; Taucher-Scholz, Gisela; Leonhardt, Heinrich & Cardoso, M. Cristina
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Peripheral re-localization of constitutive heterochromatin advances its replication timing and impairs maintenance of silencing marks. Nucleic Acids Research, 46(12), 6112-6128.
Heinz, Kathrin S; Casas-Delucchi, Corella S; Török, Timea; Cmarko, Dusan; Rapp, Alexander; Raska, Ivan & Cardoso, M Cristina
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Repli-FISH (Fluorescence in Situ Hybridization): Application of 3D-(Immuno)-FISH for the Study of DNA Replication Timing of Genetic Repeat Elements. OBM Genetics, 3(1), 1-1.
Weber, Patrick; Rausch, Cathia; Scholl, Annina & Cardoso, M. Cristina
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DNA replication dynamics of vole genome and its epigenetic regulation. Epigenetics & Chromatin, 12(1).
Heinz, Kathrin S.; Rapp, Alexander; Casas-Delucchi, Corella S.; Lehmkuhl, Anne; Romero-Fernández, Ismael; Sánchez, Antonio; Krämer, Oliver H.; Marchal, J. Alberto & Cardoso, M. Cristina
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Processive DNA synthesis is associated with localized decompaction of constitutive heterochromatin at the sites of DNA replication and repair. Nucleus, 10(1), 231-253.
Chagin, Vadim O.; Reinhart, Britta; Becker, Annette; Mortusewicz, Oliver; Jost, K. Laurence; Rapp, Alexander; Leonhardt, Heinrich & Cardoso, M. Cristina
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Targeted Manipulation/Repositioning of Subcellular Structures and Molecules. Methods in Molecular Biology (2019), 199-208. Springer New York.
Heinz, Kathrin S. & Cardoso, M. Cristina
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Are the processes of DNA replication and DNA repair reading a common structural chromatin unit?. Nucleus, 11(1), 66-82.
Mamberti, Stefania & Cardoso, M. Cristina
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Cohesin depleted cells rebuild functional nuclear compartments after endomitosis. Nature Communications, 11(1).
Cremer, Marion; Brandstetter, Katharina; Maiser, Andreas; Rao, Suhas S. P.; Schmid, Volker J.; Guirao-Ortiz, Miguel; Mitra, Namita; Mamberti, Stefania; Klein, Kyle N.; Gilbert, David M.; Leonhardt, Heinrich; Cardoso, M. Cristina; Aiden, Erez Lieberman; Harz, Hartmann & Cremer, Thomas
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Developmental differences in genome replication program and origin activation. Nucleic Acids Research, 48(22), 12751-12777.
Rausch, Cathia; Weber, Patrick; Prorok, Paulina; Hörl, David; Maiser, Andreas; Lehmkuhl, Anne; Chagin, Vadim O; Casas-Delucchi, Corella S; Leonhardt, Heinrich & Cardoso, M Cristina
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Cytosine base modifications regulate DNA duplex stability and metabolism. Nucleic Acids Research, 49(22), 12870-12894.
Rausch, Cathia; Zhang, Peng; Casas-Delucchi, Corella S; Daiß, Julia L; Engel, Christoph; Coster, Gideon; Hastert, Florian D; Weber, Patrick & Cardoso, M Cristina
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Nuclear organisation and replication timing are coupled through RIF1–PP1 interaction. Nature Communications, 12(1).
Gnan, Stefano; Flyamer, Ilya M.; Klein, Kyle N.; Castelli, Eleonora; Rapp, Alexander; Maiser, Andreas; Chen, Naiming; Weber, Patrick; Enervald, Elin; Cardoso, M. Cristina; Bickmore, Wendy A.; Gilbert, David M. & Buonomo, Sara C. B.
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Analysis of Cell Cycle and DNA Compaction Dependent Subnuclear Distribution of Histone Marks. Methods in Molecular Biology (2022, 10, 19), 225-239. Springer US.
Pradhan, Sunil Kumar & Cardoso, M. Cristina
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The Chromatin Architectural Protein CTCF Is Critical for Cell Survival upon Irradiation-Induced DNA Damage. International Journal of Molecular Sciences, 23(7), 3896.
Mamberti, Stefania; Pabba, Maruthi K.; Rapp, Alexander; Cardoso, M. Cristina & Scholz, Michael
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Replisome loading reduces chromatin motion independent of DNA synthesis. eLife, 12(2023, 10, 31).
Pabba, Maruthi Kumar; Ritter, Christian; Chagin, Vadim O; Meyer, Janis; Celikay, Kerem; Stear, Jeffrey H; Loerke, Dinah; Kolobynina, Ksenia; Prorok, Paulina; Schmid, Alice Kristin; Leonhardt, Heinrich; Rohr, Karl & Cardoso, M Cristina
