Organization of sister chromatids in replicated chromosomes

Eukaryotic cells encode their genetic information, called the genome, within large DNA molecules packaged into the tiny volumes of the cell nucleus. The three-dimensional organization of these DNA molecules is mediated by motor-driven extrusion of DNA loops and by weak physical and chemical interactions that organize large compartments via phase separation. This results in a highly structured folding of the genome, which ensures the proper expression, maintenance and inheritance of genetic information. DNA looping brings regulatory DNA sequences close to their target genes, and phase separation mediates the segregation of active and repressed genes into distinct compartments; both processes contributing to the regulated translation of genetic information into functional proteins. In proliferating cells, the genome is replicated during the cell cycle giving rise to identical DNA copies, called sister chromatids. Following replication, sister chromatids remain linked to each other to ensure error-free repair of damaged DNA molecules. During cell division, the folding of sister chromatids into discrete bodies is essential for their transport to daughter cells. Although sister chromatid interactions are crucial for both DNA repair and faithful inheritance of the genome, the three-dimensional organization of sister chromatids is poorly understood. How the links between sister chromatids are distributed along DNA molecules, and how loop extrusion and phase separation contribute to the folding of sister chromatids are unexplored. Moreover, whether sister chromatid interactions contribute to the regulation of gene expression is unknown. Investigating the three-dimensional organization of replicated genomes in a high-throughput manner was not feasible until recently due to the identical DNA sequences of sister chromatids. This limitation has been overcome by the development of a novel sequencing-based methodology in the Gerlich Laboratory that makes the distinction of sister chromatids possible and enables to investigate the folding of replicated genomes. Using this novel technique, the project funded by the FWF within the context of the Hertha-Firnberg Programme is aimed at understanding the three-dimensional organization of sister chromatids, the contribution of loop extrusion and phase separation in the folding of sister chromatids, and the role of sister chromatid interactions in the regulation of gene expression.

Eukaryotic cells store their genetic information within DNA molecules, which can be up to several centimeters long. These long DNA molecules need to be packaged into the tiny volume of the cell nucleus. The DNA molecules fold into chromosomes, which must be highly regulated so that the genome remains accessible, and the genetic information can be read, repaired, and passed on to next generations during cell division. Key organizers of DNA folding are cohesin protein complexes, which actively extrude DNA into loops, thereby promoting interactions between distant DNA regions vital for gene expression. During cell division, the genome is duplicated, resulting in two identical copies of each chromosomal DNA, called sister chromatids. Following duplication, a subset of cohesin complexes physically links sister chromatids together, establishing cohesion. Cohesion between sister chromatids is essential for accurate segregation of the genetic information into daughter cells and ensures error-free DNA repair prior to cell division. The co-existence of extruded DNA loops on individual DNA molecules and physical linkages between DNA molecules following genome duplication suggests a complex organization of sister chromatids. However, the precise arrangement of DNA loops and cohesion sites to support the physiological functions of both loop extrusion and cohesion has remained elusive. Funded by the Firnberg Programme of the Austrian Science Fund, my project is aimed at unveiling the organization principles of replicated chromosomes. Through innovative DNA sequencing techniques and advanced bioinformatics analyses, I uncovered the diverse 3D conformations of sister chromatids and elucidated the underlying molecular mechanisms. My findings reveal that loop extrusion on individual DNA molecules relocates cohesive linkages to specific regions, where they accumulate to maintain sister chromatid cohesion. The separation of sister chromatids by loop extrusion may be crucial to support proper gene regulation, while maintaining sister chromatid proximity at specific sites may facilitate efficient DNA repair. My project hence provides insights into the organization of replicated genomes.