MicroChrom · Revealing the architecture of replicated human chromosomes by sister-chromatid-sensitive fluorescence in situ hybridisation

The extremely long DNA molecules encoding complex genomes of higher eukaryotes must be organised inside the tiny volume of cells so that the genetic information can be read, maintained, and propagated across cell generations. Genomic DNA is folded into loops such that contacts between distant DNA elements can mediate transcriptional regulation or assembly of mechanically stiff mitotic chromosomes. Following DNA replication, each chromosome contains two DNA molecules, which interact with each other to support homology-directed repair during interphase and DNA compaction during mitosis.Advances in sequencing technology and super-resolution microscopy have revealed the 3D structure of chromosomes with unprecedented resolution, but conventional methods cannot distinguish between the identical sequence present in replicated sister-chromatids. The Gerlich lab has recently developed a new sequencing-based technique termed sister-chromatid-sensitive (scs)Hi-C, which allows the discrimination of intra- from inter-molecular contacts in replicated chromosomes. However, scsHi-C can only inform on interaction probabilities and does not reveal the actual 3D structure in single cells.Therefore, the aim of my proposed project is to establish a sister-chromatid-sensitive fluorescence in situ hybridisation (scsFISH) method for the highly multiplexed and automated detection of the 3D architecture of replicated genomes within individual cells. The ring-shaped cohesin complex is a key regulator of chromosome structure by forming loops within DNA molecules and by linking sister-chromatids. By depleting cohesin core subunits/regulators, I will use scsFISH to understand how cohesin structures replicated chromosomes. I will further investigate how sister-chromatid conformation influences the efficiency of homology-directed repair at double-strand breaks. My project will hence provide insights into the organisation of replicated genomes, with important implications for genomic integrity.