Investigating the Function of the ORC-associated Protein LRWD1 in Linking DNA Replication and Chromatin Folding

• Applicant: Dr. Till Bartke
• Subject Area: General Genetics and Functional Genome Biology ; Cell Biology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 450084515

Faithful inheritance of the genetic material from one cell to another is essential for the development and survival of all organisms. During this process cells must ensure that their DNA is duplicated only once per cell cycle and that the resulting copies of the genome are evenly segregated between the mother and daughter cells. In higher eukaryotes DNA synthesis is initiated at numerous origins of replication that are co-regulated in so-called replication domains. It was suggested that selected origins within a replication domain are brought into close spatial proximity through chromatin looping and activated simultaneously in a coordinated fashion. However, the evidence leading to this conclusion is still only indirect, and the molecular mechanisms by which origins are selected and coordinated are unknown. The goal of this proposal is to investigate this link between DNA replication and chromatin topology.Origins are bound by a multi-subunit protein complex termed the origin recognition complex (ORC) that also contains the ORC-associated protein LRWD1/ORCA. LRWD1 was shown to play a role in recruiting ORC to chromatin. In an effort to investigate the function of LRWD1 in origin formation we identified the cohesin subunit Smc3 alongside ORC as a LRWD1 interacting protein. ChIP-seq analyses to identify genomic binding sites of LRWD1 revealed that most genomic LRWD1 binding sites are co-occupied by Smc3. In addition to its role in sister chromatid cohesion cohesin functions as a looping factor that mediates chromatin interactions between distant genomic loci. Comparison of our LRWD1 ChIP-seq data with chromosome conformation capture studies indicated that LRWD1 marks cohesin binding sites that specify short-range chromatin loops, and that are distinct from the long-range contacts between domain boundaries that cohesin specifies together with the boundary factor CTCF. Our results, therefore, point towards a functional relationship between LRWD1, the ORC complex, and cohesin and thus a direct link between DNA replication initiation and chromatin topology.Based on these findings we propose a detailed mechanistic investigation of the function of LRWD1 in linking origin formation and chromatin folding. We will establish a tissue culture-based dCas9 recruitment system for LRWD1 in order to direct LRWD1 to specific chromatin sites, and inducible LRWD1 and cohesin depletion models that allow removal of these factors during specific cell cycle stages. We will combine these experimental systems with chromosome conformation capture, DNA replication, and ChIP assays for ORC and pre-replication complex subunits, LRWD1, and cohesin to test how tethering or removal of LRWD1 influences origin formation and chromatin folding. This targeted approach will allow us to describe the relationship between origin formation and chromosome topology in unprecedented detail, and will provide novel insights into how these processes are intertwined at the molecular level.

• DFG Programme: Research Grants