Evolution of 3D Genome Folding Mechanisms and Gene Regulatory Strategies in Metazoans Abstract: The overarching goal of this project is to elucidate the role that 3D genome folding plays in shaping transcriptional regulatory strategies in animals. It has been proposed that organisms use multiple mechanisms, notably loop extrusion and phase separation, to fold nuclear DNA into a variety of structures including compartments and topologically associating domains (TADs). These features have been shown to have functional consequences on the transcriptional regulation of genes. As 3D features differ between lineages and have different regulatory properties, it is likely that changes to 3D folding mechanisms could have profound consequences on the transcriptional regulation strategies used in distinct lineages. Despite rapid progress, largely driven by development of chromosomal conformation capture techniques, in identifying the mechanisms of genome folding in major animal model organisms such as flies, nematodes and mouse, several barriers exist to our broader understanding of the relationship between evolution of 3D genome folding and evolution of transcriptional regulatory strategies including 1) 3D genome studies cover only a fraction of the diversity of animal genomes and many critical transitional lineages are not sampled 2) 3D genome maps generated by HiC represent an averaged view of the interplay between several co-existing mechanisms, making it challenging to link specific transcriptional processes to 3D architecture and disentangle their contribution to gene regulation. To overcome these challenges, we will employ a novel phylogenetic comparative approach using chromatin technologies, functional genetics and single-cell approaches to 1) generate 3D genome maps in outgroups to the vertebrates and to the Bilateria using approaches we have established in non-model marine invertebrates 2) functionally assay the role of chromatin-interacting proteins we have isolated from these lineages to determine the mechanistic basis of changes to genome folding 3) characterize the transcriptional regulatory strategies used in these animals via approaches we have developed to study gene regulatory mechanisms. This work will generate a fuller view of the evolution of 3D genome folding mechanisms in animals and will provide insight into the global drivers that shape gene regulatory strategies, which ultimately form the basis of changes to body plan complexity and phenotypic evolution. Furthermore, by contributing to our understanding of the basic mechanisms that shape 3D genome folding and gene regulation, insight generated through this comparative work will better inform our understanding of the dysregulation of gene expression that occurs in pathological contexts in humans such as cancer. The flexibility and perspective of the MIRA is ideally suited to support this integrated, multi-faceted program whereby rapidly evolving technologies can be readily integrat...