Topologically Associating Domains: At the Crossroads of Genome Structure and Function

Abstract

Understanding the 3D architecture of the genome is crucial for elucidating its role in gene regulation and expression. The complex organization of the genome within the cell nucleus plays a pivotal role in gene expression. Topologically associating domains (TADs) are a prominent and ubiquitous architectural feature of genomes in higher organisms, defined as genomic regions that interact more frequently with each other than with their neighboring regions. It has been suggested that TADs function to facilitate the interaction of regulatory elements with their target genes located in the same TAD. However, the relationship between the structure of TADs and the function of the genes they contain remains unclear. This thesis explores the relationship between chromatin domain architecture and gene expression by application of single-cell and single-allele imaging approaches. I have developed innovative single-allele, high-throughput imaging assays, combining DNA and RNA fluorescence in situ hybridization (FISH) to simultaneously probe the structure of individual TADs and the transcriptional activity of their genes. My analysis revealed that transcriptional activity at the allele level is independent of TAD boundary pairing. Notably, variations in TAD boundary distances between alleles within the same nucleus did not correlate with gene activity. Moreover, my results show that global transcription inhibition does not alter TAD structure, whereas the degradation of cohesin, a key TAD architectural protein complex, leads to reduced transcriptional activity alongside the loss of TAD boundary interactions. These findings challenge prevailing assumptions about the functional roles of TAD structure. They underscore the complexity of genomic regulation and open avenues for further research on the mechanisms governing gene expression and the potential of targeting genome architecture for therapeutic purposes.