The vast morphological variability observed across the animal kingdom is thought to be mainly achieved by modifying the expression patterns of a common toolbox of genes during development. Cis regulatory elements (CREs) are small DNA sequences scattered throughout the non-coding genome and are responsible for the spatio-temporal control of transcription through physical interaction with core promoters. They harbor binding sites for proteins such as transcription factors and chromatin organizers like CTCF and cohesin. The chromatin of multiple bilaterian species is compartmentalized in topologically associating domains (TADs), genomic regions within which sequences preferentially contact each other. This organization has been proposed to be essential to organize the regulatory information contained in animal genomes, bringing together CREs and their target promoters and avoiding promiscuous contacts. However, the impact of this spatial chromatin organization on gene expression, its degree of evolutionary conservation and the molecular mechanisms responsible for its formation are still poorly understood. In this thesis we have tried to contribute to the understanding of these aspects of chromatin spatial organization through the analysis of chromatin architecture of a specific locus: the Six cluster. Six homeobox genes are essential developmental regulators organized in gene clusters conserved during evolution. By using 4C-seq technologies on the Six genes presents in zebrafish, mouse and sea urching we reveal that the Six clusters share a deeply evolutionarily conserved 3D chromatin organization that predates the Cambrian explosion. This chromatin architecture generates two largely independent regulatory landscapes (RLs) contained in two adjacent TADs and it is maintained stable during development. By disrupting the conserved TAD border in one of the zebrafis Six clusters, we demonstrate that this border is critical for preventing competition between promoters and enhancers located in separated RLs, thereby generating different expression patterns in genes located in close genomic proximity. Moreover, evolutionary comparison of Six-associated TAD borders reveals the presence of CCCTC-binding factor (CTCF) sites with diverging orientations in all studied deuterostomes. Genome-wide examination of mammalian HiC data reveals that this conserved CTCF configuration is a general signature of TAD borders, underscoring that common organizational principles underlie TAD compartmentalization in deuterostome evolution.

Peer Reviewed