Detailed and mechanistic characterization of Topologically Associating Domain (TAD) boundaries using complementary single-molecule sequencing and super-resolution imaging approaches – TADwalker

Topologically Associated Domains (TADs) compartmentalize vertebrate genomes into functional neighbourhoods for gene regulation, DNA replication, recombination and repair. Both structural variation in the genome and perturbed protein function can cause the reorganization of TAD structure. In the context of disease, TAD restructuration has been reported in a range of different cancers and embryonic defects, which until now has mostly been linked to transcriptional deregulation and gene-enhancer contact rewiring.
TADs are formed by a continuously ongoing mechanism of Cohesin-mediated loop extrusion, which requires blocking at defined TAD boundaries to maintain separation between neighboring TADs. In vertebrate cells, the large majority of TAD boundaries bind the CTCF insulator protein. Experimental studies and in silico models for TAD formation have generally assumed that a single static CTCF binding site is sufficient to create a functional TAD boundary. Partner 1 in the project has recently reported that most TAD boundaries have a modular nature where multiple CTCF binding sites cluster in extended transition zones. We speculate that this clustering counters against the dynamic DNA binding kinetics of CTCF. Partner 2, using super-resolution oligopainting, has reported that domains on either side of a TAD boundary can variably intermingle in individual cells, confirming the dynamic capacity of TAD boundaries. More recently, Partner 1 has developed Nano-C, a multi-contact 3C assay that allows simultaneous targeting of multiple viewpoints, to confirm that individual CTCF sites within modular TAD boundaries additively contribute to insulation. Partner 2 has recently developed a 100-fold more efficient version of Oligopaints that are compatible with multicolor sequential FISH used to reconstruct chromatin folding at single allele resolution. Until now, a quantitative and molecular characterization how modular and dynamic TAD boundaries interact with the loop extrusion machinery to insulate TADs has not been reported.
In our TADwalker project, we will capitalize on our complementary expertise in Nano-C and super-resolution Oligopaint imaging to perform a molecular characterization of TAD boundaries in mouse embryonic stem cells. Practically, we will combine the explorative capacity of Nano-C to identify elements with insulating function and the capacity of super-resolution imaging to quantitatively analyze large numbers of individual cells.
To achieve our overall goal, the project is divided into three work packages. First, we will generate an in-depth description of a representative set of modular TAD boundaries in normal cells, which will serve as a reference for our mechanistic studies. Second, we will determine the molecular interactions of modular TAD boundaries with the loop extrusion machinery. For this purpose, we will remove, on-by-one, the major components of the loop extrusion machinery or the CTCF protein, followed by determination of how the structure and insulation of TAD boundaries are affected. Third, we will precisely dissect the function of the modular nature of TAD boundaries by systematically removing or inversing individual CTCF binding sites within two selected TAD boundaries. Again, we will measure how those perturbations to the modularity of TAD boundaries will influence the structure and insulating function of those boundaries.
The outcome of our project will provide a highly detailed and molecular characterization of how modular TAD boundaries engage with the loop extrusion machinery to create stable TADs. Its results will allow an important refinement of the existing models for TAD structure and function. Moreover, it will provide new leads to explain how distant structural variation, located within the extended transition zones that are formed by modular TAD boundaries, cause (moderate) disease-associated perturbations to gene regulation, DNA replication, recombination and repair.