Distinct topologically associated domains underlie the regulatory logic in lymphatic endothelial cells contributing to proper cell differentiation

The activation and repression of genes is a fundamental part of proper embryonic development and functional tissue formation, ensuring that unique molecular codes are set up to orchestrate cell differentiation. Changes in chromatin organisation dictate accessibility to gene regulatory elements and control gene expression. Several molecular factors regulating lymphatic endothelial cell (LEC) specification and differentiation have been identified. However, it remains to be defined how chromatin is organised in lymphatic endothelium and how it orchestrates lymphatic vessel network formation. In this study, we combined Hi-C and ATAC-sequencing to characterise 3D chromatin architecture and accessibility in LECs and blood endothelial cells (BECs). We have identified cell type-specific topologically associated domains (TADs) in LECs and BECs. Specifically, our data revealed changes in the TAD boundaries and differentially segregating enhancers regions in lymphatic-associated loci, such as prox1a and tbx1. This multi-omic approach also defined the regulatory logic for nine genes whose expression is enriched in LECs. In vivo validation of their short- and long-range enhancers confirmed their LEC-confined activity. Leveraging these datasets, we reconstructed mafba tissue-specific regulatory networks and identified a genetic interaction with tfe3a in vivo necessary to limit ectopic vessel formation. Overall, our work provides a powerful resource of multi-omic datasets that can be used to systematically determine the regulatory networks governing LEC identity and genes linked to lymphatic disease.