Synthetic biology of long-range gene regulation
Prof. Dr. Stefan Mundlos
Recent studies have shown that the genome is organized in a specific three-dimensional (3D) configuration which has a major influence on gene regulation. Studies using chromosome conformation capture technologies such as HiC have shown that mammalian genomes are organized in distinctly folded chromatin modules, called topologically associated domains (TADs) that are separated from each other by boundary regions. TADs subdivide the genome into discrete genomic units that restrict the possible contacts enhancers can establish with their target genes. We use a CRISPR/Cas9 based strategy to investigate the effect of variations in this configuration in vivo in mice. Deletions, duplications and inversions, for example, can result in the fusion of TADs and the re-wiring of enhancer-promoter contacts and consecutive alterations in gene expression. Such changes can cause disease by gene misexpression (Lupianez et al. 2015), but can also be the origin of evolutionary novelty, as shown by us for the mole (Real et al. 2020). Thus, alterations in the 3D chromatin configuration can result in major shifts of gene expression. In an evolutionary context such changes in TAD-landscapes need to be incorporated in the existing regulatory context (Ringel et al. 2022). Our findings provide a framework for interpreting the effect of structural variations in a disease and evolutionary context.
Based on our previous findings we now want to combine cutting edge genomic engineering with synthetic genomics to manipulate the mouse genome at an unprecedented scale. TADs and regulatory landscapes in general cover large regions of the genome. In vitro cellar systems and/or short artificial constructs are inherently incapable of modelling the full functionality of such landscapes in developmental gene regulation in its full complexity. Genomic engineering and the de novo synthesis of large (up to 150 kb) DNA fragments using yeast assembly approaches make it possible to re-engineer complex rearrangements and entire regulatory landscapes. Technologies for single copy targeting and CRISPR-Cas9 genome editing allow the insertion of these synthetic DNA fragments into mouse embryonic stem cells (mESCs) to produce mice that carry altered regulatory sequences in their genome. This approach's modularity and versatility will help identify and characterize the regulatory effect of individual components and how they work together, giving fundamental insights into gene regulation and how differences in sequence give rise to morphological adaptation and diversity. We will focus on alterations in 3D genomic conformation identified in genome-genome comparisons between species and on patient-derived disease-associated changes.
Lupiáñez DG et al. Disruptions of topological chromatin domains cause pathogenic rewiring of gene-enhancer interactions. Cell. 2015 May 21;161(5):1012-1025.
M Real F et al. The mole genome reveals regulatory rearrangements associated with adaptive intersexuality. Science. 2020 Oct 9;370(6513):208-214.
Ringel AR et al. Repression and 3D-restructuring resolves regulatory conflicts in evolutionarily rearranged genomes. Cell. 2022 Sep 29;185(20):3689-3704.e21.
For more information have a look at the website of the Development & Disease group.