The three-dimensional regulatory landscapes of the globin genes

One of the most important outstanding questions in biology involves the precise spatial and temporal regulation of gene activity, which enables different cell types to express the specific set of genes required for their function and is therefore a cornerstone for complex biological life. Cis-regulatory elements, such as gene promoters and enhancers, play a key role in controlling gene activity. These elements physically interact with the genes they regulate within structural chromatin domains. The organisation of chromosomes into these domains is critical for specific regulation of gene expression and disruption of these structures underlies common human disease. However, it is not understood how chromatin domains form, how interactions between the cis-regulatory elements contained within them are established, or how such interactions influence gene expression. The major hurdles in addressing these questions are to determine chromatin structures with high resolution and sensitivity and to examine their dynamic nature within single cells. To overcome these, I have developed Tri-C, a new chromosome conformation capture assay that can analyse concurrent chromatin interactions at single alleles at high resolution. By combining Tri-C with conventional chromosome conformation capture techniques, I have analysed the three-dimensional regulatory landscapes of the well-characterised murine globin loci at unprecedented depth. Additionally, to examine the roles of cis-regulatory elements in establishing chromatin architecture, I have analysed how engineered deletions in enhancers and CTCF-binding elements in the murine alpha-globin locus disrupt its chromatin landscape. These analyses reveal that the chromatin domains containing the globin genes represent compartmentalised structures, which are delimited by CTCF boundaries. The heterogeneity of interactions in these domains between individual cells is indicative for a dynamic process of loop extrusion underlying their formation. Within chromatin domains, preferential structures are formed in which multiple enhancers and promoters interact simultaneously. These complexes provide a structural basis for understanding how multiple cis-regulatory elements cooperate to establish robust regulation of gene expression. Importantly, these complex, tissue-specific structures, cannot be explained by loop extrusion alone and indicate other, independent mechanisms contributing to chromosome architecture, likely involving interactions mediated by multi-protein complexes. Together, these analyses show that the current view of genome organisation, in which chromosomes are organised by stable loops and domains that self-assemble into hierarchical structures, is not correct. Rather, chromatin architecture reflects a complex interplay between distinct molecular mechanisms contributing to the formation of regulatory landscapes that facilitate precise, robust control of gene expression.