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Chromosome conformation capture (3C) provides an adaptable tool for studying diverse biological questions. Current 3C methods generally provide either low-resolution interaction profiles across the entire genome, or high-resolution interaction profiles at limited numbers of loci. Due to technical limitations, generation of reproducible high-resolution interaction profiles has not been achieved at genome-wide scale. Here, to overcome this barrier, we systematically test each step of 3C and report two improvements over current methods. We show that up to 30% of reporter events generated using the popular in situ 3C method arise from ligations between two individual nuclei, but this noise can be almost entirely eliminated by isolating intact nuclei after ligation. Using Nuclear-Titrated Capture-C, we generate reproducible high-resolution genome-wide 3C interaction profiles by targeting 8055 gene promoters in erythroid cells. By pairing high-resolution 3C interaction calls with nascent gene expression we interrogate the role of promoter hubs and super-enhancers in gene regulation.

The promoters of mammalian genes are commonly regulated by multiple distal enhancers, which physically interact within discrete chromatin domains. How such domains form and how the regulatory elements within them interact in single cells is not understood. To address this we developed Tri-C, a new chromosome conformation capture (3C) approach, to characterize concurrent chromatin interactions at individual alleles. Analysis by Tri-C identifies heterogeneous patterns of single-allele interactions between CTCF boundary elements, indicating that the formation of chromatin domains likely results from a dynamic process. Within these domains, we observe specific higher-order structures that involve simultaneous interactions between multiple enhancers and promoters. Such regulatory hubs provide a structural basis for understanding how multiple cis-regulatory elements act together to establish robust regulation of gene expression.

Self-interacting chromatin domains encompass genes and their cis-regulatory elements; however, the three-dimensional form a domain takes, whether this relies on enhancer-promoter interactions, and the processes necessary to mediate the formation and maintenance of such domains, remain unclear. To examine these questions, here we use a combination of high-resolution chromosome conformation capture, a non-denaturing form of fluorescence in situ hybridisation and super-resolution imaging to study a 70 kb domain encompassing the mouse α-globin regulatory locus. We show that this region forms an erythroid-specific, decompacted, self-interacting domain, delimited by frequently apposed CTCF/cohesin binding sites early in terminal erythroid differentiation, and does not require transcriptional elongation for maintenance of the domain structure. Formation of this domain does not rely on interactions between the α-globin genes and their major enhancers, suggesting a transcription-independent mechanism for establishment of the domain. However, absence of the major enhancers does alter internal domain interactions. Formation of a loop domain therefore appears to be a mechanistic process that occurs irrespective of the specific interactions within.

The three-dimensional (3D) organization of chromosomes can be probed using methods like Capture-C. However, it is unclear how such population-level data relate to the organization within a single cell, and the mechanisms leading to the observed interactions are still largely obscure. We present a polymer modeling scheme based on the assumption that chromosome architecture is maintained by protein bridges, which form chromatin loops. To test the model, we perform FISH experiments and compare with Capture-C data. Starting merely from the locations of protein binding sites, our model accurately predicts the experimentally observed chromatin interactions, revealing a population of 3D conformations.