Chromosome conformation capture (3C) methods are central to understanding the link between nuclear structure and function, and the physical interactions between distal regulatory elements and promoters. However, no one method is appropriate to address all biological questions, as each variant differs markedly in resolution, reproducibility, throughput and biases. A thorough appreciation of the strengths and weaknesses of each technique is critical when choosing the correct method for a specif...

Many genes determining cell identity are regulated by clusters of Mediator-bound enhancer elements collectively referred to as super-enhancers. These super-enhancers have been proposed to manifest higher-order properties important in development and disease. Here we report a comprehensive functional dissection of one of the strongest putative super-enhancers in erythroid cells. By generating a series of mouse models, deleting each of the five regulatory elements of the α-globin super-enhancer...

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 interacti...

The genome is organized via CTCF-cohesin-binding sites, which partition chromosomes into 1-5 megabase (Mb) topologically associated domains (TADs), and further into smaller sub-domains (sub-TADs). Here we examined in vivo an ∼80 kb sub-TAD, containing the mouse α-globin gene cluster, lying within a ∼1 Mb TAD. We find that the sub-TAD is flanked by predominantly convergent CTCF-cohesin sites that are ubiquitously bound by CTCF but only interact during erythropoiesis, defining a self-interactin...

It has been known for over a century that chromatin is not randomly distributed within the nucleus. However, the question of how DNA is folded and the influence of such folding on nuclear processes remain topics of intensive current research. A longstanding, unanswered question is whether nuclear organization is simply a reflection of nuclear processes such as transcription and replication, or whether chromatin is folded by independent mechanisms and this per se encodes function? Evidence is ...

Chromosome conformation capture (3C) techniques are crucial to understanding tissue-specific regulation of gene expression, but current methods generally require large numbers of cells. This hampers the investigation of chromatin architecture in rare cell populations. We present a new low-input Capture-C approach that can generate high-quality 3C interaction profiles from 10 000-20 000 cells, depending on the resolution used for analysis. We also present a PCR-free, sequencing-free 3C techniq...

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 stud...