Single-allele chromatin interactions identify regulatory hubs in dynamic compartmentalized domains

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

Dynamics of the 4D genome during in vivo lineage specification and differentiation

Mammalian gene expression patterns are controlled by regulatory elements, which interact within topologically associating domains (TADs). The relationship between activation of regulatory elements, formation of structural chromatin interactions and gene expression during development is unclear. Here, we present Tiled-C, a low-input chromosome conformation capture (3C) technique. We use this approach to study chromatin architecture at high spatial and temporal resolution through in vivo mouse erythroid differentiation. Integrated analysis of chromatin accessibility and single-cell expression data shows that regulatory elements gradually become accessible within pre-existing TADs during early differentiation. This is followed by structural re-organization within the TAD and formation of specific contacts between enhancers and promoters. Our high-resolution data show that these enhancer-promoter interactions are not established prior to gene expression, but formed gradually during differentiation, concomitant with progressive upregulation of gene activity. Together, these results provide new insight into the close, interdependent relationship between chromatin architecture and gene regulation during development

A gain-of-function single nucleotide variant creates a new promoter which acts as an orientation-dependent enhancer-blocker

Many single nucleotide variants (SNVs) associated with human traits and genetic diseases are thought to alter the activity of existing regulatory elements. Some SNVs may also create entirely new regulatory elements which change gene expression, but the mechanism by which they do so is largely unknown. Here we show that a single base change in an otherwise unremarkable region of the human α-globin cluster creates an entirely new promoter and an associated unidirectional transcript. This SNV downregulates α-globin expression causing α-thalassaemia. Of note, the new promoter lying between the α-globin genes and their associated super-enhancer disrupts their interaction in an orientation-dependent manner. Together these observations show how both the order and orientation of the fundamental elements of the genome determine patterns of gene expression and support the concept that active genes may act to disrupt enhancer-promoter interactions in mammals as in Drosophila. Finally, these findings should prompt others to fully evaluate SNVs lying outside of known regulatory elements as causing changes in gene expression by creating new regulatory elements

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.</p

The relationship between genome structure and function

Precise patterns of gene expression in metazoans are controlled by three classes of regulatory elements: promoters, enhancers and boundary elements. During differentiation and development, these elements form specific interactions in dynamic higher-order chromatin structures. However, the relationship between genome structure and its function in gene regulation is not completely understood. Here we review recent progress in this field and discuss whether genome structure plays an instructive role in regulating gene expression or is a reflection of the activity of the regulatory elements of the genome

Analysis of sub-kilobase chromatin topology reveals nano-scale regulatory interactions with variable dependence on cohesin and CTCF

Chromosome conformation capture (3 C) techniques have captured largescale 3D genome architecture. Here the authors present their “Tiled-MCC” approach for generation of 3 C data across megabase-scale loci at very high (up to 20 bp) resolution, which allowed them to observe nano-scale chromatin structures and investigate how these structures depend on cohesin and CTCF

Single-allele chromatin interactions identify regulatory hubs in dynamic compartmentalized domains

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

Reactivation of a developmentally silenced embryonic globin gene

The α- and β-globin loci harbor developmentally expressed genes, which are silenced throughout post-natal life. Reactivation of these genes may offer therapeutic approaches for the hemoglobinopathies, the most common single gene disorders. Here, we address mechanisms regulating the embryonically expressed α-like globin, termed ζ-globin. We show that in embryonic erythroid cells, the ζ-gene lies within a ~65 kb sub-TAD (topologically associating domain) of open, acetylated chromatin and interacts with the α-globin super-enhancer. By contrast, in adult erythroid cells, the ζ-gene is packaged within a small (~10 kb) sub-domain of hypoacetylated, facultative heterochromatin within the acetylated sub-TAD and that it no longer interacts with its enhancers. The ζ-gene can be partially re-activated by acetylation and inhibition of histone de-acetylases. In addition to suggesting therapies for severe α-thalassemia, these findings illustrate the general principles by which reactivation of developmental genes may rescue abnormalities arising from mutations in their adult paralogues