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

Repulsive Forces Between Looping Chromosomes Induce Entropy-Driven Segregation

One striking feature of chromatin organization is that chromosomes are compartmentalized into distinct territories during interphase, the degree of intermingling being much smaller than expected for linear chains. A growing body of evidence indicates that the formation of loops plays a dominant role in transcriptional regulation as well as the entropic organization of interphase chromosomes. Using a recently proposed model, we quantitatively determine the entropic forces between chromosomes. This Dynamic Loop Model assumes that loops form solely on the basis of diffusional motion without invoking other long-range interactions. We find that introducing loops into the structure of chromatin results in a multi-fold higher repulsion between chromosomes compared to linear chains. Strong effects are observed for the tendency of a non-random alignment; the overlap volume between chromosomes decays fast with increasing loop number. Our results suggest that the formation of chromatin loops imposes both compartmentalization as well as order on the system without requiring additional energy-consuming processes

Investigating the three-dimensional architecture of genomes by polymer physics

In mammalian cell nuclei, chromatin has a spatial organization that is strictly related to cellular biological functions, such as regulation of gene transcription and expression. However, still today, chromatin structure is currently poorly understood despite being subjected to intense investigation. Recent findings have revealed that chromatin has a complex, hierarchical organization spanning from the sub-Mb scale up to the entire chromosome length. To shed light on this intricate pattern of interactions revealed by experimental data, polymer physics models have been introduced. In this work, we focused on the “String&Binders Switch” (SBS) model, where non-random chromatin conformations are established through specific interaction of chromatin with diffusing DNA-binding molecules, driving folding by formation of loops. The SBS model recapitulates several features of chromatin organization, such as the large-scale average behavior of experimental data, the mechanisms underlying the self-assembly of TADs and the hierarchical organization of genome, as emerging from experimental data. Moreover, by the SBS model, it is possible to reconstruct the 3D architecture of real genomic regions with high accuracy, without any a-priori knowledge of the molecular factors responsible for chromatin folding. Importantly, our polymer models are able to predict the effects of structural variants in the genomic sequence on the 3D architecture, with a very good accuracy. In this scenario, our polymer modeling methods emerge as a powerful approach to predict pathogenic effects, facilitating the interpretation and diagnosis of this type of genomic rearrangements

Heterogeneity and Intrinsic Variation in Spatial Genome Organization [preprint]

The genome is hierarchically organized in 3D space and its architecture is altered in differentiation, development and disease. Some of the general principles that determine global 3D genome organization have been established. However, the extent and nature of cell-to-cell and cell-intrinsic variability in genome architecture are poorly characterized. Here, we systematically probe the heterogeneity in genome organization in human fibroblasts by combining high-resolution Hi-C datasets and high-throughput genome imaging. Optical mapping of several hundred genome interaction pairs at the single cell level demonstrates low steady-state frequencies of colocalization in the population and independent behavior of individual alleles in single nuclei. Association frequencies are determined by genomic distance, higher-order chromatin architecture and chromatin environment. These observations reveal extensive variability and heterogeneity in genome organization at the level of single cells and alleles and they demonstrate the coexistence of a broad spectrum of chromatin and genome conformations in a cell population

Modelling genome-wide topological associating domains in mouse embryonic stem cells

Chromosome conformation capture (3C)-based techniques such as chromosome conformation capture carbon copy (5C) and Hi-C revealed that the folding of mammalian chromosomes is highly hierarchical. A fundamental structural unit in the hierarchy is represented by topologically associating domains (TADs), sub-megabase regions of the genome within which the chromatin fibre preferentially interacts. 3C-based methods provide the mean contact probabilities between chromosomal loci, averaged over a large number of cells, and do not give immediate access to the single-cell conformations of the chromatin fibre. However, coarse-grained polymer models based on 5C data can be used to extract the single-cell conformations of single TADs. Here, we extend this approach to analyse around 2500 TADs in murine embryonic stem cells based on high-resolution Hi-C data. This allowed to predict the cell-to-cell variability in single contacts within genome-wide TADs and correlations between them. Based on these results, we predict that TADs are more similar to ideal chains than to globules in terms of their physical size and three-dimensional shape distribution. Furthermore, we show that their physical size and the degree of structural anisotropy of single TADs are correlated with the level of transcriptional activity of the genes that it harbours. Finally, we show that a large number of multiplets of genomic loci co-localize more often than expected by random, and these loci are particularly enriched in promoters, enhancers and CTCF-bound sites. These results provide the first genome-wide structural reconstruction of TADs using polymeric models obeying the laws of thermodynamics and reveal important universal trends in the correlation between chromosome structure and transcription

Forces driving the three-dimensional folding of eukaryotic genomes

The last decade has radically renewed our understanding of higher order chromatin folding in the eukaryotic nucleus. As a result, most current models are in support of a mostly hierarchical and relatively stable folding of chromosomes dividing chromosomal territories into A- (active) and B- (inactive) compartments, which are then further partitioned into topologically associating domains (TADs), each of which is made up from multiple loops stabilized mainly by the CTCF and cohesin chromatin-binding complexes. Nonetheless, the structure-to-function relationship of eukaryotic genomes is still not well understood. Here, we focus on recent work highlighting the biophysical and regulatory forces that contribute to the spatial organization of genomes, and we propose that the various conformations that chromatin assumes are not so much the result of a linear hierarchy, but rather of both converging and conflicting dynamic forces that act on it

The physics of DNA folding: polymer models and phase-separation

Within cell nuclei, several biophysical processes occur in order to allow the correct activities of the genome such as transcription and gene regulation. To quantitatively investigate such processes, polymer physics models have been developed to unveil the molecular mechanisms underlying genome functions. Among these, phase-separation plays a key role since it controls gene activity and shapes chromatin spatial structure. In this paper, we review some recent experimental and theoretical progress in the field and show that polymer physics in synergy with numerical simulations can be helpful for several purposes, including the study of molecular condensates, gene-enhancer dynamics, and the three-dimensional reconstruction of real genomic regions

Polymer Physics and structural organization of chromosomes in mammalian genomes

Chromosomes in mammalian cell nucleus have a very complex spatial organization, linked to important functional purposes. In this PhD thesis, we show that the organization is hierarchical from very low length scales up to entire chromosomes scale. Then, we use polymer physics to describe general aspects of chromosome architecture. Finally, we presents polymer physics based computational methods to accurately reconstruct the 3D structure of real loci of the genome

The impact of HTLV-1 on the cellular genome.

Human T-lymphotropic virus type-1 (HTLV-1) is the causative agent of adult T-cell leukaemia/lymphoma (ATL), an aggressive CD4+ T-cell malignancy. The mechanisms of leukaemogenesis in ATL are incompletely understood. Insertional mutagenesis has not previously been thought to contribute to the pathogenesis of ATL. However, the recent discovery that HTLV-1 binds the key chromatin architectural protein CTCF raises the hypothesis that HTLV-1 deregulates host gene expression by causing abnormal chromatin looping, bringing the strong HTLV-1 promoter-enhancer near to host genes that lie up to 2Mb from the integrated provirus. Here we review current opinion on the mechanisms of oncogenesis in ATL, with particular emphasis on the local and distant impact of HTLV-1 on the structure and expression of the host genome