The three-dimensional (3D) structure of chromatin is intrinsically associated with gene regulation and cell function
. Methods based on chromatin conformation capture have mapped chromatin structures ...in neuronal systems such as in vitro differentiated neurons, neurons isolated through fluorescence-activated cell sorting from cortical tissues pooled from different animals and from dissociated whole hippocampi
. However, changes in chromatin organization captured by imaging, such as the relocation of Bdnf away from the nuclear periphery after activation
, are invisible with such approaches
. Here we developed immunoGAM, an extension of genome architecture mapping (GAM)
, to map 3D chromatin topology genome-wide in specific brain cell types, without tissue disruption, from single animals. GAM is a ligation-free technology that maps genome topology by sequencing the DNA content from thin (about 220 nm) nuclear cryosections. Chromatin interactions are identified from the increased probability of co-segregation of contacting loci across a collection of nuclear slices. ImmunoGAM expands the scope of GAM to enable the selection of specific cell types using low cell numbers (approximately 1,000 cells) within a complex tissue and avoids tissue dissociation
. We report cell-type specialized 3D chromatin structures at multiple genomic scales that relate to patterns of gene expression. We discover extensive 'melting' of long genes when they are highly expressed and/or have high chromatin accessibility. The contacts most specific of neuron subtypes contain genes associated with specialized processes, such as addiction and synaptic plasticity, which harbour putative binding sites for neuronal transcription factors within accessible chromatin regions. Moreover, sensory receptor genes are preferentially found in heterochromatic compartments in brain cells, which establish strong contacts across tens of megabases. Our results demonstrate that highly specific chromatin conformations in brain cells are tightly related to gene regulation mechanisms and specialized functions.
Human chromosomes have a complex 3D spatial organization in the cell nucleus, which comprises a hierarchy of physical interactions across genomic scales. Such an architecture serves important ...functional roles, as genes and their regulators have to physically interact to control gene regulation. However, the molecular mechanisms underlying the formation of those contacts remain poorly understood. Here, we describe a polymer-physics-based approach to investigate the machinery shaping genome folding and function. In silico model predictions on DNA single-molecule 3D structures are validated against independent super-resolution single-cell microscopy data, supporting a scenario whereby chromosome architecture is controlled by thermodynamics mechanisms of phase separation. Finally, as an application of our methods, the validated single-polymer conformations of the theory are used to benchmark powerful technologies to probe genome structure, such as Hi-C, SPRITE, and GAM.
The mammalian genome has a complex, functional 3D organization. However, it remains largely unknown how DNA contacts are orchestrated by chromatin organizers. Here, we infer from only Hi-C the ...cell-type-specific arrangement of DNA binding sites sufficient to recapitulate, through polymer physics, contact patterns genome wide. Our model is validated by its predictions in a set of duplications at Sox9 against available independent data. The binding site types fall in classes that well match chromatin states from segmentation studies, yet they have an overlapping, combinatorial organization along chromosomes necessary to accurately explain contact specificity. The chromatin signatures of the binding site types return a code linking chromatin states to 3D architecture. The code is validated by extensive de novo predictions of Hi-C maps in an independent set of chromosomes. Overall, our results shed light on how 3D information is encrypted in 1D chromatin via the specific combinatorial arrangement of binding sites.
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•Polymer physics identifies a code linking 3D chromatin structure to 1D chromatin states•The code is shown to be sufficient to explain Hi-C data genome wide•DNA-DNA contact binding sites have a genomic overlapping, combinatorial organization•The code is validated by de novo predictions of contact maps in wild-types and mutants
Esposito et al. identify a code linking 3D chromatin structure to 1D chromatin states by combining polymer physics and machine learning. The code is sufficient to produce 3D conformations consistent with Hi-C genome wide. That sheds light on how the multitude of specific regulatory DNA contacts is orchestrated by chromatin organizing factors.
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.
In eukaryotic cell nuclei, a variety of DNA interactions with nuclear elements occur, which, in combination with intra- and inter-chromosomal cross-talks, shape a functional 3D architecture. In some ...cases they are organized by active, i.e. actin/myosin, motors. More often, however, they have been related to passive diffusion mechanisms. Yet, the crucial questions on how DNA loci recognize their target and are reliably shuttled to their destination by Brownian diffusion are still open. Here, we complement the current experimental scenario by considering a physics model, in which the interaction between distant loci is mediated by diffusing bridging molecules. We show that, in such a system, the mechanism underlying target recognition and colocalization is a thermodynamic switch-like process (a phase transition) that only occurs if the concentration and affinity of binding molecules is above a threshold, or else stable contacts are not possible. We also briefly discuss the kinetics of this `passive-shuttling' process, as produced by random diffusion of DNA loci and their binders, and derive predictions based on the effects of genomic modifications and deletions.
Gene expression states influence the 3D conformation of the genome through poorly understood mechanisms. Here, we investigate the conformation of the murine HoxB locus, a gene-dense genomic region ...containing closely spaced genes with distinct activation states in mouse embryonic stem (ES) cells. To predict possible folding scenarios, we performed computer simulations of polymer models informed with different chromatin occupancy features that define promoter activation states or binding sites for the transcription factor CTCF. Single-cell imaging of the locus folding was performed to test model predictions. While CTCF occupancy alone fails to predict the in vivo folding at genomic length scale of 10 kb, we found that homotypic interactions between active and Polycomb-repressed promoters co-occurring in the same DNA fiber fully explain the HoxB folding patterns imaged in single cells. We identify state-dependent promoter interactions as major drivers of chromatin folding in gene-dense regions.
Granular materials are of substantial importance in many industrial and natural processes, yet their complex behaviours, ranging from mechanical properties of static packing to their dynamics, ...rheology and instabilities, are still poorly understood. Here we focus on the dynamics of compaction and its 'jamming' phenomena, outlining recent statistical mechanics approaches to describe it and their deep correspondence with thermal systems such as glass formers. In fact, granular media are often presented as ideal systems for studying complex relaxation towards equilibrium. Granular compaction is defined as an increase of the bulk density of a granular medium submitted to mechanical perturbation. This phenomenon, relevant in many industrial processes and widely studied by the soil mechanics community, is simple enough to be fully investigated and yet reveals all the complex nature of granular dynamics, attracting considerable attention in a broad range of disciplines ranging from chemical to physical sciences.
In higher eukaryotes, chromosomes have a complex three‐dimensional (3D) conformation in the cell nucleus serving vital functional purposes, yet their folding principles remain poorly understood at ...the single‐molecule level. Here, we summarize recent approaches from polymer physics to comprehend the physical mechanisms underlying chromatin architecture. In particular, we focus on two models that have been supported by recent, growing experimental evidence, the Loop Extrusion model and the Strings&Binders phase separation model. We discuss their key ingredients, how they compare to experimental data and some insight they provide on chromatin architecture and gene regulation. Progress in that research field are opening the possibility to predict how genomic mutations alter the network of contacts between genes and their regulators and how that is linked to genetic diseases, such as congenital disorders and cancer.
We review two main classes of polymer models employed to understand how the complex, functional 3D architecture of chromosomes is shaped within the cell nucleus. In the first class, represented by the SBS model, chromatin conformation is established via phase separation mechanisms by interactions between distal DNA sites. In the second class, represented by the LE model, contacts are determined by active motors that extrude DNA loops between distal anchoring sites.
We review the picture of chromatin large-scale 3D organization emerging from the analysis of Hi-C data and polymer modeling. In higher mammals, Hi-C contact maps reveal a complex higher-order ...organization, extending from the sub-Mb to chromosomal scales, hierarchically folded in a structure of domains-within-domains (metaTADs). The domain folding hierarchy is partially conserved throughout differentiation, and deeply correlated to epigenomic features. Rearrangements in the metaTAD topology relate to gene expression modifications: in particular, in neuronal differentiation models, topologically associated domains (TADs) tend to have coherent expression changes within architecturally conserved metaTAD niches. To identify the nature of architectural domains and their molecular determinants within a principled approach, we discuss models based on polymer physics. We show that basic concepts of interacting polymer physics explain chromatin spatial organization across chromosomal scales and cell types. The 3D structure of genomic loci can be derived with high accuracy and its molecular determinants identified by crossing information with epigenomic databases. In particular, we illustrate the case of the
Sox9
locus, linked to human congenital disorders. The model
in-silico
predictions on the effects of genomic rearrangements are confirmed by available 5C data. That can help establishing new diagnostic tools for diseases linked to chromatin mis-folding, such as congenital disorders and cancer.
Complex architectural rearrangements are associated to the control of the HoxD genes in different cell types; yet, how they are implemented in single cells remains unknown. By use of polymer models, ...we dissect the locus 3D structure at the single DNA molecule level in mouse embryonic stem and cortical neuronal cells, as the HoxD cluster changes from a poised to a silent state. Our model describes published Hi-C, 3-way 4C, and FISH data with high accuracy and is validated against independent 4C data on the Nsi-SB 0.5-Mb duplication and on triple contacts. It reveals the mode of action of compartmentalization on the regulation of the HoxD genes that have gene- and cell-type-specific multi-way interactions with their regulatory elements and high cell-to-cell variability. It shows that TADs and higher-order 3D structures, such as metaTADs, associate with distinct combinations of epigenetic factors, including but not limited to CCCTC-binding factor (CTCF) and histone marks.
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•Single-molecule 3D conformations of the HoxD locus are inferred by polymer models•Model predictions are validated against independent data in wild-type and mutants•Cell-type- and gene-specific multi-way contacts occur across HoxD genes and regulators•The locus 3D architecture exhibits a cell-type-specific, high cell-to-cell variability
Bianco et al. reconstruct the 3D structure of the murine HoxD locus by using polymer models at the single DNA molecule level. The locus architecture rearranges upon differentiation from embryonic stem cells to cortical neurons in connection to epigenetic changes, as cell-type- and gene-specific multi-way contacts are established with regulatory elements.
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