Repression and 3D-restructuring resolves regulatory conflicts in evolutionarily rearranged genomes.

Regulatory landscapes drive complex developmental gene expression, but it remains unclear how their integrity is maintained when incorporating novel genes and functions during evolution. Here, we investigated how a placental mammal-specific gene, Zfp42, emerged in an ancient vertebrate topologically associated domain (TAD) without adopting or disrupting the conserved expression of its gene, Fat1. In ESCs, physical TAD partitioning separates Zfp42 and Fat1 with distinct local enhancers that drive their independent expression. This separation is driven by chromatin activity and not CTCF/cohesin. In contrast, in embryonic limbs, inactive Zfp42 shares Fat1's intact TAD without responding to active Fat1 enhancers. However, neither Fat1 enhancer-incompatibility nor nuclear envelope-attachment account for Zfp42's unresponsiveness. Rather, Zfp42's promoter is rendered inert to enhancers by context-dependent DNA methylation. Thus, diverse mechanisms enabled the integration of independent Zfp42 regulation in the Fat1 locus. Critically, such regulatory complexity appears common in evolution as, genome wide, most TADs contain multiple independently expressed genes.

Cell-type specialization is encoded by specific chromatin topologies.

The three-dimensional (3D) structure of chromatin is intrinsically associated with gene regulation and cell function1-3. 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 hippocampi4-6. However, changes in chromatin organization captured by imaging, such as the relocation of Bdnf away from the nuclear periphery after activation7, are invisible with such approaches8. Here we developed immunoGAM, an extension of genome architecture mapping (GAM)2,9, 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 dissociation2,10. 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.

Multiplex-GAM: genome-wide identification of chromatin contacts yields insights overlooked by Hi-C.

Technology for measuring 3D genome topology is increasingly important for studying gene regulation, for genome assembly and for mapping of genome rearrangements. Hi-C and other ligation-based methods have become routine but have specific biases. Here, we develop multiplex-GAM, a faster and more affordable version of genome architecture mapping (GAM), a ligation-free technique that maps chromatin contacts genome-wide. We perform a detailed comparison of multiplex-GAM and Hi-C using mouse embryonic stem cells. When examining the strongest contacts detected by either method, we find that only one-third of these are shared. The strongest contacts specifically found in GAM often involve 'active' regions, including many transcribed genes and super-enhancers, whereas in Hi-C they more often contain 'inactive' regions. Our work shows that active genomic regions are involved in extensive complex contacts that are currently underestimated in ligation-based approaches, and highlights the need for orthogonal advances in genome-wide contact mapping technologies.

Comparison of the Hi-C, GAM and SPRITE methods using polymer models of chromatin.

Hi-C, split-pool recognition of interactions by tag extension (SPRITE) and genome architecture mapping (GAM) are powerful technologies utilized to probe chromatin interactions genome wide, but how faithfully they capture three-dimensional (3D) contacts and how they perform relative to each other is unclear, as no benchmark exists. Here, we compare these methods in silico in a simplified, yet controlled, framework against known 3D structures of polymer models of murine and human loci, which can recapitulate Hi-C, GAM and SPRITE experiments and multiplexed fluorescence in situ hybridization (FISH) single-molecule conformations. We find that in silico Hi-C, GAM and SPRITE bulk data are faithful to the reference 3D structures whereas single-cell data reflect strong variability among single molecules. The minimal number of cells required in replicate experiments to return statistically similar contacts is different across the technologies, being lowest in SPRITE and highest in GAM under the same conditions. Noise-to-signal levels follow an inverse power law with detection efficiency and grow with genomic distance differently among the three methods, being lowest in GAM for genomic separations >1 Mb.

Reversibly Tuning the Viscosity of Peptide-Based Solutions Using Visible Light.

Light can be used to design stimuli-responsive systems. We induce transient changes in the assembly of a low molecular weight gelator solution using a merocyanine photoacid. Through our approach, reversible viscosity changes can be achieved via irradiation, delivering systems where flow can be controlled non-invasively on demand.

Investigating multigelator systems across multiple length scales.

Preparation of multicomponent systems provides a method for changing the properties of low molecular weight gelator (LMWG)-based systems. Here we have prepared a variety of multicomponent systems where both components are N-functionalised dipeptide-based LMWGs that may either co-assemble or self-sort when mixed. We exemplify how varying the concentration ratio of the two components can be used to tune the properties of the multicomponent systems pre-gelation, during gelation and in the gel state using viscosity and rheology measurements, circular dichroism, NMR and small angle neutron scattering. We also investigate the effect of changing the chirality of a single component on the properties of these systems. While predicting the outcome of multicomponent assembly is a challenge, the preparation of a variety of systems allows us to probe the factors affecting their design. This work provides insights into how the properties of multicomponent systems composed of two gelators with the same basic structural design can be tuned by varying the chirality and the concentration ratio of the two components and considering the behaviour of the two components when alone.

Unveiling the Machinery behind Chromosome Folding by Polymer Physics Modeling.

Understanding the mechanisms underlying the complex 3D architecture of mammalian genomes poses, at a more fundamental level, the problem of how two or multiple genomic sites can establish physical contacts in the nucleus of the cells. Beyond stochastic and fleeting encounters related to the polymeric nature of chromatin, experiments have revealed specific, privileged patterns of interactions that suggest the existence of basic organizing principles of folding. In this review, we focus on two major and recently proposed physical processes of chromatin organization: loop-extrusion and polymer phase-separation, both supported by increasing experimental evidence. We discuss their implementation into polymer physics models, which we test against available single-cell super-resolution imaging data, showing that both mechanisms can cooperate to shape chromatin structure at the single-molecule level. Next, by exploiting the comprehension of the underlying molecular mechanisms, we illustrate how such polymer models can be used as powerful tools to make predictions in silico that can complement experiments in understanding genome folding. To this aim, we focus on recent key applications, such as the prediction of chromatin structure rearrangements upon disease-associated mutations and the identification of the putative chromatin organizing factors that orchestrate the specificity of DNA regulatory contacts genome-wide.

Controlled Annealing in Adaptive Multicomponent Gels.

We use a pH-driven annealing process to convert between co-assembled and self-sorted networks in multicomponent gels. The initially formed gels at low pH are co-assembled, with the two components coexisting within the same self-assembled structures. We use an enzymatic approach to increase the pH, resulting in a gel-to-sol transition, followed by a hydrolysis to lower the pH once again. As the pH decreases, a self-sorted network is formed by a two-stage gelation process determined by the pKa of each component. This approach can be expanded to layered systems to generate many varied systems by changing composition and rates of pH change, adapting their microstructure and so allowing access to a far greater range of morphologies and complexity than can be achieved in single component systems.

A novel complex genomic rearrangement affecting the KCNJ2 regulatory region causes a variant of Cooks syndrome.

Cooks syndrome (CS) is an ultrarare limb malformation due to in tandem microduplications involving KCNJ2 and extending to the 5' regulatory element of SOX9. To date, six CS families were resolved at the molecular level. Subsequent studies explored the evolutionary and pathological complexities of the SOX9-KCNJ2/Sox9-Kcnj2 locus, and suggested a key role for the formation of novel topologically associating domain (TAD) by inter-TAD duplications in causing CS. Here, we report a unique case of CS associated with a de novo 1;17 translocation affecting the KCNJ2 locus. On chromosome 17, the breakpoint mapped between KCNJ16 and KCNJ2, and combined with a ~ 5 kb deletion in the 5' of KCNJ2. Based on available capture Hi-C data, the breakpoint on chromosome 17 separated KCNJ2 from a putative enhancer. Gene expression analysis demonstrated downregulation of KCNJ2 in both patient's blood cells and cultured skin fibroblasts. Our findings suggest that a complex rearrangement falling in the 5' of KCNJ2 may mimic the developmental consequences of in tandem duplications affecting the SOX9-KCNJ2/Sox9-Kcnj2 locus. This finding adds weight to the notion of an intricate role of gene regulatory regions and, presumably, the related three-dimensional chromatin structure in normal and abnormal human morphology.

Preformed chromatin topology assists transcriptional robustness of Shh during limb development.

Long-range gene regulation involves physical proximity between enhancers and promoters to generate precise patterns of gene expression in space and time. However, in some cases, proximity coincides with gene activation, whereas, in others, preformed topologies already exist before activation. In this study, we investigate the preformed configuration underlying the regulation of the Shh gene by its unique limb enhancer, the ZRS, in vivo during mouse development. Abrogating the constitutive transcription covering the ZRS region led to a shift within the Shh-ZRS contacts and a moderate reduction in Shh transcription. Deletion of the CTCF binding sites around the ZRS resulted in the loss of the Shh-ZRS preformed interaction and a 50% decrease in Shh expression but no phenotype, suggesting an additional, CTCF-independent mechanism of promoter-enhancer communication. This residual activity, however, was diminished by combining the loss of CTCF binding with a hypomorphic ZRS allele, resulting in severe Shh loss of function and digit agenesis. Our results indicate that the preformed chromatin structure of the Shh locus is sustained by multiple components and acts to reinforce enhancer-promoter communication for robust transcription.

Further Delineation of Duplications of ARX Locus Detected in Male Patients with Varying Degrees of Intellectual Disability.

The X-linked gene encoding aristaless-related homeobox (ARX) is a bi-functional transcription factor capable of activating or repressing gene transcription, whose mutations have been found in a wide spectrum of neurodevelopmental disorders (NDDs); these include cortical malformations, paediatric epilepsy, intellectual disability (ID) and autism. In addition to point mutations, duplications of the ARX locus have been detected in male patients with ID. These rearrangements include telencephalon ultraconserved enhancers, whose structural alterations can interfere with the control of ARX expression in the developing brain. Here, we review the structural features of 15 gain copy-number variants (CNVs) of the ARX locus found in patients presenting wide-ranging phenotypic variations including ID, speech delay, hypotonia and psychiatric abnormalities. We also report on a further novel Xp21.3 duplication detected in a male patient with moderate ID and carrying a fully duplicated copy of the ARX locus and the ultraconserved enhancers. As consequences of this rearrangement, the patient-derived lymphoblastoid cell line shows abnormal activity of the ARX-KDM5C-SYN1 regulatory axis. Moreover, the three-dimensional (3D) structure of the Arx locus, both in mouse embryonic stem cells and cortical neurons, provides new insight for the functional consequences of ARX duplications. Finally, by comparing the clinical features of the 16 CNVs affecting the ARX locus, we conclude that-depending on the involvement of tissue-specific enhancers-the ARX duplications are ID-associated risk CNVs with variable expressivity and penetrance.

Polymer models are a versatile tool to study chromatin 3D organization.

The development of new experimental technologies is opening the way to a deeper investigation of the three-dimensional organization of chromosomes inside the cell nucleus. Genome architecture is linked to vital functional purposes, yet a full comprehension of the mechanisms behind DNA folding is still far from being accomplished. Theoretical approaches based on polymer physics have been employed to understand the complexity of chromatin architecture data and to unveil the basic mechanisms shaping its structure. Here, we review some recent advances in the field to discuss how Polymer Physics, combined with numerical Molecular Dynamics simulation and Machine Learning based inference, can capture important aspects of genome organization, including the description of tissue-specific structural rearrangements, the detection of novel, regulatory-linked architectural elements and the structural variability of chromatin at the single-cell level.

Inference of chromosome 3D structures from GAM data by a physics computational approach.

The combination of modelling and experimental advances can provide deep insights for understanding chromatin 3D organization and ultimately its underlying mechanisms. In particular, models of polymer physics can help comprehend the complexity of genomic contact maps, as those emerging from technologies such as Hi-C, GAM or SPRITE. Here we discuss a method to reconstruct 3D structures from Genome Architecture Mapping (GAM) data, based on PRISMR, a computational approach introduced to find the minimal polymer model best describing Hi-C input data from only polymer physics. After recapitulating the PRISMR procedure, we describe how we extended it for treating GAM data. We successfully test the method on a 6 Mb region around the Sox9 gene and, at a lower resolution, on the whole chromosome 7 in mouse embryonic stem cells. The PRISMR derived 3D structures from GAM co-segregation data are finally validated against independent Hi-C contact maps. The method results to be versatile and robust, hinting that it can be similarly applied to different experimental data, such as SPRITE or microscopy distance data.

Molecular Dynamics simulations of the Strings and Binders Switch model of chromatin.

In recent years interest has grown on the applications of polymer physics to model chromatin folding in order to try to make sense of the complexity of experimental data emerging from new technologies such as Hi-C or GAM, in a principled way. Here we review the methods employed to efficiently implement Molecular Dynamics computer simulations of polymer models, focusing in particular on the String&Binders Switch (SBS) model. The constant improvement of such methods and computer power is returning increasingly more accurate insights on the structure and molecular mechanisms underlying the spatial organization of chromosomes in the cell nucleus. We aim to provide an account of the state of the art of computational techniques employed in this type of investigations and to review recent applications of such methods to the description of real genomic loci, such as the Sox9 locus in mESC.

Predicting chromatin architecture from models of polymer physics.

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.

Multiscale modelling of chromatin 4D organization in SARS-CoV-2 infected cells.

SARS-CoV-2 can re-structure chromatin organization and alter the epigenomic landscape of the host genome, but the mechanisms that produce such changes remain unclear. Here, we use polymer physics to investigate how the chromatin of the host genome is re-organized upon infection with SARS-CoV-2. We show that re-structuring of A/B compartments can be explained by a re-modulation of intra-compartment homo-typic affinities, which leads to the weakening of A-A interactions and the enhancement of A-B mixing. At the TAD level, re-arrangements are physically described by a reduction in the loop extrusion activity coupled with an alteration of chromatin phase-separation properties, resulting in more intermingling between different TADs and a spread in space of the TADs themselves. In addition, the architecture of loci relevant to the antiviral interferon response, such as DDX58 or IFIT, becomes more variable within the 3D single-molecule population of the infected model, suggesting that viral infection leads to a loss of chromatin structural specificity. Analysing the time trajectories of pairwise gene-enhancer and higher-order contacts reveals that this variability derives from increased fluctuations in the chromatin dynamics of infected cells. This suggests that SARS-CoV-2 alters gene regulation by impacting the stability of the contact network in time.

Atomic structures of naphthalene dipeptide micelles unravel mechanisms of assembly and gelation.

Peptide-based biopolymers have gained increasing attention due to their versatile applications. A naphthalene dipeptide (2NapFF) can form chirality-dependent tubular micelles, leading to supramolecular gels. The precise molecular arrangement within these micelles and the mechanism governing gelation have remained enigmatic. We determined, at near-atomic resolution, cryoelectron microscopy structures of the 2NapFF micelles LL-tube and LD-tube, generated by the stereoisomers (l,l)-2NapFF and (l,d)-2NapFF, respectively. The structures reveal that the fundamental packing of dipeptides is driven by the systematic π-π stacking of aromatic rings and that same-charge repulsion between the carbonyl groups is responsible for the stiffness of both tubes. The structural analysis elucidates how a single residue's altered chirality gives rise to markedly distinct tubular structures and sheds light on the mechanisms underlying the pH-dependent gelation of LL- and LD-tubes. The understanding of dipeptide packing and gelation mechanisms provides insights for the rational design of 2NapFF derivatives, enabling the modulation of micellar dimensions.

A single dose of cocaine rewires the 3D genome structure of midbrain dopamine neurons.

Midbrain dopamine neurons (DNs) respond to a first exposure to addictive drugs and play key roles in chronic drug usage1-3. As the synaptic and transcriptional changes that follow an acute cocaine exposure are mostly resolved within a few days4,5, the molecular changes that encode the long-term cellular memory of the exposure within DNs remain unknown. To investigate whether a single cocaine exposure induces long-term changes in the 3D genome structure of DNs, we applied Genome Architecture Mapping and single nucleus transcriptomic analyses in the mouse midbrain. We found extensive rewiring of 3D genome architecture at 24 hours past exposure which remains or worsens by 14 days, outlasting transcriptional responses. The cocaine-induced chromatin rewiring occurs at all genomic scales and affects genes with major roles in cocaine-induced synaptic changes. A single cocaine exposure triggers extensive long-lasting changes in chromatin condensation in post-synaptic and post-transcriptional regulatory genes, for example the unfolding of Rbfox1 which becomes most prominent 14 days post exposure. Finally, structurally remodeled genes are most expressed in a specific DN sub-type characterized by low expression of the dopamine auto-receptor Drd2, a key feature of highly cocaine-sensitive cells. These results reveal an important role for long-lasting 3D genome remodelling in the cellular memory of a single cocaine exposure, providing new hypotheses for understanding the inception of drug addiction and 3D genome plasticity.