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.

RNA-mediated symmetry breaking enables singular olfactory receptor choice.

Olfactory receptor (OR) choice provides an extreme example of allelic competition for transcriptional dominance, where every olfactory neuron stably transcribes one of approximately 2,000 or more OR alleles1,2. OR gene choice is mediated by a multichromosomal enhancer hub that activates transcription at a single OR3,4, followed by OR-translation-dependent feedback that stabilizes this choice5,6. Here, using single-cell genomics, we show formation of many competing hubs with variable enhancer composition, only one of which retains euchromatic features and transcriptional competence. Furthermore, we provide evidence that OR transcription recruits enhancers and reinforces enhancer hub activity locally, whereas OR RNA inhibits transcription of competing ORs over distance, promoting transition to transcriptional singularity. Whereas OR transcription is sufficient to break the symmetry between equipotent enhancer hubs, OR translation stabilizes transcription at the prevailing hub, indicating that there may be sequential non-coding and coding mechanisms that are implemented by OR alleles for transcriptional prevalence. We propose that coding OR mRNAs possess non-coding functions that influence nuclear architecture, enhance their own transcription and inhibit transcription from their competitors, with generalizable implications for probabilistic cell fate decisions.

Physics meets biology: The joining of two forces to further our understanding of cellular function.

Some biological questions are tough to solve through standard molecular and cell biological methods and naturally lend themselves to investigation by physical approaches. Below, a group of formally trained physicists discuss, among other things, how they apply physics to address biological questions and how physical approaches complement conventional biological approaches.

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.

8-oxodG accumulation within super-enhancers marks fragile CTCF-mediated chromatin loops.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a major product of the DNA oxidization process, has been proposed to have an epigenetic function in gene regulation and has been associated with genome instability. NGS-based methodologies are contributing to the characterization of the 8-oxodG function in the genome. However, the 8-oxodG epigenetic role at a genomic level and the mechanisms controlling the genomic 8-oxodG accumulation/maintenance have not yet been fully characterized. In this study, we report the identification and characterization of a set of enhancer regions accumulating 8-oxodG in human epithelial cells. We found that these oxidized enhancers are mainly super-enhancers and are associated with bidirectional-transcribed enhancer RNAs and DNA Damage Response activation. Moreover, using ChIA-PET and HiC data, we identified specific CTCF-mediated chromatin loops in which the oxidized enhancer and promoter regions physically associate. Oxidized enhancers and their associated chromatin loops accumulate endogenous double-strand breaks which are in turn repaired by NHEJ pathway through a transcription-dependent mechanism. Our work suggests that 8-oxodG accumulation in enhancers-promoters pairs occurs in a transcription-dependent manner and provides novel mechanistic insights on the intrinsic fragility of chromatin loops containing oxidized enhancers-promoters interactions.

Complex multi-enhancer contacts captured by genome architecture mapping.

The organization of the genome in the nucleus and the interactions of genes with their regulatory elements are key features of transcriptional control and their disruption can cause disease. Here we report a genome-wide method, genome architecture mapping (GAM), for measuring chromatin contacts and other features of three-dimensional chromatin topology on the basis of sequencing DNA from a large collection of thin nuclear sections. We apply GAM to mouse embryonic stem cells and identify enrichment for specific interactions between active genes and enhancers across very large genomic distances using a mathematical model termed SLICE (statistical inference of co-segregation). GAM also reveals an abundance of three-way contacts across the genome, especially between regions that are highly transcribed or contain super-enhancers, providing a level of insight into genome architecture that, owing to the technical limitations of current technologies, has previously remained unattainable. Furthermore, GAM highlights a role for gene-expression-specific contacts in organizing the genome in mammalian nuclei.

Hmga2 protein loss alters nuclear envelope and 3D chromatin structure.

BACKGROUND: The high-mobility group Hmga family of proteins are non-histone chromatin-interacting proteins which have been associated with a number of nuclear functions, including heterochromatin formation, replication, recombination, DNA repair, transcription, and formation of enhanceosomes. Due to its role based on dynamic interaction with chromatin, Hmga2 has a pathogenic role in diverse tumors and has been mainly studied in a cancer context; however, whether Hmga2 has similar physiological functions in normal cells remains less explored. Hmga2 was additionally shown to be required during the exit of embryonic stem cells (ESCs) from the ground state of pluripotency, to allow their transition into epiblast-like cells (EpiLCs), and here, we use that system to gain further understanding of normal Hmga2 function. RESULTS: We demonstrated that Hmga2 KO pluripotent stem cells fail to develop into EpiLCs. By using this experimental system, we studied the chromatin changes that take place upon the induction of EpiLCs and we observed that the loss of Hmga2 affects the histone mark H3K27me3, whose levels are higher in Hmga2 KO cells. Accordingly, a sustained expression of polycomb repressive complex 2 (PRC2), responsible for H3K27me3 deposition, was observed in KO cells. However, gene expression differences between differentiating wt vs Hmga2 KO cells did not show any significant enrichments of PRC2 targets. Similarly, endogenous Hmga2 association to chromatin in epiblast stem cells did not show any clear relationships with gene expression modification observed in Hmga2 KO. Hmga2 ChIP-seq confirmed that this protein preferentially binds to the chromatin regions associated with nuclear lamina. Starting from this observation, we demonstrated that nuclear lamina underwent severe alterations when Hmga2 KO or KD cells were induced to exit from the naïve state and this phenomenon is accompanied by a mislocalization of the heterochromatin mark H3K9me3 within the nucleus. As nuclear lamina (NL) is involved in the organization of 3D chromatin structure, we explored the possible effects of Hmga2 loss on this phenomenon. The analysis of Hi-C data in wt and Hmga2 KO cells allowed us to observe that inter-TAD (topologically associated domains) interactions in Hmga2 KO cells are different from those observed in wt cells. These differences clearly show a peculiar compartmentalization of inter-TAD interactions in chromatin regions associated or not to nuclear lamina. CONCLUSIONS: Overall, our results indicate that Hmga2 interacts with heterochromatic lamin-associated domains, and highlight a role for Hmga2 in the crosstalk between chromatin and nuclear lamina, affecting the establishment of inter-TAD interactions.

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.

RNA polymerase II primes Polycomb-repressed developmental genes throughout terminal neuronal differentiation.

Polycomb repression in mouse embryonic stem cells (ESCs) is tightly associated with promoter co-occupancy of RNA polymerase II (RNAPII) which is thought to prime genes for activation during early development. However, it is unknown whether RNAPII poising is a general feature of Polycomb repression, or is lost during differentiation. Here, we map the genome-wide occupancy of RNAPII and Polycomb from pluripotent ESCs to non-dividing functional dopaminergic neurons. We find that poised RNAPII complexes are ubiquitously present at Polycomb-repressed genes at all stages of neuronal differentiation. We observe both loss and acquisition of RNAPII and Polycomb at specific groups of genes reflecting their silencing or activation. Strikingly, RNAPII remains poised at transcription factor genes which are silenced in neurons through Polycomb repression, and have major roles in specifying other, non-neuronal lineages. We conclude that RNAPII poising is intrinsically associated with Polycomb repression throughout differentiation. Our work suggests that the tight interplay between RNAPII poising and Polycomb repression not only instructs promoter state transitions, but also may enable promoter plasticity in differentiated cells.

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.

Hierarchical folding and reorganization of chromosomes are linked to transcriptional changes in cellular differentiation.

Mammalian chromosomes fold into arrays of megabase-sized topologically associating domains (TADs), which are arranged into compartments spanning multiple megabases of genomic DNA. TADs have internal substructures that are often cell type specific, but their higher-order organization remains elusive. Here, we investigate TAD higher-order interactions with Hi-C through neuronal differentiation and show that they form a hierarchy of domains-within-domains (metaTADs) extending across genomic scales up to the range of entire chromosomes. We find that TAD interactions are well captured by tree-like, hierarchical structures irrespective of cell type. metaTAD tree structures correlate with genetic, epigenomic and expression features, and structural tree rearrangements during differentiation are linked to transcriptional state changes. Using polymer modelling, we demonstrate that hierarchical folding promotes efficient chromatin packaging without the loss of contact specificity, highlighting a role far beyond the simple need for packing efficiency.