The role of chromosome domains in shaping the functional genome.

The genome must be highly compacted to fit within eukaryotic nuclei but must be accessible to the transcriptional machinery to allow appropriate expression of genes in different cell types and throughout developmental pathways. A growing body of work has shown that the genome, analogously to proteins, forms an ordered, hierarchical structure that closely correlates and may even be causally linked with regulation of functions such as transcription. This review describes our current understanding of how these functional genomic "secondary and tertiary structures" form a blueprint for global nuclear architecture and the potential they hold for understanding and manipulating genomic regulation.

Transcriptional regulation and chromatin architecture maintenance are decoupled functions at the Sox2 locus.

How distal regulatory elements control gene transcription and chromatin topology is not clearly defined, yet these processes are closely linked in lineage specification during development. Through allele-specific genome editing and chromatin interaction analyses of the Sox2 locus in mouse embryonic stem cells, we found a striking disconnection between transcriptional control and chromatin architecture. We traced nearly all Sox2 transcriptional activation to a small number of key transcription factor binding sites, whose deletions have no effect on promoter-enhancer interaction frequencies or topological domain organization. Local chromatin architecture maintenance, including at the topologically associating domain (TAD) boundary downstream from the Sox2 enhancer, is widely distributed over multiple transcription factor-bound regions and maintained in a CTCF-independent manner. Furthermore, partial disruption of promoter-enhancer interactions by ectopic chromatin loop formation has no effect on Sox2 transcription. These findings indicate that many transcription factors are involved in modulating chromatin architecture independently of CTCF.

Three-dimensional folding and functional organization principles of the Drosophila genome.

Chromosomes are the physical realization of genetic information and thus form the basis for its readout and propagation. Here we present a high-resolution chromosomal contact map derived from a modified genome-wide chromosome conformation capture approach applied to Drosophila embryonic nuclei. The data show that the entire genome is linearly partitioned into well-demarcated physical domains that overlap extensively with active and repressive epigenetic marks. Chromosomal contacts are hierarchically organized between domains. Global modeling of contact density and clustering of domains show that inactive domains are condensed and confined to their chromosomal territories, whereas active domains reach out of the territory to form remote intra- and interchromosomal contacts. Moreover, we systematically identify specific long-range intrachromosomal contacts between Polycomb-repressed domains. Together, these observations allow for quantitative prediction of the Drosophila chromosomal contact map, laying the foundation for detailed studies of chromosome structure and function in a genetically tractable system.

Transcription induces context-dependent remodeling of chromatin architecture during differentiation.

Metazoan chromosomes are organized into discrete spatial domains (TADs), believed to contribute to the regulation of transcriptional programs. Despite extensive correlation between domain organization and gene activity, a direct mechanistic link is unclear, with perturbation studies often showing little effect. To follow chromatin architecture changes during development, we used Capture Hi-C to interrogate the domains around key differentially expressed genes during mouse thymocyte maturation, uncovering specific remodeling events. Notably, one TAD boundary was broadened to accommodate RNA polymerase elongation past the border, and subdomains were formed around some activated genes without changes in CTCF binding. The ectopic induction of some genes was sufficient to recapitulate domain formation in embryonic stem cells, providing strong evidence that transcription can directly remodel chromatin structure. These results suggest that transcriptional processes drive complex chromosome folding patterns that can be important in certain genomic contexts.

Gene-to-gene coordinated regulation of transcription and alternative splicing by 3D chromatin remodeling upon NF-κB activation.

The NF-κB protein p65/RelA plays a pivotal role in coordinating gene expression in response to diverse stimuli, including viral infections. At the chromatin level, p65/RelA regulates gene transcription and alternative splicing through promoter enrichment and genomic exon occupancy, respectively. The intricate ways in which p65/RelA simultaneously governs these functions across various genes remain to be fully elucidated. In this study, we employed the HTLV-1 Tax oncoprotein, a potent activator of NF-κB, to investigate its influence on the three-dimensional organization of the genome, a key factor in gene regulation. We discovered that Tax restructures the 3D genomic landscape, bringing together genes based on their regulation and splicing patterns. Notably, we found that the Tax-induced gene-gene contact between the two master genes NFKBIA and RELA is associated with their respective changes in gene expression and alternative splicing. Through dCas9-mediated approaches, we demonstrated that NFKBIA-RELA interaction is required for alternative splicing regulation and is caused by an intragenic enrichment of p65/RelA on RELA. Our findings shed light on new regulatory mechanisms upon HTLV-1 Tax and underscore the integral role of p65/RelA in coordinated regulation of NF-κB-responsive genes at both transcriptional and splicing levels in the context of the 3D genome.

The NF-κB pathway is essential for coordinating gene expression in response to various stimuli, including viral infections. Most studies have focused on the role of NF-κB in transcriptional regulation. In the present study, the impact of the potent NF-κB activator HTLV-1 Tax oncoprotein on the three-dimensional organization of the genome was investigated. Tax-mediated NF-κB activation was found to restructure the 3D genomic landscape in cells and to bring genes together in multigene complexes that are coordinately regulated either transcriptionally or through alternative splicing by NF-κB. Induced coordinate changes in transcription and alternative splicing included the two master genes of NF-κB pathway NFKBIA and RELA. The findings have significant implications for understanding cell fate determination and disease development associated with HTLV-1 infection, as well as chronic NF-κB activation in various human inflammatory diseases and cancer.

RNA polymerase II CTD is dispensable for transcription and required for termination in human cells.

The largest subunit of RNA polymerase (Pol) II harbors an evolutionarily conserved C-terminal domain (CTD), composed of heptapeptide repeats, central to the transcriptional process. Here, we analyze the transcriptional phenotypes of a CTD-Δ5 mutant that carries a large CTD truncation in human cells. Our data show that this mutant can transcribe genes in living cells but displays a pervasive phenotype with impaired termination, similar to but more severe than previously characterized mutations of CTD tyrosine residues. The CTD-Δ5 mutant does not interact with the Mediator and Integrator complexes involved in the activation of transcription and processing of RNAs. Examination of long-distance interactions and CTCF-binding patterns in CTD-Δ5 mutant cells reveals no changes in TAD domains or borders. Our data demonstrate that the CTD is largely dispensable for the act of transcription in living cells. We propose a model in which CTD-depleted Pol II has a lower entry rate onto DNA but becomes pervasive once engaged in transcription, resulting in a defect in termination.

Short tandem repeats are important contributors to silencer elements in T cells.

The action of cis-regulatory elements with either activation or repression functions underpins the precise regulation of gene expression during normal development and cell differentiation. Gene activation by the combined activities of promoters and distal enhancers has been extensively studied in normal and pathological contexts. In sharp contrast, gene repression by cis-acting silencers, defined as genetic elements that negatively regulate gene transcription in a position-independent fashion, is less well understood. Here, we repurpose the STARR-seq approach as a novel high-throughput reporter strategy to quantitatively assess silencer activity in mammals. We assessed silencer activity from DNase hypersensitive I sites in a mouse T cell line. Identified silencers were associated with either repressive or active chromatin marks and enriched for binding motifs of known transcriptional repressors. CRISPR-mediated genomic deletions validated the repressive function of distinct silencers involved in the repression of non-T cell genes and genes regulated during T cell differentiation. Finally, we unravel an association of silencer activity with short tandem repeats, highlighting the role of repetitive elements in silencer activity. Our results provide a general strategy for genome-wide identification and characterization of silencer elements.

Myod1 and GR coordinate myofiber-specific transcriptional enhancers.

Skeletal muscle is a dynamic tissue the size of which can be remodeled through the concerted actions of various cues. Here, we investigated the skeletal muscle transcriptional program and identified key tissue-specific regulatory genetic elements. Our results show that Myod1 is bound to numerous skeletal muscle enhancers in collaboration with the glucocorticoid receptor (GR) to control gene expression. Remarkably, transcriptional activation controlled by these factors occurs through direct contacts with the promoter region of target genes, via the CpG-bound transcription factor Nrf1, and the formation of Ctcf-anchored chromatin loops, in a myofiber-specific manner. Moreover, we demonstrate that GR negatively controls muscle mass and strength in mice by down-regulating anabolic pathways. Taken together, our data establish Myod1, GR and Nrf1 as key players of muscle-specific enhancer-promoter communication that orchestrate myofiber size regulation.

Chromosome topology guides the Drosophila Dosage Compensation Complex for target gene activation.

X chromosome dosage compensation in Drosophila requires chromosome-wide coordination of gene activation. The male-specific lethal dosage compensation complex (DCC) identifies and binds to X-chromosomal high-affinity sites (HAS) from which it boosts transcription. A sub-class of HAS, PionX sites, represent first contacts on the X. Here, we explored the chromosomal interactions of representative PionX sites by high-resolution 4C and determined the global chromosome conformation by Hi-C in sex-sorted embryos. Male and female X chromosomes display similar nuclear architecture, concordant with clustered, constitutively active genes. PionX sites, like HAS, are evenly distributed in the active compartment and engage in short- and long-range interactions beyond compartment boundaries. Long-range, inter-domain interactions between DCC binding sites are stronger in males, suggesting that the complex refines chromatin organization. By de novo induction of DCC in female cells, we monitored the extent of activation surrounding PionX sites. This revealed a remarkable range of DCC action not only in linear proximity, but also at megabase distance if close in space, suggesting that DCC profits from pre-existing chromosome folding to activate genes.

Distinct polymer physics principles govern chromatin dynamics in mouse and Drosophila topological domains.

BACKGROUND: In higher eukaryotes, the genome is partitioned into large "Topologically Associating Domains" (TADs) in which the chromatin displays favoured long-range contacts. While a crumpled/fractal globule organization has received experimental supports at higher-order levels, the organization principles that govern chromatin dynamics within these TADs remain unclear. Using simple polymer models, we previously showed that, in mouse liver cells, gene-rich domains tend to adopt a statistical helix shape when no significant locus-specific interaction takes place. RESULTS: Here, we use data from diverse 3C-derived methods to explore chromatin dynamics within mouse and Drosophila TADs. In mouse Embryonic Stem Cells (mESC), that possess large TADs (median size of 840 kb), we show that the statistical helix model, but not globule models, is relevant not only in gene-rich TADs, but also in gene-poor and gene-desert TADs. Interestingly, this statistical helix organization is considerably relaxed in mESC compared to liver cells, indicating that the impact of the constraints responsible for this organization is weaker in pluripotent cells. Finally, depletion of histone H1 in mESC alters local chromatin flexibility but not the statistical helix organization. In Drosophila, which possesses TADs of smaller sizes (median size of 70 kb), we show that, while chromatin compaction and flexibility are finely tuned according to the epigenetic landscape, chromatin dynamics within TADs is generally compatible with an unconstrained polymer configuration. CONCLUSIONS: Models issued from polymer physics can accurately describe the organization principles governing chromatin dynamics in both mouse and Drosophila TADs. However, constraints applied on this dynamics within mammalian TADs have a peculiar impact resulting in a statistical helix organization.

Cohesins form chromosomal cis-interactions at the developmentally regulated IFNG locus.

Cohesin-mediated sister chromatid cohesion is essential for chromosome segregation and post-replicative DNA repair. In addition, evidence from model organisms and from human genetics suggests that cohesin is involved in the control of gene expression. This non-canonical role has recently been rationalized by the findings that mammalian cohesin complexes are recruited to a subset of DNase I hypersensitive sites and to conserved noncoding sequences by the DNA-binding protein CTCF. CTCF functions at insulators (which control interactions between enhancers and promoters) and at boundary elements (which demarcate regions of distinct chromatin structure), and cohesin contributes to its enhancer-blocking activity. The underlying mechanisms remain unknown, and the full spectrum of cohesin functions remains to be determined. Here we show that cohesin forms the topological and mechanistic basis for cell-type-specific long-range chromosomal interactions in cis at the developmentally regulated cytokine locus IFNG. Hence, the ability of cohesin to constrain chromosome topology is used not only for the purpose of sister chromatid cohesion, but also to dynamically define the spatial conformation of specific loci. This new aspect of cohesin function is probably important for normal development and disease.

A chromatin insulator driving three-dimensional Polycomb response element (PRE) contacts and Polycomb association with the chromatin fiber.

Regulation of gene expression involves long-distance communication between regulatory elements and target promoters, but how this is achieved remains unknown. Insulator elements have been proposed to modulate the communication between regulatory elements and promoters due to their ability to insulate genes from regulatory elements or to take part in long-distance interactions. Using a high-resolution chromatin conformation capture (H3C) method, we show that the Drosophila gypsy insulator behaves as a conformational chromatin border that is able to prohibit contacts between a Polycomb response element (PRE) and a distal promoter. On the other hand, two spaced gypsy elements form a chromatin loop that is able to bring an upstream PRE in contact with a downstream gene to mediate its repression. Chromatin immunoprecipitation (ChIP) profiles of the Polycomb protein and its associated H3K27me3 histone mark reflect this insulator-dependent chromatin conformation, suggesting that Polycomb action at a distance can be organized by local chromatin topology.

Competition between transcription and loop extrusion modulates promoter and enhancer dynamics.

The spatiotemporal configuration of genes with distal regulatory elements, and the impact of chromatin mobility on transcription, remain unclear. Loop extrusion is an attractive model for bringing genetic elements together, but how this functionally interacts with transcription is also largely unknown. We combine live tracking of genomic loci and nascent transcripts with molecular dynamics simulations to assess the spatiotemporal arrangement of the Sox2 gene and its enhancer, in response to a battery of perturbations. We find a close link between chromatin mobility and transcriptional status: active elements display more constrained mobility, consistent with confinement within specialized nuclear sites, and alterations in enhancer mobility distinguish poised from transcribing alleles. Strikingly, we find that whereas loop extrusion and transcription factor-mediated clustering contribute to promoter-enhancer proximity, they have antagonistic effects on chromatin dynamics. This provides an experimental framework for the underappreciated role of chromatin dynamics in genome regulation.

Competition between transcription and loop extrusion modulates promoter and enhancer dynamics.

The spatiotemporal configuration of genes with distal regulatory elements, and the impact of chromatin mobility on transcription, remain unclear. Loop extrusion is an attractive model for bringing genetic elements together, but how this functionally interacts with transcription is also largely unknown. We combine live tracking of genomic loci and nascent transcripts with molecular dynamics simulations to assess the 4D arrangement of the Sox2 gene and its enhancer, in response to a battery of perturbations. We find that alterations in chromatin mobility, not promoter-enhancer distance, is more informative about transcriptional status. Active elements display more constrained mobility, consistent with confinement within specialized nuclear sites, and alterations in enhancer mobility distinguish poised from transcribing alleles. Strikingly, we find that whereas loop extrusion and transcription factor-mediated clustering contribute to promoter-enhancer proximity, they have antagonistic effects on chromatin dynamics. This provides an experimental framework for the underappreciated role of chromatin dynamics in genome regulation.

Drought Tolerance Strategies and Autophagy in Resilient Wheat Genotypes.

Drought resiliency strategies combine developmental, physiological, cellular, and molecular mechanisms. Here, we compare drought responses in two resilient spring wheat (Triticum aestivum) genotypes: a well-studied drought-resilient Drysdale and a resilient genotype from the US Pacific North-West Hollis. While both genotypes utilize higher water use efficiency through the reduction of stomatal conductance, other mechanisms differ. First, Hollis deploys the drought escape mechanism to a greater extent than Drysdale by accelerating the flowering time and reducing root growth. Second, Drysdale uses physiological mechanisms such as non-photochemical quenching (NPQ) to dissipate the excess of harvested light energy and sustain higher Fv/Fm and ϕPSII, whereas Hollis maintains constant NPQ but lower Fv/Fm and ϕPSII values. Furthermore, more electron donors of the electron transport chain are in the oxidized state in Hollis than in Drysdale. Third, many ROS homeostasis parameters, including peroxisome abundance, transcription of peroxisome biogenesis genes PEX11 and CAT, catalase protein level, and enzymatic activity, are higher in Hollis than in Drysdale. Fourth, transcription of autophagy flux marker ATG8.4 is upregulated to a greater degree in Hollis than in Drysdale under drought, whereas relative ATG8 protein abundance under drought stress is lower in Hollis than in Drysdale. These data demonstrate the activation of autophagy in both genotypes and a greater autophagic flux in Hollis. In conclusion, wheat varieties utilize different drought tolerance mechanisms. Combining these mechanisms within one genotype offers a promising strategy to advance crop resiliency.

Genomic interactions: chromatin loops and gene meeting points in transcriptional regulation.

The chromosome conformation capture (3C) technique and its genome-wide applications ('4C') have identified a plethora of distal DNA sequences that are frequently in close spatial proximity. In many cases, these have been correlated with transcriptional regulation of the interacting genes, but the functional significance of many of the extreme long-range and interchromosomal interactions remains unclear. This review summarises our current understanding of how chromatin conformation can impinge on gene expression, the major questions that need to be addressed to understand this more fully, and how these questions may be answered in the near future.

Assessment of 3D Interactions Between Promoters and Distal Regulatory Elements with Promoter Capture Hi-C (PCHi-C).

Chromosome conformation capture and its variants interrogate population-average chromatin structure at a higher resolution and throughput than microscopic methods. Capture Hi-C is a variant tailored for the simultaneous assessment of all interactions with thousands of specific bait sequences, so is particularly suited to genome-wide studies of promoter interactions with distal regulatory elements, such as enhancers. We present the principles and methods for Promoter Capture Hi-C (PCHi-C), from experimental design to data analysis.

4C-Seq: Interrogating Chromatin Looping with Circular Chromosome Conformation Capture.

Chromosome conformation capture and its variants have allowed chromatin topology to be interrogated at a superior resolution and throughput than by microscopic methods. Among the method derivatives, 4C-seq (circular chromosome conformation capture, coupled to high-throughput sequencing) is a versatile, cost-effective means of assessing all chromatin interactions with a specific genomic region of interest, making it particularly suitable for interrogating chromatin looping events. We present the principles and procedures for designing and implementing successful 4C-seq experiments.

Precise measurements of chromatin diffusion dynamics by modeling using Gaussian processes.

The spatiotemporal organization of chromatin influences many nuclear processes: from chromosome segregation to transcriptional regulation. To get a deeper understanding of these processes, it is essential to go beyond static viewpoints of chromosome structures, to accurately characterize chromatin's diffusion properties. We present GP-FBM: a computational framework based on Gaussian processes and fractional Brownian motion to extract diffusion properties from stochastic trajectories of labeled chromatin loci. GP-FBM uses higher-order temporal correlations present in the data, therefore, outperforming existing methods. Furthermore, GP-FBM allows to interpolate incomplete trajectories and account for substrate movement when two or more particles are present. Using our method, we show that average chromatin diffusion properties are surprisingly similar in interphase and mitosis in mouse embryonic stem cells. We observe surprising heterogeneity in local chromatin dynamics, correlating with potential regulatory activity. We also present GP-Tool, a user-friendly graphical interface to facilitate usage of GP-FBM by the research community.

Defining Functionally Relevant Spatial Chromatin Domains: It is a TAD Complicated.

Chromosome conformation capture and orthologous methods uncovered the spatial organization of metazoan chromosomes into autonomously folded substructures, often termed topologically associated domains (TADs). There is a striking correlation between TAD organization and hallmarks of genome function, such as histone modifications or gene expression, and disruptions of specific TAD structures have been associated with pathological misexpression of underlying genes. However, complete disruption of TADs seems to have mild effects on the transcriptome, raising questions as to the importance of chromatin topology in regulating the expression of most genes. Furthermore, despite a growing number of genetic perturbation studies, it is still largely unclear how TAD-like domains are defined, maintained, or potentially reorganized. This perspective article discusses the recent work exploring the complexity of the relationship between TADs and transcription, arguing that it is not satisfactorily explained by any of the "rules" that have been previously described.