Systematic assessment of ISWI subunits shows that NURF creates local accessibility for CTCF.

Catalytic activity of the imitation switch (ISWI) family of remodelers is critical for nucleosomal organization and DNA binding of certain transcription factors, including the insulator protein CTCF. Here we define the contribution of individual subcomplexes by deriving a panel of isogenic mouse stem cell lines, each lacking one of six ISWI accessory subunits. Individual deletions of subunits of either CERF, RSF, ACF, WICH or NoRC subcomplexes only moderately affect the chromatin landscape, while removal of the NURF-specific subunit BPTF leads to a strong reduction in chromatin accessibility and SNF2H ATPase localization around CTCF sites. This affects adjacent nucleosome occupancy and CTCF binding. At a group of sites with reduced chromatin accessibility, CTCF binding persists but cohesin occupancy is reduced, resulting in decreased insulation. These results suggest that CTCF binding can be separated from its function as an insulator in nuclear organization and identify a specific role for NURF in mediating SNF2H localization and chromatin opening at bound CTCF sites.

Intra- and interchromosomal contact mapping reveals the Igh locus has extensive conformational heterogeneity and interacts with B-lineage genes.

To produce a diverse antibody repertoire, immunoglobulin heavy-chain (Igh) loci undergo large-scale alterations in structure to facilitate juxtaposition and recombination of spatially separated variable (VH), diversity (DH), and joining (JH) genes. These chromosomal alterations are poorly understood. Uncovering their patterns shows how chromosome dynamics underpins antibody diversity. Using tiled Capture Hi-C, we produce a comprehensive map of chromatin interactions throughout the 2.8-Mb Igh locus in progenitor B cells. We find that the Igh locus folds into semi-rigid subdomains and undergoes flexible looping of the VH genes to its 3' end, reconciling two views of locus organization. Deconvolution of single Igh locus conformations using polymer simulations identifies thousands of different structures. This heterogeneity may underpin the diversity of V(D)J recombination events. All three immunoglobulin loci also participate in a highly specific, developmentally regulated network of interchromosomal interactions with genes encoding B cell-lineage factors. This suggests a model of interchromosomal coordination of B cell development.

Integrative approaches to study enhancer-promoter communication.

The spatiotemporal control of gene expression in complex multicellular organisms relies on noncoding regulatory sequences such as enhancers, which activate transcription of target genes often over large genomic distances. Despite the advances in the identification and characterization of enhancers, the principles and mechanisms by which enhancers select and control their target genes remain largely unknown. Here, we review recent interdisciplinary and quantitative approaches based on emerging techniques that aim to address open questions in the field, notably how regulatory information is encoded in the DNA sequence, how this information is transferred from enhancers to promoters, and how these processes are regulated in time.

Cohesin and CTCF control the dynamics of chromosome folding.

In mammals, interactions between sequences within topologically associating domains enable control of gene expression across large genomic distances. Yet it is unknown how frequently such contacts occur, how long they last and how they depend on the dynamics of chromosome folding and loop extrusion activity of cohesin. By imaging chromosomal locations at high spatial and temporal resolution in living cells, we show that interactions within topologically associating domains are transient and occur frequently during the course of a cell cycle. Interactions become more frequent and longer in the presence of convergent CTCF sites, resulting in suppression of variability in chromosome folding across time. Supported by physical models of chromosome dynamics, our data suggest that CTCF-anchored loops last around 10 min. Our results show that long-range transcriptional regulation might rely on transient physical proximity, and that cohesin and CTCF stabilize highly dynamic chromosome structures, facilitating selected subsets of chromosomal interactions.

Design, Labeling, and Application of Probes for RNA smFISH.

Visualization of single mRNA molecules in fixed cells can be achieved using single molecule fluorescent in situ hybridization (smFISH). This approach enables accurate quantification of mRNA numbers and localization at a single-cell level. To ensure reliable results using smFISH, it is critical to use fluorescent probes that are highly specific to their RNA target. To facilitate probe design, we have created anglerFISH, a user-friendly command-line based pipeline. In this chapter, we present how to perform a smFISH experiment using user-designed and labeled probes.

VisuStatR: visualizing motility and morphology statistics on images in R.

MOTIVATION: Live-cell microscopy has become an essential tool for analyzing dynamic processes in various biological applications. Thereby, high-throughput and automated tracking analyses allow the simultaneous evaluation of large numbers of objects. However, to critically assess the influence of individual objects on calculated summary statistics, and to detect heterogeneous dynamics or possible artifacts, such as misclassified or -tracked objects, a direct mapping of gained statistical information onto the actual image data would be necessary. RESULTS: We present VisuStatR as a platform independent software package that allows the direct visualization of time-resolved summary statistics of morphological characteristics or motility dynamics onto raw images. The software contains several display modes to compare user-defined summary statistics and the underlying image data in various levels of detail. AVAILABILITY AND IMPLEMENTATION: VisuStatR is a free and open-source R-package, containing a user-friendly graphical-user interface and is available via GitHub at https://github.com/grrchrr/VisuStatR/ under the MIT+ license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Inversion of a topological domain leads to restricted changes in its gene expression and affects interdomain communication.

The interplay between the topological organization of the genome and the regulation of gene expression remains unclear. Depletion of molecular factors (e.g. CTCF) underlying topologically associating domains (TADs) leads to modest alterations in gene expression, whereas genomic rearrangements involving TAD boundaries disrupt normal gene expression and can lead to pathological phenotypes. Here, we targeted the TAD neighboring that of the noncoding transcript Xist, which controls X-chromosome inactivation. Inverting 245 kb within the TAD led to expected rearrangement of CTCF-based contacts but revealed heterogeneity in the 'contact' potential of different CTCF sites. Expression of most genes therein remained unaffected in mouse embryonic stem cells and during differentiation. Interestingly, expression of Xist was ectopically upregulated. The same inversion in mouse embryos led to biased Xist expression. Smaller inversions and deletions of CTCF clusters led to similar results: rearrangement of contacts and limited changes in local gene expression, but significant changes in Xist expression in embryos. Our study suggests that the wiring of regulatory interactions within a TAD can influence the expression of genes in neighboring TADs, highlighting the existence of mechanisms of inter-TAD communication.

Nonlinear control of transcription through enhancer-promoter interactions.

Chromosome structure in mammals is thought to regulate transcription by modulating three-dimensional interactions between enhancers and promoters, notably through CTCF-mediated loops and topologically associating domains (TADs)1-4. However, how chromosome interactions are actually translated into transcriptional outputs remains unclear. Here, to address this question, we use an assay to position an enhancer at large numbers of densely spaced chromosomal locations relative to a fixed promoter, and measure promoter output and interactions within a genomic region with minimal regulatory and structural complexity. A quantitative analysis of hundreds of cell lines reveals that the transcriptional effect of an enhancer depends on its contact probabilities with the promoter through a nonlinear relationship. Mathematical modelling suggests that nonlinearity might arise from transient enhancer-promoter interactions being translated into slower promoter bursting dynamics in individual cells, therefore uncoupling the temporal dynamics of interactions from those of transcription. This uncovers a potential mechanism of how distal enhancers act from large genomic distances, and of how topologically associating domain boundaries block distal enhancers. Finally, we show that enhancer strength also determines absolute transcription levels as well as the sensitivity of a promoter to CTCF-mediated transcriptional insulation. Our measurements establish general principles for the context-dependent role of chromosome structure in long-range transcriptional regulation.

Polymer Folding Simulations from Hi-C Data.

In the absence of a clear molecular understanding of the mechanism that stabilizes specific contacts in interphasic chromatin, we resort to the principle of maximum entropy to build a polymeric model based on the Hi-C data of the specific system one wants to study. The interactions are set by an iterative Monte Carlo algorithm to reproduce the average contacts summarized by the Hi-C map. The study of the ensemble of conformations generated by the algorithm can report a much richer set of information than the experimental map alone, including colocalization of multiple sites, fluctuations of the contacts, and kinetical properties.

deepBlink: threshold-independent detection and localization of diffraction-limited spots.

Detection of diffraction-limited spots in single-molecule microscopy images is traditionally performed with mathematical operators designed for idealized spots. This process requires manual tuning of parameters that is time-consuming and not always reliable. We have developed deepBlink, a neural network-based method to detect and localize spots automatically. We demonstrate that deepBlink outperforms other state-of-the-art methods across six publicly available datasets containing synthetic and experimental data.

HP1 drives de novo 3D genome reorganization in early Drosophila embryos.

Fundamental features of 3D genome organization are established de novo in the early embryo, including clustering of pericentromeric regions, the folding of chromosome arms and the segregation of chromosomes into active (A-) and inactive (B-) compartments. However, the molecular mechanisms that drive de novo organization remain unknown1,2. Here, by combining chromosome conformation capture (Hi-C), chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq), 3D DNA fluorescence in situ hybridization (3D DNA FISH) and polymer simulations, we show that heterochromatin protein 1a (HP1a) is essential for de novo 3D genome organization during Drosophila early development. The binding of HP1a at pericentromeric heterochromatin is required to establish clustering of pericentromeric regions. Moreover, HP1a binding within chromosome arms is responsible for overall chromosome folding and has an important role in the formation of B-compartment regions. However, depletion of HP1a does not affect the A-compartment, which suggests that a different molecular mechanism segregates active chromosome regions. Our work identifies HP1a as an epigenetic regulator that is involved in establishing the global structure of the genome in the early embryo.

Chromosome Conformation Capture and Beyond: Toward an Integrative View of Chromosome Structure and Function.

Rapidly developing technologies have recently fueled an exciting era of discovery in the field of chromosome structure and nuclear organization. In addition to chromosome conformation capture (3C) methods, new alternative techniques have emerged to study genome architecture and biological processes in the nucleus, often in single or living cells. This sets an unprecedented stage for exploring the mechanisms that link chromosome structure and biological function. Here we review popular as well as emerging approaches to study chromosome organization, focusing on the contribution of complementary methodologies to our understanding of structures revealed by 3C methods and their biological implications, and discuss the next technical and conceptual frontiers.

A Conserved Noncoding Locus Regulates Random Monoallelic Xist Expression across a Topological Boundary.

cis-Regulatory communication is crucial in mammalian development and is thought to be restricted by the spatial partitioning of the genome in topologically associating domains (TADs). Here, we discovered that the Xist locus is regulated by sequences in the neighboring TAD. In particular, the promoter of the noncoding RNA Linx (LinxP) acts as a long-range silencer and influences the choice of X chromosome to be inactivated. This is independent of Linx transcription and independent of any effect on Tsix, the antisense regulator of Xist that shares the same TAD as Linx. Unlike Tsix, LinxP is well conserved across mammals, suggesting an ancestral mechanism for random monoallelic Xist regulation. When introduced in the same TAD as Xist, LinxP switches from a silencer to an enhancer. Our study uncovers an unsuspected regulatory axis for X chromosome inactivation and a class of cis-regulatory effects that may exploit TAD partitioning to modulate developmental decisions.

Coarse Graining of a Giant Molecular System: The Chromatin Fiber.

The chromatin fiber is a complex polymer whose conformational properties are quite important to regulate gene transcription. One cannot but resort to coarse-grained models to describe the structure and the dynamics of this system on the length scale of the cellular nucleus. Bulk biological data can be used within the framework of the principle of maximum entropy to generate a realistic interaction potential that can be used to sample the equilibrium state of the fiber. The analysis of the structure and of the dynamics of the fiber can be correlated with its biological function, thus providing interesting results about transcriptional regulation.

DamC reveals principles of chromatin folding in vivo without crosslinking and ligation.

Current understanding of chromosome folding is largely reliant on chromosome conformation capture (3C)-based experiments, where chromosomal interactions are detected as ligation products after chromatin crosslinking. To measure chromosome structure in vivo, quantitatively and without crosslinking and ligation, we implemented a modified version of DNA adenine methyltransferase identification (DamID) named DamC, which combines DNA methylation-based detection of chromosomal interactions with next-generation sequencing and biophysical modeling of methylation kinetics. DamC performed in mouse embryonic stem cells provides the first in vivo validation of the existence of topologically associating domains (TADs), CTCF loops and confirms 3C-based measurements of the scaling of contact probabilities. Combining DamC with transposon-mediated genomic engineering shows that new loops can be formed between ectopic and endogenous CTCF sites, which redistributes physical interactions within TADs. DamC provides the first crosslinking- and ligation-free demonstration of the existence of key structural features of chromosomes and provides novel insights into how chromosome structure within TADs can be manipulated.

The bipartite TAD organization of the X-inactivation center ensures opposing developmental regulation of Tsix and Xist.

The mouse X-inactivation center (Xic) locus represents a powerful model for understanding the links between genome architecture and gene regulation, with the non-coding genes Xist and Tsix showing opposite developmental expression patterns while being organized as an overlapping sense/antisense unit. The Xic is organized into two topologically associating domains (TADs) but the role of this architecture in orchestrating cis-regulatory information remains elusive. To explore this, we generated genomic inversions that swap the Xist/Tsix transcriptional unit and place their promoters in each other's TAD. We found that this led to a switch in their expression dynamics: Xist became precociously and ectopically upregulated, both in male and female pluripotent cells, while Tsix expression aberrantly persisted during differentiation. The topological partitioning of the Xic is thus critical to ensure proper developmental timing of X inactivation. Our study illustrates how the genomic architecture of cis-regulatory landscapes can affect the regulation of mammalian developmental processes.

A symmetric toggle switch explains the onset of random X inactivation in different mammals.

Gene-regulatory networks control the establishment and maintenance of alternative gene-expression states during development. A particular challenge is the acquisition of opposing states by two copies of the same gene, as in the case of the long non-coding RNA Xist in mammals at the onset of random X-chromosome inactivation (XCI). The regulatory principles that lead to stable mono-allelic expression of Xist remain unknown. Here, we uncover the minimal regulatory network that can ensure female-specific and mono-alleleic upregulation of Xist, by combining mathematical modeling and experimental validation of central model predictions. We identify a symmetric toggle switch as the basis for random mono-allelic upregulation of Xist, which reproduces data from several mutant, aneuploid and polyploid mouse cell lines with various Xist expression patterns. Moreover, this toggle switch explains the diversity of strategies employed by different species at the onset of XCI. In addition to providing a unifying conceptual framework with which to explore XCI across mammals, our study sets the stage for identifying the molecular mechanisms needed to initiate random XCI.

Challenges and guidelines toward 4D nucleome data and model standards.

Due to recent advances in experimental and theoretical approaches, the dynamic three-dimensional organization (3D) of the nucleus has become a very active area of research in life sciences. We now understand that the linear genome is folded in ways that may modulate how genes are expressed during the basic functioning of cells. Importantly, it is now possible to build 3D models of how the genome folds within the nucleus and changes over time (4D). Because genome folding influences its function, this opens exciting new possibilities to broaden our understanding of the mechanisms that determine cell fate. However, the rapid evolution of methods and the increasing complexity of data can result in ambiguity and reproducibility challenges, which may hamper the progress of this field. Here, we describe such challenges ahead and provide guidelines to think about strategies for shared standardized validation of experimental 4D nucleome data sets and models.

The Dynamics of mRNA Turnover Revealed by Single-Molecule Imaging in Single Cells.

RNA degradation plays a fundamental role in regulating gene expression. In order to characterize the spatiotemporal dynamics of RNA turnover in single cells, we developed a fluorescent biosensor based on dual-color, single-molecule RNA imaging that allows intact transcripts to be distinguished from stabilized degradation intermediates. Using this method, we measured mRNA decay in single cells and found that individual degradation events occur independently within the cytosol and are not enriched within processing bodies. We show that slicing of an mRNA targeted for endonucleolytic cleavage by the RNA-induced silencing complex can be observed in real time in living cells. This methodology provides a framework for investigating the entire life history of individual mRNAs from birth to death in single cells.

Reciprocal insulation analysis of Hi-C data shows that TADs represent a functionally but not structurally privileged scale in the hierarchical folding of chromosomes.

Understanding how regulatory sequences interact in the context of chromosomal architecture is a central challenge in biology. Chromosome conformation capture revealed that mammalian chromosomes possess a rich hierarchy of structural layers, from multi-megabase compartments to sub-megabase topologically associating domains (TADs) and sub-TAD contact domains. TADs appear to act as regulatory microenvironments by constraining and segregating regulatory interactions across discrete chromosomal regions. However, it is unclear whether other (or all) folding layers share similar properties, or rather TADs constitute a privileged folding scale with maximal impact on the organization of regulatory interactions. Here, we present a novel algorithm named CaTCH that identifies hierarchical trees of chromosomal domains in Hi-C maps, stratified through their reciprocal physical insulation, which is a single and biologically relevant parameter. By applying CaTCH to published Hi-C data sets, we show that previously reported folding layers appear at different insulation levels. We demonstrate that although no structurally privileged folding level exists, TADs emerge as a functionally privileged scale defined by maximal boundary enrichment in CTCF and maximal cell-type conservation. By measuring transcriptional output in embryonic stem cells and neural precursor cells, we show that the likelihood that genes in a domain are coregulated during differentiation is also maximized at the scale of TADs. Finally, we observe that regulatory sequences occur at genomic locations corresponding to optimized mutual interactions at the same scale. Our analysis suggests that the architectural functionality of TADs arises from the interplay between their ability to partition interactions and the specific genomic position of regulatory sequences.