Super-enhancer interactomes from single cells link clustering and transcription.

Regulation of gene expression hinges on the interplay between enhancers and promoters, traditionally explored through pairwise analyses. Recent advancements in mapping genome folding, like GAM, SPRITE, and multi-contact Hi-C, have uncovered multi-way interactions among super-enhancers (SEs), spanning megabases, yet have not measured their frequency in single cells or the relationship between clustering and transcription. To close this gap, here we used multiplexed imaging to map the 3D positions of 376 SEs across thousands of mammalian nuclei. Notably, our single-cell images reveal that while SE-SE contacts are rare, SEs often form looser associations we termed "communities". These communities, averaging 4-5 SEs, assemble cooperatively under the combined effects of genomic tethers, Pol2 clustering, and nuclear compartmentalization. Larger communities are associated with more frequent and larger transcriptional bursts. Our work provides insights about the SE interactome in single cells that challenge existing hypotheses on SE clustering in the context of transcriptional regulation.

Polycomb repression of Hox genes involves spatial feedback but not domain compaction or phase transition.

Polycomb group proteins have a critical role in silencing transcription during development. It is commonly proposed that Polycomb-dependent changes in genome folding, which compact chromatin, contribute directly to repression by blocking the binding of activating complexes. Recently, it has also been argued that liquid-liquid demixing of Polycomb proteins facilitates this compaction and repression by phase-separating target genes into a membraneless compartment. To test these models, we used Optical Reconstruction of Chromatin Architecture to trace the Hoxa gene cluster, a canonical Polycomb target, in thousands of single cells. Across multiple cell types, we find that Polycomb-bound chromatin frequently explores decompact states and partial mixing with neighboring chromatin, while remaining uniformly repressed, challenging the repression-by-compaction or phase-separation models. Using polymer simulations, we show that these observed flexible ensembles can be explained by 'spatial feedback'-transient contacts that contribute to the propagation of the epigenetic state (epigenetic memory), without inducing a globular organization.

Harmonizing the Generation and Pre-publication Stewardship of FAIR Image data.

Together with the molecular knowledge of genes and proteins, biological images promise to significantly enhance the scientific understanding of complex cellular systems and to advance predictive and personalized therapeutic products for human health. For this potential to be realized, quality-assured image data must be shared among labs at a global scale to be compared, pooled, and reanalyzed, thus unleashing untold potential beyond the original purpose for which the data was generated. There are two broad sets of requirements to enable image data sharing in the life sciences. One set of requirements is articulated in the companion White Paper entitled "Enabling Global Image Data Sharing in the Life Sciences," which is published in parallel and addresses the need to build the cyberinfrastructure for sharing the digital array data (arXiv:2401.13023 [q-bio.OT], https://doi.org/10.48550/arXiv.2401.13023). In this White Paper, we detail a broad set of requirements, which involves collecting, managing, presenting, and propagating contextual information essential to assess the quality, understand the content, interpret the scientific implications, and reuse image data in the context of the experimental details. We start by providing an overview of the main lessons learned to date through international community activities, which have recently made considerable progress toward generating community standard practices for imaging Quality Control (QC) and metadata. We then provide a clear set of recommendations for amplifying this work. The driving goal is to address remaining challenges, and democratize access to common practices and tools for a spectrum of biomedical researchers, regardless of their expertise, access to resources, and geographical location.

Boundary stacking interactions enable cross-TAD enhancer-promoter communication during limb development.

Although promoters and their enhancers are frequently contained within a topologically associating domain (TAD), some developmentally important genes have their promoter and enhancers within different TADs. Hypotheses about molecular mechanisms enabling cross-TAD interactions remain to be assessed. To test these hypotheses, we used optical reconstruction of chromatin architecture to characterize the conformations of the Pitx1 locus on single chromosomes in developing mouse limbs. Our data support a model in which neighboring boundaries are stacked as a result of loop extrusion, bringing boundary-proximal cis-elements into contact. This stacking interaction also contributes to the appearance of architectural stripes in the population average maps. Through molecular dynamics simulations, we found that increasing boundary strengths facilitates the formation of the stacked boundary conformation, counter-intuitively facilitating border bypass. This work provides a revised view of the TAD borders' function, both facilitating and preventing cis-regulatory interactions, and introduces a framework to distinguish border-crossing from border-respecting enhancer-promoter pairs.

Single-cell chromatin state transitions during epigenetic memory formation.

Repressive chromatin modifications are thought to compact chromatin to silence transcription. However, it is unclear how chromatin structure changes during silencing and epigenetic memory formation. We measured gene expression and chromatin structure in single cells after recruitment and release of repressors at a reporter gene. Chromatin structure is heterogeneous, with open and compact conformations present in both active and silent states. Recruitment of repressors associated with epigenetic memory produces chromatin compaction across 10-20 kilobases, while reversible silencing does not cause compaction at this scale. Chromatin compaction is inherited, but changes molecularly over time from histone methylation (H3K9me3) to DNA methylation. The level of compaction at the end of silencing quantitatively predicts epigenetic memory weeks later. Similarly, chromatin compaction at the Nanog locus predicts the degree of stem-cell fate commitment. These findings suggest that the chromatin state across tens of kilobases, beyond the gene itself, is important for epigenetic memory formation.

Leveraging polymer modeling to reconstruct chromatin connectivity from live images.

Chromosomal dynamics plays a central role in a number of critical biological processes, such as transcriptional regulation, genetic recombination, and DNA replication. However, visualization of chromatin is generally limited to live imaging of a few fluorescently labeled chromosomal loci or high-resolution reconstruction of multiple loci from a single time frame. To aid in mapping the underlying chromosomal structure based on parsimonious experimental measurements, we present an exact analytical expression for the evolution of the polymer configuration based on a flexible-polymer model, and we propose an algorithm that tracks the polymer configuration from live images of chromatin marked with several fluorescent marks. Our theory identifies the resolution of microscopy needed to achieve high-accuracy tracking for a given spacing of markers, establishing the statistical confidence in the assignment of genome identity to the visualized marks. We then leverage experimental data of locus-tracking measurements to demonstrate the validity of our modeling approach and to establish a basis for the design of experiments with a desired resolution. Altogether, this work provides a computational approach founded on polymer physics that vastly improves the interpretation of in vivo measurements of biopolymer dynamics.

SnapFISH: a computational pipeline to identify chromatin loops from multiplexed DNA FISH data.

Multiplexed DNA fluorescence in situ hybridization (FISH) imaging technologies have been developed to map the folding of chromatin fibers at tens of nanometers and up to several kilobases in resolution in single cells. However, computational methods to reliably identify chromatin loops from such imaging datasets are still lacking. Here we present a Single-Nucleus Analysis Pipeline for multiplexed DNA FISH (SnapFISH), to process the multiplexed DNA FISH data and identify chromatin loops. SnapFISH can identify known chromatin loops from mouse embryonic stem cells with high sensitivity and accuracy. In addition, SnapFISH obtains comparable results of chromatin loops across datasets generated from diverse imaging technologies. SnapFISH is freely available at https://github.com/HuMingLab/SnapFISH .

Spatial and temporal organization of the genome: Current state and future aims of the 4D nucleome project.

The four-dimensional nucleome (4DN) consortium studies the architecture of the genome and the nucleus in space and time. We summarize progress by the consortium and highlight the development of technologies for (1) mapping genome folding and identifying roles of nuclear components and bodies, proteins, and RNA, (2) characterizing nuclear organization with time or single-cell resolution, and (3) imaging of nuclear organization. With these tools, the consortium has provided over 2,000 public datasets. Integrative computational models based on these data are starting to reveal connections between genome structure and function. We then present a forward-looking perspective and outline current aims to (1) delineate dynamics of nuclear architecture at different timescales, from minutes to weeks as cells differentiate, in populations and in single cells, (2) characterize cis-determinants and trans-modulators of genome organization, (3) test functional consequences of changes in cis- and trans-regulators, and (4) develop predictive models of genome structure and function.

Loop stacking organizes genome folding from TADs to chromosomes.

Although population-level analyses revealed significant roles for CTCF and cohesin in mammalian genome organization, their contributions at the single-cell level remain incompletely understood. Here, we used a super-resolution microscopy approach to measure the effects of removal of CTCF or cohesin in mouse embryonic stem cells. Single-chromosome traces revealed cohesin-dependent loops, frequently stacked at their loop anchors forming multi-way contacts (hubs), bridging across TAD boundaries. Despite these bridging interactions, chromatin in intervening TADs was not intermixed, remaining separated in distinct loops around the hub. At the multi-TAD scale, steric effects from loop stacking insulated local chromatin from ultra-long range (>4 Mb) contacts. Upon cohesin removal, the chromosomes were more disordered and increased cell-cell variability in gene expression. Our data revise the TAD-centric understanding of CTCF and cohesin and provide a multi-scale, structural picture of how they organize the genome on the single-cell level through distinct contributions to loop stacking.

Structural elements promote architectural stripe formation and facilitate ultra-long-range gene regulation at a human disease locus.

Enhancer clusters overlapping disease-associated mutations in Pierre Robin sequence (PRS) patients regulate SOX9 expression at genomic distances over 1.25 Mb. We applied optical reconstruction of chromatin architecture (ORCA) imaging to trace 3D locus topology during PRS-enhancer activation. We observed pronounced changes in locus topology between cell types. Subsequent analysis of single-chromatin fiber traces revealed that these ensemble-average differences arise through changes in the frequency of commonly sampled topologies. We further identified two CTCF-bound elements, internal to the SOX9 topologically associating domain, which promote stripe formation, are positioned near the domain's 3D geometric center, and bridge enhancer-promoter contacts in a series of chromatin loops. Ablation of these elements results in diminished SOX9 expression and altered domain-wide contacts. Polymer models with uniform loading across the domain and frequent cohesin collisions recapitulate this multi-loop, centrally clustered geometry. Together, we provide mechanistic insights into architectural stripe formation and gene regulation over ultra-long genomic ranges.

Recent progress and challenges in single-cell imaging of enhancer-promoter interaction.

In the past two years, approaches relying on high-resolution microscopy and live-cell imaging have increasingly contributed to our understanding of the 3D genome organization and its importance for transcriptional control. Here, we describe recent progress that has highlighted how flexible and heterogeneous 3D chromatin structure is, on the length scales relevant to transcriptional control. We describe work that has investigated how robust transcriptional outcomes may be derived from such flexible organization without the need for clearly distinct structures in active and silent cells. We survey the latest state of the art in directly observing the dynamics of chromatin interactions, and suggest how some recent, apparently contradictory conclusions may be reconciled.

The spatial organization of transcriptional control.

In animals, the sequences for controlling gene expression do not concentrate just at the transcription start site of genes, but are frequently thousands to millions of base pairs distal to it. The interaction of these sequences with one another and their transcription start sites is regulated by factors that shape the three-dimensional (3D) organization of the genome within the nucleus. Over the past decade, indirect tools exploiting high-throughput DNA sequencing have helped to map this 3D organization, have identified multiple key regulators of its structure and, in the process, have substantially reshaped our view of how 3D genome architecture regulates transcription. Now, new tools for high-throughput super-resolution imaging of chromatin have directly visualized the 3D chromatin organization, settling some debates left unresolved by earlier indirect methods, challenging some earlier models of regulatory specificity and creating hypotheses about the role of chromatin structure in transcriptional regulation.

Polycomb-mediated genome architecture enables long-range spreading of H3K27 methylation.

Polycomb-group proteins play critical roles in gene silencing through the deposition of histone H3 lysine 27 trimethylation (H3K27me3) and chromatin compaction. This process is essential for embryonic stem cell (ESC) pluripotency, differentiation, and development. Polycomb repressive complex 2 (PRC2) can both read and write H3K27me3, enabling progressive spreading of H3K27me3 on the linear genome. Long-range Polycomb-associated DNA contacts have also been described, but their regulation and role in gene silencing remain unclear. Here, we apply H3K27me3 HiChIP, a protein-directed chromosome conformation method, and optical reconstruction of chromatin architecture to profile long-range Polycomb-associated DNA loops that span tens to hundreds of megabases across multiple topological associated domains in mouse ESCs and human induced pluripotent stem cells. We find that H3K27me3 loop anchors are enriched for Polycomb nucleation points and coincide with key developmental genes. Genetic deletion of H3K27me3 loop anchors results in disruption of spatial contact between distant loci and altered H3K27me3 in cis, both locally and megabases away on the same chromosome. In mouse embryos, loop anchor deletion leads to ectopic activation of the partner gene, suggesting that Polycomb-associated loops control gene silencing during development. Further, we find that alterations in PRC2 occupancy resulting from an RNA binding­deficient EZH2 mutant are accompanied by loss of Polycomb-associated DNA looping. Together, these results suggest PRC2 uses RNA binding to enhance long-range chromosome folding and H3K27me3 spreading. Developmental gene loci have unique roles in Polycomb spreading, emerging as important architectural elements of the epigenome.

A leukemia-protective germline variant mediates chromatin module formation via transcription factor nucleation.

Non-coding variants coordinate transcription factor (TF) binding and chromatin mark enrichment changes over regions spanning >100 kb. These molecularly coordinated regions are named "variable chromatin modules" (VCMs), providing a conceptual framework of how regulatory variation might shape complex traits. To better understand the molecular mechanisms underlying VCM formation, here, we mechanistically dissect a VCM-modulating noncoding variant that is associated with reduced chronic lymphocytic leukemia (CLL) predisposition and disease progression. This common, germline variant constitutes a 5-bp indel that controls the activity of an AXIN2 gene-linked VCM by creating a MEF2 binding site, which, upon binding, activates a super-enhancer-like regulatory element. This triggers a large change in TF binding activity and chromatin state at an enhancer cluster spanning >150 kb, coinciding with subtle, long-range chromatin compaction and robust AXIN2 up-regulation. Our results support a model in which the indel acts as an AXIN2 VCM-activating TF nucleation event, which modulates CLL pathology.

How subtle changes in 3D structure can create large changes in transcription.

Animal genomes are organized into topologically associated domains (TADs). TADs are thought to contribute to gene regulation by facilitating enhancer-promoter (E-P) contacts within a TAD and preventing these contacts across TAD borders. However, the absolute difference in contact frequency across TAD boundaries is usually less than 2-fold, even though disruptions of TAD borders can change gene expression by 10-fold. Existing models fail to explain this hypersensitive response. Here, we propose a futile cycle model of enhancer-mediated regulation that can exhibit hypersensitivity through bistability and hysteresis. Consistent with recent experiments, this regulation does not exhibit strong correlation between E-P contact and promoter activity, even though regulation occurs through contact. Through mathematical analysis and stochastic simulation, we show that this system can create an illusion of E-P biochemical specificity and explain the importance of weak TAD boundaries. It also offers a mechanism to reconcile apparently contradictory results from recent global TAD disruption with local TAD boundary deletion experiments. Together, these analyses advance our understanding of cis-regulatory contacts in controlling gene expression and suggest new experimental directions.

Deep learning connects DNA traces to transcription to reveal predictive features beyond enhancer-promoter contact.

Chromatin architecture plays an important role in gene regulation. Recent advances in super-resolution microscopy have made it possible to measure chromatin 3D structure and transcription in thousands of single cells. However, leveraging these complex data sets with a computationally unbiased method has been challenging. Here, we present a deep learning-based approach to better understand to what degree chromatin structure relates to transcriptional state of individual cells. Furthermore, we explore methods to "unpack the black box" to determine in an unbiased manner which structural features of chromatin regulation are most important for gene expression state. We apply this approach to an Optical Reconstruction of Chromatin Architecture dataset of the Bithorax gene cluster in Drosophila and show it outperforms previous contact-focused methods in predicting expression state from 3D structure. We find the structural information is distributed across the domain, overlapping and extending beyond domains identified by prior genetic analyses. Individual enhancer-promoter interactions are a minor contributor to predictions of activity.

mSWI/SNF promotes Polycomb repression both directly and through genome-wide redistribution.

The mammalian SWI/SNF complex, or BAF complex, has a conserved and direct role in antagonizing Polycomb-mediated repression. Yet, BAF also promotes repression by Polycomb in stem cells and cancer. How BAF both antagonizes and promotes Polycomb-mediated repression remains unknown. Here, we utilize targeted protein degradation to dissect the BAF-Polycomb axis in mouse embryonic stem cells on short timescales. We report that rapid BAF depletion redistributes Polycomb repressive complexes PRC1 and PRC2 from highly occupied domains, like Hox clusters, to weakly occupied sites normally opposed by BAF. Polycomb redistribution from highly repressed domains results in their decompaction, gain of active epigenomic features and transcriptional derepression. Surprisingly, through dose-dependent degradation of PRC1 and PRC2, we identify a conventional role for BAF in Polycomb-mediated repression, in addition to global Polycomb redistribution. These findings provide new mechanistic insight into the highly dynamic state of the Polycomb-Trithorax axis.

Tracing DNA paths and RNA profiles in cultured cells and tissues with ORCA.

Chromatin conformation capture (3C) methods and fluorescent in situ hybridization (FISH) microscopy have been used to investigate the spatial organization of the genome. Although powerful, both techniques have limitations. Hi-C is challenging for low cell numbers and requires very deep sequencing to achieve its high resolution. In contrast, FISH can be done on small cell numbers and capture rare cell populations, but typically targets pairs of loci at a lower resolution. Here we detail a protocol for optical reconstruction of chromatin architecture (ORCA), a microscopy approach to trace the 3D DNA path within the nuclei of fixed tissues and cultured cells with a genomic resolution as fine as 2 kb and a throughput of ~10,000 cells per experiment. ORCA can identify structural features with comparable resolution to Hi-C while providing single-cell resolution and multimodal measurements characteristic of microscopy. We describe how to use this DNA labeling in parallel with multiplexed labeling of dozens of RNAs to relate chromatin structure and gene expression in the same cells. Oligopaint probe design, primary probe making, sample collection, cryosectioning and RNA/DNA primary probe hybridization can be completed in 1.5 weeks, while automated RNA/DNA barcode hybridization and RNA/DNA imaging typically takes 2-6 d for data collection and 2-7 d for the automated steps of image analysis.

Quantitative cytogenetics reveals molecular stoichiometry and longitudinal organization of meiotic chromosome axes and loops.

During meiosis, chromosomes adopt a specialized organization involving assembly of a cohesin-based axis along their lengths, with DNA loops emanating from this axis. We applied novel, quantitative, and widely applicable cytogenetic strategies to elucidate the molecular bases of this organization using Caenorhabditis elegans. Analyses of wild-type (WT) chromosomes and de novo circular minichromosomes revealed that meiosis-specific HORMA-domain proteins assemble into cohorts in defined numbers and co-organize the axis together with 2 functionally distinct cohesin complexes (REC-8 and COH-3/4) in defined stoichiometry. We further found that REC-8 cohesins, which load during S phase and mediate sister-chromatid cohesion, usually occur as individual complexes, supporting a model wherein sister cohesion is mediated locally by a single cohesin ring. REC-8 complexes are interspersed in an alternating pattern with cohorts of axis-organizing COH-3/4 complexes (averaging 3 per cohort), which are insufficient to confer cohesion but can bind to individual chromatids, suggesting a mechanism to enable formation of asymmetric sister-chromatid loops. Indeed, immunofluorescence/fluorescence in situ hybridization (immuno-FISH) assays demonstrate frequent asymmetry in genomic content between the loops formed on sister chromatids. We discuss how features of chromosome axis/loop architecture inferred from our data can help to explain enigmatic, yet essential, aspects of the meiotic program.