Long-range regulation of transcription scales with genomic distance in a gene-specific manner.

Although critical for tuning the timing and level of transcription, enhancer communication with distal promoters is not well understood. Here, we bypass the need for sequence-specific transcription factors (TFs) and recruit activators directly using a chimeric array of gRNA oligos to target dCas9 fused to the activator VP64-p65-Rta (CARGO-VPR). We show that this approach achieves effective activator recruitment to arbitrary genomic sites, even those inaccessible when targeted with a single guide.

Enhancer activation from transposable elements in extrachromosomal DNA.

Extrachromosomal DNA (ecDNA) is a hallmark of aggressive cancer, contributing to both oncogene amplification and tumor heterogeneity. Here, we used Hi-C, super-resolution imaging, and long-read sequencing to explore the nuclear architecture of -amplified ecDNA in colorectal cancer cells. Intriguingly, we observed frequent spatial proximity between ecDNA and 68 repetitive elements which we called ecDNA-interacting elements or EIEs.

Long range regulation of transcription scales with genomic distance in a gene specific manner.

While critical for tuning the timing and level of transcription, enhancer communication with distal promoters is not well understood. Here we bypass the need for sequence-specific transcription factors and recruit activators directly using CARGO-VPR, an approach for targeting dCas9-VPR using a multiplexed array of RNA guides. We show that this approach achieves effective activator recruitment to arbitrary genomic sites, even those inaccessible by single dCas9.

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.

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.

Harmonizing the Generation and Pre-publication Stewardship of FAIR bioimage 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 bioimage 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 bioimage data sharing in the life sciences.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Advances in Chromatin Imaging at Kilobase-Scale Resolution.

It is now widely appreciated that the spatial organization of the genome is nonrandom, and its complex 3D folding has important consequences for many genome processes. Recent developments in multiplexed, super-resolution microscopy have enabled an unprecedented view of the polymeric structure of chromatin - from the loose folds of whole chromosomes to the detailed loops of cis-regulatory elements that regulate gene expression. Facilitated by the use of robotics, microfluidics, and improved approaches to super-resolution, thousands to hundreds of thousands of individual cells can now be analyzed in an individual experiment.

Atlas of Subcellular RNA Localization Revealed by APEX-Seq.

We introduce APEX-seq, a method for RNA sequencing based on direct proximity labeling of RNA using the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome as a resource, revealing extensive patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts.