Jpx RNA regulates CTCF anchor site selection and formation of chromosome loops.

Chromosome loops shift dynamically during development, homeostasis, and disease. CCCTC-binding factor (CTCF) is known to anchor loops and construct 3D genomes, but how anchor sites are selected is not yet understood. Here, we unveil Jpx RNA as a determinant of anchor selectivity. Jpx RNA targets thousands of genomic sites, preferentially binding promoters of active genes. Depleting Jpx RNA causes ectopic CTCF binding, massive shifts in chromosome looping, and downregulation of >700 Jpx target genes. Without Jpx, thousands of lost loops are replaced by de novo loops anchored by ectopic CTCF sites. Although Jpx controls CTCF binding on a genome-wide basis, it acts selectively at the subset of developmentally sensitive CTCF sites. Specifically, Jpx targets low-affinity CTCF motifs and displaces CTCF protein through competitive inhibition. We conclude that Jpx acts as a CTCF release factor and shapes the 3D genome by regulating anchor site usage.

A Well-Mixed E. coli Genome: Widespread Contacts Revealed by Tracking Mu Transposition.

The three-dimensional structures of chromosomes are increasingly being recognized as playing a major role in cellular regulatory states. The efficiency and promiscuity of phage Mu transposition was exploited to directly measure in vivo interactions between genomic loci in E. coli. Two global organizing principles have emerged: first, the chromosome is well-mixed and uncompartmentalized, with transpositions occurring freely between all measured loci; second, several gene families/regions show "clustering": strong three-dimensional co-localization regardless of linear genomic distance. The activities of the SMC/condensin protein MukB and nucleoid-compacting protein subunit HU-α are essential for the well-mixed state; HU-α is also needed for clustering of 6/7 ribosomal RNA-encoding loci. The data are explained by a model in which the chromosomal structure is driven by dynamic competition between DNA replication and chromosomal relaxation, providing a foundation for determining how region-specific properties contribute to both chromosomal structure and gene regulation.

Genome-Scale Imaging of the 3D Organization and Transcriptional Activity of Chromatin.

The 3D organization of chromatin regulates many genome functions. Our understanding of 3D genome organization requires tools to directly visualize chromatin conformation in its native context. Here we report an imaging technology for visualizing chromatin organization across multiple scales in single cells with high genomic throughput. First we demonstrate multiplexed imaging of hundreds of genomic loci by sequential hybridization, which allows high-resolution conformation tracing of whole chromosomes. Next we report a multiplexed error-robust fluorescence in situ hybridization (MERFISH)-based method for genome-scale chromatin tracing and demonstrate simultaneous imaging of more than 1,000 genomic loci and nascent transcripts of more than 1,000 genes together with landmark nuclear structures. Using this technology, we characterize chromatin domains, compartments, and trans-chromosomal interactions and their relationship to transcription in single cells. We envision broad application of this high-throughput, multi-scale, and multi-modal imaging technology, which provides an integrated view of chromatin organization in its native structural and functional context.

Physical and Functional Compartmentalization of Archaeal Chromosomes.

The three-dimensional organization of chromosomes can have a profound impact on their replication and expression. The chromosomes of higher eukaryotes possess discrete compartments that are characterized by differing transcriptional activities. Contrastingly, most bacterial chromosomes have simpler organization with local domains, the boundaries of which are influenced by gene expression. Numerous studies have revealed that the higher-order architectures of bacterial and eukaryotic chromosomes are dependent on the actions of structural maintenance of chromosomes (SMC) superfamily protein complexes, in particular, the near-universal condensin complex. Intriguingly, however, many archaea, including members of the genus Sulfolobus do not encode canonical condensin. We describe chromosome conformation capture experiments on Sulfolobus species. These reveal the presence of distinct domains along Sulfolobus chromosomes that undergo discrete and specific higher-order interactions, thus defining two compartment types. We observe causal linkages between compartment identity, gene expression, and binding of a hitherto uncharacterized SMC superfamily protein that we term "coalescin."

SMCHD1 Merges Chromosome Compartments and Assists Formation of Super-Structures on the Inactive X.

Mammalian chromosomes are partitioned into A/B compartments and topologically associated domains (TADs). The inactive X (Xi) chromosome, however, adopts a distinct conformation without evident compartments or TADs. Here, through exploration of an architectural protein, structural-maintenance-of-chromosomes hinge domain containing 1 (SMCHD1), we probe how the Xi is reconfigured during X chromosome inactivation. A/B compartments are first fused into "S1" and "S2" compartments, coinciding with Xist spreading into gene-rich domains. SMCHD1 then binds S1/S2 compartments and merges them to create a compartment-less architecture. Contrary to current views, TADs remain on the Xi but in an attenuated state. Ablating SMCHD1 results in a persistent S1/S2 organization and strengthening of TADs. Furthermore, loss of SMCHD1 causes regional defects in Xist spreading and erosion of heterochromatic silencing. We present a stepwise model for Xi folding, where SMCHD1 attenuates a hidden layer of Xi architecture to facilitate Xist spreading.

Lineage-Specific Genome Architecture Links Enhancers and Non-coding Disease Variants to Target Gene Promoters.

Long-range interactions between regulatory elements and gene promoters play key roles in transcriptional regulation. The vast majority of interactions are uncharted, constituting a major missing link in understanding genome control. Here, we use promoter capture Hi-C to identify interacting regions of 31,253 promoters in 17 human primary hematopoietic cell types. We show that promoter interactions are highly cell type specific and enriched for links between active promoters and epigenetically marked enhancers. Promoter interactomes reflect lineage relationships of the hematopoietic tree, consistent with dynamic remodeling of nuclear architecture during differentiation. Interacting regions are enriched in genetic variants linked with altered expression of genes they contact, highlighting their functional role. We exploit this rich resource to connect non-coding disease variants to putative target promoters, prioritizing thousands of disease-candidate genes and implicating disease pathways. Our results demonstrate the power of primary cell promoter interactomes to reveal insights into genomic regulatory mechanisms underlying common diseases.

The Three-Dimensional Organization of Mammalian Genomes.

Animal development depends on not only the linear genome sequence that embeds millions of cis-regulatory elements, but also the three-dimensional (3D) chromatin architecture that orchestrates the interplay between cis-regulatory elements and their target genes. Compared to our knowledge of the cis-regulatory sequences, the understanding of the 3D genome organization in human and other eukaryotes is still limited. Recent advances in technologies to map the 3D genome architecture have greatly accelerated the pace of discovery. Here, we review emerging concepts of chromatin organization in mammalian cells, discuss the dynamics of chromatin conformation during development, and highlight important roles for chromatin organization in cancer and other human diseases.

Multi-scale architecture of archaeal chromosomes.

Chromosome conformation capture (3C) technologies have identified topologically associating domains (TADs) and larger A/B compartments as two salient structural features of eukaryotic chromosomes. These structures are sculpted by the combined actions of transcription and structural maintenance of chromosomes (SMC) superfamily proteins. Bacterial chromosomes fold into TAD-like chromosomal interaction domains (CIDs) but do not display A/B compartment-type organization. We reveal that chromosomes of Sulfolobus archaea are organized into CID-like topological domains in addition to previously described larger A/B compartment-type structures. We uncover local rules governing the identity of the topological domains and their boundaries. We also identify long-range loop structures and provide evidence of a hub-like structure that colocalizes genes involved in ribosome biogenesis. In addition to providing high-resolution descriptions of archaeal chromosome architectures, our data provide evidence of multiple modes of organization in prokaryotic chromosomes and yield insights into the evolution of eukaryotic chromosome conformation.

Balancing cohesin eviction and retention prevents aberrant chromosomal interactions, Polycomb-mediated repression, and X-inactivation.

Depletion of architectural factors globally alters chromatin structure but only modestly affects gene expression. We revisit the structure-function relationship using the inactive X chromosome (Xi) as a model. We investigate cohesin imbalances by forcing its depletion or retention using degron-tagged RAD21 (cohesin subunit) or WAPL (cohesin release factor). Cohesin loss disrupts the Xi superstructure, unveiling superloops between escapee genes with minimal effect on gene repression. By contrast, forced cohesin retention markedly affects Xi superstructure, compromises spreading of Xist RNA-Polycomb complexes, and attenuates Xi silencing. Effects are greatest at distal chromosomal ends, where looping contacts with the Xist locus are weakened. Surprisingly, cohesin loss creates an Xi superloop, and cohesin retention creates Xi megadomains on the active X chromosome. Across the genome, a proper cohesin balance protects against aberrant inter-chromosomal interactions and tempers Polycomb-mediated repression. We conclude that a balance of cohesin eviction and retention regulates X inactivation and inter-chromosomal interactions across the genome.

Lamina-Dependent Stretching and Unconventional Chromosome Compartments in Early C. elegans Embryos.

Current models suggest that chromosome domains segregate into either an active (A) or inactive (B) compartment. B-compartment chromatin is physically separated from the A compartment and compacted by the nuclear lamina. To examine these models in the developmental context of C. elegans embryogenesis, we undertook chromosome tracing to map the trajectories of entire autosomes. Early embryonic chromosomes organized into an unconventional barbell-like configuration, with two densely folded B compartments separated by a central A compartment. Upon gastrulation, this conformation matured into conventional A/B compartments. We used unsupervised clustering to uncover subpopulations with differing folding properties and variable positioning of compartment boundaries. These conformations relied on tethering to the lamina to stretch the chromosome; detachment from the lamina compacted, and allowed intermingling between, A/B compartments. These findings reveal the diverse conformations of early embryonic chromosomes and uncover a previously unappreciated role for the lamina in systemic chromosome stretching.

High-Resolution Mapping of Multiway Enhancer-Promoter Interactions Regulating Pathogen Detection.

Eukaryotic gene expression regulation involves thousands of distal regulatory elements. Understanding the quantitative contribution of individual enhancers to gene expression is critical for assessing the role of disease-associated genetic risk variants. Yet, we lack the ability to accurately link genes with their distal regulatory elements. To address this, we used 3D enhancer-promoter (E-P) associations identified using split-pool recognition of interactions by tag extension (SPRITE) to build a predictive model of gene expression. Our model dramatically outperforms models using genomic proximity and can be used to determine the quantitative impact of enhancer loss on gene expression in different genetic backgrounds. We show that genes that form stable E-P hubs have less cell-to-cell variability in gene expression. Finally, we identified transcription factors that regulate stimulation-dependent E-P interactions. Together, our results provide a framework for understanding quantitative contributions of E-P interactions and associated genetic variants to gene expression.

Microscopy-Based Chromosome Conformation Capture Enables Simultaneous Visualization of Genome Organization and Transcription in Intact Organisms.

Eukaryotic chromosomes are organized in multiple scales, from nucleosomes to chromosome territories. Recently, genome-wide methods identified an intermediate level of chromosome organization, topologically associating domains (TADs), that play key roles in transcriptional regulation. However, these methods cannot directly examine the interplay between transcriptional activation and chromosome architecture while maintaining spatial information. Here we present a multiplexed, sequential imaging approach (Hi-M) that permits simultaneous detection of chromosome organization and transcription in single nuclei. This allowed us to unveil the changes in 3D chromatin organization occurring upon transcriptional activation and homologous chromosome unpairing during awakening of the zygotic genome in intact Drosophila embryos. Excitingly, the ability of Hi-M to explore the multi-scale chromosome architecture with spatial resolution at different stages of development or during the cell cycle will be key to understanding the mechanisms and consequences of the 4D organization of the genome.

Mechanisms of Interplay between Transcription Factors and the 3D Genome.

Transcription factors (TFs) bind DNA in a sequence-specific manner and thereby serve as the protein anchors and determinants of 3D genome organization. Conversely, chromatin conformation shapes TF activity, for example, by looping TF-bound enhancers to distally located target genes. Despite considerable effort, our understanding of the mechanistic relation between TFs and 3D genome organization remains limited, in large part due to this interdependency. In this review, we summarize the evidence for the diverse mechanisms by which TFs and their activity shape the 3D genome and vice versa. We further highlight outstanding questions and potential approaches for untangling the complex relation between TF activity and the 3D genome.

X-Chromosome Inactivation: A Crossroads Between Chromosome Architecture and Gene Regulation.

In somatic nuclei of female therian mammals, the two X chromosomes display very different chromatin states: One X is typically euchromatic and transcriptionally active, and the other is mostly silent and forms a cytologically detectable heterochromatic structure termed the Barr body. These differences, which arise during female development as a result of X-chromosome inactivation (XCI), have been the focus of research for many decades. Initial approaches to define the structure of the inactive X chromosome (Xi) and its relationship to gene expression mainly involved microscopy-based approaches. More recently, with the advent of genomic techniques such as chromosome conformation capture, molecular details of the structure and expression of the Xi have been revealed. Here, we review our current knowledge of the 3D organization of the mammalian X-chromosome chromatin and discuss its relationship with gene activity in light of the initiation, spreading, and maintenance of XCI, as well as escape from gene silencing.

Multiscale Dynamic Structuring of Bacterial Chromosomes.

Since the nucleoid was isolated from bacteria in the 1970s, two fundamental questions emerged and are still in the spotlight: how bacteria organize their chromosomes to fit inside the cell and how nucleoid organization enables essential biological processes. During the last decades, knowledge of bacterial chromosome organization has advanced considerably, and today, such chromosomes are considered to be highly organized and dynamic structures that are shaped by multiple factors in a multiscale manner. Here we review not only the classical well-known factors involved in chromosome organization but also novel components that have recently been shown to dynamically shape the 3D structuring of the bacterial genome. We focus on the different functional elements that control short-range organization and describe how they collaborate in the establishment of the higher-order folding and disposition of the chromosome. Recent advances have opened new avenues for a deeper understanding of the principles and mechanisms of chromosome organization in bacteria.

Improving comparative analyses of Hi-C data via contrastive self-supervised learning.

Hi-C is a widely applied chromosome conformation capture (3C)-based technique, which has produced a large number of genomic contact maps with high sequencing depths for a wide range of cell types, enabling comprehensive analyses of the relationships between biological functionalities (e.g. gene regulation and expression) and the three-dimensional genome structure. Comparative analyses play significant roles in Hi-C data studies, which are designed to make comparisons between Hi-C contact maps, thus evaluating the consistency of replicate Hi-C experiments (i.e. reproducibility measurement) and detecting statistically differential interacting regions with biological significance (i.e. differential chromatin interaction detection). However, due to the complex and hierarchical nature of Hi-C contact maps, it remains challenging to conduct systematic and reliable comparative analyses of Hi-C data. Here, we proposed sslHiC, a contrastive self-supervised representation learning framework, for precisely modeling the multi-level features of chromosome conformation and automatically producing informative feature embeddings for genomic loci and their interactions to facilitate comparative analyses of Hi-C contact maps. Comprehensive computational experiments on both simulated and real datasets demonstrated that our method consistently outperformed the state-of-the-art baseline methods in providing reliable measurements of reproducibility and detecting differential interactions with biological meanings.

Implications of the Evolutionary Trajectory of Centromeres in the Fungal Kingdom.

Chromosome segregation during the cell cycle is an evolutionarily conserved, fundamental biological process. Dynamic interaction between spindle microtubules and the kinetochore complex that assembles on centromere DNA is required for faithful chromosome segregation. The first artificial minichromosome was constructed by cloning the centromere DNA of the budding yeast Saccharomyces cerevisiae. Since then, centromeres have been identified in >60 fungal species. The DNA sequence and organization of the sequence elements are highly diverse across these fungal centromeres. In this article, we provide a comprehensive view of the evolution of fungal centromeres. Studies of this process facilitated the identification of factors influencing centromere specification, maintenance, and propagation through many generations. Additionally, we discuss the unique features and plasticity of centromeric chromatin and the involvement of centromeres in karyotype evolution. Finally, we discuss the implications of recurrent loss of RNA interference (RNAi) and/or heterochromatin components on the trajectory of the evolution of fungal centromeres and propose the centromere structure of the last common ancestor of three major fungal phyla-Ascomycota, Basidiomycota, and Mucoromycota.

Sex-specific splicing of Z- and W-borne nr5a1 alleles suggests sex determination is controlled by chromosome conformation.

Pogona vitticeps has female heterogamety (ZZ/ZW), but the master sex-determining gene is unknown, as it is for all reptiles. We show that nr5a1 (Nuclear Receptor Subfamily 5 Group A Member 1), a gene that is essential in mammalian sex determination, has alleles on the Z and W chromosomes (Z-nr5a1 and W-nr5a1), which are both expressed and can recombine. Three transcript isoforms of Z-nr5a1 were detected in gonads of adult ZZ males, two of which encode a functional protein. However, ZW females produced 16 isoforms, most of which contained premature stop codons. The array of transcripts produced by the W-borne allele (W-nr5a1) is likely to produce truncated polypeptides that contain a structurally normal DNA-binding domain and could act as a competitive inhibitor to the full-length intact protein. We hypothesize that an altered configuration of the W chromosome affects the conformation of the primary transcript generating inhibitory W-borne isoforms that suppress testis determination. Under this hypothesis, the genetic sex determination (GSD) system of P. vitticeps is a W-borne dominant female-determining gene that may be controlled epigenetically.

Chromosome-scale de novo genome assembly and annotation of three representative Casuarina species: C. equisetifolia, C. glauca, and C. cunninghamiana.

Australian pine (Casuarina spp.) is extensively planted in tropical and subtropical regions for wood production, shelterbelts, environmental protection, and ecological restoration due to their superior biological characteristics, such as rapid growth, wind and salt tolerance, and nitrogen fixation. To analyze the genomic diversity of Casuarina, we sequenced the genomes and constructed de novo genome assemblies of the three most widely planted Casuarina species: C. equisetifolia, C. glauca, and C. cunninghamiana. We generated chromosome-scale genome sequences using both Pacific Biosciences (PacBio) Sequel sequencing and chromosome conformation capture technology (Hi-C). The total genome sizes for C. equisetifolia, C. glauca, and C. cunninghamiana are 268 942 579 bp, 296 631 783 bp, and 293 483 606 bp, respectively, of which 25.91, 27.15, and 27.74% were annotated as repetitive sequences. We annotated 23 162, 24 673, and 24 674 protein-coding genes in C. equisetifolia, C. glauca, and C. cunninghamiana, respectively. We then collected branchlets from male and female individuals for whole-genome bisulfite sequencing (BS-seq) to explore the epigenetic regulation of sex determination in these three species. Transcriptome sequencing (RNA-seq) revealed differential expression of phytohormone-related genes between male and female plants. In summary, we generated three chromosome-level genome assemblies and comprehensive DNA methylation and transcriptome datasets from both male and female material for three Casuarina species, providing a basis for the comprehensive investigation of genomic diversity and functional gene discovery of Casuarina in the future.

Helical chromonema coiling is conserved in eukaryotes.

Efficient chromatin condensation is required to transport chromosomes during mitosis and meiosis, forming daughter cells. While it is well accepted that these processes follow fundamental rules, there has been a controversial debate for more than 140 years on whether the higher-order chromatin organization in chromosomes is evolutionarily conserved. Here, we summarize historical and recent investigations based on classical and modern methods. In particular, classical light microscopy observations based on living, fixed, and treated chromosomes covering a wide range of plant and animal species, and even in single-cell eukaryotes suggest that the chromatids of large chromosomes are formed by a coiled chromatin thread, named the chromonema. More recently, these findings were confirmed by electron and super-resolution microscopy, oligo-FISH, molecular interaction data, and polymer simulation. Altogether, we describe common and divergent features of coiled chromonemata in different species. We hypothesize that chromonema coiling in large chromosomes is a fundamental feature established early during the evolution of eukaryotes to handle increasing genome sizes.