How chromosomes are positioned and folded within the nucleus has implications for gene regulation. In this Review, Kempfer and Pombo describe and evaluate methods for studying chromosome architecture and outline the insights they are providing about nuclear organization.
The 3D genome
The three-dimensional configuration of the genome is complex, dynamic and crucial for gene regulation. In the past few years, technological advances in chromosome conformation capture methods and in microscopy techniques revealed how the organization of the genome is interconnected with nuclear architecture and can vary between cell types and during cell differentiation and development. This collection includes recent articles from across the Nature group of journals and showcases both the latest advances in the methodologies used to study genome organization, and our recent understanding of how genome organization and nuclear architecture regulate gene expression, cell fate and cell function in physiology and disease. The content of this collection has been chosen by the editors of Nature Reviews Molecular Cell Biology.
Different genomic regions are replicated at different times during the S phase of the cell cycle, forming early- and late-replicating domains that occupy different locations in the nucleus. The recent identification of specific DNA sequences and long non-coding RNAs that regulate DNA replication timing is providing key insights into the roles of replication timing and into timing and 3D organization.
The 3D organization of the genome is crucial for gametogenesis, embryogenesis and cell differentiation through its modulation of transcription, DNA replication and cell division. Recent studies have highlighted the roles of 3D chromatin dynamics, such as the formation of enhancer–promoter interactions in mammalian development.
For appropriate control of gene expression, enhancers must communicate with the right target genes at the right time, typically over large genomic distances. In this Review, Schoenfelder and Fraser discuss our latest understanding of long-range enhancer–promoter crosstalk, including target-gene specificity, interaction dynamics, protein and RNA architects of interactions, roles of 3D genome organization and the pathological consequences of regulatory rewiring.
Genome organization can regulate gene expression, but can gene expression regulate genome organization? Recent studies reveal that, although not required for higher-level genome organization, transcription has a role in the formation and stabilization of genomic subdomains and enhancer–promoter interactions.
High-resolution studies of chromosome conformation are revealing that the 3D genome is organized into smaller structural features than was previously supposed and is primarily composed of compartmental domains and CTCF loops. In this Perspectives article Rowley and Corces describe the latest views on the organizational drivers and principles of the 3D genome, and the interplay between genome activity and organization.
Mechanical cues from the microenvironment can be efficiently transmitted to the nucleus to engage in the regulation of genome organization and gene expression. Recent technological and theoretical progress sheds new light on the relationships between cell mechanics, nuclear and chromosomal architecture and gene transcription.
This Review summarizes our understanding of plant chromatin organization and positioning beyond the nucleosomal level, advanced by up-to-date chromatin conformation capture methods and visualization techniques, as well as discusses future directions.
The three-dimensional (3D) organization of eukaryote chromosomes regulates genome function and nuclear processes such as DNA replication, transcription and DNA-damage repair. Experimental and computational methodologies for 3D genome analysis have been rapidly expanding, with a focus on high-throughput chromatin conformation capture techniques and on data analysis.
Recent studies show that structural variation can alter the genome architecture, leading to changes in the regulation of gene expression that cause disease. The authors review the role of genetic structural variation in disease and the pathogenic potential of changes to the 3D genome.
In this Perspective, Job Dekker et al. outline the goals and strategies of the 4D Nucleome Network, a consortium of researchers that aims to map the spatial organization and dynamics of the human and mouse genomes to gain insight into the structure and biological functions of the nucleus.
Genome-wide mapping of regulatory elements will improve our understanding of how genetic variation in the noncoding genome affects disease phenotypes.
In this Review, the authors compare commonly used chromosome conformation capture techniques, describing their respective strengths and weaknesses, and provide advice for the end user on which approach and analysis method to use.
Mechanistic insights are emerging into how long non-coding RNAs (lncRNAs) regulate gene expression by coordinating regulatory proteins, localizing to genomic loci and shaping nuclear organization. Interestingly, lncRNAs can perform functions that cannot be carried out by DNA elements or proteins alone, such as amplifying regulatory signals in the nucleus.
Three-dimensional genome organization can shape gene expression by facilitating interactions between regulatory elements. The authors review the process of X-chromosome inactivation with a focus on chromatin organization and subnuclear localization of the active and inactive X chromosomes, as well as the potential roles of long non-coding RNAs.
Mutations in non-coding parts of the genome can cause disease. Technological advances are providing unprecedented detail on genome organization and folding, and have revealed that enhancer–target gene coupling is spatially restricted, as it occurs within topologically associated domains (TADs), and that disrupting such organization can lead to disease-associated gene dysregulation.
Job Dekker asserts that cases in which data from microscopy- and 3C-based methods appear discordant about genome organization will provide opportunities to improve our models of chromatin folding.
Genome-wide mapping of chromatin contacts reveals the structural and organizational changes that the metazoan genome undergoes during cell differentiation. These changes involve entire chromosomes, which are influenced by contacts with nuclear structures such as the lamina, and local interactions mediated by transcription factors and chromatin looping.
In this article the authors review current knowledge on chromatin architecture and the molecular mechanisms that underlie it. They discuss how three-dimensional (3D) organization of chromatin relates to gene expression, development and disease, and consider its effect on genome evolution.
Synthetic biology approaches to characterize gene regulation have largely used transcription factor circuits in bacteria. However, the multilayered regulation of genes by chromatin in eukaryotes provides opportunities for more sophisticated control of gene expression. This Review describes diverse approaches for engineering eukaryotic chromatin states, the insights gained into physiological gene regulation principles, and the broad potential applications throughout biomedical research and industry.
The evolution of genes is influenced by regional variation in mutation rates (RViMR). Chromatin organization affects RViMR, although the correlation between chromatin state and mutation types and rates is complex. This Review describes recent research on RViMR and chromatin organization, and the emerging findings from investigations of both germline and somatic mutations.
Promoter–enhancer loopings and other features of the 3D genome are dynamically regulated in the brain. In this Review, Akbarian and colleagues discuss how neuronal and glial gene expression is governed by the 3D genome, with implications for cognition and neuropsychiatric disease.
In this Review, Aifantis and colleagues describe recent insights into the epigenetic dysregulation of malignant blood stem cell differentiation.
Research & Methods
Transcription factors orchestrate dynamic interplay between genome topology and gene regulation during cell reprogramming
The authors analyze time-resolved changes in genome topology, gene expression, transcription-factor binding, and chromatin state during iPSC generation. They conclude that 3D genome reorganization generally precedes gene expression changes and that removal of locus-specific topological barriers explains why pluripotency genes are activated sequentially during reprogramming.
Risk loci for breast cancer have been identified by genome-wide association studies. Here, the authors use Capture Hi-C to identify 110 putative target genes at 33 loci and assessed associations of gene expression, SNP genotype, and survival, providing evidence of mechanisms that may influence the prognosis and risk of breast cancer.
Transcriptional decomposition reveals active chromatin architectures and cell specific regulatory interactions
Transcriptional regulation is coupled with chromosomal positioning and chromatin architecture. Here the authors develop a transcriptional decomposition approach to separate expression associated with genome structure from independent effects not directly associated with genomic positioning.
Super-enhancers (SEs) are important regulatory elements for gene expression, but their intrinsic properties remain poorly understood. Here the authors analyse Hi-C and ChIP-seq data and find that a significant fraction of SEs are hierarchically organized, containing both hub and non-hub enhancers.
3DNetMod identifies nested topologically associating domains (TADs) and subTADs from Hi-C data.
Knowledge of three-dimensional (3D) genome structure and how polyploidization shapes it remains poor. A study now characterizes and compares 3D genomes for diploid and tetraploid cotton, showing how allopolyploidization affects 3D genome architecture and transcriptional regulation.
Transcriptome, DNA methylome and Hi-C profiling of peri- and post-implantation mouse cell lineages identified allele- and lineage-specific methylation patterns. Global demethylation and remethylation correlate with megabase chromatin compartments.
Baarlink et al. identify a transient pool of nuclear F-actin, the dynamics of which are controlled by cofilin-1 that accumulates after mitosis and is important for chromatin reorganization in G1.
Stratification of TAD boundaries reveals preferential insulation of super-enhancers by strong boundaries
Topologically associating domains (TADs) detected by Hi-C technologies are megabase-scale areas of highly interacting chromatin. Here Gong, Lazaris et al. develop a computational approach to improve the reproducibility of Hi-C contact matrices and stratify TAD boundaries based on their insulating strength.
Analysis of the 3D genomic organization of Schizosaccharomyces pombe during the cell cycle reveals that condensin mediates formation of large domains that serve as chromosomal compaction units, whereas cohesin forms smaller, more stable domains.
Genome-wide analyses of somatic mutations across six cancer types show that mutation frequencies differ between chromosomal regions located at the nuclear core versus the periphery, and thus mutational patterns are influenced by nuclear architecture.
The nuclear organization of interphase chromosomes is thought to be mediated by architectural protein complexes such as CTCF and cohesin, which are found at loops and at the boundaries of topological domains (TADs). However, experimental depletion of these proteins has shown limited impact on chromosome organization. Here, Francois Spitz and colleagues perform an inducible deletion of the cohesin-loading factor Nipbl in liver cells in mice. They find that depletion of chromosome-associated cohesin leads to the loss of TADs and TAD-associated loops, but segregation of the genome into compartments is preserved and transcription is affected only at a subset of genes. The disappearance of TADs unmasks a finer compartment structure that reflects local transcriptional activity. Genome organization therefore seems to result from two distinct mechanisms with different requirements for cohesin.
Sub-kb Hi-C in D. melanogaster reveals conserved characteristics of TADs between insect and mammalian cells
Topologically associating domain (TAD) boundaries in flies seem to be different from those in mammals. Here, the authors use Hi-C with sub-kb resolution to identify about 4000 TADs in flies, most demarcated by the insulator complexes BEAF-32/CP190 or BEAF-32/Chromator like CTCF/cohesin in mammals.
Although topologically associating domains (TADs) have been extensively investigated, it is not clear to what extent DNA sequence contributes to their formation. Here the authors develop software to identify high-resolution TAD boundaries and reveal their relationship to underlying DNA motifs.
Enhancer connectome in primary human cells identifies target genes of disease-associated DNA elements
High-resolution contact maps of active enhancers and target genes generated by H3K27ac HiChIP in primary human cells provide rational guides to link noncoding disease-associated risk variants to candidate causal genes. Genes are validated by CRISPR activation and interference at connected enhancers and eQTL analysis, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases.
Identifying topologically associating domains and subdomains by Gaussian Mixture model And Proportion test
Spatial organization of the genome plays a crucial role in regulating gene expression. Here the authors introduce GMAP, the Gaussian Mixture model And Proportion test, to identify topologically associating domains and subdomains in Hi-C data.
Promoter-enhancer interactions identified from Hi-C data using probabilistic models and hierarchical topological domains
Proximity-ligation methods like Hi-C map DNA-DNA interactions and reveal its organization into topologically associating domains (TADs). Here the authors describe PSYCHIC, a computational approach for analysing Hi-C data that allows the identification of promoter-enhancer interactions.
3D genome of multiple myeloma reveals spatial genome disorganization associated with copy number variations
Chromosome conformation capture techniques enable the study of genome organization in cancer cells. Here, the authors use Hi-C, WGS, and RNA-seq to study the 3D genome of multiple myeloma and find that genome disorganization is associated with copy number variations and changes in gene expression.
Schalbetter et al. show by Hi-C and modelling that mitotic chromosome compaction in budding yeast occurs by cis-looping of chromatin, and reveal distinct roles for cohesin and condensin depending on chromatin context.
Lineage-specific dynamic and pre-established enhancer–promoter contacts cooperate in terminal differentiation
Adam Rubin, Brook Barajas, Mayra Furlan-Magaril and colleagues studied dynamic chromatin across the genome of differentiating human skin keratinocytes, identifying both stable and reorganizing classes of transcriptional enhancers.
Topologically associating domains are ancient features that coincide with Metazoan clusters of extreme noncoding conservation
Metazoan genomes contain many clusters of conserved noncoding elements. Here, the authors provide evidence that these clusters coincide with distinct topologically associating domains in humans and Drosophila, revealing a conserved regulatory genomic architecture.
Tissue-specific CTCF–cohesin-mediated chromatin architecture delimits enhancer interactions and function in vivo
Hanssen et al. show that CTCF–cohesin binding sites at the α-globin gene cluster function as boundaries to restrict the interaction of enhancers with the flanking chromatin, thus preventing abnormal gene expression.
Chromatin looping plays an important role in gene regulation and the ability to manipulate loops would aid in understanding how this occurs. Here the authors present CLOuD9, a system that uses dimerized Cas9 complexes to selectively and reversibly establish chromatin loops.
In mammals, chromatin undergoes reorganization after fertilization, but little is known about the molecular basis for reprogramming of higher-order chromatin structure. Here, Wei Xie and colleagues have developed a low-input Hi-C approach, which they apply to examine chromatin organization in mouse oocytes and preimplantation embryos. They find that chromatin has markedly reduced higher-order structure for both parental genomes after fertilization. Topological associated domain boundaries and chromatin compartments start to emerge in zygotes but the subsequent maturation of three-dimensional chromatin architecture is surprisingly slow.
New analyses reveal that TERRA transcripts arising from the subtelomeric pseudoautosomal (PAR) region of sex chromosomes nucleate pairing of X alleles in mouse ES cells.
Eukaryotic chromosomes undergo a cycle of compaction and decondensation during the cell cycle. Here, Peter Fraser and colleagues have developed an improved single-cell Hi-C method to characterize the 3D organization of chromosomes through the cell cycle in thousands of individual mouse embryonic stem cells. They find that chromosomal compartments, topological-associated domains and loops are each governed by distinct dynamics and reveal a continuum of dynamic chromosomal structural features throughout the cell cycle. The results will be a new point of reference for interpreting chromosome conformation Hi-C maps.
Six tools to call chromatin interactions and seven tools for topologically associating domain calling are systematically compared with real and simulated data. The strengths and weaknesses of each tool are discussed.
This Analysis explores the relationship between chromosome conformation capture (for example, Hi-C) and FISH datasets, and uses simulations to reconcile measurements from the two technologies.
Active and poised promoter states drive folding of the extended HoxB locus in mouse embryonic stem cells
Homotypic interactions between active and Polycomb-repressed promoters co-occurring in the same DNA fiber, rather than CTCF occupancy, explain the 3D HoxB folding pattern.
Cohesin and CTCF are known to spatially organize mammalian genomes into chromatin loops and topologically associated domains. CTCF binds to specific DNA sequences, but it is unclear how cohesin is recruited to these sites. Here, Jan-Michael Peters and colleagues show that the distribution of cohesin in the mouse genome depends on CTCF, transcription and the cohesin-release factor Wapl. In the absence of CTCF, cohesin accumulates at the transcription start sites of active genes, which are bound by the cohesion-loading complex. In the absence of both CTCF and Wapl, cohesin accumulates at the 3′ end of active genes. The authors propose that cohesin is loaded onto DNA at sites that are distinct from its final binding sites and can be translocated by transcription until it either encounters CTCF bound to DNA or is released by Wapl. A mechanism of transcription-mediated cohesin translocation could allow the extrusion of chromatin loops.
CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome
Regulatory elements for specific human genes are rapidly identified with CRISPR epigenome editing.
ERCC1–XPF cooperates with CTCF and cohesin to facilitate the developmental silencing of imprinted genes
Chatzinikolaou et al. show that the nucleotide excision repair complex ERCC1–XPF cooperates with the chromatin organizer CTCF, cohesin subunits and ATRX to facilitate the silencing of a subset of imprinted genes in the developing liver.
It has been difficult to investigate chromosome organization in early embryos with genomic techniques owing to the paucity of cellular material. Here, Kikuë Tachibana-Konwalski and colleagues have developed a single-nucleus Hi-C protocol, which they apply to investigate chromatin organization during the developmental transition from oocytes to zygotes in mice. They find that chromatin architecture is distinct in the paternal and maternal pronuclei within a single-cell zygote. Zygotic maternal nuclei contain topological domains and loops but no A–B compartments, whereas compartments can be observed in paternal nuclei. Clusters of contacts are variable between individual cells and do not always match topological domains across populations. The authors propose that the organization of zygotic maternal chromatin represents a transition state towards that of totipotent cells.
To understand how chromosomes are folded and organized in the nucleus, researchers have taken advantage of microscopy and molecular techniques based on chromosome conformation capture, such as Hi-C. In this paper, Ernest Laue and colleagues describe a novel approach in which they first image and then apply a single-cell Hi-C protocol to individual haploid mouse embryonic stem cells in the G1 phase of the cell cycle. This high-resolution approach allowed the authors to examine how the topological domains and looping of chromosomes vary from cell to cell, at a scale of less than 100 kilobases, and to validate the chromosome structures by imaging.
Our understanding of the three-dimensional organization of the genome in the nucleus has improved dramatically as a result of both developments in microscopy and molecular methods based on chromatin conformation capture (3C). Here, Ana Pombo and colleagues present a novel method of measuring chromatin contacts, called genome architecture mapping (GAM). GAM involves sequencing DNA from a large collection of thin nuclear cryosections and, unlike 3C methods, does not require ligation to capture contacts in an unbiased manner. It overcomes some of the limitations of 3C-based methods and reveals abundant three-way contacts across the genome.
Genome-wide mapping of long-range contacts unveils clustering of DNA double-strand breaks at damaged active genes
Capture Hi-C analysis reveals that DNA double-strand breaks within transcriptionally active regions of the human genome form clusters that exhibit delayed repair in the G1 phase of the cell cycle.
Single-cell combinatorial indexed Hi-C (sciHi-C) is a streamlined protocol for generating thousands of high-quality single-cell chromosome conformation data sets that resemble bulk Hi-C data in aggregate.
Chromosomes must be folded efficiently in the nucleus to allow compactness and to regulate access to DNA. Analytical methods used to examine chromosomal structures have included chromosome conformation capture techniques, which detect pairwise spatial proximity between genomic loci. Here, Amos Tanay and colleagues develop a chromosome conformation capture sequencing method that uses chromosomal walks (C-walks) to link multiple genomic loci together into three-dimensional proximity chains. Applying the technique to mammalian cells, the authors show that chromosomal territories and topological domains (TADs) are part of a nested hierarchical fold structure and they characterize the chromosomal conformation around active and inactive genes. These findings add to our understanding of how higher order chromosomal structures participate in genome regulation.
Chromatin is organized into hierarchical three-dimensional structures that are thought to have a role in gene regulation by defining the functional units within which cis-regulatory elements interact with their target genes. Here, Daniel Geschwind and colleagues use the Hi-C technique to generate high-resolution three-dimensional maps of chromatin contacts in human developing brain. By integrating the Hi-C maps with other datasets they identify novel enhancer–promoter contacts, many of which are associated with human cognitive function. They also integrate these chromatin contact maps with non-coding variants identified in schizophrenia genome-wide association studies to propose novel candidate risk genes and pathways for schizophrenia.
SOX9 is a developmental transcription factor with functions in chondrocyte differentiation and male sex determination, and genomic duplications in the SOX9 locus have been linked to various human diseases. Stefan Mundlos and colleagues use chromosome conformation capture techniques to look at the effect of such duplications on the chromatin partitioning units termed topologically associated domains (TADs) that surround the mouse Sox9 locus. They find that although TADs are stable genomic regulatory units, they can be rearranged by structural genomic variations to create novel chromatin regulatory domains. Duplications are generally thought to confer their phenotypic effect through an increase in gene dosage, but these results show how duplications can also affect higher order chromatin structure.
During female development, X-chromosome inactivation is triggered by upregulation of the non-coding Xist RNA from one of the two X chromosomes. Chromosome conformation capture approaches have shown a loss of local structure on the inactive X (Xi) and formation of large mega-domains, separated by a region containing the DXZ4 macrosatellite. These authors investigate the structure, chromatin accessibility and expression status of the mouse Xi using allele-specific Hi-C, ATAC–seq and RNA–seq in embryonic stem cells and neural progenitor cells (NPCs). The Xi in NPCs lacks topologically associating domains (TADs) except around genes that escape X-chromosome inactivation, suggesting that TAD formation is driven by gene activity. The DXZ4-containing region and Xist shape the mega-domain structure of the Xi.
How chromatin is folded in the nucleus has important implications for many biological processes, from the regulation of gene expression to DNA replication. Here Xiaowei Zhuang and colleagues use super-resolution imaging to directly observe the organization of Drosophila chromatin at a scale spanning the sizes of individual genes and gene regulatory domains. They find that transcriptionally active, inactive, and Polycomb-repressed chromatin states each have a distinct spatial organization. Transcriptionally inactive chromatin resembles the fractal globule state of a polymer, whereas Polycomb domains have a unique compact organization and spatial isolation from other domains, explaining why gene expression is so strongly repressed in this state.
Cancer genome sequencing studies have identified recurrent IDH mutations in brain tumours and other cancers. IDH mutant gliomas have altered DNA methylation landscapes, such as hypermethylation of CpG island promoters. Here, Brad Bernstein and colleagues show that the effects of IDH1 mutation in gliomas are not limited to CpG islands, and the binding sites of the methylation-sensitive insulator CTCF are also hypermethylated. Disruption of a CTCF boundary near the glioma oncogene PDGFRA allows a constitutive enhancer to aberrantly contact and activate it. IDH mutations can therefore promote gliomagenesis by disrupting chromosomal topology and allowing aberrant gene regulatory interactions.
Dosage compensation in the roundworm Caenorhabditis elegans is a good model for understanding the role of three-dimensional chromosome organization in regulating gene expression. Here, Barbara Meyer and colleagues use genome-wide chromosome conformation capture techniques in wild-type XX hermaphrodite embryos and those lacking the dosage compensation complex (DCC), to obtain three-dimensional maps of the C. elegans genome. The DCC remodels hermaphrodite X chromosomes into a spatial conformation of topologically associating domains that is distinct from that on autosomes.
The genomic landscape of balanced cytogenetic abnormalities associated with human congenital anomalies
Michael Talkowski and colleagues analyze balanced chromosomal abnormalities in 273 individuals by whole-genome sequencing. Their findings suggest that sequence-level resolution improves prediction of clinical outcomes for balanced rearrangements and provides insight into pathogenic mechanisms such as altered gene regulation due to changes in chromosome topology.
Sean Whalen and colleagues present a computational method, TargetFinder, for reconstructing three-dimensional regulatory landscapes using one-dimensional genomic features. TargetFinder identifies the minimal set of features necessary to predict individual interacting enhancer–promoter pairs and accurately distinguishes them from non-interacting pairs.
A single three-dimensional chromatin compartment in amphioxus indicates a stepwise evolution of vertebrate Hox bimodal regulation
José Luis Gómez-Skarmeta, Hector Escrivá, Ignacio Maeso, Damien Devos and colleagues perform 4C-seq profiling of the Hox cluster in amphioxus embryos and find that, unlike in vertebrate embryos, the cluster is organized into a single chromatin interaction domain. They suggest that the vertebrate Hox bipartite regulatory system is an evolutionary novelty.
Sarah Elderkin and colleagues show that PRC1 acts as a master regulator of genome architecture in mouse embryonic stem cells by organizing genes in three-dimensional interaction networks. They find that the strongest spatial network is composed of the four Hox clusters and key early developmental transcription factor genes, and they propose that selective release of genes from this spatial network underlies cell fate specification during embryonic development.
Nicholas Luscombe, Cameron Osborne and colleagues report the use of Capture Hi-C (CHi-C) to detect the long-range interactions of almost 22,000 promoters in 2 human cell types. They found that transcriptionally inactive genes interact with previously uncharacterized elements that may act as long-range silencers.
Douglas Higgs and colleagues functionally test the α-globin super-enhancer in mice by genetically deleting its constituent enhancers. They find that the individual regulatory elements seem to act independently and in an additive way with respect to hematological phenotype, gene expression, and chromatin structure and conformation.
Modeling disease risk through analysis of physical interactions between genetic variants within chromatin regulatory circuitry
Peter Scacheri and colleagues identify ‘outside’ SNPs that physically interact with GWAS risk SNPs as part of a target gene's regulatory circuitry. Their findings suggest a model whereby outside variants and GWAS SNPs that physically interact collude to influence target transcript levels as well as clinical risk.
Transcription factors mediate condensin recruitment and global chromosomal organization in fission yeast
Ken-ichi Noma and colleagues use ChIA-PET to identify genome-wide associations mediated by condensin and cohesin in fission yeast. They find that cohesin and condensin generate small and larger chromatin domains, respectively, and that condensin, but not cohesin, connects cell cycle–regulated genes bound by mitotic transcription factors.
The chromatin remodeler Brg1 activates enhancer repertoires to establish B cell identity and modulate cell growth
B lineage development requires the transcription factors E2A, EBF1, Foxo1 and Ikaros. Murre and colleagues show that these factors gain access to lineage-specific enhancer sites by the action of the chromatin remodeler Brg1.
Tcra and Tcrd segments are embedded within a single locus but undergo distinct temporal rearrangements. Krangel and colleagues show that chromatin looping mediated by CTCF regulates the availability of variable gene segments to the RAG recombinase.
The covalent insertion of fluorophore-labeled DNA adaptors by Tn5 transposase into open chromatin allows its imaging and subsequent analysis by sequencing from exactly the same samples.
HiChIP combines chromosome conformation capture with immunoprecipitation- and tagmentation-based library preparation to uncover the 3D chromatin architecture focused around a protein of interest.
The combination of short and long crosslinkers during chromosome conformation capture allows the interrogation of structure from the nucleosome to the chromosome-wide level in yeast.
UMI-4C is a rapid, simplified barcoding approach to targeted chromatin conformation capture that produces high-complexity libraries from low sample input, is easily multiplexed and gives a quantitative, statistically defined readout.
Targeted DNase Hi-C uses DNase I instead of restriction enzymes for chromatin fragmentation and improves the resolution of chromatin interaction maps.
Pooling barcoded 3C libraries and simultaneously capturing interactions at many loci of interest generates reproducible cis- and trans-interaction maps at high resolution from low amounts of input material. This allows for the comparison of interactions in different cell types using common software designed for differential analysis of sequence count data, rather than requiring software specifically designed for 3C experiments.
Retroviruses such as HIV integrate into the host genome as an essential step prior to their replication. Here Lelek et al. identify nuclear pore complex proteins that are essential for HIV nuclear import and productive integration, and show that the intranuclear protein Tpr influences integration into transcriptionally active chromatin.
Nuclear lamins mediate interactions between chromatin and the nuclear envelope, however they are also found throughout the nucleoplasm. By measuring the dynamics of different genomic loci, Bronshtein et al.show that lamin A is also required for the stability of the nuclear interior.
Polycomb Group (PcG) proteins regulate gene expression and genome architecture. Using super-resolution microscopy and molecular simulations, Waniet al. describe the organization of PcG proteins into hundreds of nano-scale protein clusters and suggest these clusters shape genome architecture.
The formation of chromatin loops is mainly mediated by DNA-binding proteins (DBPs) that bind to the interacting sites and form complexes in 3D space. Here, Zhang et al.present an algorithm integrating ChIP-seq and Hi-C data to systematically identify both the 1D- and 3D-cooperation between DBPs.
Chromosome conformation is a dynamic process, especially in brain. Here, Mitchell and colleagues devise a method they call NeuroDam that can prospectively tag chromosome conformation in the mouse brain in vivo, and longitudinally assess long range chromosome looping weeks and months later.
Capture of associated targets on chromatin links long-distance chromatin looping to transcriptional coordination
Chromatin architecture is a key regulator of transcriptional processes, however current methods to investigate it have technical limitations. Here, the authors describe a novel chromatin capture technique, CATCH, which can be used to identify and characterize complex genomic interaction networks.
Investigation of the spatial structure and interactions of the genome at sub-kilobase-pair resolution using T2C
This protocol describes targeted chromatin capture (T2C), a high-resolution method to interrogate 3D chromatin organization and genomic interactions at sub-kilobase-pair resolution that requires minimal cell numbers and sequencing depth.
This protocol describes how to prepare samples for labeling nuclei of cultured mammalian cells for 3D structured illumination microscopy of nuclear structures. Image acquisition, registration and downstream image analysis are also described.
Li et al. provide a protocol for long-read ChIA-PET, a technique for mapping chromatin interactions. The longer paired-end tags, which are generated by tagmentation, provide sufficient coverage to determine haplotype-specific chromatin interactions at single-nucleotide resolution.
Ramani et al. describe a protocol for in situ DNase Hi-C as an alternative to traditional Hi-C methods that use restriction enzymes. The use of DNase I for chromatin digestion circumvents the resolution limit imposed when relying on genomic restriction sites.