Topologically Associating Domains Borders Research Articles

Active enhancers strengthen insulation by RNA-mediated CTCF binding at chromatin domain boundaries.

Vertebrate genomes are partitioned into chromatin domains or topologically associating domains (TADs), which are typically bound by head-to-head pairs of CTCF binding sites. Transcription at domain boundaries correlates with better insulation; however, it is not known whether the boundary transcripts themselves contribute to boundary function. Here we characterize boundary-associated RNAs genome-wide, focusing on the disease-relevant INK4a/ARF and MYC TAD. Using CTCF site deletions and boundary-associated RNA knockdowns, we observe that boundary-associated RNAs facilitate recruitment and clustering of CTCF at TAD borders. The resulting CTCF enrichment enhances TAD insulation, enhancer-promoter interactions, and TAD gene expression. Importantly, knockdown of boundary-associated RNAs results in loss of boundary insulation function. Using enhancer deletions and CRISPRi of promoters, we show that active TAD enhancers, but not promoters, induce boundary-associated RNA transcription, thus defining a novel class of regulatory enhancer RNAs.

Comparison of the somatic TADs and lampbrush chromomere-loop complexes in transcriptionally active prophase I oocytes.

In diplotene oocyte nuclei of all vertebrate species, except mammals, chromosomes lack interchromosomal contacts and chromatin is linearly compartmentalized into distinct chromomere-loop complexes forming lampbrush chromosomes. However, the mechanisms underlying the formation of chromomere-loop complexes remain unexplored. Here we aimed to compare somatic topologically associating domains (TADs), recently identified in chicken embryonic fibroblasts, with chromomere-loop complexes in lampbrush meiotic chromosomes. By measuring 3D-distances and colocalization between linear equidistantly located genomic loci, positioned within one TAD or separated by a TAD border, we confirmed the presence of predicted TADs in chicken embryonic fibroblast nuclei. Using three-colored FISH with BAC probes, we mapped equidistant genomic regions included in several sequential somatic TADs on isolated chicken lampbrush chromosomes. Eight genomic regions, each comprising two or three somatic TADs, were mapped to non-overlapping neighboring lampbrush chromatin domains - lateral loops, chromomeres, or chromomere-loop complexes. Genomic loci from the neighboring somatic TADs could localize in one lampbrush chromomere-loop complex, while genomic loci belonging to the same somatic TAD could be localized in neighboring lampbrush chromomere-loop domains. In addition, FISH-mapping of BAC probes to the nascent transcripts on the lateral loops indicates transcription of at least 17 protein-coding genes and 2 non-coding RNA genes during the lampbrush stage of chicken oogenesis, including genes involved in oocyte maturation and early embryo development.

The role of insulators and transcription in 3D chromatin organization of flies.

The DNA in many organisms, including humans, is shown to be organized in topologically associating domains (TADs). In Drosophila, several architectural proteins are enriched at TAD borders, but it is still unclear whether these proteins play a functional role in the formation and maintenance of TADs. Here, we show that depletion of BEAF-32, Cp190, Chro, and Dref leads to changes in TAD organization and chromatin loops. Their depletion predominantly affects TAD borders located in regions moderately enriched in repressive modifications and depleted in active ones, whereas TAD borders located in euchromatin are resilient to these knockdowns. Furthermore, transcriptomic data has revealed hundreds of genes displaying differential expression in these knockdowns and showed that the majority of differentially expressed genes are located within reorganized TADs. Our work identifies a novel and functional role for architectural proteins at TAD borders in Drosophila and a link between TAD reorganization and subsequent changes in gene expression.

TADreg: a versatile regression framework for TAD identification, differential analysis and rearranged 3D genome prediction

Background/AimIn higher eukaryotes, the three-dimensional (3D) organization of the genome is intimately related to numerous key biological functions including gene expression, DNA repair and DNA replication regulations. Alteration of 3D organization, in particular topologically associating domains (TADs), is detrimental to the organism and can give rise to a broad range of diseases such as cancers.MethodsHere, we propose a versatile regression framework which not only identifies TADs in a fast and accurate manner, but also detects differential TAD borders across conditions for which few methods exist, and predicts 3D genome reorganization after chromosomal rearrangement. Moreover, the framework is biologically meaningful, has an intuitive interpretation and is easy to visualize.Result and conclusionThe novel regression ranks among top TAD callers. Moreover, it identifies new features of the genome we called TAD facilitators, and that are enriched with specific transcription factors. It also unveils the importance of cell-type specific transcription factors in establishing novel TAD borders during neuronal differentiation. Lastly, it compares favorably with the state-of-the-art method for predicting rearranged 3D genome.

Contribution of 3D genome topological domains to genetic risk of cancers: a genome-wide computational study

BackgroundGenome-wide association studies have identified statistical associations between various diseases, including cancers, and a large number of single-nucleotide polymorphisms (SNPs). However, they provide no direct explanation of the mechanisms underlying the association. Based on the recent discovery that changes in three-dimensional genome organization may have functional consequences on gene regulation favoring diseases, we investigated systematically the genome-wide distribution of disease-associated SNPs with respect to a specific feature of 3D genome organization: topologically associating domains (TADs) and their borders.ResultsFor each of 449 diseases, we tested whether the associated SNPs are present in TAD borders more often than observed by chance, where chance (i.e., the null model in statistical terms) corresponds to the same number of pointwise loci drawn at random either in the entire genome, or in the entire set of disease-associated SNPs listed in the GWAS catalog. Our analysis shows that a fraction of diseases displays such a preferential localization of their risk loci. Moreover, cancers are relatively more frequent among these diseases, and this predominance is generally enhanced when considering only intergenic SNPs. The structure of SNP-based diseasome networks confirms that localization of risk loci in TAD borders differs between cancers and non-cancer diseases. Furthermore, different TAD border enrichments are observed in embryonic stem cells and differentiated cells, consistent with changes in topological domains along embryogenesis and delineating their contribution to disease risk.ConclusionsOur results suggest that, for certain diseases, part of the genetic risk lies in a local genetic variation affecting the genome partitioning in topologically insulated domains. Investigating this possible contribution to genetic risk is particularly relevant in cancers. This study thus opens a way of interpreting genome-wide association studies, by distinguishing two types of disease-associated SNPs: one with an effect on an individual gene, the other acting in interplay with 3D genome organization.

Chromatin topology and the timing of enhancer function at the HoxD locus

The HoxD gene cluster is critical for proper limb formation in tetrapods. In the emerging limb buds, different subgroups of Hoxd genes respond first to a proximal regulatory signal, then to a distal signal that organizes digits. These two regulations are exclusive from one another and emanate from two distinct topologically associating domains (TADs) flanking HoxD, both containing a range of appropriate enhancer sequences. The telomeric TAD (T-DOM) contains several enhancers active in presumptive forearm cells and is divided into two sub-TADs separated by a CTCF-rich boundary, which defines two regulatory submodules. To understand the importance of this particular regulatory topology to control Hoxd gene transcription in time and space, we either deleted or inverted this sub-TAD boundary, eliminated the CTCF binding sites, or inverted the entire T-DOM to exchange the respective positions of the two sub-TADs. The effects of such perturbations on the transcriptional regulation of Hoxd genes illustrate the requirement of this regulatory topology for the precise timing of gene activation. However, the spatial distribution of transcripts was eventually resumed, showing that the presence of enhancer sequences, rather than either their exact topology or a particular chromatin architecture, is the key factor. We also show that the affinity of enhancers to find their natural target genes can overcome the presence of both a strong TAD border and an unfavorable orientation of CTCF sites.

Insulator-based loops mediate the spreading of H3K27me3 over distant micro-domains repressing euchromatin genes

Abstract Background Chromosomes are subdivided spatially to delimit long-range interactions into topologically associating domains (TADs). TADs are often flanked by chromatin insulators and transcription units that may participate in such demarcation. Remarkably, single-cell Drosophila TAD units correspond to dynamic heterochromatin nano-compartments that can self-assemble. The influence of insulators on such dynamic compartmentalization remains unclear. Moreover, to what extent heterochromatin domains are fully compartmentalized away from active genes remains unclear from Drosophila to human. Results Here, we identify H3K27me3 micro-domains genome-wide in Drosophila, which are attributed to the three-dimensional spreading of heterochromatin marks into euchromatin. Whereas depletion of insulator proteins increases H3K27me3 spreading locally, across heterochromatin borders, it concomitantly decreases H3K27me3 levels at distant micro-domains discrete sites. Quantifying long-range interactions suggests that random interactions between heterochromatin TADs and neighbor euchromatin cannot predict the presence of micro-domains, arguing against the hypothesis that they reflect defects in self-folding or in insulating repressive TADs. Rather, micro-domains are predicted by specific long-range interactions with the TAD borders bound by insulator proteins and co-factors required for looping. Accordingly, H3K27me3 spreading to distant sites is impaired by insulator mutants that compromise recruitment of looping co-factors. Both depletions and insulator mutants significantly reduce H3K27me3 micro-domains, deregulating the flanking genes. Conclusions Our data highlight a new regulatory mode of H3K27me3 by insulator-based long-range interactions controlling distant euchromatic genes.

Chromatin architecture reorganization during neuronal cell differentiation in Drosophila genome

The organization of the genome into topologically associating domains (TADs) was shown to have a regulatory role in development and cellular function, but the mechanism involved in TAD establishment is still unclear. Here, we present the first high-resolution contact map of Drosophila neuronal cells (BG3) and identify different classes of TADs by comparing this to genome organization in embryonic cells (Kc167). We find that only some TADs are conserved in both cell lines, whereas the rest are cell-type–specific. This is supported by a change in the enrichment of architectural proteins at TAD borders, with BEAF-32 present in embryonic cells and CTCF in neuronal cells. Furthermore, we observe strong divergent transcription, together with RNA Polymerase II occupancy and an increase in DNA accessibility at the TAD borders. TAD borders that are specific to neuronal cells are enriched in enhancers controlled by neuronal-specific transcription factors. Our results suggest that TADs are dynamic across developmental stages and reflect the interplay between insulators, transcriptional states, and enhancer activities.

Rapid reversible changes in compartments and local chromatin organization revealed by hyperosmotic shock.

Nuclear architecture is decisive for the assembly of transcriptional responses. However, how chromosome organization is dynamically modulated to permit rapid and transient transcriptional changes in response to environmental challenges remains unclear. Here we show that hyperosmotic stress disrupts different levels of chromosome organization, ranging from A/B compartment changes to reduction in the number and insulation of topologically associating domains (TADs). Concomitantly, transcription is greatly affected, TAD borders weaken, and RNA Polymerase II runs off from hundreds of transcription end sites. Stress alters the binding profiles of architectural proteins, which explains the disappearance of local chromatin organization. These processes are dynamic, and cells rapidly reconstitute their default chromatin conformation after stress removal, uncovering an intrinsic organization. Transcription is not required for local chromatin reorganization, while compartment recovery is partially transcription-dependent. Thus, nuclear organization in mammalian cells can be rapidly modulated by environmental changes in a reversible manner.

Systemic Loss and Gain of Chromatin Architecture throughout Zebrafish Development

SummaryThe spatial organization of chromosomes is critical in establishing gene expression programs. We generated in situ Hi-C maps throughout zebrafish development to gain insight into higher-order chromatin organization and dynamics. Zebrafish chromosomes segregate in active and inactive chromatin (A/B compartments), which are further organized into topologically associating domains (TADs). Zebrafish A/B compartments and TADs have genomic features similar to those of their mammalian counterparts, including evolutionary conservation and enrichment of CTCF binding sites at TAD borders. At the earliest time point, when there is no zygotic transcription, the genome is highly structured. After zygotic genome activation (ZGA), the genome loses structural features, which are re-established throughout early development. Despite the absence of structural features, we see clustering of super-enhancers in the 3D genome. Our results provide insight into vertebrate genome organization and demonstrate that the developing zebrafish embryo is a powerful model system to study the dynamics of nuclear organization.

Sub-kb Hi-C in D. melanogaster reveals conserved characteristics of TADs between insect and mammalian cells

Topologically associating domains (TADs) are fundamental elements of the eukaryotic genomic structure. However, recent studies suggest that the insulating complexes, CTCF/cohesin, present at TAD borders in mammals are absent from those in Drosophila melanogaster, raising the possibility that border elements are not conserved among metazoans. Using in situ Hi-C with sub-kb resolution, here we show that the D. melanogaster genome is almost completely partitioned into >4000 TADs, nearly sevenfold more than previously identified. The overwhelming majority of these TADs are demarcated by the insulator complexes, BEAF-32/CP190, or BEAF-32/Chromator, indicating that these proteins may play an analogous role in flies as that of CTCF/cohesin in mammals. Moreover, extended regions previously thought to be unstructured are shown to consist of small contiguous TADs, a property also observed in mammals upon re-examination. Altogether, our work demonstrates that fundamental features associated with the higher-order folding of the genome are conserved from insects to mammals.

TAD-free analysis of architectural proteins and insulators.

The three-dimensional (3D) organization of the genome is intimately related to numerous key biological functions including gene expression and DNA replication regulations. The mechanisms by which molecular drivers functionally organize the 3D genome, such as topologically associating domains (TADs), remain to be explored. Current approaches consist in assessing the enrichments or influences of proteins at TAD borders. Here, we propose a TAD-free model to directly estimate the blocking effects of architectural proteins, insulators and DNA motifs on long-range contacts, making the model intuitive and biologically meaningful. In addition, the model allows analyzing the whole Hi-C information content (2D information) instead of only focusing on TAD borders (1D information). The model outperforms multiple logistic regression at TAD borders in terms of parameter estimation accuracy and is validated by enhancer-blocking assays. In Drosophila, the results support the insulating role of simple sequence repeats and suggest that the blocking effects depend on the number of repeats. Motif analysis uncovered the roles of the transcriptional factors pannier and tramtrack in blocking long-range contacts. In human, the results suggest that the blocking effects of the well-known architectural proteins CTCF, cohesin and ZNF143 depend on the distance between loci, where each protein may participate at different scales of the 3D chromatin organization.

Widespread Rearrangement of 3D Chromatin Organization Underlies Polycomb-Mediated Stress-Induced Silencing

Chromosomes of metazoan organisms are partitioned in the interphase nucleus into discrete topologically associating domains (TADs). Borders between TADs are formed in regions containing active genes and clusters of architectural protein binding sites. The transcription of most genes is repressed after temperature stress in Drosophila. Here we show that temperature stress induces relocalization of architectural proteins from TAD borders to inside TADs, and this is accompanied by a dramatic rearrangement in the 3D organization of the nucleus. TAD border strength declines, allowing foran increase in long-distance inter-TAD interactions. Similar but quantitatively weaker effects are observed upon inhibition of transcription or depletion of individual architectural proteins. Heat shock-induced inter-TAD interactions result in increased contacts among enhancers and promoters of silenced genes, which recruit Pc and form Pc bodies in the nucleolus. These results suggest that the TAD organization of metazoan genomes is plastic and can be reconfigured quickly.