Active chromatin and transcription play a key role in chromosome partitioning into topologiсally associating domains
ABSTRACT: Recent advances enabled by Hi-C technique have unraveled principles of chromosomal folding, which were since linked to many genomic processes. In particular, Hi-C revealed that chromosomes of animals are organized into Topologically Associating Domains (TADs), evolutionary conserved compact chromatin domains that influence gene expression. However, mechanisms that underlie partitioning of the genome into TADs remain poorly understood. To explore principals of TAD folding in Drosophila melanogaster, we performed Hi-C and PolyA+ RNA-seq in four cell lines of various origins (S2, Kc167, DmBG3-c2, and OSC). Contrary to previous studies, we find that regions between TADs (i.e. the inter-TADs and TAD boundaries) in Drosophila are only weakly enriched with the insulator protein dCTCF, while another insulator protein Su(Hw) is preferentially present within TADs. However, Drosophila inter-TADs harbor active chromatin and constitutively transcribed (housekeeping) genes. Accordingly, we find that binding of insulator proteins dCTCF and Su(Hw) predict TAD boundaries much worse than the active chromatin marks (in the minimal case, H3K4me3 and total RNA) do. Moreover, inter-TADs correspond to decompacted interbands of polytene chromosomes, whereas TADs mostly correspond to densely packed bands. Collectively, our results suggest that TADs are condensed chromatin domains depleted in active chromatin marks, separated by regions of active chromatin that cannot be organized into compact structures, possibly due to high levels of histone acetylation. Finally, we test this hypothesis by polymer simulations, and find that TAD partitioning can be explained by different modes of inter-nucleosomal interactions for active and inactive chromatin. Hi-C experiments, PolyA+ RNA profiling and mapping of chromosomal rearrangements in four Drosophila melanogaster cell lines.
Project description:Recent advances enabled by the Hi-C technique have unraveled many principles of chromosomal folding that were subsequently linked to disease and gene regulation. In particular, Hi-C revealed that chromosomes of animals are organized into topologically associating domains (TADs), evolutionary conserved compact chromatin domains that influence gene expression. Mechanisms that underlie partitioning of the genome into TADs remain poorly understood. To explore principles of TAD folding in Drosophila melanogaster, we performed Hi-C and poly(A)(+) RNA-seq in four cell lines of various origins (S2, Kc167, DmBG3-c2, and OSC). Contrary to previous studies, we find that regions between TADs (i.e., the inter-TADs and TAD boundaries) in Drosophila are only weakly enriched with the insulator protein dCTCF, while another insulator protein Su(Hw) is preferentially present within TADs. However, Drosophila inter-TADs harbor active chromatin and constitutively transcribed (housekeeping) genes. Accordingly, we find that binding of insulator proteins dCTCF and Su(Hw) predicts TAD boundaries much worse than active chromatin marks do. Interestingly, inter-TADs correspond to decompacted inter-bands of polytene chromosomes, whereas TADs mostly correspond to densely packed bands. Collectively, our results suggest that TADs are condensed chromatin domains depleted in active chromatin marks, separated by regions of active chromatin. We propose the mechanism of TAD self-assembly based on the ability of nucleosomes from inactive chromatin to aggregate, and lack of this ability in acetylated nucleosomal arrays. Finally, we test this hypothesis by polymer simulations and find that TAD partitioning may be explained by different modes of inter-nucleosomal interactions for active and inactive chromatin.
Project description:Deciphering the rules of genome folding in the cell nucleus is essential to understand its functions. Recent chromosome conformation capture (Hi-C) studies have revealed that the genome is partitioned into topologically associating domains (TADs), which demarcate functional epigenetic domains defined by combinations of specific chromatin marks. However, whether TADs are true physical units in each cell nucleus or whether they reflect statistical frequencies of measured interactions within cell populations is unclear. Using a combination of Hi-C, three-dimensional (3D) fluorescent in situ hybridization, super-resolution microscopy, and polymer modeling, we provide an integrative view of chromatin folding in Drosophila. We observed that repressed TADs form a succession of discrete nanocompartments, interspersed by less condensed active regions. Single-cell analysis revealed a consistent TAD-based physical compartmentalization of the chromatin fiber, with some degree of heterogeneity in intra-TAD conformations and in cis and trans inter-TAD contact events. These results indicate that TADs are fundamental 3D genome units that engage in dynamic higher-order inter-TAD connections. This domain-based architecture is likely to play a major role in regulatory transactions during DNA-dependent processes.
Project description:A current question in the high-order organization of chromatin is whether topologically associating domains (TADs) are distinct from other hierarchical chromatin domains. However, due to the unclear TAD definition in tradition, the structural and functional uniqueness of TAD is not well studied. In this work, we refined TAD definition by further constraining TADs to the optimal separation on global intra-chromosomal interactions. Inspired by this constraint, we developed a novel method, called HiTAD, to detect hierarchical TADs from Hi-C chromatin interactions. HiTAD performs well in domain sensitivity, replicate reproducibility and inter cell-type conservation. With a novel domain-based alignment proposed by us, we defined several types of hierarchical TAD changes which were not systematically studied previously, and subsequently used them to reveal that TADs and sub-TADs differed statistically in correlating chromosomal compartment, replication timing and gene transcription. Finally, our work also has the implication that the refinement of TAD definition could be achieved by only utilizing chromatin interactions, at least in part. HiTAD is freely available online.
Project description:The spatial arrangement of interphase chromosomes in the nucleus is important for gene expression and genome function in animals and in plants. The recently developed Hi-C technology is an efficacious method to investigate genome packing. Here we present a detailed Hi-C map of the three-dimensional genome organization of the plant Arabidopsis thaliana. We find that local chromatin packing differs from the patterns seen in animals, with kilobasepair-sized segments that have much higher intrachromosome interaction rates than neighboring regions, representing a dominant local structural feature of genome conformation in A. thaliana. These regions, which appear as positive strips on two-dimensional representations of chromatin interaction, are enriched in epigenetic marks H3K27me3, H3.1, and H3.3. We also identify more than 400 insulator-like regions. Furthermore, although topologically associating domains (TADs), which are prominent in animals, are not an obvious feature of A. thaliana genome packing, we found more than 1000 regions that have properties of TAD boundaries, and a similar number of regions analogous to the interior of TADs. The insulator-like, TAD-boundary-like, and TAD-interior-like regions are each enriched for distinct epigenetic marks and are each correlated with different gene expression levels. We conclude that epigenetic modifications, gene density, and transcriptional activity combine to shape the local packing of the A. thaliana nuclear genome.
Project description:Mammalian chromatin is spatially organized at many scales showing two prominent features in interphase: (i) alternating regions (1-10 Mb) of active and inactive chromatin that spatially segregate into different compartments, and (ii) domains (<1 Mb), that is, regions that preferentially interact internally [topologically associating domains (TADs)] and are central to gene regulation. There is growing evidence that TADs are formed by active extrusion of chromatin loops by cohesin, whereas compartmentalization is established according to local chromatin states. Here, we use polymer simulations to examine how loop extrusion and compartmental segregation work collectively and potentially interfere in shaping global chromosome organization. A model with differential attraction between euchromatin and heterochromatin leads to phase separation and reproduces compartmentalization as observed in Hi-C. Loop extrusion, essential for TAD formation, in turn, interferes with compartmentalization. Our integrated model faithfully reproduces Hi-C data from puzzling experimental observations where altering loop extrusion also led to changes in compartmentalization. Specifically, depletion of chromatin-associated cohesin reduced TADs and revealed finer compartments, while increased processivity of cohesin strengthened large TADs and reduced compartmentalization; and depletion of the TAD boundary protein CTCF weakened TADs while leaving compartments unaffected. We reveal that these experimental perturbations are special cases of a general polymer phenomenon of active mixing by loop extrusion. Our results suggest that chromatin organization on the megabase scale emerges from competition of nonequilibrium active loop extrusion and epigenetically defined compartment structure.
Project description:Chromatin of mammalian nucleus folds into discrete contact enriched regions such as Topologically Associating Domains (TADs). Folding hierarchy and internal organization of TADs is highly dynamic throughout cellular differentiation, and are correlated with gene activation and silencing. To account for multiple interacting TADs, we developed a parsimonious randomly cross-linked (RCL) polymer model that maps high frequency Hi-C encounters within and between TADs into direct loci interactions using cross-links at a given base-pair resolution. We reconstruct three TADs of the mammalian X chromosome for three stages of differentiation. We compute the radius of gyration of TADs and the encounter probability between genomic segments. We found 1) a synchronous compaction and decompaction of TADs throughout differentiation and 2) high order organization into meta-TADs resulting from weak inter-TAD interactions. Finally, the present framework allows to infer transient properties of the chromatin from steady-state statistics embedded in the Hi-C/5C data.
Project description:BACKGROUND:The three-dimensional (3D) structure of the genome plays a crucial role in gene expression regulation. Chromatin conformation capture technologies (Hi-C) have revealed that the genome is organized in a hierarchy of topologically associated domains (TADs), sub-TADs, and chromatin loops. Identifying such hierarchical structures is a critical step in understanding genome regulation. Existing tools for TAD calling are frequently sensitive to biases in Hi-C data, depend on tunable parameters, and are computationally inefficient. METHODS:To address these challenges, we developed a novel sliding window-based spectral clustering framework that uses gaps between consecutive eigenvectors for TAD boundary identification. RESULTS:Our method, implemented in an R package, SpectralTAD, detects hierarchical, biologically relevant TADs, has automatic parameter selection, is robust to sequencing depth, resolution, and sparsity of Hi-C data. SpectralTAD outperforms four state-of-the-art TAD callers in simulated and experimental settings. We demonstrate that TAD boundaries shared among multiple levels of the TAD hierarchy were more enriched in classical boundary marks and more conserved across cell lines and tissues. In contrast, boundaries of TADs that cannot be split into sub-TADs showed less enrichment and conservation, suggesting their more dynamic role in genome regulation. CONCLUSION:SpectralTAD is available on Bioconductor, http://bioconductor.org/packages/SpectralTAD/ .
Project description:MOTIVATION:The three-dimensional structure of the genome is an important regulator of many cellular processes including differentiation and gene regulation. Recently, technologies such as Hi-C that combine proximity ligation with high-throughput sequencing have revealed domains of self-interacting chromatin, called topologically associating domains (TADs), in many organisms. Current methods for identifying TADs using Hi-C data assume that TADs are non-overlapping, despite evidence for a nested structure in which TADs and sub-TADs form a complex hierarchy. RESULTS:We introduce a model for decomposition of contact frequencies into a hierarchy of nested TADs. This model is based on empirical distributions of contact frequencies within TADs, where positions that are far apart have a greater enrichment of contacts than positions that are close together. We find that the increase in contact enrichment with distance is stronger for the inner TAD than for the outer TAD in a TAD/sub-TAD pair. Using this model, we develop the TADtree algorithm for detecting hierarchies of nested TADs. TADtree compares favorably with previous methods, finding TADs with a greater enrichment of chromatin marks such as CTCF at their boundaries. AVAILABILITY AND IMPLEMENTATION:A python implementation of TADtree is available at http://compbio.cs.brown.edu/software/ CONTACT:email@example.com SUPPLEMENTARY INFORMATION:Supplementary data are available at Bioinformatics online.
Project description:Topologically associating domains (TADs) are widely recognized as fundamental elements of the 3D structure of the eukaryotic genome. However, while the structural importance of the insulator protein CTCF together with cohesin at TAD borders in mammalian cells is well established, the absence of such co-localization at most TAD borders in recent Hi-C studies of D. melanogaster is enigmatic, raising the possibility that these TAD border elements are not generally conserved among metazoans. Using in situ Hi-C with sub-kb resolution, we show that the genome of D. melanogaster is almost completely partitioned into more than 4,000 TADs (median size, 13 kb), nearly 7-fold more than previously identified. The overwhelming majority of these TADs are demarcated by pairs of Drosophila-specific insulator proteins, BEAF-32/CP190 or BEAF-32/Chromator, indicating that these proteins may play an analogous role in Drosophila as that of the CTCF/cohesin pair in mammals. Moreover, we find that previously identified TADs enriched for inactive chromatin are predominantly assembled from the higher-level interactions between smaller TADs. In contrast, the closely-spaced small TADs in regions previously thought to be unstructured “inter-TADs” are organized in an open configuration with far fewer TAD-TAD interactions. Such structures can also be identified in some “inter-TAD” regions of the mammalian genome, suggesting that larger assemblages of small self-interacting TADs separated by a “burst” of adjacent small, weakly interacting TADs may be a conserved, basic characteristic of the higher order folding of the metazoan genome. Overall design: The genome-wide interactome with sub-kb resolution were generated using in-situ Hi-C method in both asychonous Drosophola cell line S2R+ and the cells that were arrest at G1/S
Project description:Recent chromosome conformation capture (3C) derived techniques have revealed that topologically associating domain (TAD) is a pervasive element in chromatin three-dimensional (3D) organization. However, there is currently no parameter to quantitatively measure the structural characteristics of TADs, thus obscuring our understanding on the structural and functional differences among TADs. Based on our finding that there exist intrinsic chromatin interaction patterns in TADs, we define a theoretical parameter, called aggregation preference (AP), to characterize TAD structures by capturing the interaction aggregation degree. Applying this defined parameter to 11 Hi-C data sets generated by both traditional and in situ Hi-C experimental pipelines, our analyses reveal that heterogeneous structures exist among TADs, and this structural heterogeneity is significantly correlated to DNA sequences, epigenomic signals and gene expressions. Although TADs can be stable in genomic positions across cell lines, structural comparisons show that a considerable number of stable TADs undergo significantly structural rearrangements during cell changes. Moreover, the structural change of TAD is tightly associated with its transcription remodeling. Altogether, the theoretical parameter defined in this work provides a quantitative method to link structural characteristics and biological functions of TADs, and this linkage implies that chromatin interaction pattern has the potential to mark transcription activity in TADs.