Project description:The three-dimensional (3D) organization of the genome within the cell nucleus contributes to cell-specific gene expression in different cell types1. High-throughput 3CM-bM-^@M-^Sderived methods have revealed that the genome is segmented into contiguous topologically associating domains (TADs), which help to orchestrate gene expression changes during differentiation and development2-5. Using ChIP-Seq, Hi-C and 3D modelling techniques, we reveal that TADs regulate the rapid gene expression changes induced by progestin in T47D breast cancer cells. In response to the hormone, TADs maintain their borders and operate as discrete regulatory units in which the majority of the genes are either transcriptionally activated or repressed. Additionally, the epigenetic signatures of the TADs are coordinately modified by hormone in correlation with the transcriptional changes. Hormone-induced changes in gene activity and chromatin remodelling are accompanied by structural changes that are distinct for activated or repressed TADs. Integrative 3D modelling revealed that TADs are structurally expanded if active and compacted if repressed, and that this is accompanied by differential changes in accessibility. We thus propose that TADs function as M-bM-^@M-^\regulonsM-bM-^@M-^] to enable spatially proximal genes to be coordinately transcribed in response to hormones. T47D-MTVL human breast cancer cells were incubated with the progestin R5020 for different times at 37M-BM-:C and prepared for ChIP-Seq or Hi-C according published protocols
Project description:Eukaryotic genomes are folded into a hierarchy of three-dimensional structures that impact nuclear functions, including transcription, replication, and repair1-3. Studies in Drosophila and mammals have revealed megabase-sized topologically associated domains (TADs) within chromosomes, which in turn are spatially restricted within the nucleus4-8. However, little is known about local physical constraints that drive higher-order folding of chromosomes. Here we performed Hi-C analysis of the fission yeast Schizosaccharomyces pombe to explore genome organization at high resolution. S. pombe comprises a small genome ideal for examining structural features of chromatin folding, and contains fundamental components present in higher eukaryotes. Large domains of heterochromatin coat centromeres and telomeres and recruit cohesin, a ring-like protein complex that binds sister chromatids and mediates long range looping of interphase chromosomes. Our analyses reveal a highly ordered chromosome organization, consistent with a Rabl configuration, which is dependent on constraints imposed at centromeres and telomeres. We find that local chromatin compaction and cohesin recruitment to centromeres mediated by heterochromatin is required for maintaining global genome territorial restraint. In addition to larger complex domains, we also observed locally interacting regions of chromatin ~50 kilobases long, which organize chromosome arms into structures referred to as “globules”. Globule boundaries are enriched in cohesin and convergent gene orientation. The role of cohesin in maintaining globule domains is independent of its role in sister chromatid cohesion, as globule domains are also a feature of G1 chromosome architecture. Defect in cohesin disrupts globule domains and results in an altered chromosome organization at larger scales, including the loss of chromosome territories. Disruption of globules also affects functional annotation of the genome, leading to impairment of borders between neighboring transcriptional units. Our analyses reveal key features of chromatin organization and folding and show that distinct mechanisms uniquely impact the hierarchy of genome organization to protect genome integrity and to coordinate nuclear functions. Comparison of HiC contact maps under various conditions reveal fundamental principles of genome organization
Project description:In eukaryotes, the nucleus is organized into a three dimensional structure consisting of both local interactions such as those between enhancers and promoters, and long-range higher-order structures such as nuclear bodies. This organization is central to many aspects of nuclear function, including DNA replication, transcription, and cell cycle progression. Nuclear structure intrinsically occurs within single cells; however, measuring such a broad spectrum of 3D DNA interactions on a genome-wide scale and at the single cell level has been a great challenge. To address this, we developed single-cell split-pool recognition of interactions by tag extension (scSPRITE), a new method that enables measurements of genome-wide maps of 3D DNA structure in thousands of individual nuclei. scSPRITE maximizes the number of DNA contacts detected per cell enabling high-resolution genome structure maps within each cells and is easy-to-use and cost-effective. scSPRITE accurately detects chromosome territories, active and inactive compartments, topologically associating domains (TADs), and higher-order structures within single cells. In addition, scSPRITE measures cell-to-cell heterogeneity in genome structure at different levels of resolution and shows that TADs are dynamic units of genome organization that can vary between different cells within a population. scSPRITE will improve our understanding of nuclear architecture and its relationship to nuclear function within an individual nucleus from complex cell types and tissues containing a diverse population of cells.
Project description:Deciphering the rules of genome folding in the cell nucleus is essential in order to understand its functions. Recent 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. Here, using a combination of Hi-C, 3D-Fluorescent In Situ Hybridization (3D-FISH), 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 nano-compartments, 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:Deciphering the rules of genome folding in the cell nucleus is essential in order to understand its functions. Recent 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. Here, using a combination of Hi-C, 3D-Fluorescent In Situ Hybridization (3D-FISH), 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 nano-compartments, 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:Deciphering the rules of genome folding in the cell nucleus is essential in order to understand its functions. Recent 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. Here, using a combination of Hi-C, 3D-Fluorescent In Situ Hybridization (3D-FISH), 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 nano-compartments, 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:The three-dimensional (3D) organization of the genome within the cell nucleus contributes to cell-specific gene expression in different cell types1. High-throughput 3C–derived methods have revealed that the genome is segmented into contiguous topologically associating domains (TADs), which help to orchestrate gene expression changes during differentiation and development2-5. Using ChIP-Seq, Hi-C and 3D modelling techniques, we reveal that TADs regulate the rapid gene expression changes induced by progestin in T47D breast cancer cells. In response to the hormone, TADs maintain their borders and operate as discrete regulatory units in which the majority of the genes are either transcriptionally activated or repressed. Additionally, the epigenetic signatures of the TADs are coordinately modified by hormone in correlation with the transcriptional changes. Hormone-induced changes in gene activity and chromatin remodelling are accompanied by structural changes that are distinct for activated or repressed TADs. Integrative 3D modelling revealed that TADs are structurally expanded if active and compacted if repressed, and that this is accompanied by differential changes in accessibility. We thus propose that TADs function as “regulons” to enable spatially proximal genes to be coordinately transcribed in response to hormones.