Simultaneous mapping of 3D structure and nascent RNAs argues against nuclear compartments that preclude transcription
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE211287 | GEO | 2022/11/15
REPOSITORIES: GEO
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Project description:The nucleus is a highly organized arrangement of RNA, DNA, and protein molecules that are compartmentalized within three-dimensional (3D) structures involved in shared functional and regulatory processes. Although RNA has long been proposed to play a global role in organizing nuclear structure, exploring the role of RNA in shaping nuclear structure has remained a challenge because no existing methods can simultaneously measure RNA-RNA, RNA-DNA, and DNA-DNA contacts within 3D structures. To address this, we developed RNA & DNA SPRITE (RD-SPRITE) to comprehensively map the location of all RNAs relative to DNA and other RNAs. Using this approach, we identify many RNAs that are localized near their transcriptional loci (RNA-DNA) together with other diffusible ncRNAs (RNA-RNA) within higher-order DNA structures (DNA-DNA). These RNA-chromatin compartments span three major classes of nuclear functions: RNA processing (including ribosome biogenesis, mRNA splicing, snRNA biogenesis, and histone mRNA processing), heterochromatin assembly, and gene regulation. More generally, we identify hundreds of ncRNAs that form stable nuclear compartments in spatial proximity to their transcriptional loci. We find that dozens of nuclear compartments require RNA to guide protein regulators into these 3D structures, and focusing on several ncRNAs, we show that these ncRNAs specifically regulate heterochromatin assembly and the expression of genes contained within these compartments. Together, our results demonstrate a unique mechanism by which RNA acts to shape nuclear structure by forming high concentration territories immediately upon transcription, binding to diffusible regulators, and guiding them into spatial compartments to regulate a wide range of essential nuclear functions.
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Project description:Chromosomes are three-dimensional structures in the nucleus that are organized at multiple levels. Studies in mammals have shown that chromosomes segregate in active and inactive compartments (A and B, resp.). A compartments have high transcription activity, high GC-content and replicate early in S-phase. B compartments on the other hand are transcriptionally inactive, have low CG content are often found at the nuclear periphery. Every compartment is further divided into megabase-sized Topologically Associated Domains (TADs), the functional units of gene regulation. Regions within such a domain preferentially interact with each other. TAD positions are conserved between mouse and human genomes. However, it is unclear if non-mammalian vertebrates also show these organizational features and how these change throughout development. We have generated Hi-C maps for zebrafish embryos and show that the structural features of mammalian genomes (A/B-compartments and TADs) are also found in this species. We find that the genes within TADs show spatial co-expression. Moreover, the order of genes within a TAD is conserved to other vertebrate species. One of the key features of development is the switch from maternally loaded mRNAs to the transcriptional activation of the zygotic genome, known as Zygotic Genome Activation (ZGA). Prior to ZGA we see both TADs and compartments. Paradoxically, after the initiation of transcription, TADs and compartments completely disappear. These features are progressively re-established in the following developmental stages. Our results show that chromosome conformation can be formed independent of transcription. Analysis of active enhancer and promoter marks after ZGA showed that promoters and enhancers are enriched at future TAD-boundaries. Later in time, when TADs are present, the enrichment of active enhancers at the TAD-boundaries is lost. These data suggest that proximal enhancer-promoter interactions are needed for gene regulation when TADs are not in place. The loss of both TADs and compartments represent an interesting avenue for the further study of factors involved in the formation of the structural features of vertebrate chromosomes.
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Project description:Chromosomes are three-dimensional structures in the nucleus that are organized at multiple levels. Studies in mammals have shown that chromosomes segregate in active and inactive compartments (A and B, resp.). A compartments have high transcription activity, high GC-content and replicate early in S-phase. B compartments on the other hand are transcriptionally inactive, have low CG content are often found at the nuclear periphery. Every compartment is further divided into megabase-sized Topologically Associated Domains (TADs), the functional units of gene regulation. Regions within such a domain preferentially interact with each other. TAD positions are conserved between mouse and human genomes. However, it is unclear if non-mammalian vertebrates also show these organizational features and how these change throughout development. We have generated Hi-C maps for zebrafish embryos and show that the structural features of mammalian genomes (A/B-compartments and TADs) are also found in this species. We find that the genes within TADs show spatial co-expression. Moreover, the order of genes within a TAD is conserved to other vertebrate species. One of the key features of development is the switch from maternally loaded mRNAs to the transcriptional activation of the zygotic genome, known as Zygotic Genome Activation (ZGA). Prior to ZGA we see both TADs and compartments. Paradoxically, after the initiation of transcription, TADs and compartments completely disappear. These features are progressively re-established in the following developmental stages. Our results show that chromosome conformation can be formed independent of transcription. Analysis of active enhancer and promoter marks after ZGA showed that promoters and enhancers are enriched at future TAD-boundaries. Later in time, when TADs are present, the enrichment of active enhancers at the TAD-boundaries is lost. These data suggest that proximal enhancer-promoter interactions are needed for gene regulation when TADs are not in place. The loss of both TADs and compartments represent an interesting avenue for the further study of factors involved in the formation of the structural features of vertebrate chromosomes.
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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.
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Project description:Radiation therapy is commonly used to treat glioblastoma multiforme (GBM) brain tumor. Ionizing radiation (IR) induces dose- specific variations in transcriptional programs, implicating they are tightly regulated and critical components in the tumor response and survival. Yet, our understanding of the downstream molecular events triggered by lethal vs sublethal IR doses is limited. Herein, we report that variations in the genetic programs are positively and functionally correlated with the exposure to lethal or sublethal IR doses. Genome architecture analysis revealed that gene regulation is spatially and temporally coordinated with DNA repair kinetics. Radiation-activated genes were pre-positioned in active sub-nuclear compartments and were up-regulated following DNA damage response, while the DNA repair activity shifted to the inactive heterochromatic spatial compartments. The IR dose affected the levels of DNA damage repair and transcription modulation, but not the order of events, which was linked to their spatial nuclear positioning. Thus, distinct coordinated temporal dynamics of DNA damage repair and transcription reprogramming in the active and inactive sub-nuclear compartments highlight the importance of high-order genome organization in synchronizing the molecular events following IR.
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