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Systemic gain and loss of chromatin architecture throughout zebrafish development [4C]

ABSTRACT: 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.

ORGANISM(S): Danio rerio

PROVIDER: GSE105014 | GEO | 2018/07/01

REPOSITORIES: GEO

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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.

Project description:Three-dimensional genome structure plays an important role in gene regulation. Globally chromosomes are organized into active and inactive compartments, while at the gene level looping interactions connect promoters to regulatory elements. Topologically Associating Domains (TADs), typically several hundred kilobases in size form an intermediate level of organization. Major questions include how TADs are formed and what their relation is with looping interactions between genes and regulatory elements. Here we performed a focused 5C analysis of a 2.8 Mb region on chromosome 7 surrounding CFTR in a panel of cell types. We find that the same TAD boundaries are present in all cell types, indicating that TADs represent a universal chromosome architecture. Further, we find that these TAD boundaries are present irrespective of expression and looping of genes located between them. In contrast looping interactions between promoters and regulatory elements are cell-type specific and occur mostly within TADs. This is exemplified by the CFTR promoter that in different cell types interacts with distinct sets of distal cell type-specific regulatory elements that are all located within the same TAD. Finally, we find that long-range associations between loci located in different TADs are also detected but these display much lower interaction frequencies than looping interactions within TADs. Interestingly, interactions between TADs are also highly cell type-specific and often involve loci clustered around TAD boundaries. These data point to key roles of invariant TAD boundaries in constraining as well as mediating cell type-specific long-range interactions and gene regulation. We investigated a 2.8 Mb region on Chromosome 7 (hg18 chr7: 115797757-118405450) containing the ENCODE region ENm001 42. The 5C experiment was designed to interrogate looping interactions between HindIII fragments containing transcription start sites (TSSs) and any other HindIII restriction fragment (distal fragments) in the target region. Libraries were generated for five cell lines: Caco2, Calu3, Capan1, GM12878 and HepG2, with two biological replicates for each line. 5C probes were designed at HindIII restriction sites (AAGCTT) using 5C primer design tools previously developed and made publicly available online at our My5C website (http://my5C.umassmed.edu). Probes were designed based on the ENCODE manual region 1 (ENM001) design 25 with additional probes placed throughout the region when appropriate. We also added probes to extend the analysis to include a 700 Kb gene desert region located directly adjacent to ENM001. Probe settings were: U-BLAST, 3; S-BLAST, 100; 15-MER, 3,000; MIN_FSIZE, 250; MAX_FSIZE, 20,000; OPT_TM, 65; OPT_PSIZE, 40. We designed 74 reverse 5C probes, and 605 forward 5C probes.

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Systemic gain and loss of chromatin architecture throughout zebrafish development [4C]

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