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Chromosome Structure II: Stem-loops and circle-loops

ABSTRACT: The chromosomes in multicellular eukaryotes are organized into a series of topologically independent loops called TADs. In flies, TADs are formed by physical interactions between neighboring boundaries. Fly boundaries exhibit distinct partner preferences, and pairing interactions between boundaries are typically orientation dependent. Pairing can be head-to-tail or head-to-head. The former generates a stem-loop TAD, while the latter gives a circle-loop TAD. The TAD that encompasses the Drosophila even skipped (eve) gene is formed by the head-to-tail pairing of the nhomie and homie boundaries. To explore the relationship between loop topology and the physical and regulatory landscape, we flanked the nhomie boundary region with two attP sites. The attP sites were then used to generate four boundary replacements: λ DNA, nhomie forward (WT orientation), nhomie reverse (opposite of WT), and homie forward (same as WT homie). The nhomie forward replacement restores the WT physical and regulatory landscape: In MicroC experiments, the eve TAD is a volcano triangle topped by a plume, and the eve gene and its regulatory elements are sequestered from interactions with neighbors. The λ DNA replacement lacks boundary function: the endpoint of the “new” eve TAD on the nhomie side is ill-defined, and eve stripe enhancers activate a nearby gene, eIF3j. While nhomie reverse and homie forward restore the eve TAD, the topology is a circle-loop, and this changes the local physical and regulatory landscape. In MicroC experiments, the eve TAD interacts with its neighbors, and the plume at the top of the eve volcano triangle is replaced by a cloud of contacts with the next-door TADs. Consistent with the loss of isolation afforded by the stem-loop topology, the eve enhancers weakly activate genes in the neighboring TADs. Conversely, eve function is partially disrupted.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE263270 | GEO | 2024/04/04

REPOSITORIES: GEO

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Project description:The genome is partitioned into Topologically Associating Domains (TADs). About half of the boundaries of these TADs exhibit transcriptional activity and are correlated with better TAD insulation. However, the association between these transcripts and TAD insulation, enhancer:promoter interactions and transcription of genes remains unknown. Here we investigate the functional roles of these bRNAs (boundary-RNA) in boundary insulation and consequent effects on enhancer-promoter interactions and TAD transcription genome-wide and more specifically on the disease relevant INK4a/ARF TAD. Using a series of CTCF site deletions and bRNA knockdown experiments at this TAD boundary, we show a direct association of CTCF with the bidirectional bRNAs where the loss of bRNA triggers the concomitant loss of: CTCF at the TAD boundary, its insulation, enhancer:promoter interactions and gene transcription within the targeted TAD. We used another series of enhancer deletions and CRISPRi on promoters within INK4a/ARF TAD to better understand the regulation and origins of bRNA itself. We observe that the enhancers interact with boundaries and positively regulate the bRNA transcription at TAD boundaries. In return, the bRNAs recruit/stabilize the CTCF even on weak motifs within these boundaries and supports CTCF binding in clusters, therefore enhancing TAD insulation. This favors the intra-TAD enhancer:promoter interactions and leads to robust gene transcription. Functionally, bRNAs are more stable than eRNAs and their knockdown exactly mimics the CTCF site deletion. Furthermore, transcribing boundaries exhibit high TAD transcription in TCGA tumour datasets. Together, these results show that active enhancers directly mediate better insulation of TADs by activating the transcription at TAD boundaries triggering CTCF clustering at the boundary which results in better insulation and favours robust intra-TAD enhancer:promoter interactions to activate the gene transcription.

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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Chromosome Structure II: Stem-loops and circle-loops

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