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High-resolution TADs reveal DNA sequences underlying genome organization in flies

ABSTRACT: Eukaryotic chromatin is partitioned into domains called TADs that are broadly conserved between species and virtually identical among cell types within the same species. Previous studies in mammals have shown that the DNA binding protein CTCF and cohesin contribute to a fraction of TAD boundaries. However, the relative importance of the role of epigenetic activity (like histone marks and transcription) over the genetic information (DNA sequence) in TAD formation is still not understood. Using our new software, HiCExplorer, we annotated high-resolution (570 bp) TAD boundaries in flies and identified eight DNA motifs enriched at boundaries. Known insulator proteins bind five of these motifs while the remaining three motifs are novel. We find that boundaries are either at core promoters of active genes or at non-promoter regions of inactive chromatin and that these two groups are characterized by different sets of DNA motifs. In contrast to mammals, the CTCF motif is not a major boundary motif in flies, while the motifs identified by M1BP and Beaf32 are highly enriched on boundaries. Using refined in-situ Hi-C experiments and analysis in cells depleted of M1BP, we show that M1BP is a new boundary protein that is important for fly genome organisation. We demonstrate that boundaries can be accurately predicted using only the motif sequences at open chromatin sites. We propose that DNA sequence itself encodes chromosome folding by allocation of boundary proteins in the genome. Finally, we present an interactive online database to access and explore the spatial organization of fly, mouse and human genomes, available at http://chorogenome.ie-freiburg.mpg.de.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE97965 | GEO | 2017/06/26

SECONDARY ACCESSION(S): PRJNA383527

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.

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High-resolution TADs reveal DNA sequences underlying genome organization in flies

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