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RNA-Chromatin Interactome Reveals ncRNA Functions for Transcription Regulation in 3D Genome Organization

ABSTRACT: RNA-Chromatin Interactome Reveals ncRNA Functions for Transcription Regulation in 3D Genome Organization

PROVIDER: PRJNA339288 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR4040050_1.fastq.gz	Fastqsanger.gz
	SRR4040050_2.fastq.gz	Fastqsanger.gz
	SRR4040060_1.fastq.gz	Fastqsanger.gz
	SRR4040060_2.fastq.gz	Fastqsanger.gz
	SRR4040051_1.fastq.gz	Fastqsanger.gz

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RNA-Chromatin Interactome Reveals ncRNA Functions for Transcription Regulation in 3D Genome Organization

Project description:Noncoding RNAs (ncRNAs) are an emerging class of regulatory molecules with a broad range of regulatory functions believed to be mediated by ncRNA-chromatin interactions. Genome-wide understanding of ncRNA functions requires precise mapping of all ncRNAs and their target loci. Current methods for studying chromatin-associated ncRNA lack specificity or are limited to singly assessing RNAs. We devised an unbiased strategy to identify all RNA Interactions with Chromatin by Paired-End-Taging (RICh-PET) and applied this approach to characterize the Drosophila RNA-chromatin interactome. We discovered that ncRNAs primarily target promoters and enhancers in open chromatin regions in colocalization with RNAPII and other TFs, suggesting combinatorial regulatory instructions for each locus. Enzymatic nuclear digestion followed by examination of specific chromatin loci indicated that ncRNAs collectively promote chromatin accessability, RNAPII-mediated interactions and overall 3D-genome organization. Our study demonstrates that RICh-PET and related methods represent a powerful suite of tools to interrogate the genome biology of ncRNAs.

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Project description:Although non-coding RNAs (ncRNAs) have been proposed to play important roles in nuclear organization with three-dimensional (3D) conformation and gene transcription regulation, exploring the targeting and diffusion patterns of ncRNAs on chromatin has been challenging because it requires a technique that can simultaneously detect high-affinity sites and their higher-order contacts in 3D structures. Here we developed a new method, RNA-associated chromatin DNA-DNA interactions (RDD) in solution, which enables genome-wide detection of genomic binding sites and high-resolution chromatin interactions for a specific ncRNA. Using RDD to investigate the well-known ncRNA roX2 that involved in Drosophila dosage compensation, we not only recapitulate roX2 targeting chromatin sites but also reveal the landscape of chromatin DNA-DNA contacts at various resolutions from loops, domains to territories. We further show that roX2 anchors at the target gene TES and spreads around as a 'boot-like' structure for maintaining gene activity, where tethered daisy-chain chromatin loops, bridging to TSS to dock RNAPII-related contact hubs to achieve a precise twofold expression of male X-linked genes, further crossing active domains to form distinct hubs to exhibit the condensed properties of roX2 in high-throughput sequencing. These results provide a genome-wide 3D model for explaining the mechanism of ncRNA roX2 involved dosage compensation in Drosophila. Together, we demonstrate the high specificity and robustness of the RDD method to capture RNA-associated chromatin DNA-DNA interactome, which provides topological insights into the mechanism of gene transcription regulation by ncRNAs.

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Widespread rearrangement of 3D chromatin organization underlies Polycomb-mediated stress-induced silencing [Hi-C]

Project description:Chromosomes of metazoan organisms are partitioned in the interphase nucleus into discrete topologically associating domains (TADs). Borders between TADs are preferentially formed in regions containing high density of active genes and clusters of architectural protein binding sites. Transcription of most genes is turned off during the heat shock response in Drosophila. Here we show that temperature stress induces relocalization of architectural proteins from TAD borders to inside TADs, and this is accompanied by a dramatic rearrangement in the 3D organization of the nucleus. TAD border strength declines, allowing for an increase in long-distance inter-TAD interactions. Similar but quantitatively weaker effects are observed upon inhibition of transcription or depletion of individual architectural proteins. New heat shock-induced inter-TAD interactions result in increased contacts among enhancers and promoters of silenced genes, which recruit Pc and form Pc bodies at the nucleolus. These results suggest that the TAD organization of metazoan genomes is plastic and can be quickly reconfigured to allow new interactions between distant sequences. Analysis of 3D chromatin organization using Hi-C in Drosophila Kc167 cells. Cells were grown at 25 C and heat shocked for 20 min at 36.5 C. Cells were also treated with flavopiridol or triptolide to inhibit transcription elongation or initiation, respectively. Cells were depeleted of Cap-H2 or Rad21 using RNAi. Finally, cells depleted of RAd21 were subjected to heat shock at 36.5 C for 20 min.

2015-02-24 | E-GEOD-63515 | biostudies-arrayexpress

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