Isogenic comparison of Airn and Xist reveals core principles of Polycomb recruitment by lncRNAs
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ABSTRACT:
PROVIDER: PRJNA961141 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR24288726.fastq.gz | Fastqsanger.gz | |||
| SRR24288727.fastq.gz | Fastqsanger.gz | |||
| SRR24288728.fastq.gz | Fastqsanger.gz | |||
| SRR24288729.fastq.gz | Fastqsanger.gz | |||
| SRR24288730.fastq.gz | Fastqsanger.gz |
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Project description:The mechanisms and biological roles of Polycomb repressive complex (PRC) recruitment by lncRNAs remain unclear. To gain insight, we expressed two lncRNAs that recruit PRCs to multi-megabase domains, Airn and Xist, from an ectopic locus and compared effects. Unexpectedly, ectopic Airn recruited PRC1 and PRC2 to chromatin with a potency resembling Xist yet did not repress genes. Compared to PRC2, PRC1 was more proximal to Airn and Xist, where its enrichment over C-rich elements required the RNA-binding protein HNRNPK. Fusing Airn to Repeat A, the domain required for gene silencing by Xist, enabled gene silencing and altered local patterns but not relative levels of PRC-directed modifications. Our data suggest that endogenously, Airn recruits PRCs to maintain rather than initiate gene silencing; that PRC recruitment occurs independently of Xist Repeat A; and that protein-bridged interactions, not direct RNA contacts, underlie PRC recruitment by Airn, Xist, and other lncRNAs.
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Project description:In the developing mouse embryo, expression of the lncRNA Airn induces gene silencing and recruits Polycomb Repressive Complexes (PRCs) to varying extents over a 15 megabase domain, but the mechanisms remain unclear. Using high-resolution approaches, we show in mouse trophoblast stem cells that Airn expression induces long-range changes to chromatin architecture centered around a network of CpG islands occupied by PRCs and SMC1A/Cohesin. Extent of contact with Airn predicts underlying intensity of Polycomb-directed chromatin modifications and Airn-induced changes to chromatin architecture. Deletion of individual CpG islands alternately increase or decrease gene silencing and PRC activity over the domain in a manner predicted by changes in chromatin architecture. We conclude that Airn is a potent repressive lncRNA whose effects are controlled by an equilibratory network of DNA regulatory elements that modulate its frequency of contact with target chromatin.
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Project description:In the developing mouse embryo, expression of the lncRNA Airn induces gene silencing and recruits Polycomb Repressive Complexes (PRCs) to varying extents over a 15 megabase domain, but the mechanisms remain unclear. Using high-resolution approaches, we show in mouse trophoblast stem cells that Airn expression induces long-range changes to chromatin architecture centered around a network of CpG islands occupied by PRCs and SMC1A/Cohesin. Extent of contact with Airn predicts underlying intensity of Polycomb-directed chromatin modifications and Airn-induced changes to chromatin architecture. Deletion of individual CpG islands alternately increase or decrease gene silencing and PRC activity over the domain in a manner predicted by changes in chromatin architecture. We conclude that Airn is a potent repressive lncRNA whose effects are controlled by an equilibratory network of DNA regulatory elements that modulate its frequency of contact with target chromatin.
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Project description:In the developing mouse embryo, expression of the lncRNA Airn induces gene silencing and recruits Polycomb Repressive Complexes (PRCs) to varying extents over a 15 megabase domain, but the mechanisms remain unclear. Using high-resolution approaches, we show in mouse trophoblast stem cells that Airn expression induces long-range changes to chromatin architecture centered around a network of CpG islands occupied by PRCs and SMC1A/Cohesin. Extent of contact with Airn predicts underlying intensity of Polycomb-directed chromatin modifications and Airn-induced changes to chromatin architecture. Deletion of individual CpG islands alternately increase or decrease gene silencing and PRC activity over the domain in a manner predicted by changes in chromatin architecture. We conclude that Airn is a potent repressive lncRNA whose effects are controlled by an equilibratory network of DNA regulatory elements that modulate its frequency of contact with target chromatin.
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Project description:To test if the imprinted long non-coding RNA (lncRNA) Airn transcription or its product silences the protein-coding Igf2r gene, we shortened the endogenous lncRNA to four different lengths by inserting polyadenylation (polyA) cassettes on the paternal chromosome via homologous recombination in ES cells. ES cell differentiation was used to recapitulate the developmental onset of Airn and Igf2r imprinted expression and polyA cassettes inserted before (T3, T16, T27) or after (T31, T51) the Igf2r promoter successfully truncated Airn, with the exception of T27. RNA hybridization to a tiling array (MIRTA) demonstrated loss of Airn upstream of the Igf2r promoter (except T27) and absence of novel spliced variants in all truncation alleles. To further test the transcriptional overlap model we moved the Airn promoter ~700bp before the Igf2r TSS in ES cells that lack an endogenous paternal Airn promoter and called this cell line FAP (Forward-Airn-Promoter). RNA hybridization to a tiling array (MIRTA) demonstrated that the moved Airn promoter expresses Airn in wildtype orientation and terminates at the wildtype 3' end. ES cell lines expressing different Airn variants were differentiated on gelatinized dishes after feeder cell depletion and LIF withdrawal by 0.27M-BM-5M retinoic acid for 5 days and Airn length was assayed.
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Project description:The role of Xist-mediated Polycomb recruitment in the initiation of X-chromosome inactivation
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Project description:To test if the imprinted long non-coding RNA (lncRNA) Airn transcription or its product silences the protein-coding Igf2r gene, we shortened the endogenous lncRNA to four different lengths by inserting polyadenylation (polyA) cassettes on the paternal chromosome via homologous recombination in ES cells. ES cell differentiation was used to recapitulate the developmental onset of Airn and Igf2r imprinted expression and polyA cassettes inserted before (T3, T16, T27) or after (T31, T51) the Igf2r promoter successfully truncated Airn, with the exception of T27. RNA hybridization to a tiling array (MIRTA) demonstrated loss of Airn upstream of the Igf2r promoter (except T27) and absence of novel spliced variants in all truncation alleles. To further test the transcriptional overlap model we moved the Airn promoter ~700bp before the Igf2r TSS in ES cells that lack an endogenous paternal Airn promoter and called this cell line FAP (Forward-Airn-Promoter). RNA hybridization to a tiling array (MIRTA) demonstrated that the moved Airn promoter expresses Airn in wildtype orientation and terminates at the wildtype 3' end.
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Project description:Long noncoding RNAs (lncRNAs) cause Polycomb Repressive Complexes (PRCs) to spread over broad regions of the mammalian genome. We report that in mouse trophoblast stem cells, the Kcnq1ot1 and Airn lncRNAs induce PRC-dependent chromatin modifications over multi-megabase domains. Throughout the Airn-targeted domain, extent of PRC-dependent modification correlated with intra-nuclear distance to the Airn locus, pre-existing genome architecture, and the abundance of Airn itself. Specific CpG islands displayed characteristics indicating that they nucleate the spread of PRCs upon exposure to Airn. Chromatin environments surrounding Xist, Airn, and Kcnq1ot1 suggest common mechanisms of PRC engagement and spreading. Our data indicate that lncRNA potency can be tightly linked to lncRNA abundance, and that within lncRNA-targeted domains, PRCs are recruited to CpG islands via lncRNA-independent mechanisms. We propose that CpG islands that autonomously recruit PRCs interact with lncRNAs and their associated proteins through 3-dimensional space to nucleate the spread of PRCs in lncRNA-targeted domains.
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Project description:Long noncoding RNAs (lncRNAs) cause Polycomb Repressive Complexes (PRCs) to spread over broad regions of the mammalian genome. We report that in mouse trophoblast stem cells, the Kcnq1ot1 and Airn lncRNAs induce PRC-dependent chromatin modifications over multi-megabase domains. Throughout the Airn-targeted domain, extent of PRC-dependent modification correlated with intra-nuclear distance to the Airn locus, pre-existing genome architecture, and the abundance of Airn itself. Specific CpG islands displayed characteristics indicating that they nucleate the spread of PRCs upon exposure to Airn. Chromatin environments surrounding Xist, Airn, and Kcnq1ot1 suggest common mechanisms of PRC engagement and spreading. Our data indicate that lncRNA potency can be tightly linked to lncRNA abundance, and that within lncRNA-targeted domains, PRCs are recruited to CpG islands via lncRNA-independent mechanisms. We propose that CpG islands that autonomously recruit PRCs interact with lncRNAs and their associated proteins through 3-dimensional space to nucleate the spread of PRCs in lncRNA-targeted domains.
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