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Neat1 is a p53-inducible lincRNA essential for transformation suppression

ABSTRACT: p53 is a transcriptional activator that induces myriad target genes. However, those p53-inducible genes most critical for tumor suppression remain elusive. Here, we identify Neat1, a ncRNA constituent of paraspeckles, as a p53 target gene induced by mouse and human p53 in different cell types and by diverse stress signals. Using fibroblasts derived from Neat1-/- mice, we examine the functional role of Neat1 in the p53 pathway. We find that Neat1 is dispensable for cell-cycle arrest and apoptosis in response to genotoxic stress, but plays a crucial role in suppressing transformation in response to oncogenic signals. To determine the effects of Neat1 deficiency on cells, we used E1A;HRasV12-expressing wild-type and Neat1-/- mouse embryonic fibroblasts (MEFs) for RNA-sequencing analysis. This analysis revealed that Neat1 deficiency impacts the regulatory networks involved in nervous system development and axon guidance programs, as well as genes of the SWI/SNF complex and components of the pancreas development network. These findings suggest that the ability of Neat1 to globally regulate gene expression, with effects on diverse transcriptional programs, provides a potential mechanism for how Neat1 acts to suppress transformation and tumor initiation.

ORGANISM(S): Mus musculus

PROVIDER: GSE100098 | GEO | 2017/07/27

SECONDARY ACCESSION(S): PRJNA390672

REPOSITORIES: GEO

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Project description:Long noncoding RNAs (lncRNAs) are important regulators of chromatin; however, the mechanistic roles for many lncRNAs are poorly understood in part because their direct interactions with genomic loci and proteins are difficult to assess. We used CHART-seq to map the genomic binding sites for two highly expressed human lncRNAs, NEAT1 and MALAT1, which localize within the nucleus to paraspeckles and nuclear speckles, respectively. We show that NEAT1 and MALAT1 localize to hundreds of genomic sites in human cells, primarily over active genes. NEAT1 and MALAT1 exhibit colocalization to many of these loci, but display distinct gene body binding patterns at these sites, suggesting independent but complementary functions for these RNAs. Protein mass spectrometry analysis of CHART-enriched material (CHART-MS) identified numerous proteins enriched by both lncRNAs, supporting complementary binding and function, in addition to unique associated proteins. Transcriptional inhibition or stimulation affects the localization of NEAT1 to active chromatin sites, implying that DNA sequence itself does not target NEAT1 to chromatin and that localization responds to cues involved in the transcription process. Paired-end CHART-seq was performed for a single replicate of each capture oligonucleotide in untreated MCF-7 cells to establish binding sites of these RNAs, for a total of 6 samples. To investigate the effects of transcriptional inhibition and E2 stimulation on the localization of these RNAs, we performed paired-end CHART-seq with each capture oligonucleotide for two biological replicates of flavopiridol- and vehicle (DMSO)-treated MCF-7 cells and for two biological replicates of E2- and vehicle (ethanol)-treated MCF-7 cells. To establish the overlap of NEAT1 and MALAT1 binding sites with a known component of paraspeckles (NEAT1-containing subnuclear body), we performed paired-end ChIP-seq for the paraspeckle component PSF in MCF-7 cells, as well as a single-end biological replicate.

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Neat1 is a p53-inducible lincRNA essential for transformation suppression

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