Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Human endothelial polyA+ RNA expression analysis

ABSTRACT: The human genome harbors a large number of sequences encoding for RNAs that are not translated but control cellular functions by distinct mechanisms. The expression and function of the longer transcripts namely the long non-coding RNAs (lncRNAs) in the vasculature is largely unknown. Here, we characterized the expression of lncRNAs in human endothelial cells and elucidated the function of the highly expressed metastasis-associated lung adenocarcinoma transcript 1 (MALAT1; also known as MALAT-1 or NEAT2). Endothelial cells of different origin express high levels of the conserved lncRNAs MALAT1, TUG1, MEG, linc00657 and linc00493. MALAT1 was significantly increased by hypoxia and controls a phenotypic switch in endothelial cells. Silencing of MALAT1 by siRNAs or GapmeRs induced a pro-migratory response and increased basal sprouting and migration, whereas proliferation of endothelial cells was inhibited. If angiogenesis was further stimulated by VEGF, MALAT1 siRNAs induced discontinuous sprouts indicative of defective proliferation of stalk cells. In vivo studies confirmed that genetic ablation of MALAT1 inhibited proliferation of endothelial cells and reduced neonatal retina vascularization. Gene expression profiling followed by confirmatory qRT-PCR demonstrated that silencing of MALAT1 impaired the expression of various cell cycle regulators. Silencing of MALAT1 tips the balance from a proliferative to a migratory endothelial cell phenotype in vitro and its genetic deletion reduces neonatal vascular growth in vivo. Human endothelial polyA+ RNA expression analysis. We used two replicate samples of human umbilical vein endothelial cells.

ORGANISM(S): Homo sapiens

SUBMITTER: Reinier Boon

PROVIDER: E-GEOD-54384 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Long noncoding RNA MALAT1 regulates endothelial cell function and vessel growth.

Michalik Katharina M KM You Xintian X Manavski Yosif Y Doddaballapur Anuradha A Zörnig Martin M Braun Thomas T John David D Ponomareva Yuliya Y Chen Wei W Uchida Shizuka S Boon Reinier A RA Dimmeler Stefanie S

Circulation research 20140306 9

<h4>Rationale</h4>The human genome harbors a large number of sequences encoding for RNAs that are not translated but control cellular functions by distinct mechanisms. The expression and function of the longer transcripts namely the long noncoding RNAs in the vasculature are largely unknown.<h4>Objective</h4>Here, we characterized the expression of long noncoding RNAs in human endothelial cells and elucidated the function of the highly expressed metastasis-associated lung adenocarcinoma transcri ...[more]

PMID: 24602777

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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.

Project description:Kaposi’s sarcoma-associated herpesvirus (KSHV) is a g-herpesvirus which persists as circular extrachromasomal DNA in the nucleus during latent infection. During latency a limited number of viral proteins are expressed, including LANA (latency-associated nuclear antigen). LANA is a multi-functional protein known to interact with transcriptional regulators and chromatin remodelers, and to regulate the LANA and RTA promoters. We hypothesized that LANA may contribute to the establishment of latency through controlling chromatinization and histone modification of KSHV episomes. We performed ChIP-seq analysis and correlated H3K4me3, H3K27me3, polII, and LANA occupancy of the KSHV genome at nucleotide resolution. Epigenetic marks were analyzed in BCBL-1 lymphoid cells and in telomerase-immortalized vein endothelial TIVE-LTC cells. We found that the transcription active mark H3K4me3, but not silencing mark H3K27me3, was enriched at KSHV regions where LANA is bound. Co-occupancy of LANA and H3K4 was also detected on 167 host genes, of which 89 are actively transcribed. By comparing LANA occupancy with the profiling of 43 transcription factors from ENCODE, a subset of transcription factors was enriched at regions where LANA is bound, including znf143, CTCF, and Stat1. These results indicate that LANA also plays a role in modulating expression of host genes. Co-immunoprecipitation of LANA with the H3K4 methyltransferase hSET1 suggests that LANA recruits hSET1 to specific loci on the viral genome, leading to H3K4 methylation and ensuring transcription of latency-associated genes. Host gene expression may be regulated by the same mechanism. LANA binding was correlated with the distribution in the latent KSHV genome of the transcription active histone modification H3K4me3, the silencing mark H3K27me3, and RNA polymerase II, using cells of lymphoid and endothelial origin. A similar analysis was done for the human genome. Biological replicates (BR) and technical replicates (TR) were included.

Dataset Information

Human endothelial polyA+ RNA expression analysis

Publications

Long noncoding RNA MALAT1 regulates endothelial cell function and vessel growth.

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