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Characterization of the imprinting signature of mouse embryo fibroblasts by RNA deep sequencing.

ABSTRACT: Mouse embryo fibroblasts (MEFs) are convenient sources for biochemical studies when cell number in mouse embryos is limiting. To derive the imprinting signature of MEFs and potentially detect novel imprinted genes we performed strand- and allele-specific RNA deep sequencing. We used sequenom allelotyping in embryo and adult organs to verify parental allele-specific expression. Thirty-two known ubiquitously imprinted genes displayed correct parental allele-specific transcripts in MEFs. Our analysis did not reveal any novel imprinted genes, but detected extended parental allele-specific transcripts in several known imprinted domains: maternal allele-specific transcripts downstream of Grb10 and downstream of Meg3, Rtl1as and Rian in the Dlk1-Dio3 cluster, an imprinted domain implicated in development and pluripotency. We detected paternal allele-specific transcripts downstream of Nespas, Peg3, Peg12 and Snurf/Snrpn. These imprinted transcript extensions were not unique to MEFs, but were also present in other somatic cells. The 5' end points of the imprinted transcript extensions did not carry opposing chromatin marks or parental allele-specific DNA methylation, suggesting that their parental allele-specific transcription is under the control of the extended imprinted genes. Based on the imprinting signature of MEFs, these cells provide valid models for understanding the biochemical aspects of genomic imprinting.

SUBMITTER: Tran DA

PROVIDER: S-EPMC3919614 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Characterization of the imprinting signature of mouse embryo fibroblasts by RNA deep sequencing.

Tran Diana A DA Bai Angela Y AY Singh Purnima P Wu Xiwei X Szabó Piroska E PE

Nucleic acids research 20131111 3

Mouse embryo fibroblasts (MEFs) are convenient sources for biochemical studies when cell number in mouse embryos is limiting. To derive the imprinting signature of MEFs and potentially detect novel imprinted genes we performed strand- and allele-specific RNA deep sequencing. We used sequenom allelotyping in embryo and adult organs to verify parental allele-specific expression. Thirty-two known ubiquitously imprinted genes displayed correct parental allele-specific transcripts in MEFs. Our analys ...[more]

PMID: 24217910

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Project description:BackgroundHypoxia is a critical aspect of the glioma microenvironment and has been associated with poor prognosis and resistance to various therapies. However, the mechanisms responsible for hypoxic survival of glioma cells remain unclear. Recent studies strongly suggest that microRNAs act as critical mediators of the hypoxic response. We thus hypothesized their prominent role in hypoxia resistance in glioblastoma (GBM) and aimed to identify those.ResultsWith this study, we present the first detailed analysis of small RNA transcriptome of cell line U87MG, a grade IV glioma cell line, and its alteration under hypoxic condition. Based on deep sequencing and microarray data, we identify a set of hypoxia regulated microRNAs, with the miR-210-3p and its isomiRs showing highest induction in GBM cell lines U87MG and U251MG. We show miR-210-3p, miR-1275, miR-376c-3p, miR-23b-3p, miR-193a-3p and miR-145-5p to be up-regulated, while miR-92b-3p, miR-20a-5p, miR-10b-5p, miR-181a-2-3p and miR-185-5p are down-regulated by hypoxia. Interestingly, certain hypoxia-induced miRNAs are also known to be over-expressed in GBM tumors, suggesting that hypoxia may be one of the factors involved in establishing the miRNA signature of GBM. Transcription factor binding sites for Hypoxia inducible factor 1 A (HIF1A) were identified in the promoter region (5 kb upstream) of 30 hypoxia-induced miRNAs. HIF-1A over-expression and silencing studies show regulation of specific miRNAs, including miR-210-3p, to be HIF1A dependent. On the other hand, miR-210-3p leads to an increase in transcriptional activity of HIF and its target genes vascular endothelial growth factor (VEGF) and carbonic anhydrase 9 (CA9). MiR-210-3p levels were found to be high in GBM patient samples and showed good correlation with the known hypoxia markers CA9 and VEGF. We show that miR-210-3p promotes hypoxic survival and chemoresistance in GBM cells and targets a negative regulator of hypoxic response, HIF3A. Additionally, a total of 139 novel miRNAs were discovered by the analysis of deep sequencing data and three of these were found to be differentially expressed under hypoxia.ConclusionsOverall, our study reveals a novel miRNA signature of hypoxia in GBM and suggests miR-210-3p to be an oncogenic player and a novel potential intrinsic marker of hypoxia in glioblastoma.

Dataset Information

Characterization of the imprinting signature of mouse embryo fibroblasts by RNA deep sequencing.

Publications

Characterization of the imprinting signature of mouse embryo fibroblasts by RNA deep sequencing.

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