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Transcriptional Regulation of Human NANOG by Alternate Promoters in Embryonic Stem Cells.

ABSTRACT: INTRODUCTION:The potential of pluripotent stem cells to be used for cell therapy depends on a comprehensive understanding of the molecular mechanisms underlying their unique ability to specify cells of all germ layers while undergoing unlimited self-renewal. Alternative splicing and alternate promoter selection contribute to this mechanism by increasing the number of transcripts generated from a single gene locus and thus enabling expression of novel protein variants which may differ in their biological role. The homeodomain-containing transcription factor NANOG plays a critical role in maintaining the pluripotency of Embryonic Stem Cells (ESC). Therefore, a thorough understanding of the transcriptional regulation of the NANOG locus in ESCs is necessary. METHODS:Regulatory footprints and transcription levels were identified for NANOG in human embryonic stem cells from data obtained using high-throughput sequencing methodologies. Quantitative real-time PCR following reverse transcription of RNA extracted human ESCs was used to validate the expression of transcripts from a region that extends upstream of the annotated NANOG transcriptional start. Promoter identification and characterization were performed using promoter reporter and electrophoretic mobility shift assays. RESULTS:Transcriptionally active chromatin marking and transcription factor binding site enrichment were observed at a region upstream of the known transcriptional start site of NANOG. Expression of novel transcripts from this transcriptionally active region confirmed the existence of NANOG alternative splicing in human ESCs. We identified an alternate NANOG promoter of significant strength at this upstream region. We also discovered that NANOG autoregulates its expression by binding to its proximal downstream promoter. CONCLUSION:Our study reveals novel transcript expression from NANOG in human ESCs, indicating that alternative splicing increases the diversity of transcripts originating from the NANOG locus and that these transcripts are expressed by an alternate promoter. Alternative splicing and alternate promoter usage collaborate to regulate NANOG, enabling its function in the maintenance of ESCs.

SUBMITTER: Das S

PROVIDER: S-EPMC4112544 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Transcriptional Regulation of Human <i>NANOG</i> by Alternate Promoters in Embryonic Stem Cells.

Das Satyabrata S Jena Snehalata S Kim Eun-Mi EM Zavazava Nicholas N Levasseur Dana N DN

Journal of stem cell research & therapy 20120801

<h4>Introduction</h4>The potential of pluripotent stem cells to be used for cell therapy depends on a comprehensive understanding of the molecular mechanisms underlying their unique ability to specify cells of all germ layers while undergoing unlimited self-renewal. Alternative splicing and alternate promoter selection contribute to this mechanism by increasing the number of transcripts generated from a single gene locus and thus enabling expression of novel protein variants which may differ in ...[more]

PMID: 25077040

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Project description:The pluripotent transcription factor NANOG is essential for maintaining embryonic stem cells and driving tumorigenesis. We previously showed that PKC activity is involved in the regulation of NANOG expression. To explore the possible involvement of microRNAs in regulating the expression of key pluripotency factors, we performed a genome-wide analysis of microRNA expression in the embryonal carcinoma cell line NT2/D1 in the presence of the PKC activator, PMA. We found that MIR630 was significantly upregulated in PMA-treated cells. Experimentally, we showed that transfection of MIR630 mimic into embryonal carcinoma cell lines directly targeted the 3'UTR of OCT4, SOX2, and NANOG and markedly suppressed their expression. RNAhybrid and RNA22 algorithms were used to predict miRNA target sites in the NANOG 3'UTR, four possible target sites of MIR630 were identified. To examine the functional interaction between MIR630 and NANOG mRNA, the predicted MIR630 target sites in the NANOG 3'UTR were deleted and the activity of the reporters were compared. After targeted mutation of the predicted MIR630 target sites, the MIR630 mimic inhibited NANOG significantly less than the wild-type reporters. It is worth noting that mutation of a single putative binding site in the 3'UTR of NANOG did not completely abolish MIR630-mediated suppression, suggesting that MIR630 in the NANOG 3'UTR may have multiple binding sites and act together to maximally repress NANOG expression. Interestingly, MIR630 mimics significantly downregulated NANOG gene transcription. Exogenous expression of OCT4, SOX2, and NANOG lacking the 3'UTR almost completely rescued the reduced transcriptional activity of MIR630. MIR630 mediated the expression of differentiation markers in NT2/D1 cells, suggesting that MIR630 leads to the differentiation of NT2/D1 cell. Our findings show that MIR630 represses NANOG through transcriptional and post-transcriptional regulation, suggesting a direct link between core pluripotency factors and MIR630.

Dataset Information

Transcriptional Regulation of Human NANOG by Alternate Promoters in Embryonic Stem Cells.

Publications

Transcriptional Regulation of Human <i>NANOG</i> by Alternate Promoters in Embryonic Stem Cells.

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