title: Transcriptional Regulation of HumanNANOGby Alternate Promoters in Embryonic Stem Cells.
abstract: BACKGROUND
  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 theNANOGlocus in ESCs is necessary.
  
  
  METHODS
  Regulatory footprints and transcription levels were identified forNANOGin 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 annotatedNANOGtranscriptional 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 ofNANOG. Expression of novel transcripts from this transcriptionally active region confirmed the existence ofNANOGalternative splicing in human ESCs. We identified an alternateNANOGpromoter of significant strength at this upstream region. We also discovered thatNANOGautoregulates its expression by binding to its proximal downstream promoter.
  
  
  CONCLUSIONS
  Our study reveals novel transcript expression fromNANOGin human ESCs, indicating that alternative splicing increases the diversity of transcripts originating from theNANOGlocus and that these transcripts are expressed by an alternate promoter. Alternative splicing and alternate promoter usage collaborate to regulateNANOG, enabling its function in the maintenance of ESCs.
authors: Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, Iowa City, USA.
authors: Satyabrata
authors: S
authors: Das
authors: Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, Iowa City, USA.
authors: Snehalata
authors: S
authors: Jena
authors: Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, Iowa City, USA.
authors: Eun-Mi
authors: EM
authors: Kim
authors: Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, Iowa City, USA.
authors: Nicholas
authors: N
authors: Zavazava
authors: Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, Iowa City, USA ; Programs in Genetics and Molecular and Cellular Biology, University of Iowa, Iowa City, Iowa 52242, USA.
authors: Dana N
authors: DN
authors: Levasseur
keywords: Alternate promoter; Alternative splicing; Differentiation; NANOG; Pluripotency; Self-renewal; Stem cells