title: Identification of a novel intronic enhancer responsible for the transcriptional regulation of musashi1 in neural stem/progenitor cells.
abstract: BACKGROUND
  The specific genetic regulation of neural primordial cell determination is of great interest in stem cell biology. The Musashi1 (Msi1) protein, which belongs to an evolutionarily conserved family of RNA-binding proteins, is a marker for neural stem/progenitor cells (NS/PCs) in the embryonic and post-natal central nervous system (CNS). Msi1 regulates the translation of its downstream targets, including m-Numb and p21 mRNAs. In vitro experiments using knockout mice have shown that Msi1 and its isoform Musashi2 (Msi2) keep NS/PCs in an undifferentiated and proliferative state. Msi1 is expressed not only in NS/PCs, but also in other somatic stem cells and in tumours. Based on previous findings, Msi1 is likely to be a key regulator for maintaining the characteristics of self-renewing stem cells. However, the mechanisms regulating Msi1 expression are not yet clear.
  
  
  RESULTS
  To identify the DNA region affecting Msi1 transcription, we inserted the fusion gene ffLuc, comprised of the fluorescent Venus protein and firefly Luciferase, at the translation initiation site of the mouse Msi1 gene locus contained in a 184-kb bacterial artificial chromosome (BAC). Fluorescence and Luciferase activity, reflecting the Msi1 transcriptional activity, were observed in a stable BAC-carrying embryonic stem cell line when it was induced toward neural lineage differentiation by retinoic acid treatment. When neuronal differentiation was induced in embryoid body (EB)-derived neurosphere cells, reporter signals were detected in Msi1-positive NSCs and GFAP-positive astrocytes, but not in MAP2-positive neurons. By introducing deletions into the BAC reporter gene and conducting further reporter experiments using a minimized enhancer region, we identified a region, "D5E2," that is responsible for Msi1 transcription in NS/PCs.
  
  
  CONCLUSIONS
  A regulatory element for Msi1 transcription in NS/PCs is located in the sixth intron of the Msi1 gene. The 595-bp D5E2 intronic enhancer can transactivate Msi1 gene expression with cell-type specificity markedly similar to the endogenous Msi1 expression patterns.
authors.affiliation: Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kawase
authors.forename: Takao
authors.initials: T
authors.lastname: Imai
authors.forename: Chikako
authors.initials: C
authors.lastname: Miyauchi-Hara
authors.forename: Kunio
authors.initials: K
authors.lastname: Yaguchi
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Nishimoto
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Fukami
authors.forename: Yumi
authors.initials: Y
authors.lastname: Matsuzaki
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyawaki
authors.forename: Shigeyoshi
authors.initials: S
authors.lastname: Itohara
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Okano
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism; D020029:Base Pairing / Q000235:genetics; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005091:Exons / Q000235:genetics; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D016678:Genome / Q000235:genetics; D007438:Introns / Q000235:genetics*; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D058953:Neural Stem Cells / Q000166:cytology / Q000378:metabolism*; D055495:Neurogenesis / Q000235:genetics; D009457:Neuroglia / Q000378:metabolism; D009928:Organ Specificity / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D014158:Transcription, Genetic; D015533:Transcriptional Activation / Q000235:genetics
chemical_list: C104786:Msi1h protein, mouse; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins; D008156:Luciferases