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Identification of a novel intronic enhancer responsible for the transcriptional regulation of musashi1 in neural stem/progenitor cells.

Kawase S, Imai T, Miyauchi-Hara C, Yaguchi K, Nishimoto Y, Fukami S, Matsuzaki Y, Miyawaki A, Itohara S, Okano H - Mol Brain (2011)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo, Japan.

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.

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The 184-kb Msi1-reporter expression corresponds to endogenous Msi1 expressed in NS/PCs in vivo. (A) The original BAC (RP24-132L16) and the position of Msi1 are shown. There was a 22-kb region of Msi1 exons and introns between the 133-kb 5' upstream and 29-kb 3' downstream regions of Msi1. The translation initiation codon (ATG) was replaced with ffLuc to produce Msi1-ffLuc. (B) GFP in the E12.5 transgenic mouse containing Msi1-ffLuc, was expressed in the central nervous system and eye (upper panel: bright-field image, lower panel: fluorescent image). (C) Immunohistochemical analysis in the E14.5 Tg mouse cortex. Msi1, Group B1 SOX [SOX1/(2)/3], and Nestin expression in undifferentiated cells in the ventricular zone co-localized with GFP expression (Scale bar: 50 μm). The images shown in the small boxes in the right column panels are magnified and shown in the left corner boxes (Scale bar in magnified image: 10 μm). GFP was also present in the ventricular zone of the midbrain (D), pontine area (E), and eye (F). An arrow shows expression of GFP in retinal ganglion cell layer and an arrowhead shows expression of GFP in lens. AQ: aqueduct of midbrain, V4: fourth ventricle. Scale bar: 100 μm.
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Figure 1: The 184-kb Msi1-reporter expression corresponds to endogenous Msi1 expressed in NS/PCs in vivo. (A) The original BAC (RP24-132L16) and the position of Msi1 are shown. There was a 22-kb region of Msi1 exons and introns between the 133-kb 5' upstream and 29-kb 3' downstream regions of Msi1. The translation initiation codon (ATG) was replaced with ffLuc to produce Msi1-ffLuc. (B) GFP in the E12.5 transgenic mouse containing Msi1-ffLuc, was expressed in the central nervous system and eye (upper panel: bright-field image, lower panel: fluorescent image). (C) Immunohistochemical analysis in the E14.5 Tg mouse cortex. Msi1, Group B1 SOX [SOX1/(2)/3], and Nestin expression in undifferentiated cells in the ventricular zone co-localized with GFP expression (Scale bar: 50 μm). The images shown in the small boxes in the right column panels are magnified and shown in the left corner boxes (Scale bar in magnified image: 10 μm). GFP was also present in the ventricular zone of the midbrain (D), pontine area (E), and eye (F). An arrow shows expression of GFP in retinal ganglion cell layer and an arrowhead shows expression of GFP in lens. AQ: aqueduct of midbrain, V4: fourth ventricle. Scale bar: 100 μm.

Mentions: To identify transcription or signalling pathway-associated factors involved in regulating Msi1 gene transcription, we generated a BAC reporter gene to detect Msi1 transcriptional activity in NS/PCs. We previously reported generating an Msi1 transcription reporter gene that contained an approximately 3-kb 5' upstream region of the mouse Msi1 TSS, which, through unknown mechanisms, could act in human cells but not in mouse cells [48]. To elucidate Msi1 transcriptional regulatory mechanisms in vivo more precisely, we prepared the RP24-132L16 BAC as a genomic element containing many of the cis-elements that direct Msi1 transcription. As shown in Figure 1A, the RP24-132L16 BAC contains 133-kb of the 5' region upstream of the Msi1 TSS, the Msi1 mRNA coding region (exons-introns coding region), and 29-kb of the 3' region downstream of the 3'-end of the Msi1 gene. We then used homologous recombination techniques to insert the ffLuc reporter gene into the Msi1 translational initiation site of the RP24-132L16 BAC (Figure 1A). The ffLuc reporter gene encodes a fusion protein of the fluorescent protein Venus and firefly Luciferase [45,46]. This reporter gene both visualized Msi1 transcriptional activity in vivo, and allowed us to use Luciferase bioluminescence to quantify the level of transcriptional activity.


Identification of a novel intronic enhancer responsible for the transcriptional regulation of musashi1 in neural stem/progenitor cells.

Kawase S, Imai T, Miyauchi-Hara C, Yaguchi K, Nishimoto Y, Fukami S, Matsuzaki Y, Miyawaki A, Itohara S, Okano H - Mol Brain (2011)

The 184-kb Msi1-reporter expression corresponds to endogenous Msi1 expressed in NS/PCs in vivo. (A) The original BAC (RP24-132L16) and the position of Msi1 are shown. There was a 22-kb region of Msi1 exons and introns between the 133-kb 5' upstream and 29-kb 3' downstream regions of Msi1. The translation initiation codon (ATG) was replaced with ffLuc to produce Msi1-ffLuc. (B) GFP in the E12.5 transgenic mouse containing Msi1-ffLuc, was expressed in the central nervous system and eye (upper panel: bright-field image, lower panel: fluorescent image). (C) Immunohistochemical analysis in the E14.5 Tg mouse cortex. Msi1, Group B1 SOX [SOX1/(2)/3], and Nestin expression in undifferentiated cells in the ventricular zone co-localized with GFP expression (Scale bar: 50 μm). The images shown in the small boxes in the right column panels are magnified and shown in the left corner boxes (Scale bar in magnified image: 10 μm). GFP was also present in the ventricular zone of the midbrain (D), pontine area (E), and eye (F). An arrow shows expression of GFP in retinal ganglion cell layer and an arrowhead shows expression of GFP in lens. AQ: aqueduct of midbrain, V4: fourth ventricle. Scale bar: 100 μm.
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Figure 1: The 184-kb Msi1-reporter expression corresponds to endogenous Msi1 expressed in NS/PCs in vivo. (A) The original BAC (RP24-132L16) and the position of Msi1 are shown. There was a 22-kb region of Msi1 exons and introns between the 133-kb 5' upstream and 29-kb 3' downstream regions of Msi1. The translation initiation codon (ATG) was replaced with ffLuc to produce Msi1-ffLuc. (B) GFP in the E12.5 transgenic mouse containing Msi1-ffLuc, was expressed in the central nervous system and eye (upper panel: bright-field image, lower panel: fluorescent image). (C) Immunohistochemical analysis in the E14.5 Tg mouse cortex. Msi1, Group B1 SOX [SOX1/(2)/3], and Nestin expression in undifferentiated cells in the ventricular zone co-localized with GFP expression (Scale bar: 50 μm). The images shown in the small boxes in the right column panels are magnified and shown in the left corner boxes (Scale bar in magnified image: 10 μm). GFP was also present in the ventricular zone of the midbrain (D), pontine area (E), and eye (F). An arrow shows expression of GFP in retinal ganglion cell layer and an arrowhead shows expression of GFP in lens. AQ: aqueduct of midbrain, V4: fourth ventricle. Scale bar: 100 μm.
Mentions: To identify transcription or signalling pathway-associated factors involved in regulating Msi1 gene transcription, we generated a BAC reporter gene to detect Msi1 transcriptional activity in NS/PCs. We previously reported generating an Msi1 transcription reporter gene that contained an approximately 3-kb 5' upstream region of the mouse Msi1 TSS, which, through unknown mechanisms, could act in human cells but not in mouse cells [48]. To elucidate Msi1 transcriptional regulatory mechanisms in vivo more precisely, we prepared the RP24-132L16 BAC as a genomic element containing many of the cis-elements that direct Msi1 transcription. As shown in Figure 1A, the RP24-132L16 BAC contains 133-kb of the 5' region upstream of the Msi1 TSS, the Msi1 mRNA coding region (exons-introns coding region), and 29-kb of the 3' region downstream of the 3'-end of the Msi1 gene. We then used homologous recombination techniques to insert the ffLuc reporter gene into the Msi1 translational initiation site of the RP24-132L16 BAC (Figure 1A). The ffLuc reporter gene encodes a fusion protein of the fluorescent protein Venus and firefly Luciferase [45,46]. This reporter gene both visualized Msi1 transcriptional activity in vivo, and allowed us to use Luciferase bioluminescence to quantify the level of transcriptional activity.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo, Japan.

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.

Show MeSH
Related in: MedlinePlus