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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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D5E2-P1 expression corresponds with Msi1-positive NS/PCs and astrocytes. (A), (B) D5E2-P1 was active in primary neurospheres. (A) Primary neurospheres derived from ESC lines, on day 6. Scale bar: 100 μm. (B) Three cell lines with intense reporter expression were selected from 47 ES cell lines; EB(+RA) were selected and primary neurospheres were formed in each clone. The reporter activity was analysed on day 6. (C) Cells from secondary neurospheres were differentiated and stained with each antibody shown. D5E2-P1 showed intense GFP expression. GFP was not induced by P1, and was only slightly induced by D2E-P1. Vertical arrowheads show Marker(+)/GFP(+) cells. D5E2-P1 induced GFP expression in Msi1(+) and Nestin(+)/GFAP(+) cells. Scale bar: 50 um. (D) Immunofluorescence intensity was visualized by scatter plots. The vertical axis shows the intensity of Msi1 and each cell-type-specific marker. The horizontal axis shows GFP intensity. The ratio of marker(+) GFP(+)/GFP(+) (%) cells is indicated at the bottom of the boxes.
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Figure 5: D5E2-P1 expression corresponds with Msi1-positive NS/PCs and astrocytes. (A), (B) D5E2-P1 was active in primary neurospheres. (A) Primary neurospheres derived from ESC lines, on day 6. Scale bar: 100 μm. (B) Three cell lines with intense reporter expression were selected from 47 ES cell lines; EB(+RA) were selected and primary neurospheres were formed in each clone. The reporter activity was analysed on day 6. (C) Cells from secondary neurospheres were differentiated and stained with each antibody shown. D5E2-P1 showed intense GFP expression. GFP was not induced by P1, and was only slightly induced by D2E-P1. Vertical arrowheads show Marker(+)/GFP(+) cells. D5E2-P1 induced GFP expression in Msi1(+) and Nestin(+)/GFAP(+) cells. Scale bar: 50 um. (D) Immunofluorescence intensity was visualized by scatter plots. The vertical axis shows the intensity of Msi1 and each cell-type-specific marker. The horizontal axis shows GFP intensity. The ratio of marker(+) GFP(+)/GFP(+) (%) cells is indicated at the bottom of the boxes.

Mentions: We next examined whether the activities of the minimized Msi1 transcriptional enhancers corresponded with endogenous Msi1 expression, which was high in NS/PCs and astrocytes and low in the neuronal linage. For this purpose, EB (+RA)s were taken from the three cell lines showing the most intense Venus expression for each integrated construct (P1-, D2E-P1- and D5E2-P1-integrated EBs) and placed in floating culture for 6 days to form primary neurospheres (Figure 4). GFP-fluorescence was detected in primary neurospheres containing the D5E2 construct (Figure 5A). Primary spheres containing P1, D2E-P1, or D5E2-P1 were also subjected to Luciferase assays. The Luciferase activity was 18.2-fold stronger in the primary neurospheres with D5E2-P1 than in those with P1 alone (Figure 5B), and 1.4-fold greater in the primary neurospheres with D2E-P1 than in those with P1 alone (Figure 5B).


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)

D5E2-P1 expression corresponds with Msi1-positive NS/PCs and astrocytes. (A), (B) D5E2-P1 was active in primary neurospheres. (A) Primary neurospheres derived from ESC lines, on day 6. Scale bar: 100 μm. (B) Three cell lines with intense reporter expression were selected from 47 ES cell lines; EB(+RA) were selected and primary neurospheres were formed in each clone. The reporter activity was analysed on day 6. (C) Cells from secondary neurospheres were differentiated and stained with each antibody shown. D5E2-P1 showed intense GFP expression. GFP was not induced by P1, and was only slightly induced by D2E-P1. Vertical arrowheads show Marker(+)/GFP(+) cells. D5E2-P1 induced GFP expression in Msi1(+) and Nestin(+)/GFAP(+) cells. Scale bar: 50 um. (D) Immunofluorescence intensity was visualized by scatter plots. The vertical axis shows the intensity of Msi1 and each cell-type-specific marker. The horizontal axis shows GFP intensity. The ratio of marker(+) GFP(+)/GFP(+) (%) cells is indicated at the bottom of the boxes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3108301&req=5

Figure 5: D5E2-P1 expression corresponds with Msi1-positive NS/PCs and astrocytes. (A), (B) D5E2-P1 was active in primary neurospheres. (A) Primary neurospheres derived from ESC lines, on day 6. Scale bar: 100 μm. (B) Three cell lines with intense reporter expression were selected from 47 ES cell lines; EB(+RA) were selected and primary neurospheres were formed in each clone. The reporter activity was analysed on day 6. (C) Cells from secondary neurospheres were differentiated and stained with each antibody shown. D5E2-P1 showed intense GFP expression. GFP was not induced by P1, and was only slightly induced by D2E-P1. Vertical arrowheads show Marker(+)/GFP(+) cells. D5E2-P1 induced GFP expression in Msi1(+) and Nestin(+)/GFAP(+) cells. Scale bar: 50 um. (D) Immunofluorescence intensity was visualized by scatter plots. The vertical axis shows the intensity of Msi1 and each cell-type-specific marker. The horizontal axis shows GFP intensity. The ratio of marker(+) GFP(+)/GFP(+) (%) cells is indicated at the bottom of the boxes.
Mentions: We next examined whether the activities of the minimized Msi1 transcriptional enhancers corresponded with endogenous Msi1 expression, which was high in NS/PCs and astrocytes and low in the neuronal linage. For this purpose, EB (+RA)s were taken from the three cell lines showing the most intense Venus expression for each integrated construct (P1-, D2E-P1- and D5E2-P1-integrated EBs) and placed in floating culture for 6 days to form primary neurospheres (Figure 4). GFP-fluorescence was detected in primary neurospheres containing the D5E2 construct (Figure 5A). Primary spheres containing P1, D2E-P1, or D5E2-P1 were also subjected to Luciferase assays. The Luciferase activity was 18.2-fold stronger in the primary neurospheres with D5E2-P1 than in those with P1 alone (Figure 5B), and 1.4-fold greater in the primary neurospheres with D2E-P1 than in those with P1 alone (Figure 5B).

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