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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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Msi1 BAC deletion studies suggest that both the 55-65 kb 5' upstream region and the exon-intron coding region have Msi1 transcriptional activity. (A) Schematic representation of our deletion method. Targeted Msi1 enhancer/promoter regions were replaced with pgk pro.-Neor-bGH pA to select for successfully recombined clones. (B) The conserved genomic region between cox6a1 and pla2g1b in Msi1-ffLuc is shown. VISTA plots comparing the alignment of mouse versus human, horse, rat, and chicken. Full-length Msi1-ffLuc (D0) and deletion constructs (D1-D5) are also indicated. Blanks represent deleted regions replaced with pgk pro.-Neor-bGH pA. The positions of candidate enhancer sites used in later experiments are also shown (see details in Results). The exon-intron coding region of Msi1 gene is magnified. Red circles indicate candidate enhancer sites, white boxes indicate exons. (C) Gene expression in EBs derived from the deletion-reporter ES cell lines. All 21 cell lines were analysed for Luciferase expression in +RA neural induction cultures for 6 days (blue bars). Clones are presented in rank order for neural cultures. The red bars show the activity in the same clone under non-neural inducing conditions (-RA, not in rank order). Luciferase activity was normalized to the cell-viability fluorescence intensity using CellTiter-Blue (CTBF). The Msi1 expression in D0 and D1 was greatly increased under the neural-inducing conditions. Note that D5 showed decreased Msi1 expression; D2-D4, which included a 55-65-kb deletion, showed an even greater decrease.
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Figure 3: Msi1 BAC deletion studies suggest that both the 55-65 kb 5' upstream region and the exon-intron coding region have Msi1 transcriptional activity. (A) Schematic representation of our deletion method. Targeted Msi1 enhancer/promoter regions were replaced with pgk pro.-Neor-bGH pA to select for successfully recombined clones. (B) The conserved genomic region between cox6a1 and pla2g1b in Msi1-ffLuc is shown. VISTA plots comparing the alignment of mouse versus human, horse, rat, and chicken. Full-length Msi1-ffLuc (D0) and deletion constructs (D1-D5) are also indicated. Blanks represent deleted regions replaced with pgk pro.-Neor-bGH pA. The positions of candidate enhancer sites used in later experiments are also shown (see details in Results). The exon-intron coding region of Msi1 gene is magnified. Red circles indicate candidate enhancer sites, white boxes indicate exons. (C) Gene expression in EBs derived from the deletion-reporter ES cell lines. All 21 cell lines were analysed for Luciferase expression in +RA neural induction cultures for 6 days (blue bars). Clones are presented in rank order for neural cultures. The red bars show the activity in the same clone under non-neural inducing conditions (-RA, not in rank order). Luciferase activity was normalized to the cell-viability fluorescence intensity using CellTiter-Blue (CTBF). The Msi1 expression in D0 and D1 was greatly increased under the neural-inducing conditions. Note that D5 showed decreased Msi1 expression; D2-D4, which included a 55-65-kb deletion, showed an even greater decrease.

Mentions: To elucidate the regulatory mechanisms of Msi1 gene transcription, we performed a deletion study to determine whether there are cis-elements in the 184-kb region of genomic DNA containing the Msi1 loci that are sufficient for inducing Msi1 transcription. Accordingly, a module containing the neomycin-resistance gene was inserted into the Msi1 5'-upstream region and the exon-intron coding region (D1-D5). The BAC constructs were generated by homologous recombination, as shown in Figure 3A and 3B. The length and location of the deleted regions were designed by considering evolutionarily conserved regions among species--mouse, human, horse, rat, and chicken. This homology alignment was performed with the VISTA homology search program [50]. It was also taken into consideration that the adjacent genes Cox6A1 and Pla2G1b are located 80-kb upstream and 54-kb downstream of the Msi1 TSS, respectively (Figure 3B).


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)

Msi1 BAC deletion studies suggest that both the 55-65 kb 5' upstream region and the exon-intron coding region have Msi1 transcriptional activity. (A) Schematic representation of our deletion method. Targeted Msi1 enhancer/promoter regions were replaced with pgk pro.-Neor-bGH pA to select for successfully recombined clones. (B) The conserved genomic region between cox6a1 and pla2g1b in Msi1-ffLuc is shown. VISTA plots comparing the alignment of mouse versus human, horse, rat, and chicken. Full-length Msi1-ffLuc (D0) and deletion constructs (D1-D5) are also indicated. Blanks represent deleted regions replaced with pgk pro.-Neor-bGH pA. The positions of candidate enhancer sites used in later experiments are also shown (see details in Results). The exon-intron coding region of Msi1 gene is magnified. Red circles indicate candidate enhancer sites, white boxes indicate exons. (C) Gene expression in EBs derived from the deletion-reporter ES cell lines. All 21 cell lines were analysed for Luciferase expression in +RA neural induction cultures for 6 days (blue bars). Clones are presented in rank order for neural cultures. The red bars show the activity in the same clone under non-neural inducing conditions (-RA, not in rank order). Luciferase activity was normalized to the cell-viability fluorescence intensity using CellTiter-Blue (CTBF). The Msi1 expression in D0 and D1 was greatly increased under the neural-inducing conditions. Note that D5 showed decreased Msi1 expression; D2-D4, which included a 55-65-kb deletion, showed an even greater decrease.
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Figure 3: Msi1 BAC deletion studies suggest that both the 55-65 kb 5' upstream region and the exon-intron coding region have Msi1 transcriptional activity. (A) Schematic representation of our deletion method. Targeted Msi1 enhancer/promoter regions were replaced with pgk pro.-Neor-bGH pA to select for successfully recombined clones. (B) The conserved genomic region between cox6a1 and pla2g1b in Msi1-ffLuc is shown. VISTA plots comparing the alignment of mouse versus human, horse, rat, and chicken. Full-length Msi1-ffLuc (D0) and deletion constructs (D1-D5) are also indicated. Blanks represent deleted regions replaced with pgk pro.-Neor-bGH pA. The positions of candidate enhancer sites used in later experiments are also shown (see details in Results). The exon-intron coding region of Msi1 gene is magnified. Red circles indicate candidate enhancer sites, white boxes indicate exons. (C) Gene expression in EBs derived from the deletion-reporter ES cell lines. All 21 cell lines were analysed for Luciferase expression in +RA neural induction cultures for 6 days (blue bars). Clones are presented in rank order for neural cultures. The red bars show the activity in the same clone under non-neural inducing conditions (-RA, not in rank order). Luciferase activity was normalized to the cell-viability fluorescence intensity using CellTiter-Blue (CTBF). The Msi1 expression in D0 and D1 was greatly increased under the neural-inducing conditions. Note that D5 showed decreased Msi1 expression; D2-D4, which included a 55-65-kb deletion, showed an even greater decrease.
Mentions: To elucidate the regulatory mechanisms of Msi1 gene transcription, we performed a deletion study to determine whether there are cis-elements in the 184-kb region of genomic DNA containing the Msi1 loci that are sufficient for inducing Msi1 transcription. Accordingly, a module containing the neomycin-resistance gene was inserted into the Msi1 5'-upstream region and the exon-intron coding region (D1-D5). The BAC constructs were generated by homologous recombination, as shown in Figure 3A and 3B. The length and location of the deleted regions were designed by considering evolutionarily conserved regions among species--mouse, human, horse, rat, and chicken. This homology alignment was performed with the VISTA homology search program [50]. It was also taken into consideration that the adjacent genes Cox6A1 and Pla2G1b are located 80-kb upstream and 54-kb downstream of the Msi1 TSS, respectively (Figure 3B).

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