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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 is a transcriptional enhancer that functions in NS/PCs. Candidate enhancer alignments were selected using information from the UCSC genome data base and by looking at p300-binding sites [52,53] in the expected enhancer regions of introns and 55-65 Kb upstream. Each enhancer reporter gene used in the experiments is shown in (A). Nestin second intron-Tkp was used as a positive control. (B), (C) Candidate enhancer activities in ESCs and NS/PCs. Dissociated E14.5 cortex cells were cultured on a poly-D-Ornithine/fibronectin coated dish in an FGF-2/EGF mixed selective NS/PC conditioned medium. Enhancer reporter constructs were transfected into EB3 tg14 and NS/PCs, and the Luciferase activity was detected after 48 hours. The D2E and D5E2 sites enhanced the transcriptional activity in ESCs, and the D5E2 site enhanced it in NS/PCs [4.3-fold, 5.1-fold in ESCs, 2.2-fold, 15.2-fold in NS/PCs, respectively, compared with P1 (P1 = 1)], but D5E1 could not enhance the activity in either cell type. The data represent the mean ±SEM of three independent experiments. The data were subjected to non-repeated-measures ANOVA tests, and p values were calculated by Bonferroni multiple comparison tests. *p < 0.05: P1 to D2E-P1, D5E1-P1, or D5E2-P1, ns: not significant. FLU/RLU: firefly luciferase light unit/renilla luciferase light unit. (D), (E) The D2E and D5E2 enhancer activities were confirmed in EBs: 47 stable ES cell lines for P1, D2E-P1, and D5E2-P1were established and cultured in EB-formation conditions with or without RA. (D) Day 6 of neural-induced EB (+RA). Left panel shows bright field and right panel shows fluorescent images. Scale bar: 100 μm. (E) All cell lines were analysed for each condition on day 6. Average intensity is shown in comparison to RA-treated P1 (P1: +RA = 1, -RA = 0.33, D2E-P1: +RA = 3.47, -RA = 0.26, D5E2-P1: +RA = 16.7, -RA = 3.18.). Note that D5E2-P1 showed potent enhancer activity in the neural induced EBs.
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Figure 4: D5E2 is a transcriptional enhancer that functions in NS/PCs. Candidate enhancer alignments were selected using information from the UCSC genome data base and by looking at p300-binding sites [52,53] in the expected enhancer regions of introns and 55-65 Kb upstream. Each enhancer reporter gene used in the experiments is shown in (A). Nestin second intron-Tkp was used as a positive control. (B), (C) Candidate enhancer activities in ESCs and NS/PCs. Dissociated E14.5 cortex cells were cultured on a poly-D-Ornithine/fibronectin coated dish in an FGF-2/EGF mixed selective NS/PC conditioned medium. Enhancer reporter constructs were transfected into EB3 tg14 and NS/PCs, and the Luciferase activity was detected after 48 hours. The D2E and D5E2 sites enhanced the transcriptional activity in ESCs, and the D5E2 site enhanced it in NS/PCs [4.3-fold, 5.1-fold in ESCs, 2.2-fold, 15.2-fold in NS/PCs, respectively, compared with P1 (P1 = 1)], but D5E1 could not enhance the activity in either cell type. The data represent the mean ±SEM of three independent experiments. The data were subjected to non-repeated-measures ANOVA tests, and p values were calculated by Bonferroni multiple comparison tests. *p < 0.05: P1 to D2E-P1, D5E1-P1, or D5E2-P1, ns: not significant. FLU/RLU: firefly luciferase light unit/renilla luciferase light unit. (D), (E) The D2E and D5E2 enhancer activities were confirmed in EBs: 47 stable ES cell lines for P1, D2E-P1, and D5E2-P1were established and cultured in EB-formation conditions with or without RA. (D) Day 6 of neural-induced EB (+RA). Left panel shows bright field and right panel shows fluorescent images. Scale bar: 100 μm. (E) All cell lines were analysed for each condition on day 6. Average intensity is shown in comparison to RA-treated P1 (P1: +RA = 1, -RA = 0.33, D2E-P1: +RA = 3.47, -RA = 0.26, D5E2-P1: +RA = 16.7, -RA = 3.18.). Note that D5E2-P1 showed potent enhancer activity in the neural induced EBs.

Mentions: To investigate whether D2E and D5E2 function as localized enhancer regions to regulate Msi1 transcription, we generated constructs containing a minimal enhancer (D2E, D5E2, or none), the P1 promoter 1-kb upstream of the Msi1 TSS, and ffLuc (Figure 4A). With the original locations on the genome in mind, D2E was placed at the 5'-end of the P1 promoter, and D5E2 was placed at the 3'-end of the ffLuc-SV40 poly(A) additional signal element (Figure 4A). D5E1, which was included in the fourth intron and was well-conserved in the mouse, human, horse, and rat species (but not chicken), was not included in the p300 ChIP-sequence tags, and was used as a negative control [53] (Figure 3B). The Nestin-TKp construct contained a rat Nestin second intron enhancer combined with a thymidine kinase minimal promoter element, and ffLuc. This Nestin enhancer is known to induce transcriptional activity in NS/PCs [35,54]. These constructs were transiently introduced with an internal control Renilla luciferase-expressing plasmid into ESCs or NS/PCs derived from the mouse brain cortex at E14.5 and cultured in the presence of FGF-2 and EGF. The transfected cells were lysed after 2 days in culture, and their Luciferase activity was measured. The Luciferase activity was low in ESCs or NS/PCs with the D5E1-P1 construct or with only the P1-promoter construct. We also observed weak Luciferase activity in ESCs with D2E-P1 or D5E2-P1; reporter signals in these ESCs showed 4-5-fold increases compared to the signals in cells with only the P1 construct. Interestingly, the Luciferase activity in NS/PCs with a transfected D5E2-P1 construct increased markedly, 15.2-fold, compared with the signals of cells with the P1 construct. When D2E-P1 was introduced into NS/PCs, a 2.2-fold increase in the reporter signal was observed compared to P1 alone, however, there were no stastical difference between them. These findings together indicated that D5E2 can work efficiently as an Msi1 transcription enhancer in terms of the strength and specificity of transactivation in NS/PCs.


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 is a transcriptional enhancer that functions in NS/PCs. Candidate enhancer alignments were selected using information from the UCSC genome data base and by looking at p300-binding sites [52,53] in the expected enhancer regions of introns and 55-65 Kb upstream. Each enhancer reporter gene used in the experiments is shown in (A). Nestin second intron-Tkp was used as a positive control. (B), (C) Candidate enhancer activities in ESCs and NS/PCs. Dissociated E14.5 cortex cells were cultured on a poly-D-Ornithine/fibronectin coated dish in an FGF-2/EGF mixed selective NS/PC conditioned medium. Enhancer reporter constructs were transfected into EB3 tg14 and NS/PCs, and the Luciferase activity was detected after 48 hours. The D2E and D5E2 sites enhanced the transcriptional activity in ESCs, and the D5E2 site enhanced it in NS/PCs [4.3-fold, 5.1-fold in ESCs, 2.2-fold, 15.2-fold in NS/PCs, respectively, compared with P1 (P1 = 1)], but D5E1 could not enhance the activity in either cell type. The data represent the mean ±SEM of three independent experiments. The data were subjected to non-repeated-measures ANOVA tests, and p values were calculated by Bonferroni multiple comparison tests. *p < 0.05: P1 to D2E-P1, D5E1-P1, or D5E2-P1, ns: not significant. FLU/RLU: firefly luciferase light unit/renilla luciferase light unit. (D), (E) The D2E and D5E2 enhancer activities were confirmed in EBs: 47 stable ES cell lines for P1, D2E-P1, and D5E2-P1were established and cultured in EB-formation conditions with or without RA. (D) Day 6 of neural-induced EB (+RA). Left panel shows bright field and right panel shows fluorescent images. Scale bar: 100 μm. (E) All cell lines were analysed for each condition on day 6. Average intensity is shown in comparison to RA-treated P1 (P1: +RA = 1, -RA = 0.33, D2E-P1: +RA = 3.47, -RA = 0.26, D5E2-P1: +RA = 16.7, -RA = 3.18.). Note that D5E2-P1 showed potent enhancer activity in the neural induced EBs.
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Figure 4: D5E2 is a transcriptional enhancer that functions in NS/PCs. Candidate enhancer alignments were selected using information from the UCSC genome data base and by looking at p300-binding sites [52,53] in the expected enhancer regions of introns and 55-65 Kb upstream. Each enhancer reporter gene used in the experiments is shown in (A). Nestin second intron-Tkp was used as a positive control. (B), (C) Candidate enhancer activities in ESCs and NS/PCs. Dissociated E14.5 cortex cells were cultured on a poly-D-Ornithine/fibronectin coated dish in an FGF-2/EGF mixed selective NS/PC conditioned medium. Enhancer reporter constructs were transfected into EB3 tg14 and NS/PCs, and the Luciferase activity was detected after 48 hours. The D2E and D5E2 sites enhanced the transcriptional activity in ESCs, and the D5E2 site enhanced it in NS/PCs [4.3-fold, 5.1-fold in ESCs, 2.2-fold, 15.2-fold in NS/PCs, respectively, compared with P1 (P1 = 1)], but D5E1 could not enhance the activity in either cell type. The data represent the mean ±SEM of three independent experiments. The data were subjected to non-repeated-measures ANOVA tests, and p values were calculated by Bonferroni multiple comparison tests. *p < 0.05: P1 to D2E-P1, D5E1-P1, or D5E2-P1, ns: not significant. FLU/RLU: firefly luciferase light unit/renilla luciferase light unit. (D), (E) The D2E and D5E2 enhancer activities were confirmed in EBs: 47 stable ES cell lines for P1, D2E-P1, and D5E2-P1were established and cultured in EB-formation conditions with or without RA. (D) Day 6 of neural-induced EB (+RA). Left panel shows bright field and right panel shows fluorescent images. Scale bar: 100 μm. (E) All cell lines were analysed for each condition on day 6. Average intensity is shown in comparison to RA-treated P1 (P1: +RA = 1, -RA = 0.33, D2E-P1: +RA = 3.47, -RA = 0.26, D5E2-P1: +RA = 16.7, -RA = 3.18.). Note that D5E2-P1 showed potent enhancer activity in the neural induced EBs.
Mentions: To investigate whether D2E and D5E2 function as localized enhancer regions to regulate Msi1 transcription, we generated constructs containing a minimal enhancer (D2E, D5E2, or none), the P1 promoter 1-kb upstream of the Msi1 TSS, and ffLuc (Figure 4A). With the original locations on the genome in mind, D2E was placed at the 5'-end of the P1 promoter, and D5E2 was placed at the 3'-end of the ffLuc-SV40 poly(A) additional signal element (Figure 4A). D5E1, which was included in the fourth intron and was well-conserved in the mouse, human, horse, and rat species (but not chicken), was not included in the p300 ChIP-sequence tags, and was used as a negative control [53] (Figure 3B). The Nestin-TKp construct contained a rat Nestin second intron enhancer combined with a thymidine kinase minimal promoter element, and ffLuc. This Nestin enhancer is known to induce transcriptional activity in NS/PCs [35,54]. These constructs were transiently introduced with an internal control Renilla luciferase-expressing plasmid into ESCs or NS/PCs derived from the mouse brain cortex at E14.5 and cultured in the presence of FGF-2 and EGF. The transfected cells were lysed after 2 days in culture, and their Luciferase activity was measured. The Luciferase activity was low in ESCs or NS/PCs with the D5E1-P1 construct or with only the P1-promoter construct. We also observed weak Luciferase activity in ESCs with D2E-P1 or D5E2-P1; reporter signals in these ESCs showed 4-5-fold increases compared to the signals in cells with only the P1 construct. Interestingly, the Luciferase activity in NS/PCs with a transfected D5E2-P1 construct increased markedly, 15.2-fold, compared with the signals of cells with the P1 construct. When D2E-P1 was introduced into NS/PCs, a 2.2-fold increase in the reporter signal was observed compared to P1 alone, however, there were no stastical difference between them. These findings together indicated that D5E2 can work efficiently as an Msi1 transcription enhancer in terms of the strength and specificity of transactivation in NS/PCs.

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