From the Department of Biological Sciences, Kyoto University Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606, Japan
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ABSTRACT |
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ath3, a vertebrate basic helix-loop-helix gene homologous to Drosophila proneural gene atonal, can directly convert non-neural cells into neurons with the anterior features. In the mouse, ath3 expression initially occurs widely in the developing nervous system and then gradually becomes restricted to the neural retina. Here, we characterized the genomic organization and promoter activity of mouse ath3 (Math3). Math3 gene consists of two exons separated by an 8-kilobase intron, and the whole protein-coding region is located in the second exon. Transcription starts at two sites, which are 75 nucleotides apart from each other, and there is no typical TATA box in the upstream region of either start site. Transient transfection analysis showed that the 5'-region of Math3 can direct efficient expression in neuroblastoma cells but not in glioma or fibroblast cells. Deletion studies revealed that the proximal 193-base pair region, which contains the downstream transcription initiation site but not the upstream site, is essential for the Math3 promoter activity and can direct efficient expression in neuroblastoma cells. In contrast, retrovirus-mediated promoter analysis demonstrated that a region further upstream is additionally necessary for retinal expression. These results indicate that Math3 promoter contains two essential regulatory regions, the proximal 193-base pair region, which confers efficient neural-specific expression, and a region further upstream, required for retinal expression.
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INTRODUCTION |
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Recent studies demonstrated that vertebrate neuronal differentiation is controlled positively and negatively by multiple basic helix-loop-helix (bHLH)1 genes in an analogous way to Drosophila regulators (1, 2). For example, in Drosophila the bHLH genes of achaete-scute complex promote neuronal differentiation, whereas other bHLH genes, hairy and Enhancer of split, functionally antagonize achaete-scute complex and inhibit neuronal differentiation (3). Likewise, in mammals the bHLH gene Mash1, a mammalian homolog of Drosophila achaete-scute complex, promotes neuronal differentiation (4), whereas the bHLH gene Hes1, a mammalian homolog of Drosophila hairy and Enhancer of split, antagonizes Mash1 and inhibits neuronal differentiation (5, 6). Thus, the structures and functions of bHLH genes have been well conserved during evolution. Balance between these positive and negative bHLH genes is critical for normal neural development (4, 7, 8), and particularly, transcriptional regulation of these neural bHLH genes is very important because both ectopic expression and loss of expression cause severe abnormalities in the nervous system (4, 7-12).
Multiple neural bHLH genes homologous to Drosophila proneural gene atonal, which is essential for generation of photoreceptor and chordotonal organ neurons (13, 14), have been characterized from several vertebrate species (ath/neuroD/neurogenin family) (9-12, 15-22). Some of them are shown to promote neuronal differentiation in Xenopus (9-12). Among them, ath3 is unique because it is specifically expressed in the anterior neural tissues such as the forebrain, cranial ganglions, and retina and can generate neurons with the anterior features in Xenopus embryos (12). Thus, ath3 exhibits not only the anterior-specific expression but also the anterior-specific neurogenic activity. Furthermore, ath3 can induce expression of the photoreceptor-specific gene opsin (12), suggesting that ath3 may play an important role in retinal differentiation. In the mouse, ath3 expression occurs widely in the developing nervous system at early stages but then gradually becomes restricted to the anterior region like Xenopus ath3 (12). After birth, ath3 expression is detected only in the neural retina (12). Thus, ath3 shows two modes of the expression patterns during neural development; the initial general expression in the nervous system and the subsequent retina-specific expression.
In this study, to understand the molecular mechanism of neural-specific gene expression, we cloned mouse ath3 (Math3) gene and characterized its promoter activity. We found that the 5'-region of Math3 confers neural-specific gene expression. Furthermore, deletion study revealed that the proximal 193-bp region of Math3 promoter can direct efficient expression in neuroblastoma cells, whereas a region further upstream is necessary for retinal expression. Thus, these results suggest that the two modes of Math3 expression are controlled by two separate regulatory elements in the 5'-region of Math3 gene.
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EXPERIMENTAL PROCEDURES |
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Isolation and Characterization of Math3 Gene-- The mouse genomic library (Stratagene) was screened by hybridization with the Math3 cDNA as a probe. Nine clones were isolated from 9 × 105 plaques. The fragments hybridized positively were subcloned into pBluescript and subjected to sequence analysis.
For Southern blot analysis, the tail DNA was digested by restriction enzymes, electrophoresed on 0.7% agarose gel, and transferred to a nylon membrane filter. The 32P-labeled Math3 cDNA was hybridized at 65 °C in solution containing 0.2× SSC (1× SSC = 0.15 M NaCl, 0.015 M sodium citrate) and 0.1% SDS.Primer Extension and Reverse Transcription-mediated Polymerase Chain Reaction (PCR)-- For the primer extension experiment, primer 4, 5'-CTCTTTCCCGGGGTCAGCTCCCGCGAGTAG-3' (corresponding to the region from +175 to +204), was labeled at the 5'-end, hybridized to the mouse retina poly(A) RNA, and subjected to reverse transcription, as described previously (23).
For the reverse transcription-mediated PCR, the following primers in addition to primer 4 were used; primer 1, 5'-ACACGCAGTGGCAAAGCTGG-3' (Transient Transfection Analysis-- The reporter plasmids contained the firefly luciferase gene under the control of various lengths of Math3 promoter or the SV40 promoter. 1 µg of a reporter plasmid was transfected with 10 µl of LipofectAMINE reagent (Life Technologies, Inc.) into Neuro2a, NCB20 neuroblastoma brain hybrid cells, C6 glioma, or C3H10T1/2 cells, which were plated in 6-multiwell plates at the density of 2-4 × 105/well. 0.1 µg of the plasmid containing Renilla luciferase gene under the control of the herpes simplex virus thymidine kinase promoter (pHSVtk-RL) was also transfected as an internal standard to normalize the transfection efficiency. Medium was changed after incubation with the transfection complex at 37 °C for 6 h, and cells were further incubated at 37 °C. After 42-48 h, the cells were harvested, and the luciferase activity was measured.
Retrovirus-mediated Promoter Analysis--
For construction of
pLNSZ, which directs lacZ expression from the SV40 promoter
and neo expression from the upstream long terminal repeat,
the bacterial lacZ reporter gene was ligated into the
HindIII site of pLNSX (24). For the Math3
promoter constructs (pLNMZ), the SV40 promoter region was removed from pLNSZ by BamHI and HindIII digestion, and various
lengths of the Math3 promoter fragments were ligated
into the BamHI and HindIII sites.
Retrovirus was produced by transfecting the retroviral DNA constructs
into the packaging cell line 2mp34 (a kind gift of Dr. Kazuhiro
Ikenaka). Retrovirus solution was passed through a 0.45-µm filter and
concentrated, as described previously (6). The viral titer determined
by neo-resistance was usually 1 × 105
colony-forming units/ml.
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RESULTS |
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Structural Organization of Math3 Gene-- To understand the molecular mechanism of neural-specific gene expression, we cloned Math3 gene. Nine overlapping genomic clones were isolated from 9 × 105 plaques of a mouse genomic library by using the Math3 cDNA as a probe. Sequence comparison with the full-length Math3 cDNA revealed that Math3 gene encompassed a 12-kb region and consisted of two exons; the first exon contained only the 5'-noncoding region, whereas the second exon contained the whole protein-coding region (Fig. 1). The feature that the whole coding region is present in a single exon is also observed in Math1, Math2, and Mash1 genes (16, 17, 27), suggesting that these neural bHLH genes originated from a common ancestral gene. The two exons of Math3 were separated by an intron with the size of approximately 8 kb (Fig. 1B). Southern blot analysis using the tail DNA showed that the sizes of the hybridized DNA bands were identical to those of the cloned fragments (data not shown).
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Determination of the Transcription Initiation Site-- To determine the transcription initiation site, we first performed a primer extension experiment. The labeled antisense primer corresponding to the region from the nucleotide residue +175 to +204 was hybridized to retinal RNA and subjected to reverse transcription. This analysis demonstrated two specific bands with the sizes of 204 and 129 nucleotides (Fig. 2A, lane 1, arrows), suggesting that transcription initiates at two sites, the nucleotide residues +1 and +76.
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Transcription from Math3 Promoter in Neural Cells--
To
characterize the mechanism for neural-specific gene expression, the
promoter activity of Math3 gene was examined by a transient transfection method. A reporter plasmid containing the luciferase gene
under the control of the 5'-region of Math3 gene (from 2.8 kb to +196) was transfected into neuroblastoma, glioma, and fibroblast cell lines. The control SV40 promoter showed 30- to 40-fold activation in these cells when compared with the promoter-less construct (Fig.
3). The Math3 promoter
directed a higher level of expression than the SV40 promoter in
neuroblastoma cells, NCB20 and Neuro2a: 2.5-fold higher in NCB20 and
1.7-fold higher in Neuro2a (Fig. 3, A and B). In
contrast, the Math3 promoter exhibited much lower activity
than the SV40 promoter in other cell types: only 20% activity in C6
glioma cells and 15% activity in C3H10T1/2 fibroblast cells as
compared with the SV40 promoter (Fig. 3, C and
D). These results indicated that the 5'-region of
Math3 gene can direct neuronal-specific expression. The
intron region was also tested by a transient transfection method, but
the addition of the intron did not up-regulate neuronal expression
(data not shown).
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Transcription from Math3 Promoter in Retinal Cells-- Math3 expression initially occurs in various regions of the developing nervous system but later becomes restricted to the neural retina (12). In the adult retina, Math3 is expressed at a high level in the outer region of the inner nuclear layer (INL), where bipolar and horizontal cells are present (12). To determine the promoter regions necessary for retinal cell type-specific expression, retrovirus-mediated promoter analysis was performed (28). We generated recombinant retroviruses that direct lacZ expression under the control of the SV40 promoter or various lengths of Math3 promoter (Fig. 5, A and D and Table I). The explants of the developing retina, known to well mimic the in vivo development (8, 25, 26), were prepared from mouse embryos at day 17.5 or 18.5 and infected with these retroviruses. Only mitotic cells are infected with retrovirus, and once infected, cells precisely transmit the virus genome to their daughter cells. After 14 days of culture, at which time neuronal differentiation was finished, the retinal explants were stained with X-gal. If the promoter was functional, virus-infected cells should become blue after X-gal staining. It has been shown that during the postnatal period, the newly differentiating cells are mostly rods (almost 80%) and bipolar cells (~10%) (29, 30).
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DISCUSSION |
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The Promoter Region of Math3 Directs Neural- and
retinal-specific Expression--
In this study, we isolated and
characterized Math3 gene and showed that the 5'-region of
Math3 gene confers the cell type-specific expression. The
5'-region can direct efficient expression in neuroblastoma cells but
not in other cell types. Interestingly, the proximal 193-bp region
(from nucleotide residue +4 to +196), which consists of the 72-bp
upstream region, the second transcription initiation site (+76), and
the 121-bp 5'-noncoding region, which lacks the first transcription
initiation site (+1), is sufficient for efficient expression in
neuroblastoma cells. In addition, this 193-bp region is essential for
Math3 expression, since the region further upstream that
contains the first transcription initiation site but lacks the proximal
193-bp region cannot direct expression in neuroblastoma cells. Even the
region from 2.8 kb to +5 did not show the promoter activity. Thus,
the upstream regulatory region seems to depend upon the proximal 193-bp
region.
Negative Regulation and Retinal Development--
We previously
showed that continuous expression of Hes1 inhibits neuronal
differentiation and that, conversely, Hes1-null mutation
leads to up-regulation of Mash1 and premature neuronal differentiation in the retina (6-8). Thus, it is likely that Hes1
regulates the timing of differentiation by inhibiting Mash1 activity.
These data raise the interesting possibility that Hes1 could also
target to Math3 for inhibition of differentiation in the retina. For
example, there is an N box sequence at 107 that can be recognized by
Hes1. However, in transient transfection assay, Hes1 failed to repress
Math3 promoter activity in neuroblastoma cells.2
Thus, Hes1 does not functionally antagonize Math3 at the
transcriptional level, but it is still possible that Hes1 could inhibit
the activity of Math3 at the protein level, because Hes1 can inhibit
the activity of other bHLH factors such as Mash1 and MyoD through
protein-protein interaction (5).
Math3 and Retinal Development-- Characterization of the Math3 function is another important issue. In Xenopus, ath3 can induce retinal neuronal differentiation, and it is likely that Math3 also regulates retinal differentiation. Interestingly, Xenopus and mouse ath3, both, contain a possible phosphorylation site in the basic region, and in Xenopus, mutation of this site into Asp, which mimics the phosphorylation of this site, retains a general neurogenic activity but severely impairs the retinal differentiation activity (12). We speculate that in mice, ath3 activity is also regulated by phosphorylation of the basic region and that retinal differentiation may be induced by a nonphosphorylated form of Math3.
We previously showed that Math3 (locus symbol: Atoh3) is located on chromosome 10 (40) and closely links to eye blebs (eb) mutation, which shows eye anomalies (41). However, Southern blot analysis indicated that there is no major insertion or deletion in Math3 gene of eb mutant mice.2 Furthermore, there are many more defects in eb, including the kidney and limb, which are different from the regions expressing Math3. Therefore, the two genes Math3 and eb may be different. Now that the structure of Math3 was characterized, we can proceed to in vivo functional analysis such as loss-of-function assay in mice by targeted gene disruption. ![]() |
ACKNOWLEDGEMENT |
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We thank Kazuhiro Ikenaka for the
packaging cell line 2mp34.
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FOOTNOTES |
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* This work was supported by research grants from the Ministry of Education, Science, and Culture of Japan and Special Coordination Funds for Promoting Science and Technology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF036257 and AF034778.
Present address: The Burnham Institute, La Jolla Cancer Research
Foundation, 10901 N. Torrey Pines Rd., La Jolla, CA 92037.
§ To whom correspondence should be addressed: Tel.: 81-75-753-4438; Fax: 81-75-753-4404.
1
The abbreviations used are: bHLH, basic
helix-loop-helix; bp, base pair(s); kb, kilobase(s); INL, inner nuclear
layer; Math3, mouse atonal homolog 3; ONL, outer
nuclear layer; PCR, polymerase chain reaction; X-gal,
5-bromo-4-chloro-3-indolyl -D-galactopyranoside.
2 H. Tsuda, K. Takebayashi, S. Nakanishi, and R. Kageyama, unpublished data.
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REFERENCES |
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