©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure and Promoter Analysis of the Gene Encoding the Mouse Helix-Loop-Helix Factor HES-5
IDENTIFICATION OF THE NEURAL PRECURSOR CELL-SPECIFIC PROMOTER ELEMENT (*)

(Received for publication, October 17, 1994)

Koichi Takebayashi Chihiro Akazawa (§) Shigetada Nakanishi Ryoichiro Kageyama (¶)

From the Institute for Immunology, Kyoto University Faculty of Medicine, Kyoto 606, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

HES-5 is a mammalian basic helix-loop-helix factor that has a distant sequence homology to the product of the Drosophila pair-rule gene hairy. HES-5 mRNA is present exclusively in the developing nervous system, but its level decreases as neural differentiation proceeds. In this study, to characterize the molecular mechanism of the neural-specific expression of HES-5 we isolated the mouse HES-5 gene. This gene consists of three exons, and Southern blot analysis shows that it is a single copy gene. The transcription initiation site, determined by primer extension and reverse transcriptase-mediated polymerase chain reaction, is located 26 nucleotides downstream of a TATA box. Transient transfection analysis shows that the upstream region of the HES-5 gene can direct efficient expression in neural precursor cells and moderate expression in undifferentiated NCB20 neuroblastoma-brain hybrid cells but not in glioma or fibroblast cells. The moderate level of expression in NCB20 cells decreases when differentiation into neuron-like cells is induced. Further promoter analysis shows that this undifferentiated neural-specific expression is mediated by the multiple GC stretches present in the HES-5 promoter. Gel mobility shift analysis suggests the presence of a neural precursor cell-specific protein that binds to the GC stretches. These results raise the possibility that HES-5 expression in the developing nervous system is regulated by the GC stretch-binding protein.


INTRODUCTION

In the developing mammalian central nervous system (CNS), (^1)neural precursor cells are proliferating in the ventricular zone next to the lumen (Jacobson, 1991). When their fate is determined to become neurons or glial cells, these precursor cells stop cell division, migrate out of the ventricular zone, and undergo terminal differentiation. The underlying molecular mechanisms are not yet well understood, but recent evidence suggests that transcription factors with a helix-loop-helix (HLH) domain may be involved in these developmental processes (Johnson et al., 1990; Sasai et al., 1992; Guillemot et al., 1993; Ishibashi et al., 1994). For example, null mutation of the basic HLH (bHLH) factor MASH1, a mammalian homologue of the Drosophila achaete-scute complex (Johnson et al., 1990), results in severe loss of olfactory and autonomic neurons (Guillemot et al., 1993), suggesting that MASH1 is a positive regulator of neurogenesis. HES-1, another bHLH factor structurally related to the product of the Drosophila pair-rule gene hairy (Sasai et al., 1992), is a negative regulator of mammalian neurogenesis (Ishibashi et al., 1994). Thus, the balance between the positive and negative regulatory factors may be important for normal neurogenesis.

Recent studies show that many other HLH factors, such as other members of HES family (Akazawa et al., 1992; Sasai et al., 1992; Ishibashi et al., 1993), Id-1 (Duncan et al., 1992), and Id-2 (Neuman et al., 1993), are expressed in the developing nervous system, suggesting that they are also involved in neural differentiation. Among these HLH factors, HES-5 has a unique transcriptional activity. Unlike most other bHLH factors, HES-5 does not bind to the CANNTG sequence (the E box), but it binds to the consensus sequence CACNAG (the N box) and represses transcription (Akazawa et al., 1992). Furthermore, like Id proteins, HES-5 also represses E box-dependent transcription by preventing other bHLH activators from binding to the E box (Benezra et al., 1990; Sun et al., 1991; Christy et al., 1991; Akazawa et al., 1992). Thus, HES-5 represses transcription in two different manners, depending on the motifs.

HES-5 also shows a unique expression pattern in late embryos (Akazawa et al., 1992): 1) HES-5 is expressed exclusively in the developing nervous system, and 2) HES-5 is expressed at high levels throughout the ventricular zone where neural precursor cells are present, but the level decreases as neural differentiation proceeds. Thus, HES-5 expression correlates well to the undifferentiated stages of the neural cells. Here, we first examined HES-5 expression patterns in earlier embryos and showed that HES-5 is specific to the developing nervous system. To understand the molecular mechanism of this neural precursor cell-specific expression, we cloned mouse HES-5 gene and characterized its promoter. We found that the upstream region of the HES-5 gene directs specific expression in neural precursor cells. Further promoter analysis identified a regulatory element that activates undifferentiated neural-specific expression on a heterologous promoter.


EXPERIMENTAL PROCEDURES

Whole Mount in Situ Hybridization Analysis

Mouse embryos of embryonic day 11.5 (E11.5) were fixed by 4% paraformaldehyde and subjected to in situ hybridization analysis, as described previously (Wilkinson, 1993). Digoxygenin-labeled antisense RNA corresponding to the 1057-nucleotide ApaI fragment of pHES-5 (Akazawa et al., 1992) was used as a probe. Hybridized RNA was visualized by overnight treatment with alkaline phosphatase-conjugated anti-digoxygenin antibody at 4 °C followed by incubation in 0.34 mg/ml nitroblue tetrazolium and 0.18 mg/ml 5-bromo-4-chloro-3-indolyl phosphate.

Isolation of the Mouse HES-5 Gene

The mouse genomic library (Stratagene) was screened by hybridization in situ, as described previously (Takebayashi et al., 1994). The 1.3-kilobase (kb) EcoRI fragment of the rat HES-5 cDNA (Akazawa et al., 1992) was used as a probe. Two positive clones were obtained from 1 times 10^6 plaques, and the positive fragments were subcloned into pBluescript and subjected to sequence analysis.

Southern Blot Analysis

Mouse liver DNA digested by restriction enzymes was electrophoresed on 0.7% agarose gel and transferred to nylon membrane filter. The P-labeled 0.6-kb BglII-SpeI fragment of the mouse genomic fragment was hybridized to the DNA at 65 °C in a solution containing 0.75 M NaCl, 75 mM sodium citrate, and 0.5% SDS.

Reverse Transcriptase-mediated Polymerase Chain Reaction (RT-PCR) and Primer Extension Analysis

RT-PCR was carried out, as described previously (Lee et al., 1988; Saiki et al., 1988). Total RNA prepared from E13 CNS was subjected to reverse transcription using oligo(dT) as a primer. Primers used for PCR were as follows: P1 (corresponding to nucleotide residues -16 to +2), 5`-TGGCGTGCTGGGGTCCAG-3`; P2 (+3 to +21), 5`-GTCGCGCCAGTCCGGGACG-3`; P3 (+113 to +132), 5`-TCGGTTTTTCTCCTTGGGAC-3`. P1 and P3 primers were used to detect the 148-base pair (bp) band, and P2 and P3 were used to detect the 130-bp band. After 30 cycles of the reaction (94 °C 1 min 60 °C 1 min 74 °C 1 min), the products were electrophoresed on 2% agarose gel.

For the primer extension experiment, the 52-nucleotide SacI-EcoT14I fragment labeled at the EcoT14I site was hybridized to the mouse E13 CNS poly(A) RNA (20 µg) at 42 °C in a solution containing 80% (v/v) formamide and 0.4 M NaCl and subjected to the reverse transcription reaction as described previously (Takebayashi et al., 1994).

Transient Transfection Analysis

The reporter plasmids contained the luciferase gene under the control of either various lengths of the HES-5 promoter, the enhancer-less SV40 promoter (SV40 basic promoter, SV40b), three repeats of the HES-5 region (-120/-96) linked to the SV40b, five repeats of the GC stretch (GGCCGGCGCC) linked to the SV40b (GC5-SV40b), five repeats of the TG sequence (CTTTGTGC) linked to the SV40b, or no promoter sequence (promoterless). The GC5-SV40b was constructed by inserting the double-stranded oligonucleotide fragment (5`-CGCGTCGACGGCCGGCGCCAAGGCCGGCGCCTTGGCCGGCGCCACCAAGGCCGGCGCCTTGGCCGGCGCCA-3`) into the upstream portion of the SV40b-luciferase plasmid.

Neural precursor cells were isolated from E10.5 mouse fetal heads, as described previously (Kitani et al., 1991). Cells were seeded at 2 times 10^6 cells/well in 6-multiwell dishes coated with poly-D-lysine. C6 glioma (Benda et al., 1968), NCB20 neuroblastoma-brain hybrid (MacDermot et al., 1979), and C3H10T1/2 fibroblast cells were seeded in 6-multiwell dishes at 2 times 10^5, 1.5 times 10^5, and 2 times 10^5 cells/well, respectively. 20-24 h later, 1.8 µg of the reporter plasmids was transfected by using 10-15 µl lipofectamine reagent (Life Technologies, Inc.) per well. 0.2 µg of the cytomegalovirus promoter-directed beta-galactosidase expression (CMV-betagal) vector was also transfected as an internal standard to normalize the transfection efficiency. Cells were incubated with the transfection complexes for 6 h at 37 °C. Then, the complexes were removed, and cells were harvested 42-48 h after the start of transfection. When NCB20 cells were induced to differentiate, the medium with 5% fetal bovine serum was changed to the medium containing 1% fetal bovine serum with 1 mM dibutyryl cAMP after the transfection complexes were removed (Nirenberg et al., 1983). Luciferase activity was measured, as described previously (Ow et al., 1986).

For X-gal staining and immunostaining analysis, 2 µg of the vector containing the beta-galactosidase gene under the control of the HES-5 promoter was transfected into the neural precursor cell culture, as described above. After 48 h, the cells were fixed in 2% paraformaldehyde and stained in 1 mg/ml X-gal, 5 mM K(3)Fe(CN)(6), K(4)Fe(CN)(6), and 2 mM MgCl(2) at 37 °C for 6 h. After refixation in 4% paraformaldehyde, the cells were incubated at room temperature for 1 h with the rabbit polyclonal anti-nestin serum (1:500 dilution with phosphate-buffered saline) (Tomooka et al., 1993), which is kindly provided by Dr. Y. Tomooka. Nestin-positive cells were visualized by Vectastain ABC kit (Vector Laboratories, Burlingame, CA) with diaminobenzidine.

DNA Binding Analysis

For preparation of the probe DNA, the double-stranded oligonucleotide fragment (one strand, 5`-CGCGTCGACGGCCGGCGCCAAGGCCGGCGCCTTGGCCGGCGCCA-3`; its complementary strand, 5`-GATCTGGCGCCGGCCAAGGCGCCGGCCTTGGCGCCGGCCGTCGA-3`) was labeled at both ends by T4 polynucleotide kinase. The wild-type competitor contained the same sequence as that of the probe. The mutant probe contained three repeats of the sequence GGAAATTGCC instead of GGCGCCGGCC. Preparation of nuclear extracts and the binding reactions were done according to the procedure described previously (Kageyama et al., 1991; Sasai et al., 1992). 4 µg each of nuclear proteins was incubated with the probe (10^4 counts/min) at 0 °C for 20 min.


RESULTS

HES-5 Expression in Mouse Embryos

We previously showed that HES-5 is specifically expressed in the developing nervous system of late embryos (Akazawa et al., 1992). To examine earlier expression patterns of HES-5, we performed whole mount in situ hybridization analysis using mouse embryos of E11.5, at which time more abundant neural precursor cells are proliferating. As shown in Fig. 1, HES-5 was expressed at high levels throughout the developing CNS. Prominent expression was observed in the wall of the brain vesicles as well as in the developing spinal cord (Fig. 1). No expression was detected in the other tissues. Thus, HES-5 expression seemed specific to the developing nervous system, making HES-5 a developing neural-specific marker.


Figure 1: Whole mount in situ hybridization analysis of HES-5. Mouse embryos of E11.5 were fixed by 4% paraformaldehyde and subjected to in situ hybridization analysis. Digoxygenin-labeled antisense RNA prepared from the ApaI fragment of pHES-5 was used as a probe. Hybridized RNA was visualized by treatment with alkaline phosphatase-conjugated anti-digoxygenin antibody followed by incubation in 0.34 mg/ml nitroblue tetrazolium and 0.18 mg/ml 5-bromo-4-chloro-3-indolyl phosphate. A, ventral view of the embryo. Strong signals were observed in the telencephalon (T), mesencephalon (Ms), and the spinal cord (S) of the tail portion. B, lateral view of the embryo. Strong signals were detected throughout the developing CNS. C, upper dorsal view of the embryo. HES-5 mRNA was observed in the mesencephalon (Ms), metencephalon (Mt), and myelencephalon (My).



Structural Organization of Mouse HES-5 Gene

To understand the molecular mechanism of the neural-specific expression, we cloned the mouse HES-5 gene. Two genomic clones were isolated by screening 1 times 10^6 plaques of a mouse genomic library with the rat HES-5 cDNA probe. Both clones contained a 4.3-kb BglII fragment hybridized positively to the rat HES-5 cDNA, and the nucleotide sequence of this fragment was determined. Comparison with the full-length rat HES-5 cDNA sequence revealed that the mouse HES-5 gene consisted of three exons and two introns and that both introns were located within the protein coding region (Fig. 2). The exon-intron boundaries all possessed the consensus splicing signal conforming to the GT-AG rule. The deduced amino acid sequence of mouse HES-5 showed a complete match in the bHLH domain and 96% identity in the whole region to that of rat HES-5. On Southern blot analysis, a single band that matched the size of the mouse HES-5 gene was detected, suggesting that the mouse HES-5 gene is a single copy gene (Fig. 3B).


Figure 2: The nucleotide sequence of the mouse HES-5 gene. The nucleotide sequence of the coding strand together with the deduced amino acid sequence of HES-5 is indicated. The upper- and lower-case letters represent the exon sequence and the flanking and intron sequences, respectively. The transcription initiation site, designated as +1, is shown by an arrow. The canonical TATA box (tatata) is boxed. The bHLH region is indicated by a bar above the amino acid sequence. The stop codon is depicted by asterisks. The GC stretches (ggccggcgcc or similar sequences, solid line) and a polyadenylation signal (AATAAA, broken line) are underlined.




Figure 3: Genomic organization of mouse HES-5. A, schematic structures of the mouse HES-5, HES-1, and Drosophila hairy genes. Closed and open boxes represent the coding and noncoding regions, respectively. Thin lines indicate the flanking and intron regions. Restriction sites of the cloned mouse HES-5 gene are shown. B, Southern blot analysis of the mouse HES-5 gene. DNA (10 µg each) isolated from the mouse liver was digested by a restriction enzyme indicated above each lane. The 0.6-kb BglII-SpeI fragment containing the 5`-flanking region was used as a probe. The marker sizes (kb) are indicated on the left.



Comparison between mouse HES-5 and HES-1 (Takebayashi et al., 1994) and Drosophila hairy genes (Rushlow et al., 1989) showed that they had similar genomic organization (Fig. 3A). The first intron of the HES-5 gene was located within the bHLH region, and its position was well conserved when compared with that of the mouse HES-1 and Drosophila hairy genes. An intron equivalent to the second intron of the HES-1 gene was absent from the HES-5 gene, but the second intron of the HES-5 gene was present just downstream of the bHLH region, which is almost the same as that of the third intron of the HES-1 gene. These results suggest that mouse HES-5 and HES-1 and Drosophila hairy genes are related to each other and thus originated from the same or related ancestral gene.

Determination of Transcription Initiation Site

To determine the transcription initiation site, we first performed primer extension analysis (Fig. 4A). The SacI-EcoT14I fragment labeled at the EcoT14I site (41 nucleotides downstream of the translation initiation site) was used as a primer. This analysis demonstrated three specific bands with the sizes of 116, 118, and 120 nucleotides when RNA prepared from the CNS of E13 was used (Fig. 4A, lane 1), suggesting that the 5` terminus of the HES-5 mRNA is 78 nucleotides upstream of the translation initiation site.


Figure 4: Determination of the transcription initiation site. A, primer extension analysis of the 5` terminus of the HES-5 mRNA. Primer extension analysis was performed by using the 52-nucleotide SacI-EcoT14I fragment labeled at the EcoT14I site as a primer (shown by an arrow). 20 µg each of poly(A) RNAs isolated from the CNS of mouse embryos (lane 1) and adult liver (lane 2), and tRNA (lane 3) was used. The specific product was shown by an asterisk. The marker sizes (nucleotides) are indicated on the left. B, RT-PCR analysis. P1 primer (-16/+2): 5`-TGGCGTGCTGGGGTCCAG-3`, P2 primer (+3/+21): 5`-GTCGCGCCAGTCCGGGACG-3`, and P3 primer (+113/+132): 5`-TCGGTTTTTCTCCTTGGGAC-3` are shown in the scheme. Total RNA of mouse E13 CNS was subjected to PCR either after reverse transcription (lanes 3 and 4) or without reverse transcription (lanes 1 and 2). As a positive control, subcloned genomic DNA was used as a template (lanes 5 and 6).



To confirm the above result, we next carried out RT-PCR by using RNA of E13 CNS (Fig. 4B). When primers 2 (corresponding to nucleotide residues +3 to +21) and 3 (+113 to +132) were used, a 130-bp single band was clearly observed (lane 4). This band was not detected when reverse transcriptase was omitted (lane 2), suggesting that the region between +3 and +132 was amplified from the cDNA. However, when primers 1 (-16 to +2) and 3 were used, no band was detected (lane 3), suggesting that most of the region corresponding to primer 1 was not transcribed. Thus, the position 78 nucleotides upstream of the translation initiation site was most likely a transcription start site and therefore designated as nucleotide residue 1 (see Fig. 2).

Sequence examination of the promoter region revealed that there is a TATA motif (tatata) at nucleotide -26, which may direct precise transcription initiation (Fig. 2, boxed). Another feature is that there are multiple GC elements (Fig. 2, underlined).

Transcriptional Analysis of HES-5 Promoter

We next analyzed the promoter activity of the HES-5 gene by a transient transfection method. A reporter plasmid containing the luciferase gene under the control of the 5`-region of the HES-5 gene (from -1324 to +73) was transfected into various types of cells. As shown in Fig. 5A, HES-5 promoter directed the highest expression in neural precursor cells prepared from E10.5 mouse embryos (lane 1). Whereas undifferentiated NCB20 neuroblastoma-brain hybrid cells showed a moderate level of expression from the HES-5 promoter (lane 2), C6 glioma and C3H10T1/2 fibroblast cells exhibited low levels of expression (lanes 3 and 4). These results suggest that the 5`-region of the HES-5 gene (from -1324 to +73) is able to direct specific expression in undifferentiated neural cells.


Figure 5: Analysis of tissue specificity of the HES-5 promoter. A, luciferase assay. The reporter plasmid containing the luciferase gene under the control of the HES-5 promoter (-1324/+73) was transfected into neural precursor cells (NPC), NCB20 neuroblastoma-brain cell hybrid, C6 glioma, and C3H10T1/2 fibroblast cells using lipofectamine reagent. The relative luciferase activities of the HES-5 promoter to that of the SV40 basic promoter were measured. Each value is the average of at least four independent experiments. The CMV-betagal vector was also transfected as an internal standard to normalize the transfection efficiency. B, staining of neural precursor cells with X-gal and anti-nestin serum. 2 µg of the plasmid containing the beta-galactosidase gene under the control of the HES-5 promoter (-1324/+73) was transfected into neural precursor cells in a 6-well plate. 48 h after the transfection, cells were fixed and stained with X-gal and anti-nestin serum. X-gal-staining indicated by arrowheads was observed only in nestin-positive (brown) neural precursor cells.



Because the neural precursor cell culture prepared from mouse embryos contained some differentiating neural cells as well as a background level of fibroblasts, we next examined whether strong expression from the HES-5 promoter occurred in neural precursor cells. The culture was transfected with a plasmid containing the beta-galactosidase gene under the control of the HES-5 promoter and subsequently stained with X-gal. The culture was also immunostained with antiserum to nestin, a neural precursor cell-specific intermediate filament (Lendahl et al., 1990). As shown in Fig. 5B, X-gal staining (blue, arrowheads) was observed only in nestin-positive neural precursor cells, which formed aggregates. No X-gal staining was detected in differentiating neural cells, which extended processes, or fibroblasts. These results confirmed that expression from the HES-5 promoter occurred specifically in undifferentiated neural cells.

To determine a regulatory element responsible for HES-5 expression, reporter plasmids of the luciferase gene under the control of various lengths of HES-5 promoter were transfected into the neural precursor cell culture. As shown in Fig. 6, deletion of the sequence between -1324 and -800 showed no obvious change (lanes 1 and 2), whereas further deletion from -800 to -453, from -453 to -248, from -248 to -179, and from -179 to -141 serially reduced expression by 20-30% each (lanes 3-6). Another deletion from -121 to -95 led to a 2.9-fold reduction in activity, resulting in a quite low promoter activity (lane 8). These results suggest that HES-5 expression in neural precursor cells is controlled by multiple upstream promoter regions.


Figure 6: Deletion analysis of the HES-5 promoter. The luciferase reporter plasmid containing either various lengths of the HES-5 promoter (lanes 1-9), no promoter (lane 10), the SV40 basic promoter (lane 11), or the SV40 basic promoter with three repeats of the HES-5 promoter region (-120/-96) (lane 12) was transfected into neural precursor cells. The 5`- and 3`-end points of the HES-5 promoter used in the luciferase reporter plasmids are shown on the left. Open and striped boxes represent the regions upstream and downstream of the transcription initiation site, respectively. The luciferase activity of the SV40 basic promoter was taken to be 1, and the relative luciferase activity of each promoter was measured. Each value of relative luciferase activities is the average of at least four independent experiments. The CMV-betagal vector was also transfected as an internal standard to normalize the transfection efficiency.



Sequence examination of the multiple upstream regulatory regions revealed that these regions contained multiple copies of GC stretches (see Fig. 2, underlined), raising the possibility that these GC stretches may be involved in HES-5 expression. To examine this possibility, we made an artificial promoter containing three repeats of the region between -120 and -96 linked to the SV40 basic promoter. This region (-120/-96) contained two GC stretches and showed the most efficient activation (2.9-fold activation) (compare lanes 7 and 8). As shown in Fig. 6, the addition of this region significantly activated transcription from the SV40 basic promoter in neural precursor cells (compare lanes 11 and 12). This enhanced activity was almost comparable to that of the intact HES-5 promoter. Because the sequence between -120 and -96 also contained a TG sequence (TTTGTG) between the two GC stretches, we next examined which sequence was responsible for the transcriptional activation. We constructed two synthetic promoters: the SV40 basic promoter with five repeats of either the GC stretch or the TG sequence. As shown in Fig. 7A, the addition of the GC stretches significantly activated transcription in neural precursor cells (lane 1), while the addition of the TG sequence exhibited no significant change (lane 2). These results suggest that the multiple GC stretches play an important role in the HES-5 promoter activity in neural precursor cells.


Figure 7: Transcriptional analysis of the GC stretches of the HES-5 promoter. A, transcriptional analysis of the GC stretch and the TG sequence in neural precursor cells. The luciferase reporter gene was under the control of the SV40 basic promoter (SV40b) linked to either five repeats of GGCCGGCGCC (lane 1), five repeats of CTTTGTGC (lane 2), or no additional sequence (lane 3). Plasmids were transfected into neural precursor cells, and relative luciferase activities were measured. The luciferase activity of SV40b was taken to be 1, and relative activities were measured. B, transcriptional analysis of the GC stretches of the HES-5 promoter. Luciferase activities from the SV40 basic promoter (SV40b) (lanes 2, 4, and 6) and the SV40 basic promoter linked to five repeats of the GC stretches (GC5-SV40b) (lanes 1, 3, and 5) were measured by transfection into undifferentiated NCB20 (lanes 1 and 2), C6 (lanes 3 and 4), and C3H10T1/2 cells (lanes 5 and 6). In each cell type, the luciferase activity of SV40b was taken to be 1, and relative activities were measured. C, effect of differentiation on transcriptional activities of the HES-5 and GC5-SV40b promoters. The reporter plasmid containing the luciferase gene under the control of either the HES-5 promoter (lanes 1 and 2) or GC5-SV40b promoter (lanes 3 and 4) was transfected into NCB20 cells. The cells were either kept undifferentiated in a growth medium (lanes 1 and 3) or induced to differentiate (lanes 2 and 4). The relative luciferase activity of each promoter to that of the SV40b promoter was measured. In all these experiments, each value is the average of at least four independent experiments normalized by the beta-galactosidase activity of the co-transfected CMV-betagal vector.



To see that the multiple GC stretches also show tissue specificity, we transfected the reporter plasmid of the luciferase gene under the control of the GC stretch-containing promoter into other types of cells. As shown in Fig. 7B, the GC stretches exhibited a moderate activation in undifferentiated NCB20 neuroblastoma-brain hybrid cells (lane 1). However, these stretches showed only a weak activation in C6 glioma (lane 3) and C3H10T1/2 fibroblast cells (lane 5). Thus, these results suggest that the GC stretch can act as a positive element specifically in undifferentiated neural cells.

NCB20 cells can differentiate into neuron-like cells under a low serum medium with dibutyryl cAMP. We thus examined whether HES-5 promoter activity changes in NCB20 after induction of differentiation. As shown in Fig. 7C, HES-5 promoter activity was reduced when differentiation was induced (lane 2), agreeing well with the observation that HES-5 expression decreases as neural differentiation proceeds. Furthermore, the multiple GC stretch-containing promoter also exhibited lower expression when differentiation was induced (lane 4). These results thus further support the notion that the GC stretch activates transcription specifically in undifferentiated neural cells.

DNA Binding Analysis with the GC Stretch

To investigate whether a GC stretch-binding protein exists in neural precursor cells, we next performed a gel mobility shift assay by using the GC stretch DNA as a probe. As shown in Fig. 8, two bands were detected with nuclear extracts of neural precursor cells (lane 2, arrowheads). Both bands were competed by an excess amount of the cold GC stretch DNA (lane 4) but not by an excess amount of the cold mutant DNA which contained the AT sequence (lane 3), indicating that both bands represent specific binding. The upper band was also detected in the liver nuclear extract (lane 5), suggesting that the upper complex may be formed by a ubiquitous factor such as Sp1 (Kadonaga et al., 1986). However, the lower band was detected only in the extracts of the neural precursor cells. These results raise the possibility that the protein of the lower band may be involved in neural precursor cell-specific expression through the multiple GC stretches.


Figure 8: Gel mobility shift analysis. The P-labeled synthetic probe containing three repeats of 5`-GGCCGGCGCC-3` sequence was mixed with 4 µg each of nuclear extracts of neural precursor cells (NPC, lanes 2-4) and E13.5 mouse liver (lane 5). Reactions were carried out in the presence of 1 µg of poly(dI-dC). Lane 1 shows the probe only. Lanes 3 and 4 show the reactions with 25 ng each of the mutant competitor (AT, three repeats of 5`-GGAAATTGCC-3`) and the wild-type competitor (GC, three repeats of 5`-GGCCGGCGCC-3`), respectively. The two retarded bands are indicated by arrowheads.




DISCUSSION

Neural Precursor Cell-specific Expression of HES-5

In this study, we showed that HES-5 is specifically expressed in the developing nervous system. This specific expression is directed by the multiple GC stretches present in the HES-5 promoter. Furthermore, we detected a specific GC stretch-binding protein in neural precursor cells. These results suggest that a neural precursor cell-specific factor regulates transcription of the HES-5 gene by interacting with the GC stretch sequences.

The GC stretch sequence used in this study was GGCCGGCGCC. Multiple copies of the same or quite similar GC sequences are present in the HES-5 promoter (Fig. 2, underlined). The consensus sequence would be GGCCSGCGCC (S = G or C) although we do not have any evidence that all the GC stretches present in the HES-5 promoter show neural precursor cell-specific transcriptional activation. These GC stretches are very similar to the GC box (GGCGGG), and thus it is possible that they are recognized by Sp1, a ubiquitous transcriptional activator that regulates housekeeping genes (Kadonaga et al., 1986). Transient transfection assays showed that the addition of the GC stretches to a heterologous promoter resulted in a weak activation in cells other than undifferentiated neural cells. Thus, it seems that, at least in some cells, the GC stretches of the HES-5 promoter are recognized by a ubiquitous factor such as Sp1 when transfected transiently. In this regard, the slowly migrating band detected by the GC stretch probe on gel mobility shift analysis seems to exist ubiquitously and therefore could represent Sp1. However, HES-5 is not expressed in non-neural cells even though Sp1 is ubiquitously present (Kadonaga et al., 1986). Thus, in physiological conditions Sp1 may not interact with these GC stretches, or transcriptional repressors could antagonize the activity of Sp1. Therefore, a neural precursor cell-specific transcriptional activator, but not Sp1, may be responsible for the GC stretch-mediated HES-5 gene expression. We speculate that the neural precursor cell-specific protein detected on gel mobility shift analysis (the lower band) is the most likely candidate for the specific activator. Because HES-5 is an early neural-specific marker, the GC stretch-binding protein could be involved in early neural differentiation or neural fate determination.

The addition of three repeats of the region between -120 and -96 (a total of six repeats of GC stretches) showed more than 10-fold activation in neural precursor cells (Fig. 6, compare lanes 11 and 12), while the addition of five repeats of the GC stretches led to about 5-fold activation (Fig. 7A, compare lanes 1 and 3). This different efficiency in activation could be due to the difference in number of the GC stretches, but the spacing between each GC stretch may be also important.

Down-regulation of HES-5 Expression in the Course of Neural Differentiation

Previously, we showed that HES-5 expression in the nervous system is down-regulated in the course of neural differentiation (Akazawa et al., 1992). In NCB20 cells, transcription from the intact HES-5 promoter as well as the synthetic promoter containing the multiple GC stretches was also reduced when differentiation was induced in low serum with dibutyryl cAMP (Fig. 7C). Thus, these results suggest that the down-regulation of HES-5 expression in the course of differentiation may be mediated by the GC stretches. This down-regulation in NCB20 cells is not the result of the direct effect of cAMP because transcription was also reduced when differentiation was induced only by low serum (data not shown). One simple mechanism for the down-regulation would be that the GC stretch-binding activator decreases as neural differentiation proceeds. Further studies will be necessary to determine whether the amount of the GC stretch-binding protein correlates with the level of HES-5 expression.

In the upstream region (between -1.6 and -0.7 kb) of the HES-5 gene, there are five N box sequences (data not shown). Our previous data suggest that HES-5 represses transcription by binding to the N box (Akazawa et al., 1992). Thus, HES-5 may negatively regulate its own expression by binding to these N boxes. These results raise another possibility that down-regulation of HES-5 in the course of neurogenesis is mediated by negative autoregulation through the N box. However, when the HES-5 expression vector was cotransfected with the HES-5 promoter-directed luciferase plasmid, only a weak negative autoregulation was observed (25% reduction, data not shown). Thus, it remains to be determined whether negative autoregulation of HES-5 occurs in vivo.

Possible Functions of HES-5 in Neural Differentiation

Whereas HES-5 acts as a transcriptional repressor, its exact function in neurogenesis is not yet known. Another member of HES family, HES-1, is also a transcriptional repressor, and its expression pattern in the nervous system is quite similar to that of HES-5. Persistent expression of HES-1 prevents neuronal and glial differentiation, indicating that HES-1 is a negative regulator of neural differentiation (Ishibashi et al., 1994). Thus, it is possible that HES-5 also acts as a negative regulator of neurogenesis. However, whereas HES-1 efficiently antagonizes the activity of E47, a ubiquitous activator with a bHLH domain, HES-5 only partially antagonizes it (Akazawa et al., 1992). Thus, it is likely that HES-5 and HES-1 target different factors. Further studies about the functions of HES-5 as well as the mechanism of HES-5 expression will help understand the molecular nature of mammalian neurogenesis.


FOOTNOTES

*
This work was supported by research grants from the Ministry of Education, Science, and Culture of Japan, the Sankyo Foundation, and the Inamori Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 GenBank(TM)/EMBL Data Bank with accession number(s) D32132[GenBank].

§
Present address: The Salk Institute for Biological Studies, La Jolla, CA 92037.

To whom correspondence should be addressed: Institute for Immunology, Kyoto University Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606, Japan. Tel.: 81-75-753-4438; Fax: 81-75-753-4404.

(^1)
The abbreviations used are: CNS, central nervous system; HLH, helix-loop-helix; bHLH, basic HLH; kb, kilobases; bp, base pairs; E, embryonic day; RT-PCR, reverse transcriptase-mediated polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank Dr. C. Shimizu for technical help with in situ hybridization analysis, Dr. Y. Tomooka for anti-nestin serum, Dr. H. Higashida for NCB20 cells, and Dr. M. Ishibashi for initial help with genomic characterization.


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