(Received for publication, October 17, 1994)
From the
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.
In the developing mammalian central nervous system (CNS), ()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.
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).
Neural
precursor cells were isolated from E10.5 mouse fetal heads, as
described previously (Kitani et al., 1991). Cells were seeded
at 2 10
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
10
, 1.5
10
, and 2
10
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
-galactosidase
expression (CMV-
gal) 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
-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
Fe(CN)
,
K
Fe(CN)
, and 2 mM MgCl
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.
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).
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.
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).
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-gal 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
-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
-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-gal 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 -galactosidase activity of the co-transfected
CMV-
gal 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.
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.
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.
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D32132[GenBank].