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INTRODUCTION |
The lysosomal acid
-glucosidase
(GAA,1 acid
maltase, EC 3.2.1.20) hydrolyzes 1,4-
-glucosidic and
1,6-
-glucosidic linkages in glycogen, maltose, and isomaltose
(1, 2). Recessively inherited deficiency of the enzyme leading to
abnormal glycogen accumulation in lysosomes causes glycogen storage
disease type II, which in its most severe form presents as a
rapidly progressive myopathy and cardiomyopathy (Pompe disease) (3).
The GAA gene has been localized to chromosome 17q21-23. The
cDNA sequence has been reported, and the gene sequence has been
partially characterized (4, 5). The gene spans ~20 kb and
is composed of 20 exons including a 5'-noncoding exon 1 that is
separated by an ~2.7-kb intron from exon 2, the site of the initiator
(ATG) codon. Although its promoter region has characteristics of
housekeeping gene promoters (5), the GAA gene expression
varies extensively during development and maturation (6). To date, very
little is known regarding the transcriptional control of this gene or
the majority of lysosomal enzyme genes.
In a mini-gene model system, we have previously demonstrated that an
~2.7-kb intron 1 of the human GAA gene contains a negative regulatory element (7). In the experiments reported here, we have
employed an in vitro transfection assay in human hepatoma cells (Hep G2) and DNase I footprinting to localize the negative regulatory element within intron 1 to a 25-bp region ~1.7 kb
downstream from the exon 1/intron 1 boundary. Within this region there
are two tandem E boxes and a potential core Ying Yang 1 (YY1) binding site in juxtaposition. Electrophoretic mobility shift assay (EMSA) with
specific antibodies showed that a transcriptional repressor Hes-1, the
mammalian homologue 1 of D. hairy and Enhancer of
split (E(spl)), binds to the downstream E box
(CACGCG) and in collaboration with YY1 contributes to the repressive effect.
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EXPERIMENTAL PROCEDURES |
Plasmid Constructs for Deletional Analysis of Intron 1 Fragment--
The various intron 1 fragments of the human
GAA gene were generated by polymerase chain reaction. The
wild type oligonucleotide corresponding to the 25-bp silencer element
was 5'-ATCTCATCTGCACGCGACATCCTTG-3'. Double-stranded wild type and
mutant oligonucleotides were generated by heating the primers at
90 °C for 10 min followed by slow cooling at room temperature; the
oligonucleotides were purified on a 20% polyacrylamide gel. The DNA
fragments were subcloned into the expression vector pBLCAT2 upstream of
the TK promoter and CAT gene in both
orientations. For cotransfection with Hes-1 expression plasmid, four
copies of wild type or mutant 25-bp element were subcloned into pBLCAT2
to generate 4×wtHes-1/TK-CAT and 4×mutHes-1/TK-CAT. The integrity of
the plasmids was verified by restriction endonuclease digestion and sequencing.
Cell Culture and Transient Transfection Assay--
Hep G2 cells,
human hepatoma cell line (ATCC, Manassas, VA), were cultured in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with heat-inactivated 10% fetal calf serum (HyClone,
Logan, UT) and 100 µg/ml penicillin-streptomycin (Life Technologies,
Inc.). Hep G2 cells were transfected with 4.0 µg of purified DNA
(Qiagen, Valencia, CA) with 10 µl of LipofectAMINE reagent (Life
Technologies, Inc.) in serum-free medium for 5 h. Then the
cells were re-fed with original medium and harvested for assay 48 h posttransfection. In all experiments, cells were cotransfected with
0.5 µg of pXGH5 (human growth hormone gene expression plasmid) as an
internal control of transfection efficiency. Plasmid pUC19 was used to
compensate the amount of DNA. For cotransfection experiments, the ratio
of reporter gene constructs 4×wtHes-1/TK-CAT or 4×mutHes-1/TK-CAT and
pcDNA3 or pcDNA3-WT Hes-1 was 1:3. Cell culture medium was
collected for hGH activity assay (hGH enzyme-linked immunosorbent assay
kit, Roche Molecular Biochemicals), and cell lysates were
assayed for CAT activity (CAT enzyme-linked immunosorbent assay kit,
Roche Molecular Biochemicals). Protein concentration was determined
using a dye binding assay (Bio-Rad).
DNase I Footprinting Analysis--
Nuclear extracts from
cultured cells were prepared as described (8) and kept at
80 °C.
DNase I footprinting experiments were carried out as described (9). The
noncoding strand of the 90-bp DNA fragment (1711-1800 bp from
the exon 1/intron 1 boundary) was 3'-end-labeled with
[32P]dCTP using the Klenow fragment of DNA polymerase.
The binding reaction was carried out in a 50-µl volume with 1.0 ng of
probe and 60 µg of nuclear extract of Hep G2 cells for 30 min at room temperature. After incubation, 50 µl of cofactor buffer (10 mM MgCl2, 5 mM CaCl2)
was added, and the reaction mixtures were digested with a different
amount of DNase I (Sigma) for 2 min at room temperature. The reaction
was stopped with 100 µl of stop buffer (200 mM NaCl, 20 mM EDTA, pH 8.0, 1% SDS, 40 µg/ml tRNA). The mixture was
extracted once with phenol and pelleted with ethanol. DNA pellet was
resuspended in 4.0 µl of loading buffer, denatured at 95 °C for 3 min, and subjected to 8% polyacrylamide sequencing gel electrophoresis.
Electrophoretic Mobility Shift Assay--
The 25-bp
oligonucleotides were end-labeled with [32P]dCTP using
the Klenow fragment of DNA polymerase, and EMSA was performed as
described (10). 1.0 ng of labeled probe was incubated with 5.0 µg of
nuclear extracts of Hep G2 cells in a 20-µl volume reaction containing 100 mM Hepes, pH 7.9, 200 mM KCl, 5 mM MgCl2, 10 µg of bovine serum albumin, 0.5 mM EDTA, 2.5 mM dithiothreitol, and 30%
glycerol for 30 min at room temperature. For competition experiments, the nuclear extract was incubated with competitors at 100-fold molar
excess at room temperature for 30 min before adding the probe.
For immunosupershift assay, the nuclear extract was incubated with 1.0 µl of 1:500 diluted anti-Hes-1 antiserum or 2.0 µg of anti-YY1 and
anti-MyoD antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at room
temperature for 30 min before adding the probe. The anti-Hes-1 antisera
have been successfully used to recognize overexpressed Hes-1 in human
lung cancer cells (11). The protein-DNA complexes were run on a 5%
polyacrylamide gel and visualized by autoradiography.
Statistical Analysis--
Statistical analyses were performed
using the standard t test.
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RESULTS |
Deletion Analysis of the Intron 1 Fragment--
To localize the
silencer element, a series of intron 1 deletion fragments was
introduced into a TK-CAT reporter plasmid, and the reporter
activity was assayed in human Hep G2 cells. As expected, the 2.6-kb
intron 1 fragment significantly repressed the TK promoter activity (Fig. 1). All the constructs
(intron 1, F1-F4, F6, and F8) containing a 90-bp fragment of the
intron 1 (1711-1800 bp from the exon 1/intron 1 boundary) showed a
significant repressive effect on TK-CAT gene expression in
both forward and reverse orientations. In contrast, the constructs
without the 90-bp fragment (F5 and F7) did not repress the CAT
activities (Fig. 1). Therefore, the results indicate that the 90-bp
region contains the putative silencer element and that the
transcriptional activity of the element is independent of position and
orientation.

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Fig. 1.
Deletional analysis of intron 1 of the human
GAA gene. The positions and sizes of the
fragments within the intron 1 are marked on the graph, and the first
base pair from exon 1/intron 1 boundary is taken as position 1. The
orientations of intron 1 fragments in plasmids are indicated with
arrows. CAT activities were standardized relative to human
growth hormone activity as an internal control for transfection
efficiency. The CAT activities are expressed relative to pBLCAT2, which
is assigned a value of 100%. The bars represent mean ± S.E. *, p < 0.05; **, p < 0.01 compared with pBLCAT2. Numbers in
parentheses indicate the numbers of repeated experiments.
The dashed line indicates a change in scale.
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Characterization of the Silencer Element within the 90-bp
Region--
To further localize the functional silencer element, DNase
I footprinting analysis of the 90-bp region was performed with nuclear
extracts from Hep G2 cells. A 25-bp protected region
(5'-ATCTCATCTGCACGCGACATCCTTG-3') was revealed including two E boxes
(CANNTG) and a potential YY1 binding site (ACAT), which abuts the
second E box (Fig. 2). The second E box
is noncanonical (CACGCG) and is a preferred site for hairy
in Drosophila where it functions as a repressor (12). The
YY1 site within the element is one of the two core sequences of YY1
binding sites (ACAT and CCAT) (13).

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Fig. 2.
DNase I footprinting analysis of the 90-bp
DNA region. The 90-bp DNA region was 3'-end-labeled with
[32P]dCTP on noncoding strand and incubated with 60 µg
of nuclear extract from Hep G2 cells. Bovine serum albumin was used as
control (BSA control). Lane 1, marker,
Maxam-Gilbert G+A reaction (G+A); lane 2, bovine
serum albumin control; lane 3, Hep G2 nuclear extract.
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To test the transcriptional activity of the protected region, the
element was introduced into pBLCAT2, and transient transfection analysis was performed. As expected, it had a significant repressive effect on TK promoter activity in both orientations similar to that of
the 90-bp region (Fig. 3C).
Thus, the silencer element was localized to the 25-bp element.

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Fig. 3.
Site-directed mutagenesis and functional
analysis of the 25-bp element. A, DNA sequences of wild
type and mutants of the 25-bp element. The mutant nucleotides are in
small letters, and YY1 is in italics.
B, competition EMSA of the 25-bp element. The 25-bp
oligonucleotide probes were end-labeled with [32P]dCTP
and mixed with Hep G2 nuclear extracts. 100-fold molar excess of wild
type or mutant DNA was used as a competitor. Hes-1 and YY1 bands are
indicated with arrows. Comp., competitor;
NS, nonspecific DNA. C, functional analysis of
the wild type and mutant 25-bp element. The legend is the same as in
Fig. 1. The bars represent mean ± S.E. Each
bar represents an average of at least four independent
transfection assays. +, DNA fragment in forward orientation in plasmid;
, DNA fragment in reverse orientation; *, p < 0.01.
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Identification of the Proteins Binding to the 25-bp
Element--
To detect the transcriptional repressor(s), the 25-bp
element was used as a probe and incubated with nuclear extracts from Hep G2 cells. In EMSA, two proteins were shown to bind specifically to
the DNA. The binding of both proteins was completely competed by
unlabeled 25-bp oligonucleotide (Fig. 3B, lane
3) but not at all by a nonspecific DNA fragment (Fig.
3B, lane 8). Immunosupershift assay with rabbit
polyclonal antisera to the Hes-1 C-terminal and N-terminal peptides
showed that one DNA-protein complex band disappeared (Fig. 4,
lanes 3 and 4). In the same way, EMSA with anti-YY1 antibody or YY1 consensus
oligonucleotide revealed that the second specific DNA-protein complex
band was completely competed, and the second band disappeared (Fig. 4,
lanes 5 and 6). In contrast, the control
anti-MyoD antibody did not change the binding pattern (Fig. 4,
lane 7). These results indicate that the proteins binding to
the 25-bp element are the transcriptional factors Hes-1 and YY1.

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Fig. 4.
Identification of the proteins binding to the
25-bp element. The 32P-end-labeled 25-bp
oligonucleotides were incubated with 10 µg of Hep G2 nuclear extract.
The antibodies to the Hes-1 C-terminal and N-terminal peptides (1.0 µl, 1:500 dilution, lane 4) or anti-YY1 antibody (2.0 µ g, lane 5) or 100 ng of YY1 oligonucleotides (lane
6) were used in the experiment. Normal rabbit serum (1.0 µl,
1:500 dilution, lane 3) was used as control for anti-Hes-1
antibodies. Anti-MyoD antibody (2.0 µ g, lane 7) was used
as control for anti-YY1 antibody.
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Mutagenesis and Functional Analysis--
To determine whether both
Hes-1 and YY1 act as transcriptional repressors that bind to the
element, site-directed mutagenesis analysis was carried out. The wild
type and mutant sequences of the 25-bp element are shown in Fig.
3A. In EMSA, mutations in the element will lead to a
decreased binding of transcriptional factors, and thus these mutants
would no longer compete with the wild type probe. As shown in Fig.
3B (lanes 5 and 6), the element with
the mutation in the second E box (M2) was not able to compete with the
wild type probe for Hes-1 binding. Similarly, the element with mutation
in the YY1 site (M3) could not compete with the wild type probe for YY1
binding. Moreover, the element with both the second E box mutation and
the YY1 site mutation (M4) no longer competed for Hes-1 or YY1 binding
(Fig. 3B, lane 7). In contrast, an
oligonucleotide with a mutation of the first E box (M1) still competed
with the wild type probe (Fig. 3B, lane 4).
To further examine the transcriptional activity of Hes-1 and YY1, the
mutants of the element were subcloned into pBLCAT2 in both
orientations, and transient transfection assays were carried out in Hep
G2 cells. As shown in Fig. 3C, the mutation of the second E
box (M2) or of the YY1 binding site (M3) diminished, and the mutation
of both (M4) abolished the repressive effect of the element. Consistent
with the EMSA results, the mutation of the first E box (M1) did not
alter the repressive effect (Fig. 3C). Taken together, the
results indicate that Hes-1 binds to the second E box (CACGCG) within
the 25-bp element, whereas the activator-repressor YY1 binds to one of
the most frequent core YY1 sequences (ACAT). Neither Hes-1 nor YY1 acts
fully alone, but together they act as a transcriptional repressor.
Cotransfection Analysis with Expression Plasmid for Hes-1--
To
confirm that Hes-1 binds to the element and functions as a repressor,
the plasmids pBLCAT2, 4×wtHes-1/TK-CAT, and 4×mutHes-1/TK-CAT were
transfected alone or cotransfected with empty vector pcDNA3 or
expression plasmid-pcDNA3-WT Hes-1. Compared with transfection alone, overexpression of Hes-1 resulted in a significant decrease of
reporter gene expression of the plasmid 4×wtHes-1/TK-CAT with four
copies of wild type Hes-1 binding site (p < 0.01)
(Fig. 5). In contrast, the overexpression
of Hes-1 did not affect the reporter gene expression of the plasmid
4×mutHes-1/TK-CAT containing four copies of the mutant Hes-1 binding
site (p > 0.05) (Fig. 5). Cotransfected empty plasmid
pcDNA3 affected the reporter gene expression of neither plasmid
4×wtHes-1/TK-CAT (p > 0.05) nor 4×mutHes-1/TK-CAT (p > 0.05) (Fig. 5). These results showed that
reporter gene expression was regulated by overexpressed Hes-1.

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Fig. 5.
The effect of Hes-1 on reporter gene
expression in Hep G2 cells. Plasmids pBLCAT2, 4×wtHes-1/TK-CAT,
and 4×mutHes-1/TK-CAT were transfected alone or cotransfected with
empty vector pcDNA3 or pcDNA3-WT Hes-1 at an amount ratio of
1:3 in Hep G2 cells. The bars on the graph
represent mean ± S.E. from at least three transfection
experiments. *, p < 0.01 compared with plasmid
alone.
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DISCUSSION |
In this study, we have identified and characterized a negative
regulatory element of 25 bp within intron 1 of the human GAA gene, which is position- and orientation-independent. Site-directed mutagenesis and functional analysis showed that transcriptional factors
Hes-1 and YY1 collaborate to act as a transcriptional repressor of the
human GAA gene in the Hep G2 cell line by binding to the
element. Hes-1, a basic helix-loop-helix (bHLH) factor, binds to a C
class E box site (CACGCG), and YY1 binds to a core YY1 binding site
(ATAC) within the element.
Hes-1 is a mammalian bHLH transcription factor related to D. hairy and E(spl). It belongs to a repressor gene family
(12, 14) that acts in opposition to the activator bHLH genes. The bHLH
transcription factors have been divided into three classes based on the
recognized E box sequences (CANNTG): canonical class A (CACCTG or
CAGCTG), canonical class B (CACGTG or CATGTG), and noncanonical class C
(CACGCG or CACGAG ) (12). In Drosophila, the C
class E box (CACGCG) is the optimal binding site for the hairy
protein. E(spl) repressors prefer a canonical B class E box
(CACGTG) over the class C core and a slightly different C class E box,
which has been termed N box (CACNAG) (15-19). Furthermore, hairy mediates transcriptional repression over a distance of
more than 1.0 kb (20). In mice, Hes-1 can bind to C class E box
(CACGCG), but it prefers the N box (CACNAG). Targeted disruption of the Hes-1 gene in mice is lethal and leads to severe defects of
the neural tube and arrest of T-cell development in the thymus (21, 22), whereas persistent expression of the Hes-1 gene
prevents differentiation of mammalian neurons in the central nervous
system and neural retina (23, 24).
Although Hes-1 is essential to several developmental processes, few
target genes have been identified particularly in humans. In mice, it
has been shown that Hes-1 blocks neurogenesis by suppressing the
expression of the MASH-1 gene (25), the mammalian homologue 1 of Drosophila achaete-scute gene that is essential for
neural development (26). In human hASH1 (human
achaete-scute homolog-1) expressing small cell lung cancers
that do not express Hes-1, the introduction of Hes-1 results in a
dramatic reduction of hASH1 mRNA through a direct
binding of Hes-1 to a class C site within its promoter (11).
Here we present evidence that a completely different gene, the human
lysosomal acid
-glucosidase gene, that is unrelated to the
transcriptional activators is a downstream target of Hes-1. Furthermore, the C class E box binding site is located in the intron 1 of the GAA gene rather than in the promoter as in other targets. Hes-1 binds to a single C class E box site of the protected DNA region (5'-ATCTCATCTGCACGCGACATCCTTG-3') within intron
1; the underlined E box and flanking sequences match closely with the
hairy binding sequences (5'-GGCACGCGAC-3') in the D. achaete gene promoter (18). These results indicate that the
binding sequences of Hes-1, hairy, and E(spl) are
conserved from Drosophila to humans. In addition, in the
human GAA gene the Hes-1 binding site (1761-1785 bp from
the exon 1/intron 1 boundary) is located more than 1.8 kb from the
promoter region (5), indicating that Hes-1 is a long range repressor in
human cells.
It has been demonstrated that the Hes-1 gene and its
homologues are immediate downstream genes of the Notch signaling
pathway from invertebrates to vertebrates (27, 28). Notch is a
transmembrane protein and is related to the proliferation and
differentiation of many cell types (29, 30). Whether or not the human
GAA is involved in the signaling pathway needs to be further investigated.
YY1, a C2H2-type zinc finger DNA-binding protein, is a multifunctional
transcriptional factor that can act as an activator, a repressor, or an
initiator-binding protein. The two most common binding core sequences
are CCAT and ACAT (13). Several studies have shown that YY1 binds to
the promoter and intron regions of housekeeping genes and can regulate
their expression (31, 32). In this study, we have shown that YY1 binds
to one of the conserved sites (ACAT) within intron 1 of the human
GAA gene and functions as a partner of Hes-1 for repressive
effect. The established mechanisms of repression by YY1 include
activator replacement, interference with activator function, and
recruitment of a corepressor (13). Our data suggest that YY1 is acting
in a new way by collaborating with Hes-1.
In summary, we have demonstrated that the regulation and expression of
the human GAA gene is in part controlled at the transcriptional level
by a silencer element within its first intron. The identification of
the transcription factors affecting the GAA expression may have another practical application. A majority of patients (~80%) with a milder adult form of glycogen storage disease type II have a
markedly reduced level of an entirely normal gene product as a
consequence of a point mutation in the polypyrimidine tract of intron 1 (-13t-g) (7, 33). Pharmacological or genetic interference with
either the binding of the transcriptional factors or the interaction
between them may be used to up-regulate the gene expression.