From the
Human cytosolic aldehyde dehydrogenase 1 (ALDH1) plays a role in
the biosynthesis of retinoic acid that is a modulator for gene
expression and cell differentiation. Northern blot analysis showed that
liver tissue, pancreas tissue, hepatoma cells, and genital skin
fibroblast cells expressed high levels of ALDH1. Sequence analysis
showed that the 5`-flanking region contains a number of putative
regulatory elements, such as NF-IL6, HNF-5, GATA binding sites, and
putative response elements for interleukin-6, phenobarbital and
androgen, in addition to a noncanonical TATA box (ATAAA) and a CCAAT
box. Functional characterization of the 5`-regulatory region of the
human ALDH1 gene was carried out by a fusion to the
chloramphenicol acetyltransferase gene. A construct containing 2.6
kilobase pairs of the 5`-flanking region was efficiently expressed in
hepatoma Hep3B cells, but not in erythroleukemic K562 cells or in
fibroblast LTK
Aldehyde dehydrogenases (aldehyde:NAD
Human ALDH1
isozyme is expressed at different levels in various tissues examined,
with the highest level in the liver and the lowest or undetectable
level in the heart(8) , suggesting that the expression of ALDH1 is tissue-specific. During embryonic development, ALDH1
is detectable at an early stage(9) . The position- and
developmental stage-specific expression of ALDH1 were observed
in the mouse and chick retina, suggesting that the level of ALDH1 is
correlated with the biosynthesis of retinoic
acid(10, 11) . However, little is known regarding the
molecular mechanism of the tissue and developmental stage-specific
expression of the ALDH1 gene. Recent studies indicated that
human ALDH1 was produced in the normal genital skin fibroblast cells
but not in the cells obtained from X-linked androgen receptor-negative
testicular feminization patients(12, 13) . These
findings suggest that ALDH1 is induced by androgen receptor-androgen
complex in genital cells, and retinoic acid produced by ALDH1 plays an
important role in testicular differentiation(13) .
ALDH1 expression is modulated by phenobarbital and cyclophosphamide.
Phenobarbital induces ALDH1 activity in cultured human hepatoma cells (14) and in the liver of some rat strains(15) .
Transcriptional activation of ALDH1 was observed in
cyclophosphamide-resistant human carcinoma cells (16) and mouse
leukemic cell line L121O(17) .
To elucidate the regulatory
mechanisms of ALDH1 expression and to define the promoter
regions that are essential for its tissue-specific and inducible
expression, we have characterized the 5`-flanking region of the human ALDH1 gene.
Using an EcoRI/EcoRI fragment -673/+350 of the
clone as a template, a fragment -673/+53 with artificial HindIII site at the 5` end and XbaI site at the 3`
end was created by PCR, and subcloned into pBluescript II
KS
pUMSVOCAT
vector was digested by SmaI, tailed with dideoxythymidine
triphosphate and ligated with a PCR product (-673/+53) which
has a HindIII site at the 5` and XbaI site at the 3`
end. Digestion of this product by HindIII and XbaI
yielded CAT-assay constructs with a series of deleted ALDH1 promoter region. The fragments -2536/+53,
-1726/+53, -673/+53, -266/+53,
-149/+53, -120/+53, -91/+53 and
-50/+53 were subcloned into the HindIII/XbaI site of pUMSVOCAT vector. These products
are designated as pCAT-2536, pCAT-1726, pCAT-673, pCAT-266, pCAT-149,
pCAT-120, pCAT-91, and pCAT-50, respectively.
The SV40 promoter
region prepared from pCAT-promoter vector (Promega) ligated with
pUMSVOCAT, and the resultant pCAT-SV40 vector was used as an internal
reference for CAT-assay.
CAT assays were performed by the phase-extraction
method (21) using [
For supershift assay, anti-NF-YB
antibody (23) or anti-Oct-1 or -Oct-2 antibodies (Santa Cruz
Biotech, Inc.) were incubated with the probe-nuclear extract mixtures
for 30 min more prior to gel electrophoresis. The oligonucleotides NF-Y (24) CTF/NF1(25) , SP1(26) , and OCT (27) used for competition assays are: NF-Y:
5`-CGGTTGGCAGCCAATGAAATACAAAGATGA-3`; CTF/NF1:
5`-CCTTTGGCATGCTGCCAATATG-3`; Sp1: 5`-ATTCGATCGGGGCGGGGCGAGC-3`; OCT:
5` TGTCGAATGCAAATCACTAGAA-3`.
Figure 1:
Analysis of ALDH1 mRNA
expression in various cultured cell lines. Total RNA (20 µg)
prepared from each cell line was resolved by agarose gel
electrophoresis in the presence of formaldehyde, transferred to
nitrocellulose membrane, and hybridized with a
Figure 2:
Nucleotide sequence of the 5`-flanking
region of the human ALDH1 gene. Nucleotides are numbered
relative to the transcription start site (+1). Potential consensus
sequences for regulatory elements and transcriptional factor binding
sites are underlined. The consensus elements are abbreviated
as follows: AP-3, activator protein-3 binding site; Ets-1, Ets-1 binding site; GATA, GATA binding site; GHF-1, growth hormone factor 1 binding site; H-APF-1,
H-APF-1 binding site; IRBP, inverted repeat-binding protein
binding site; HNF-5, hepatocyte nuclear factor-5
binding site; LyF-1, LyF-1 binding site; MCBF,
M-CAT-binding protein binding site; NF-IL6, NF-IL6 binding
site; Oct, octamer factor binding site; PEA3, PEA3
binding site; SIF, sis-inducible factor binding site.
References for consensus sequences are found in (28) .
Figure 3:
Comparison of nucleotide sequences of ALDH1 5`-flanking region among human, marmoset, rat, and
mouse. Identity between nucleotides is indicated by dots, and
nucleotide deletions are indicated by the dash symbol. The
transcription start site (+1) is indicated by an asterisk, and nucleotides are counted from this position.
CCAAT box and consensus sequences for the putative regulatory elements
(Ets-1, Oct, and GATA) are boxed.
Figure 4:
Human ALDH1-CAT fusion
constructs. A partial restriction map of the human ALDH1 gene is shown
at the top. Restriction enzymes are abbreviated as follows: H, HindIII; E, EcoRI; P, PvuII; D, DraI. The ATAAA box(-32) and
CCAAT box(-74) are indicated by solid and hatchedboxes, respectively. Schematic diagrams of various human
ALDH1-CAT fusion constructs are shown below the restriction map. The
numbers are counted from the transcription start site (+1). UMS, upstream mouse sequence; SV40P, promoter of the
simian virus 40; CAT, chloramphenicol
acetyltransferase.
Figure 5:
Expression of CAT activities in Hep3B,
K562, and LTK
The
progressive removal of the 5`-sequences from -2536 to -673
resulted in augmented promoter activities, suggesting that the
5`-flanking region (-2536/-673) of the ALDH1 gene
may contain mild negative elements. Further stepwise deletions of the
sequences from -673 to -91 did not significantly affect the
reporter gene expression. However, a drastic decrease of CAT activity
occurred by deletion from -91 to -50. The CAT activity of
the deletion construct pCAT-50 was about 8-fold higher than that of the
promoterless plasmid pUMSVOCAT. These results suggest that at least two
positive cis-acting regulatory elements exist in the region between
-91 to +53. In Hep3B cells, the region from
-93/+53, containing ATAAA and CCAAT boxes, functions as a
promoter with activity similar to or even greater than the SV40
promoter (Fig. 5).
The 5`-flanking region also stimulated the
CAT expression in other cell lines. However, in comparison with that in
Hep3B cells, the degree of stimulation is very low in these cells,
which do not express the ALDH1 (Fig. 5). The construct
containing the proximal promoter (pCAT-91) exhibited the CAT activity
65-fold of that of the promoterless pUMSVOCAT in Hep3B cells, but only
6-fold in LTK
Figure 6:
CAT activities of internal deletion
mutants of the human ALDH1 promoter. The deletion mutants of
pCAT-120 were constructed by PCR-directed mutagenesis and were
transfected into Hep3B cells. The relative CAT activities are presented
as percentage of pCAT-120 activity.
Figure 7:
Gel shift assays of CCAAT motifs or
octamer motifs in the presence of Hep3B or K562 nuclear protein
extracts. A, end-labeled Oligo I, representing nucleotides
-88 to -56 of the promoter, was incubated with Hep3B crude
nuclear extract in the absence or presence of unlabeled competing
oligonucleotides. Lane1, control without added
nuclear extract; lanes 2-14, with Hep3B nuclear extract.
The following lanes were obtained with 20-, 100-, and 500-fold molar
excess of unlabeled competing oligonucleotides in the presence of Hep3B
nuclear extract: lanes 3-5, with Oligo I; lanes
6-8, with NF-Y oligonucleotide; lanes 9-11,
with CTG/NF1 oligonucleotide; lanes 12-14, with sP1
oligonucleotide. B, end-labeled Oligo II, representing
nucleotides -68 to -41 of the promoter, was incubated with
Hep3B or K562 nuclear extract in the absence or presence of competing
oligonucleotides. Lane1, control without added
nuclear extract; lanes 2-8, with Hep3B nuclear extract; lanes 9-15, with K562 nuclear extract. The following
lanes were obtained with 25- and 250-fold molar excess of unlabeled
oligonucleotides: lanes 3, 4, 10, and 11, with Oligo II; lanes5, 6, 12, and 13, with OCT oligonucleotide; lanes7, 8, 14, and 15, with Sp1
oligonucleotide.
Figure 8:
Antibody recognition of NF-Y and
octamer-binding proteins. A, end-labeled Oligo I was incubated
with Hep3B nuclear extracts (lanes 1-3) or K562 nuclear
extracts (lanes 4-6) in the presence or absence of the
specific antibody and subjected to gel shift assay. Lanes1 and 4, no antibody; lanes2 and 5, with anti-NF-YB antibody; lanes3 and 6, with rabbit Ig G. The supershifted band is
indicated by S, and NF-Y
Figure 9:
Schematic representation of the fragments
and oligonucleotides used for mapping the binding sites of the ALDH1 promoter. Nucleotide positions are shown at the top. CCAAT box, octamer motif, and ATAAA box are indicated by openboxes.
Figure 10:
Binding of nuclear factors at the minimal
promoter element (-91 to -1) with Hep3B nuclear extract. A
Frag I (region -91 to -1) was labeled and used in a gel
mobility shift assay. Lane 1, control without Hep3B nuclear
extract; lanes 2-8, with Hep3B nuclear extract. The
following lanes were obtained in the presence of unlabeled competitive
oligonucleotides; lane3, 50-fold excess of Frag I; lane4, 500-fold excess of Oligo I; lane5, 500-fold excess of Oligo II; lane6,
500-fold excess of Oligo III; lane7, 500-fold excess
of Oligo IV; lane8, 500-fold excess of
Sp1.
Figure 11:
Gel
shift assay. A, end-labeled Frag II (region -50 to
+53) was incubated in the absence (lane1) or
presence of Hep3B nuclear extract (lanes 2-9) or K562
nuclear extract (lanes10 and 11). Lane2, labeled Frag II without competitor; lanes
3-5, with 100-fold excess unlabeled Frag II, Frag III, or
Frag I, respectively; lanes 6-9, with 100-fold excess
unlabeled Oligo III, Oligo IV, Oligo V, or Sp1 respectively; lane10, labeled Frag II without competitor; lane11, with 100-fold excess unlabeled Frag II. B,
end-labeled Oligo V (region -14 to +20) was incubated in the
absence (lane1) or presence of K562 nuclear extract (lanes 2-4) or Hep3B nuclear extract (lanes
5-7). Lanes2 and 5, labeled
Oligo V without competitor; lanes3 and 6 and lanes4 and 7, with 100-fold excess
unlabeled Oligo V or NF-Y, respectively.
Northern blot analysis indicated that the level of ALDH1 in
various types of cells is regulated at the transcriptional level (Fig. 1).
Sequence analysis revealed that the extended
5`-region (-2539 to transcription start site) contains various
putative regulatory elements (Fig. 2). The putative binding
sites for transcription factors such as liver-specific HNF-5 and
NF-IL6, muscle-specific MCBF, and hematopoietic cell-specific GATA and
Est-1 may be involved in tissue- and cell type-specific expression of ALDH1.
Although human ALDH1 is constitutively
expressed at a high level in the liver, it is also inducible by
phenobarbital in human hepatic cells and the liver of some rat
strains(14, 15) . A conserved 17-bp phenobarbital
response element has been identified in phenobarbital-inducible rodent
cytochrome P450(31) , mouse glutathione S-transferase
Ya(32) , rat ALDH(15) , rat epoxide hydrolase (33) , and rabbit cytochrome P450 11C(34) . A similar
sequence (77% homology) exists in human AHLD1 (Fig. 2).
Human ALDH1 is expressed in genital skin fibroblast cells
from normal subjects, but not in the cells obtained from androgen
receptor-negative testicular feminization
patients(12, 13, 35) . Two putative
androgen-responsive elements exist at positions -688/-674
and -322/-306, suggesting the possibility of
androgen-dependent regulation of the ALDH1 expression in
genital skin cells.
It has been suggested that NF-IL6 element and
HAPE-1 element are cooperatively involved in the expression of several
acute phase genes (36) . These two elements are closely located
in the 5`-region from -2200 to -2182 of ALDH1. It
is of interest to examine whether or not the two elements are involved
in modulation of ALDH1 expression by interleukin-6 cytokine.
The present functional studies of the promoter region revealed
several regulatory elements and nuclear proteins involved in the cell
type-specific expression of ALDH1.
The deletion analysis
demonstrated that two elements, one between -91 and -51,
and the other between -50 and +53, drive the CAT expression
in Hep3B cells. Deletion of CCAAT sequence (-74/-70), but
not deletion of ATAAA sequence (-32/-28), decreased the
promoter activity (Fig. 6). Therefore, CCAAT box-binding protein
is critical for promoter activity in Hep3B cells. Previous studies
showed that mutations of CCAAT sequence in the albumin and major
histocompatibility class II promoters could result in decrease of
promoter activity(37, 38) .
In contrast with other
mammalian species, ALDH1 is hardly expressed in the liver
without an inducer in various rat strains, including
Long-Evans(15) . A part of the octamer motif and adjacent
sequences (13 bp) are deleted in rat Long-Evans strain (Fig. 3)
and in other common rat strains,(
Although multiple factors existing in Hep3B
cells, such as C/EBP, CTF/NF1, and NF-Y, could interact with CCAAT
motif(39, 40, 41, 42) , the mobility
shift assay strongly supports NF-Y as the primary factor interacting
with the CCAAT region ( Fig. 8and Fig. 10). Moreover, the
octamer region adjacent to the CCAAT box binds to nuclear proteins.
Interestingly, only an ubiquitous transcription factor, Oct-1, was
found in Hep3B nuclear extracts, while another octamer binding factor,
termed Oct-X, was detected in K562 nuclear extracts in addition to
Oct-1. It was reported that neuronal Oct-2 does not activate reporter
constructs containing an octamer motif, but it could interfere with the
activation of such constructs by Oct-1(43) . It is conceivable
that Oct-X, like Oct-2, is an inhibiting factor, competing with Oct-1
at the octamer binding site of ALDH1 in K562 cells.
Both
NF-Y and octamer factor(s) appeared to bind with the promoter region
(-91/-1, Frag I), producing a complex B (Fig. 10).
Synergy between NF-Y motif and an adjacent C/EBP site was observed in
the expression of albumin gene(44) , between Oct-1 and
glucocorticoid receptor in activation of the mouse mammary tumor virus
promoter (45) and between Oct-1 and SP-1 in activation of U2
small nuclear RNA gene (46) . A similar synergism may exist
between NF-1 and Oct-1 motifs in ALDH1 expression.
CAT
assay indicates that the ALDH1 promoter region
-91/-50 is functionally active not only in Hep3B cells but
also in LTK
Alternatively, the cell type-specific expression could be due to the
presence of different NF-Y related proteins or octamer factors in
different cell types(47) . It is also conceivable that the
concentration and ratio of ubiquitous transcription factors in
particular cell types affect the gene
expression(48, 49, 50) .
Based on the
present structural and functional analysis, the following possible
molecular mechanism can be proposed for the cell type-specific
expression of ALDH1 gene. In ALDH1-positive Hep3B cells, the
factors involved in the ALDH1 expression are NF-Y and Oct-1
binding to the promoter region -91/-50, and L1F and L2F
acting on the promoter region -50/+20. In ALDH1-negative
K562 cells, NF-Y, Oct-1, and Oct-X act on the promoter region
-91/-50, but L1F and L2F factors are missing and thus ALDH1 cannot be strongly expressed in the cells. Oct-X might
interfere with the binding of Oct -1 to the octamer sequence and
suppress the promoter activity in K562 cells.
The molecular
mechanism of the androgen-receptor mediated expression of ALDH1 remains to be elucidated.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank®/EMBL Data Bank with accession number(s)
U28416[GenBank® Link].
We thank Dr. K. Kurachi for providing us with the CAT
vector and Dr. D. Mathis for the anti-NF-Y antibody. We are also
grateful to Vibha Davé for assistance and to Dr. S. Tamura for
encouragement.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
cells, which do not express ALDH1. Within this region, we define a minimal promoter
(-91 to +53) that contains positive regulatory elements. The
study using site-directed mutagenesis demonstrated that the CCAAT box
region is the major cis-acting element involved in basal ALDH1 promoter activity in Hep3B cells. Gel mobility shift assays showed
that NF-Y and other octamer factors bound CCAAT box and an octamer
motif sequence, but not GATA site existing in the minimal promoter
region. Two additional DNA binding activities associated with the
minimal promoter were found in the nuclear extract from Hep3B cells,
but not from K562 cells. These results offer the possible molecular
mechanism of the cell type-specific expression of ALDH1 gene.
oxidoreductase, EC 1.2.1.3, ALDH)(
)
play a
role in the detoxification of alcohol-derived acetaldehyde and the
metabolism of corticosteroids, biogenic amines, and lipid peroxidation
(reviewed in (1) ). Many human ALDH isozymes are
distinguishable on the basis of separation by physicochemical methods,
tissue and subcellular distributions, and enzymatic
properties(1) . Cytosolic ALDH1 is active for retinalaldehyde
oxidation and is considered to play a major role in the synthesis of
retinoic acid(2) , which is a potent modulator for gene
expression and cell differentiation (reviewed in (3) ). The
characterization of human ALDH1 cDNA and gene has shown that this gene
spans 53 kilobase pairs, and contains 13 exons, which encode 501 amino
acid residues including the chain initiation
Met(4, 5) . The location of human ALDH1 was
assigned to chromosome 9q21(6, 7) .
Northern Blot Analysis
Total cellular RNAs were
prepared from various cultured cell lines by the established
method(18) . Twenty micrograms of total RNAs were
electrophoresed, transferred onto nitrocellulose membrane, and
hybridized with a human ALDH1 cDNA probe (4) and with a human
-actin cDNA probe, which served as an internal reference.
Plasmid Construction
A low background promoterless
CAT expression vector, PUMSVOCAT, with an unique SmaI cloning
site(19) , and the genomic clone (DASH-19) of human ALDH1 gene containing the extended 5`-region (5) were
used for preparation of the expression constructs.
. The ligation of the HindIII/EcoRI fragment -2536/-673 with
the -673/+53 fragment yielded the 5` region of ALDH1 gene covering -2536/+53. A fragment from -1736 (PvuII site) to -673 (EcoRI site) generated
from the clone -2536/+53 was ligated to a fragment
-673/+53 described above. Two fragments, i.e. -266/+53 and -149/+53, were obtained from
the -673/+53 clone by PvuII and DraI
digestion respectively. The truncated fragments from -120,
-91 and -50 to +53 were obtained by PCR using the
-673/+53 fragment as a template. The nucleotide sequences of
these truncated clones were confirmed by sequencing.
Construction of Plasmids Containing Deletions
The
deleted vectors, i.e. pCAT-120C and pCAT120
A
containing the deletion of CCAAT box (-74/-70) and the
deletion of ATAAA box (-32/-28), respectively, were
generated by a two-step PCR directed mutagenesis by the following
procedures. First, a fragment -673/-60 was prepared using
-673/-655 as 5` primer and 5`-CTCGGATACGATGAACAAACTCAG-3`
as 3` primer, and a fragment -84/+53 was prepared using
5`-GTTTGTTCATCGTATCCGAGTATG-3` as 5` primer and +53/+36 as 3`
primer. Subsequently these two fragments were mixed and used for
another round of PCR in the presence of -120/-103 as 5`
primer and +53/+36 as 3` primer. The final PCR product was
subcloned into pBluescript vector, and the internal deletion was
confirmed by sequencing. pCAT-120
A was constructed by the same
procedure except for using -673/-655 and
5`-TTGTTCCTTTCTGCACGGGCTAAA-3` as primers for amplification of
-673/-12, and 5`-GCCCGTGCAGAAAGGAACAAATAAAG-3` and
+53/+36 as primers for amplification of -43/+53.
Cell Culture
All cell lines were obtained from
American Type Culture Collection. Human non-genital fibroblast cells
(GM8447) were cultured in minimal essential medium under 5%
CO. Human genital skin fibroblast cells(9024), human
hepatoma cells (HepG2 and Hep3B), human breast cancer cells (MCF-7),
and mouse fibroblast cells (LTK
) were cultured in
Dulbecco's modified Eagle's medium under 5% CO
.
Human prostate cancer cells (LNCaP), human erythroleukemic cells
(K562), and human promyelocytic leukemia cells (HL60) were grown in
RPMI 1640 under 5% CO
. All media were supplemented with
penicillin (100 units/ml), streptomycin (100 µg/ml), and 10% fetal
bovine serum (Life Technologies, Inc.).
Transfection and CAT Assay
Human hepatoma Hep3B
cells and mouse fibroblast cells LTK were transfected
by the use of Lipofectin (Life Technologies, Inc.), and human
erythroleukemic cells (K562) were transfected by the use of Transfectam
(Promega). The cells were cotransfected with 10 µg of the test
plasmid and 2 µg of pCMV
gal control plasmid. After 48 h, the
treated cells were washed with phosphate-buffered saline and harvested
from the plate by scraping. The cells were then pelleted by
centrifugation, resuspended in 0.25 M Tris chloride (pH 8.O),
and disintegrated by freezing/thawing four times. Aliquots of the
centrifuge supernatant of the lysate were used for assay of
-galactosidase activity(20) . The remainder of the
supernatant was heated to 60 °C for 10 min to inactivate
deacetylase and stored at -70 °C prior to determination of
CAT activity.
C]chloramphenicol as
substrate. The CAT activity was normalized to
-galactosidase
activity and expressed as -fold increase in activity over that of the
simian virus 40 (SV40) early promoter.
Gel Retardation and Competition Assays
Nuclear
extracts prepared by the method of Shapiro et al.(22) were preincubated in a 20-µl reaction mixture
containing 20 mM HEPES (pH 7.9), 0.5 mM
dithiothreitol, 0.5 mM EDTA, 7% glycerol, 1 µg of
poly(dI-dC), 25 mM KCl, and 25 mM NaCl at 25 °C.
After 10 min, approximately 2 10
cpm of a
P-end-labeled nucleotide probe was added and the
incubation continued for 20 min. The mixtures, together with 2 µl
of loading buffer (50% glycerol, 1 mM EDTA, 0.25% xylene
cyanole, and 0.25% bromphenol blue), were electrophoresed in a 4%
polyacrylamide gel in 0.5
TBE buffer (45 mM Tris
borate, pH 8.4, 0.1 mM EDTA). In competition assays, a large
excess of unlabeled double-strand oligonucleotide competitor was
incubated together with the nuclear extract prior to adding the
P-labeled probe.
Cell and Tissue Specificity of ALDH1
Expression
Northern blot analysis demonstrated that ALDH1 mRNA
is abundant in human genital skin fibroblast cells(9024) and in
hepatoma cells (HepG2 and Hep3B). ALDH1 mRNA is undetectable in
non-genital skin fibroblast cells (GM8447 and LTK)
and in other cancer cell lines examined (MCF-7, LNCaP, K562, and HL60) (Fig. 1). The
-actin gene is expressed at comparable levels
in all cell types. In a variety of human tissues examined, ALDH1 gene is expressed highly in liver and pancreas, moderately in
skeletal muscle and kidney, at low levels in brain, heart, and lung,
and is undetectable in placenta (data not shown).
P-labeled
human ALDH1 cDNA probe (top) and a
P-labeled
human
-actin cDNA probe (bottom). The size of the marker
is indicated on the left in kilobases (Kb). Lane1, GM8447 human non-genital skin fibroblast cells; lane2, 9024 human genital skin fibroblast cells; lane3, HepG2 human hepatoma cells; lane4, Hep3B human hepatoma cells; lane5,
MCF-7 human breast cancer cells; lane6, LNCaP human
bladder carcinoma cells; lane7, K562 human
erythroleukemia cells; lane8, HL60 human
promyelocytic leukemia cells; lane9, LTK
mouse fibroblast cells.
5`-Flanking Region of the Human ALDH1 Gene
Two
overlapping clones for the 5`-region of the gene were obtained by
screening human genomic libraries. The nucleotide sequence of the
region (starting -2536 counting from the transcription start site
numbered +1) is shown in Fig. 2. In comparison with the
reported sequence from -676 to exon 1(5) , the present
sequence displays a T/G transversion at -396 and a C/T transition
at -100. The sequence of the entire 5`-flanking region was
scanned on both strands for the search of potential protein binding
motifs. Based on the published libraries of such motifs(28) , a
noncanonical TATA box exists at -32, and a potential CCAAT box
exists at -74. A series of potential NF-IL6-responsive elements
are scattered through the entire sequence. Several other potential
liver-specific sequences also exist in the region (Fig. 2).
In an attempt to delineate the DNA elements that are important for
the activity of the ALDH1 promoter, the 5`-sequences of the
human, marmoset (sequenced in this laboratory), mouse(29) , and
rat (15) ALDH1 genes are compared (Fig. 3).
The sequence of the human ALDH1 proximal promoter region is
very similar to that of marmoset, rat, and mouse. Within the upstream
100-bp region from the transcription start site, a 10-bp deletion
(-14/-5) exists in the mouse and rat genes, and a 13-bp
deletion (-53/-41) exists in the rat gene. However, CCAAT
and ATAAA boxes are well conserved in all species. An octamer binding
site is also conserved in the promoter regions of human, marmoset, and
mouse genes, suggesting the functional importance of these elements for
the transcriptional regulation of the ALDH1 gene.
Cell Type-dependent Transcriptional Control and
Regulatory Elements
The role of the 5` promoter region in cell
type-dependent expression of the ALDH1 gene was examined by
expressing CAT constructs containing progressive deletions of the
2536-bp fragments (Fig. 4).
In hepatoma (Hep3B) cells, which
constitutively expressed ALDH1, the CAT activity with the
vector carrying -2536/+53 (pCAT-2536) was 32-fold higher
than that of the cells transfected with the promoter less pUMSVOCAT
vector (Fig. 5). By contrast, the expression of pCAT-2536 was
similar to that of pUMSVOCAT in fibroblast LTK cells
and erythroleukemic K-562 cells, which did not express ALDH1.
cells. Deletion constructs containing
different length of the ALDH1 5`-flanking region were
transfected into Hep3B cells (whitebox), K562
fibroblast cells (stripedbox), and LTK
cells (blackbox). The position numbers are
counted from the transcription start site. The plasmids pUMSVOCAT and
pCAT-SV40 were used as a negative and a positive control, respectively.
CAT activities are calculated as percentages of pCAT-SV40 activities in
each cell line.
The cell type-specific activity of the ALDH1 promoter was
further evidenced by comparison of the CAT activity yielded by the ALDH1 promoter and that by the SV40 early promoter in
different cell lines. The relative activity of the ALDH1 promoter (measured using pCAT-SV40 as reference) was found to be
61.2 in Hep3B, 2.9 in LTK, and 7.7 in K562 cells,
implying that pCAT-2536 stimulates transcription 21 and 8 times more
efficiently in Hep3B than LTK
and K562 cells,
respectively (Fig. 5). These results indicate that the
5`-flanking region can direct the cell type-specific expression.
, and 7-fold in K562 cells (Fig. 5). These results indicate that the promoter element
(-91/+53) directs the cell type-specific expression of the ALDH1 gene.
Characterization of the Human ALDH1 Promoter
Region
In order to identify the positive regulatory elements
within the proximal promoter region (-120/+53), the basal
promoter activity of the internal deletion mutants was examined.
Deletion from -74 to -70 of the CCAAT box (pCAT-120C)
resulted in a 12.5-fold decrease of the CAT activity compared to that
expressed by the undeleted pCAT-120, indicating that the binding of a
nuclear factor(s) to the CCAAT box is essential for the basal promoter
activity in Hep3B cells (Fig. 6). On the other hand, deletion
from -32 to -28 of the ATAAA box (pCAT-120
A) did not
significantly affect the CAT activity, suggesting that the ATAAA box is
not a primary regulatory element in Hep3B cells.
Gel retardation
analysis showed that unlabeled NF-Y, a well characterized human albumin
promoter(24) , but not CTF/NFI and Sp1 sequences, competed with
labeled Oligo I (Fig. 7A, lanes 3-14).
Similarly, unlabeled Oligo I but not CTF/NFI and Sp1 oligonucleotides,
prevented the binding of the labeled NF-Y to the Hep3B nuclear protein
(data not shown). Furthermore, the antibody specific to NF-YB protein,
a member of NF-Y transcription factors(23) , disturbed the
formation of Oligo I-nuclear factor complex (Fig. 8A, bandNB). These results indicate that the nuclear
factor interacting with the CCAAT box region is identical to the
nuclear factor NF-Y.
DNA complex is indicated by NB. B, end-labeled Oligo II was incubated
with Hep3B nuclear extracts (lanes 1-4) or K562 nuclear
extracts (lanes 5-8) in the presence or absence of the
specific antibody, and subjected to gel shift assay. Lanes 1 and 5, no antibody; lanes2 and 6, with anti-Oct-1; lanes3 and 7,
with anti-Oct-2; lanes4 and 8, with rabbit
IgG. The supershift band is indicated by S.
The consensus octamer sequence, ATGCAAT, exists
adjacent to the CCAAT box in the ALDH1 promoter (Fig. 2). When Oligo II (-68/-41) was incubated with
the nuclear extracts from a slow moving complex, OB1 was observed in
the presence of the Hep3B extract, whereas two complexes, OB1 and OB2,
were detected in the presence of the K562 extract (Fig. 7B, lanes2 and 9).
The unlabeled Oligo II and Oct oligonucleotides abolished the formation
of the labeled OB1 and OB2 complexes (Fig. 7B, lanes 3-6 and 11-13). The Sp1
oligonucleotide did not affect the formation of OB1 and OB2 complexes (Fig. 7B, lanes 7, 8, 14,
and 15). Similarly, Oligo II and Oct oligonucleotides, but not
Sp1, prevented the binding of the labeled Oct oligonucleotide to the
nuclear extracts from Hep3B or K562 (data not shown). The formation of
OB1 complex was inhibited by the anti-Oct-1 antibody, and a supershift
band was produced in the gel retardation analysis (Fig. 8B, lanes2 and 6).
The formation of OB2 complex was not disturbed by the anti-Oct-1
antibody and anti-Oct-2 antibody (Fig. 8B, lanes
6-8). From these results, one can conclude that the nuclear
protein producing the slow moving OB1 is Oct-1, ubiquitously expressed
in various cells (30) and that the second complex, OB2, is
produced by another octamer factor, tentatively designated as Oct-X,
existing in K562 cells but not in Hep 3B cells.
Cooperative Binding of NF-Y and Octamer
Factor(s)
Cooperative binding of these factors to the ALDH1 promoter was examined by the mobility shift analysis using several
nucleotide fragments shown in Fig. 9. Four complexes (A, B, C,
and D) were produced by incubating Frag I (-91/-1) with the
Hep3B nuclear extract (Fig. 10). Competitive binding assay using
Oligos I, II, III, and IV revealed that complex B is related to both
Oligo I (i.e. NF-Y binding site) and Oligo II (i.e. octamer binding site) (Fig. 10, lanes4 and 5). Oligo I strongly interfered with the formation of
complex D, but not complex C, whereas Oligo II disturbed the formation
of complex C, but not complex D (Fig. 10, lanes3 and 4). Oligos III and IV and Sp1 did not compete with
Frag I in the formation of complexes A, B, C, and D. The results
suggest that complex C results from the binding of the octamer factor
to the probe, while complex D is a product of NF-Y and the probe.
Complex B consists of the probe to which both NF-Y and octamer factors
have bound.
The formation of complex A was abolished by
self-competitor, Frag I, and an excess of a truncated oligonucleotide
Frag II, but not other oligonucleotides. Complex A was not detectable
using K565 nuclear extracts (data not shown). These results indicate
that the protein involved in the formation of complex A is cell
type-specific and reacts to the region -50 to -1 of the ALDH1 promoter.
Identification of Cell Type-specific DNA-binding
Proteins
In order to further elucidate the hepatocyte-specific
transcription factor(s), the labeled nucleotide region
-50/+53 was incubated with the nuclear extracts from Hep3B
or K562 cells and subjected to the mobility shift analysis. Complex L1
was produced by the Hep3B extract but not by the K562 extract (Fig. 11A). The unlabeled Frag I, Frag II, and Frag III
abolished the formation of complex L1 (Fig. 10A, lanes 3-5), suggesting that the region
-50/-1 bind to the hepatocyte-specific transcription
factor(s). However, Oligo III, Oligo IV, and Sp1 did not strongly
interfere with the formation of L1 complex (Fig. 10A, lanes6, 7, and 9).
To find out
a possible involvement of GATA site in the formation of L1, Frag II
with altered GATA site (-34/-29, AGATAA ACCGAA) was
used for the gel shift assay. The mutated Frag II abolished the
formation of L1 complex (data not shown), suggesting that GATA binding
site may not be important for the formation. Interestingly, the
unlabeled Oligo V (-14/+20), reduced the formation of
complex L1 (Fig. 11A, lane8),
suggesting the presence of two binding factors, one for the sequence
from -51 to -1, and the other for the sequence from
-14 to +20. To confirm this possibility, labeled Oligo V
(-14/+20) was used for the mobility shift analysis. When
Oligo V was incubated with the Hep3B nuclear extract, a unique complex,
L2, was produced (Fig. 11B). Complex L2 was not
produced by the K562 nuclear extract. The hepatocyte-specific nuclear
factors involved in formation of complexes L1 and L2 are designated as
L1F and L2F, respectively.
)
indicating
the importance of this region for constitutive expression of ALDH1 in the liver.
and in K562 cells (Fig. 5).
However, judging from the fact that the stimulation is much higher (2
orders of magnitude) in Hep3B cells compared with LTK
and K562 cells, the promoter region -91/-50 is
essential but is not sufficient to direct the hepatocyte-specific
expression of ALDH1. The mobility shift analysis demonstrated
the existence of two unique nuclear proteins (L1F and L2F) producing L1
and L2 complexes, in Hep3B cells but not in K562 cells (Fig. 11). Competitive mobility shift studies indicated that L1F
binding was only partially suppressed by Oligo V, which completely
abolished L2F binding. The two nuclear proteins, L1F and L2F, might
belong to the same family of transcription factors and play a major
role in the high level expression of ALDH1 in Hep3B cells.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.