©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Transcriptional Regulation of Human Aldehyde Dehydrogenase I Gene
THE STRUCTURAL AND FUNCTIONAL ANALYSIS OF THE PROMOTER (*)

Yuchio Yanagawa , James C. Chen , Lily C. Hsu , Akira Yoshida

From the Department of Biochemical Genetics, Beckman Research Institute of the City of Hope, Duarte, California 91010

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

Aldehyde dehydrogenases (aldehyde:NAD 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) .

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.


MATERIALS AND METHODS

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.

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. 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.

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.

Construction of Plasmids Containing Deletions

The deleted vectors, i.e. pCAT-120C and pCAT120A 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-120A 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 pCMVgal 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.

CAT assays were performed by the phase-extraction method (21) using [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.

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`.


RESULTS

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).


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 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).


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) .



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.


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.



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).


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.



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.


Figure 5: Expression of CAT activities in Hep3B, K562, and LTK 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.

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, 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-120A) did not significantly affect the CAT activity, suggesting that the ATAAA box is not a primary regulatory element in Hep3B cells.


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.



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.


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-YDNA 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.


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.



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).


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.



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.


DISCUSSION

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,() indicating the importance of this region for constitutive expression of ALDH1 in the liver.

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 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.

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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL-29515 and AAO 5763. 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®/EMBL Data Bank with accession number(s) U28416[GenBank® Link].

The abbreviations used are: ALDH, aldehyde dehydrogenase; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; bp, base pair(s).

Y. Yanagawa, J. C. Chen, L. C. Hsu, and A. Yoshida, unpublished observation.


ACKNOWLEDGEMENTS

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.


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