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Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3436-3444
Differential Regulation of Coproporphyrinogen Oxidase Gene Between
Erythroid and Nonerythroid Cells
By
Shinichiro Takahashi,
Shigeru Taketani,
Jun-etsu Akasaka,
Akira Kobayashi,
Norio Hayashi,
Masayuki Yamamoto, and
Tadashi Nagai
From the Department of Biochemistry, Tohoku University School of
Medicine, Sendai, Japan; Department of Hygiene, Kansai Medical
University, Moriguchi, Japan; and the Center for Tsukuba Advanced
Research Alliance, (TARA) and Institute of Basic Medical Sciences,
University of Tsukuba, Tsukuba, Japan.
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ABSTRACT |
Coproporphyrinogen oxidase (CPO) catalyzes the sixth step of the
heme biosynthetic pathway. To assess the tissue-specific regulation of
the CPO gene promoter, mouse genomic DNA clones for CPO were isolated.
Structural analysis demonstrated that the mouse CPO gene spans
approximately 11 kb and consists of seven exons, just like its human
counterpart. Functional analysis of the promoter by transient
transfection assays indicated that synergistic action between an
SP-1-like element at  21/12, a GATA site at 59/54, and a
novel regulatory element, CPRE (-GGACTACAG-) at 49/41, is
essential for the promoter activity in murine
erythroleukemia (MEL) cells. In nonerythroid NIH3T3 cells, however, the
GATA site is not required. Gel mobility shift assays demonstrated that
specific DNA-protein complexes can be formed with each element, and
that there are cell-specific differences in factors, which bind to the
SP-1-like element between MEL and NIH3T3 cells. These results provide
evidence for differential regulation of the promoter function of CPO
gene between erythroid and nonerythroid cells.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE BIOSYNTHESIS OF HEME is differently
regulated between erythroid and nonerythroid cells.1 During
erythroid differentiation, the expression of each enzyme increases
sequentially, beginning with the first enzyme of the pathway,
erythroid-specific  -aminolevulinate synthase
(ALAS-E).2-5 In liver, on the other hand, under conditions that increase the rate of heme synthesis, only the first enzyme, nonspecific (housekeeping) ALAS-N is upregulated, while the level of
other enzymes remains unchanged.1 It is likely that this latter type of regulation may also take place in other nonerythroid tissues.
There are two principal reasons that account for the distinct
tissue-specific regulation of heme synthesis. The first is the finding
that the two ALAS isozymes6,7 are encoded by distinct genes, erythroid-specifically expressed, ALAS-E, and nonspecific, ALAS-N. The second is that both -aminolevulinate dehydratase and
porphobilinogen deaminase (PBGD), the second and third enzymes in the
pathway, respectively, are transcribed from two distinct promoters in a
tissue-specific manner with 5 -alternative
splicing.8-10 However, it is unclear whether there is
distinct tissue-specific regulation of other enzymes in the heme
biosynthetic pathway.
Coproporphyrinogen oxidase (CPO, EC 1.3.3.3), the sixth enzyme in the
heme biosynthetic pathway, catalyzes the removal of the carboxyl group
and two hydrogen atoms from the propionate groups at positions 2 and 4 of coproporphyrinogen III, resulting in the formation of vinyl groups
at these positions.11 The mRNA level and activity of CPO
have been shown to be significantly increased in murine
erythroleukemia (MEL) cells during dimethyl sulfoxide (DMSO)-induced
erythroid cell differentiation,12 while they are unchanged
in mouse liver by treatment with 2-allyl-2-isopropylacetamide, despite
a potent induction of ALAS-N by the chemical.13 These findings suggested that CPO expression might be regulated in a different manner between erythroid and nonerythroid cells. Furthermore, CPO has been reported to become rate-limiting for heme synthesis during
erythroid differentiation.12 Thus, it is important to clarify the regulatory mechanism of CPO gene expression and its role in
erythroid differentiation. Recently, mouse and human cDNA clones and a
human genomic DNA clone of CPO were isolated.14,15 Structural analysis of the human CPO gene showed that it consists of
seven exons spanning approximately 14 kb, and that CPO mRNA is
transcribed from a single promoter both in erythroid and in nonerythroid cells.14 Thus, it appears that a single
promoter may regulate both inducible and constitutive expression of the CPO gene.
To examine the regulation of the CPO gene between erythroid and
nonerythroid cells, we performed structural analysis of the mouse CPO
gene and compared its promoter function in MEL and NIH3T3 cells. Our
results indicate that CPRE, a novel regulatory element found at
49/41, plays an essential role in CPO gene expression both in MEL and NIH3T3 cells, while interaction with GATA-1 and SP-1-like element-binding protein is additionally necessary for the
maximal upregulation of the CPO gene in MEL cells.
| |
MATERIALS AND METHODS |
Isolation and characterization of mouse CPO genomic clones.
The 129 SVJ mouse genomic library in the  FIXII vector (Stratagene,
La Jolla, CA) was screened using a 1.4-kb EcoRI-digested fragment of the mouse CPO cDNA clone containing the entire coding region as a probe.15 Seven independent clones were obtained and mapped. Because two overlapping clones, B6 and B8, were found to
cover the entire mouse CPO gene, they were used for further analysis.
Nucleotide sequences were determined with a cycle sequencing system
using ABI 377 sequencer (Perkin-Elmer Corp, Foster City, CA).
Identification of the first exon and the transcription initiation
site.
The transcription start site was estimated by polymerase chain reaction
(PCR) amplification of the 5-end of cDNA. A total of 2 µg of
poly (A)+ RNA prepared from mouse liver and MEL cells were
used as starting materials, with 5-rapid amplification of cDNA
end (RACE) amplification kit (GIBCO-BRL, Rockville,
MD).16 Oligonucleotide primers complementary to nucleotides 270/251 and 273/254 of the
mouse CPO cDNA15 were used for the first strand cDNA
synthesis and PCR amplification. PCR products were isolated and
sequenced.
Cell culture.
MEL cells, clone 745A, and NIH3T3 cells were grown in Dulbecco's
Modified Eagle Medium (DMEM; Nissui, Tokyo, Japan), supplemented with
10% fetal bovine serum, and split every 3 days. For preparation of
nuclear extracts, cells from 24- to 48-hour-old cultures were resuspended in the fresh medium at a density of 5 × 104 cells/mL and incubated for 16 hours, to ascertain a
logarithmic cell growth. Then DMSO was added at a concentration of 2%
(vol/vol) and incubation was continued for 24 hours. While these cells
did not show an increase in benzidine-positive cells at 24 hours, over
90% of cells became benzidine-positive when incubation was continued
for 4 days.
Plasmids.
A series of deletion mutants of the mouse CPO gene promoter were
constructed, which were also fused to a luciferase reporter gene.
First, various DNA fragments of the mouse CPO gene promoter region were
obtained by PCR using the following forward primers: 5-CGTTGGTACCCTTCATTTATCACC-3 (spanning
positions 958 to 935), 5-ACAGCCGGTACCGAAGCTTGTG-3 (660 to 639),
5-TAGTTAAAGGGGTACCGAGCCC-3 (579 to 558),
5-CTTTGGGTACCCCAAGCACAGG-3 (374 to 353),
5-CCACAGGTACCACGAAGACAAG-3 (330 to 309),
5-CTAACGGTACCTTTGCTCACTG-3 (262 to
241), 5-AAGGGGTACCTGACTAAAGCGC-3 (217 to
196), 5-GCAGCGGTACCCACCTCTGTGC-3 (135 to
114), 5-TTTCCGGTACCGGTCAGGCGAG-3 (90 to
69), 5-CGAGGGTACCAGGCGATAGGAC-3 (72 to
51), 5-GGAGGGTACCGGACGGGACTAC-3 (64 to
43), 5-GGACGGTACCACAGTTCCCAG-3 (54 to
34), 5-GCCTGGTACCTACACTCGCAGC-3 (9 to +13),
and the acceptor primer, 5-ATCTCCAAGGTCCTCGAGGCCC-3 (+50
to +71). The amplified DNA fragments were digested with Kpn I
and Xho I and inserted into the Kpn I/Xho I
site of the pGL3-Basic plasmid (Promega, Madison, WI), which yielded
pGL946, pGL648, pGL563, pGL363, pGL319, pGL251, pGL207, pGL124, pGL79,
pGL62, pGL54, pGL44, and pGL2. pGL79-Gm, -Sm, -GXm, -51m, -49m, -47m, -45m, and -42m were created with Transformer site-directed mutagenesis system (Clontech, Palo Alto, CA) using the following
mutagenic primers, respectively, and nucleotide sequences of mutants
were verified by sequencing:
5-CGAGTGCAGGAGGCGTAAGGACGGGACTACAG-3, 5-CCTCCTCGGCTCCTTCCAGCAGCCTGCG-3,
5-CGAGTGCAGGAGGCGTAAGGACGGGTTTACAG-3, 5-GCAGGAGGCGATAGGATTGGACTACAGTTCCC-3,
5-GGAGGCGATAGGACGTTACTACAGTTCCCAGC-3, 5-GAGGCGATAGGACGGGTTTACAGTTCCCAGCAG-3,
5-GCGATAGGACGGGACACCAGTTCCCAGCAGCC-3, and
5-GATAGGACGGGACTACTATTCCCAGCAGCCTCC-3.
Transfection and luciferase assays.
A total of 2 µg of each reporter plasmid and 1 µg of
pENL17 (as an internal control) were cotransfected into MEL
cells using DOTAP
(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate) (Boehringer Mannheim Corp, Indianapolis, IN) according to the manufacturer's protocol. Briefly, a total of 5 × 105
cells were incubated with the plasmids and DOTAP in 1 mL of DMEM without serum for 6 hours. Then 2 mL of fresh DMEM with 10% fetal bovine serum was added. DMSO was added to cultures at a concentration of 2% (vol/vol) and incubation was continued for 20 hours. Cells were
then harvested, rinsed with phosphate-buffered saline (PBS) once, and
lysed in 75 µL of 1× Reporter Lysis Buffer (Toyo Ink, Ltd,
Tokyo, Japan). Luciferase activities were determined using Luciferase
Assay System (Promega) and normalized on the basis of  -galactosidase
activities, which were determined using chlorophenol red--galactopyranoside as substrate.18 NIH3T3 cells were
also transfected with the reporter plasmids similarly. NIH3T3 cells were seeded at a density of 2.5 × 105 per 3 mL of
DMEM in a 40-mm dish and incubated for 18 hours before transfection.
Preparation of nuclear extracts.
Nuclear extracts were prepared according to the method described by
Lassar et al19 with minor modification. Briefly, 1 × 107 cells were collected, washed once with PBS, pelleted,
resuspended in 1 mL of the lysis buffer containing 10 mmol/L Tris-HCl
(pH7.4), 10 mmol/L NaCl, 3 mmol/L MgCl2, and 0.5% Nonidet
P-40 and placed on ice for 5 minutes. After centrifugation, nuclear
pellets were resuspended in 100 µL of 20 mmol/L Tris-HCl (pH7.9), 420 mmol/L KCl, 0.2 mmol/L EDTA, 10% glycerol, 2 mmol/L dithiothreitol
(DTT) and 0.1 mmol/L phenylmethylsulfonyl fluoride, placed
on ice for 10 minutes, and centrifuged. The supernatants thus obtained
were used as nuclear extracts for gel mobility shift assays. Protein concentration was determined by using a Protein Assay Kit (Bio-Rad, Hercules, CA).
DNA gel mobility shift assays.
The following oligomers were used as probes for gel mobility shift
assays: CPRE, 5-ATAGGACGGGACTACAGTTCC-3; GATA,
5-TGCAGGAGGCGATAGGACGGGACT-3; SP-1-like,
5-CTCCTCGGCTCCCGCCAGCAGCCTGC-3, corresponding to nucleotides 57 to 36, 68 to 44 and
29 to 4 of the mouse CPO gene, respectively. For SP-1,
5-GGACTTTATGGGGGCGGGGCTAAAGGAGGG-3 was used, which
included the consensus SP-1 binding site. Oligomers were end-labeled
with [ -32P] adenosine triphosphate (ATP)
by T4 polynucleotide kinase (Boehringer Mannheim Corp). The antisense
oligomer was then added to the 32P-end-labeled oligomer to
yield a double-stranded probe. A total of 5 µg of nuclear extracts
was incubated in a reaction mixture containing 20 mmol/L HEPES buffer
(pH 7.8), 60 mmol/L KCl, 0.2 mmol/L EDTA, 6 mmol/L MgCl2,
0.5 mmol/L DTT, 10% (vol/vol) glycerol, and 1.5 µg of an equimolar
mixture of poly (dI-dC) and poly (dA-dT) with a 32P-labeled
oligomer for 15 minutes on ice. For competition assays, the following
oligomers were used as double-stranded forms: AEGATA, 5-TTTGGGTTTTATCTCTAGCAAGG-3 (corresponding to nucleotides
135 to 113 of the human ALAS-E gene); AECACCC,
5-CCGCAGAAGGCAGGGTGGGTGGG-3 (corresponding to nucleotides
73 to 51 of the human ALAS-E gene); and CPREm,
5-ATAGGACGAGTTAATAATTCC-3 (mutated CPRE containing 6 transversions in 49/41). For supershift assays, 3 µL of
anti-GATA-1 (N6) (Santa Cruz Biotechnology, Inc, Santa Cruz, CA) was
added to the reaction mixture after the addition of the probe. The
mixture was then loaded onto a 4% polyacrylamide gel and
electrophoresed at 150 V at 4°C.
| |
RESULTS |
Isolation of mouse CPO gene.
To clarify the regulatory mechanism of CPO gene expression, we first
isolated genomic DNA clones, which encode mouse CPO protein from a
mouse liver genomic DNA library. As shown in
Fig 1, two overlapping clones, termed B6
and B8, were isolated, which contained the entire gene including
approximately up to 4 kb of the 5-upstream region. The
organization of the mouse CPO gene was determined by Southern blot
analysis using cDNA probes corresponding to various parts of the mouse
CPO mRNA, and the sequence of each exon-intron junction was also
determined. This analysis showed that the mouse CPO gene consists of a
total of seven exons, which spans approximately 11 kb. The last exon
(exon 7) includes a large 3-untranslated region (Fig 1). The
length of the introns varies from 652 bp to 2.4 kb, and all exon-intron
boundaries conform completely to the GT/AG rule
(Table 1). Genomic DNA blot hybridization
analysis demonstrated that the mouse CPO is specified by a single
genomic locus (data not shown).
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| Fig 1.
Structure and organization of the mouse CPO gene. Two
overlapping l phage clones (B6 and B8) were analyzed. The boxes show
the relative sizes and positions of each exon in the mouse genome.
Solid and shaded boxes indicate protein coding and untranslated
regions, respectively. Recognition sites for enzymes HindIII,
EcoRI, and SacI are shown. Lower lines show restriction
endonuclease cleavage sites, as indicated.
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|
Sequencing analysis of the upstream region of the mouse CPO gene.
To investigate the regulatory mechanism of the mouse CPO gene, we
determined the sequence of the 5-upstream region up to  1,200
bp from the transcription start site (Fig
2). Notable features in this region are 12 putative GATA motives that
contain the sequence nGATAn, and four CACCC elements with no obvious
TATA or CAAT boxes. An SP-1 consensus-like sequence, CTCCCGCCAG, was
found near the transcription start site. The presence of these
cis-elements as well as their spatial arrangement are similar to those
observed in the promoter region of the human CPO gene,20
which contains four GATA motives, six putative SP-1 binding sites, and
two CACCC elements within a 786-bp region upstream from the
transcription start site.
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| Fig 2.
The 5-upstream region of the mouse CPO gene. The
nucleotide sequence of the first exon (uppercase letters) and 5
-upstream regions (1.2 kb) is demonstrated. White and black arrows
indicate the major and the minor transcription initiation sites,
respectively. The putative cis-acting GATA, CACCC, and SP-1-like
elements are boxed and underlined. The initiating codon is shown in
bold and underlined. The intronic sequence is expressed in lowercase
letters.
|
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A novel regulatory element found at 49/41 is critical
for the mouse CPO promoter activity in NIH3T3 cells.
To clarify the regulatory elements for constitutive CPO gene expression
in nonerythroid cells, transient transfection assays were performed in
NIH3T3 cells using a nested series of promoter deletion constructs
fused to a luciferase reporter gene. As shown in
Fig 3, p946 demonstrated a high level of
the reporter activity. The removal of the sequence between 946
and 54, where all GATA and CACCC sites are found, did not
significantly change the activity. A further deletion of the promoter
region from 54 to 44, however, caused a markedly
decreased activity. Essentially similar results were obtained when
H4IIE cells, a rat hepatocellular carcinoma-derived cell line, were
used instead of NIH3T3 cells in the transfection assay (data not
shown). These findings suggest that there may be a hitherto
unrecognized regulatory element in the region between 54 and
44.
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| Fig 3.
Transient mouse CPO gene promoter activity in NIH3T3
cells. A series of 5-deletion mutants of the mouse CPO gene
promoter were assayed for luciferase activity. The results are
expressed as ratios to that obtained with a pGL3-Basic plasmid. Data
are mean values from three separate experiments.
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To investigate this question, various constructs in which point
mutations were inserted into this region were generated and transfected
into NIH3T3 cells. Nucleotide substitutions at 49 and 48
(p49m) and at 42 and 41 (p42m) resulted in a moderate decrease in the reporter activity (60% compared with that of p79)
(Fig 4A). In contrast, mutation insertions
at 47 and 46 (p47m) and at 45 and 44 (p45m)
resulted in a marked decrease in the reporter activity (to 20% and
30% of that represented by p79, respectively), while mutations at
51 and 50 had no effect (Fig 4A). These results indicate
that the nucleotide sequence, GGACTACAG, which we termed CPRE, is
essential for the promoter function in NIH3T3 cells. The removal of a
region between 44 and 2, which contained an SP-1-like
element, CTCCCGCCAG, resulted in a further decrease in the promoter
activity (Fig 3). The importance of the SP-1-like element for the
promoter activity was substantiated by the finding that p79Sm, a
mutated p79, had a significantly decreased reporter activity (60%
compared with the wild-type p79) (Fig 4B). p79SXm, double mutations in
the CPRE, and the SP-1-like element depressed the promoter activity
more than did p79Xm, and p79Sm, a single mutation construct of CPRE and
SP-1-like element, respectively (Fig 4B). These findings indicate that
CPRE at 49/41 is essential for the promoter activity, and
that the SP-1-like element at 21/12 also additionally
contributes to the promoter function.
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| Fig 4.
(A) Identification of CPRE by functional assays in NIH3T3
cells. The effects of nucleotide transversions in the region between
51 and 40 were examined. The results are expressed as the ratios
of luciferase activities to that obtained with a pGL3-Basic plasmid.
Data are the means of triplicate determinations. (B) Various constructs
with the indicated mutations were transfected into NIH3T3 cells.
Results are the means of three separate experiments.
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Functional studies of the mouse CPO gene promoter in MEL cells.
CPO gene expression has been reported to increase significantly during
erythroid differentiation in MEL cells.20 As shown in
Fig 5, p946 conferred a 70-fold higher
transcription of the reporter gene compared with pGL3Basic, a
promoter-less luciferase reporter construct. Treatment with DMSO, a
potent inducer of erythroid differentiation of MEL cells, elicited a
significant increase in luciferase activity (Fig 5), while the same
treatment had no effect on the activity in NIH3T3 cells (data not
shown). These findings indicate that the promoter activity is increased
during erythroid differentiation of MEL cells, but not in nonerythroid cells such as NIH3T3 cells.
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| Fig 5.
Transient mouse CPO gene promoter activity in MEL cells
in the presence or absence of DMSO. MEL cells were transfected with the
series of 5-deletion mutants shown in Fig 3 and incubated in the
presence or absence of 2% DMSO for 20 hours. The results are expressed
as ratios to the luciferase activity of pGL3-Basic plasmid in the cells
without DMSO treatment. Data are means of three separate experiments.
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The levels of both basal and DMSO-induced luciferase activities
remained unaffected by the removal of a sequence between 946 and
54, which contains 12 GATA sites and four CACCC elements. Further deletion from 54 to 44, however, resulted in a
significant decrease both in the basal and in DMSO-induced luciferase
activities, and the removal of a sequence between 44 and
2 caused a further loss of activity (Fig 5). These results
suggest that CPRE at 49/41 and the SP-1-like element at
21/12 may also be important for the promoter activity in MEL
cells.
Because our previous studies showed that a GATA site near the
SP-1-like element may be involved in the human CPO gene
upregulation,20 this question was examined in detail. For
this purpose, various mutated constructs containing nucleotide
substitutions were generated from p79, transfected into MEL cells, and
luciferase activities were determined (Fig
6A). A mutation in the GATA site (p79Gm) had no effect on the promoter
activity, although it abrogated formation of the specific DNA-protein
complex (data not shown). In contrast, a mutation in the CPRE (p79Xm)
decreased luciferase activity (60% of that generated by the
wild-type p79), and inhibition was markedly pronounced when the GATA
site was also mutated. p79Sm, a mutation in the SP-1-like element
alone, significantly decreased the activity, which was similar to those
observed with p79GSm and p79SXm, a second mutation in the GATA site,
and CPRE, respectively. These results indicate that the SP-1-like
element is essential for basic expression, but not sufficient for full
activation of the promoter, and that there may be synergistic
regulation of the promoter function among these elements in erythroid
cells.
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| Fig 6.
Effect of disruption of individual or paired cis-elements
on luciferase activities. (A) MEL cells were transfected with various
constructs with the indicated mutations and incubated for 20 hours in
the absence of DMSO. Results are the means of three separate
experiments. (B) MEL cells were transfected with various mutation
constructs and incubated for 20 hours in the presence of DMSO. Fold
induction is expressed as the ratio of luciferase activities of cells
transfected with indicated constructs in the figure to that obtained
with PGL3-Basic. Results are expressed as the means of three separate
experiments.
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The role of the three regulatory elements was then examined during
erythroid cell differentiation of MEL cells by DMSO treatment. p79GXm
and p79GSm, which did not contain GATA, but contained the SP-1-like
element and CPRE, respectively, demonstrated only a slight increase in
luciferase activity after DMSO treatment. In contrast, p79SXm, which
contained the GATA site, but not SP-1-like element and CPRE, showed a
twofold increase in the luciferase activity as compared with that by
p79 (Fig 6B). These results indicate that the GATA site at
59/54 plays also a significant role in DMSO-mediated
induction of the promoter activity, consistent with our earlier finding
in human CPO gene promoter activity.20
A specific DNA-protein complex is formed at CPRE at
49/41 in both MEL and NIH3T3 cells.
Our results demonstrated that CPRE plays a critical role in mouse CPO
promoter function both in erythroid and in nonerythroid cells. To
identify nuclear proteins that bind to this element, gel mobility shift
assays were performed using a CPRE double-stranded oligomer. When the
radiolabeled oligomer was incubated with nuclear extracts from MEL
cells, a single major retarded band was found (Fig 7, lane 1). This band disappeared by
the addition of a 125-fold molar excess of the unlabelled CPRE (Fig 7,
lane 2), while the addition of CPREm, a mutated CPRE oligomer, had no
effect (Fig 7, lane 3). A single band with a similar gel retardation
property was also observed when nuclear extracts from NIH3T3 cells were used (Fig 7 lane 4 and 6), indicating that there may be a common element between erythroid and nonerythroid cells, which may be recognized by their nuclear proteins. The amount of DNA-protein complex
at this site was unaffected by DMSO treatment of MEL cells (data not
shown), however, suggesting that upregulation of the promoter activity
by DMSO treatment is likely due to an increase in the transactivation
activity of CPRE-binding protein.
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| Fig 7.
Formation of a DNA-protein complex with CPRE of the mouse
promoter. Five-microgram aliquots of nuclear extracts from MEL or
NIH3T3 cells were incubated with the end-labeled oligomers
corresponding to CPRE in the absence () or presence of a 125-fold
(MEL) or 175-fold (NIH3T3) molar excess of the indicated
oligonucleotides. The specific DNA-protein complex band is shown by the
arrow.
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The GATA site at 59/54 is recognized by GATA-1 in MEL
cells.
Our studies show that GATA site at 59/54 is also
important for the CPO promoter function (Fig 6A and B). Because GATA-1 is known to be abundantly expressed in MEL cells,21-23 it
is possible that GATA-1 may contribute to the CPO promoter function via
binding to GATA site(s). Gel retardation assays showed that there is a single major retardation band by the addition of an oligomer
corresponding to the GATA site (59/54), and that it
disappeared by the addition of a 250-fold molar excess of the unlabeled
oligomer, or an oligomer containing the GATA consensus sequence
(Fig 8, lane 3 and 4). Furthermore,
addition of anti-GATA-1 antibody entirely supershifted this band (Fig
8, lane 6). These results clearly indicate that GATA-1 specifically
binds to this site in MEL cells. However, the amount of DNA-protein
complex was less in DMSO-treated than in untreated cells (Fig 8, lane 1 and 2). While this finding seems paradoxical, it is consistent with a
previous report, which showed that GATA-1 mRNA level was transiently
decreased after DMSO treatment, but recovered with time.24
Thus, it is possible that GATA-1 may become transiently either active,
or an interaction between GATA-1 and other nuclear proteins may be
induced by DMSO treatment.
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| Fig 8.
Gel mobility shift/supershift assay of MEL cell extracts.
Five-microgram aliquots of nuclear extracts from untreated (lanes 1, 3, 4, 5, 6) or DMSO-treated (lane 2) 745A cells were incubated with
end-labeled oligomers corresponding to the GATA site (59/54) in
the absence (lanes 1 and 2) or presence of a 250-fold molar excess of
the indicated oligonucleotides (lanes 3, 4, 5) or anti-GATA-1 antibody
(lane 6). The arrowhead indicates the complex of the probe and GATA-1,
and the arrow represents the ternary complex of the probe, GATA-1, and
the antibody.
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SP-1 does not bind to the SP-1-like element (21/12) in
MEL and NIH3T3 cells.
The SP-1-like element at 21/12 was also shown to be
important for the promoter function both in MEL and NIH3T3 cells, which also functions synergistically with GATA-1 and CPRE binding proteins in
MEL cells (Fig 6A). As shown in Fig 9, when
a nuclear extract prepared from MEL cells was incubated with the
radiolabeled oligomer containing the SP-1-like element, a single
retarded band that migrated faster than that with SP-1 was observed
(Fig 9, lane 1). This band could not be competed by the addition of a
cold oligomer containing SP-1 consensus sequence (Fig 9, lane 3). This was not due to a difference in the affinity of SP-1 for these elements,
as the band representing SP-1 was not suppressed by the addition of an
excess amount of the cold oligomer containing an SP-1-like element
(Fig 9, lane 6). Furthermore, the addition of anti-SP-1 antibody had
no effect on the migration of the band (data not shown), indicating
that the nuclear protein(s), which forms a complex with SP-1 is
different from that which forms a complex with the SP-1-like element.
A specific DNA-protein complex was also formed when a nuclear extract
from NIH3T3 cells was used. It migrated similarly as did the SP-1-DNA
complex, but its formation was not inhibited by the addition of an
excess amount of the cold oligomer corresponding to the SP-1 consensus
sequence (Fig 9, lane 11). These results indicate, as in the case with
MEL cells, that SP-1 also does not bind to this element in NIH3T3
cells. It should also be noted that there is difference in the
migration of bands between MEL and NIH3T3 cells, suggesting that
different factors may bind to the SP-1-like element in each type of
cells. Alternatively, the same factor may bind with different
affinities in a different cellular environment, eg, proteolytic
modifications in MEL cells, and such a possibility requires further
investigation.
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| Fig 9.
Formation of a DNA-protein complex with the SP-1-like
element of the mouse CPO promoter. Five-microgram aliquots of nuclear
extracts from 745A (lanes 1 to 8) or NIH3T3 (lanes 9 to 16) cells were
incubated with the end-labeled oligomers corresponding to the
SP-1-like element (-21/-11) (lanes 1 to 4, 9 to 12) or the SP-1
consensus sequence (lanes 5 to 8, 13 to 16). Competition assays were
performed with a 250-fold molar excess of unlabeled oligonucleotides
containing the SP-1-like element (21/11) (lanes 2, 6, 10, 14),
SP-1 consensus sequence (lanes 3, 7, 11, 15), and GATA consensus
sequence from the human ALAS-E promoter (lanes 4, 8, 12, 16). The
arrow and the arrowheads indicate the complexes of the probe and
nuclear proteins.
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|
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DISCUSSION |
In this study, we examined the regulatory mechanism of the mouse CPO
gene expression in erythroid and nonerythroid cells. We first examined
the structure of the mouse CPO gene and identified that the mouse CPO
gene structure is highly similar to the human CPO gene. Namely, the
5-upstream region of the mouse CPO gene contains several
putative cis-regulatory elements including GATA sites, CACCC sites, and
SP-1-like element, just like the human CPO gene. The spatial
arrangement of these elements is also similar for both
genes,14,20 suggesting that CPO gene expression may be
regulated in a similar manner between mice and humans. By examining the
mouse CPO gene promoter function, we identified a novel regulatory element (GGACTACAG, termed CPRE) that is involved in the induction of
luciferase activity in NIH3T3 cells, whereas an SP-1-like element at
21/12 was also required for maximal promoter activity in DMSO-induced MEL cells (Fig 4B and 6A). Disruption of CPRE resulted in
a marked decrease in the promoter activity in NIH3T3 cells, while it
decreased the promoter activity only moderately in MEL cells (Fig 4B
and 6A), suggesting a different role of CPRE between erythroid and
nonerythroid cells. Gel mobility shift assays demonstrated that a
specific DNA-protein complex was formed at CPRE both in MEL and NIH3T3
cells (Fig 7). Our search in the GenBank database showed that CPRE is
also present in the human -globin gene promoter,25 and
CPRE-related sequences are also found in genes of several heme
biosynthetic enzymes, such as uroporphyrinogen
decarboxylase,26 CPO,14,20 rat
ALAS-N,27 and in the erythroid and nonerythroid mouse
PBGD10,28 (Table 2). A
CPRE-related sequence is also present in the human ALAS-E (our
unpublished data). It is unclear at present whether these sequences are
functional or not, but it is possible that CPRE-binding factor(s) may
contribute to heme synthesis by exerting its effect on gene expression
of these enzymes. Identification of the factor(s) that interacts with
this element may therefore be important for better understanding of
tissue-specific regulation of heme pathway genes.
Our previous studies showed that an SP-1 site adjacent to the GATA
sequence in the human CPO gene contributes to basic gene expression in
MEL cells.20 Therefore, we suspected that an SP-1-like element at 21/12 in the mouse CPO gene may also be
involved in the promoter function. Indeed, its disruption resulted in a moderate decrease in the promoter activity in NIH3T3 cells, indicating its contribution (Fig 4B). In MEL cells, the SP-1-like element alone,
however, demonstrated only a very low activity, but its combination
with CPRE or GATA site (59/54) markedly enhanced the
promoter activity (Fig 6B). Gel mobility shift assays demonstrated that
this element may be recognized by different nuclear factors between MEL
and NIH3T3 cells (Fig 9), suggesting that transcriptional regulation by
SP-1-like element may be different between erythroid and nonerythroid
cells. A similar GC-rich element also exists in the proximal promoter
region of human ferrochelatase gene.29 Thus, there may be a
role of this element in the expression of other heme biosynthetic
enzymes, and this question should be clarified.
The GATA site at 59/54 was shown to bind to GATA-1,
consistent with our previous findings for its role in human CPO gene expression (Fig 8).20 Because GATA factors are key
contributors to the differentiation process of hematopoietic
cells21-23,30 and because GATA binding sites have been
widely found in the gene promoter of several heme synthetic pathway
enzymes,31 GATA-1 may also be essential for upregulation of
the CPO gene during erythroid differentiation. In the present study,
the GATA site alone or in combination with CPRE, showed only a low
reporter activity, while together with the SP-1-like element, it
markedly stimulated the activity in MEL cells (Fig 6A). Synergistic
effects of various combinations of transcription factors, including
GATA-1, on TATA-less promoters have been reported.32-34 Our
results also suggest that interaction between GATA-1 and SP-1-like
element-binding proteins, as well as CPRE and SP-1-like
element-binding-proteins may be important for CPO gene expression in
erythroid cells.
GATA-1 significantly contributes to the DMSO-induced promoter activity
in MEL cells (Fig 6B). On the other hand, the amount of GATA-1-DNA
complex formed at the 59/54 GATA site was slightly decreased by DMSO treatment of MEL cells (Fig 8). DMSO treatment also
did not increase the formation of DNA-protein complexes at CPRE and
SP-1-like element (data not shown). Thus, GATA-1-mediated increase in
transactivation and a decrease in DNA binding in DMSO-treated MEL cells
may seem paradoxical. However, our findings are consistent with an
earlier report, which showed that MEL cells contain two distinct
GATA-binding proteins that have different patterns of expression during
erythroid differentiation, and that GATA-1 mRNA levels decreased
transiently during DMSO-induced differentiation.24 One
possible explanation may be that GATA-1 may become an active form by
certain modification such as phosphorylation during erythroid differentiation.35 Alternatively, DMSO treatment may induce interaction among transcription factors, resulting in the activation of
the promoter function.
To account for tissue-specific regulation, we provide a hypothetical
scheme for differential CPO regulation between erythroid and
nonerythroid cells (Fig 10). Namely, in
erythroid cells, binding proteins to the SP-1-like element, CPRE and
GATA-1, cooperatively function in CPO gene expression. In contrast, in
nonerythroid cells, CPRE-binding protein by itself plays a principal
role in basic expression, while GATA-1 plays little role and the
SP-1-like element may exert only a moderate activity. The
tissue-specific regulation of CPO gene promoter function may also be
influenced by differences in the affinity of tissue-specific factors to
the SP-1-like element, Factor X and Y in MEL and NIH3T3 cells,
respectively. While the exact nature of the regulatory mechanism of CPO
gene expression is yet to be clarified, this model should serve as a
useful hypothesis for evaluation for tissue-specific regulation of heme
biosynthetic enzymes.
View larger version (26K):
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| Fig 10.
Model of the differential regulation of the mouse CPO
gene promoter function in erythroid and nonerythroid cells.
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FOOTNOTES |
Submitted March 12, 1998;
accepted June 24, 1998.
Supported in part by Grants-in-Aid from the Ministry of Education,
Science, Sports and Culture, Japan (to N.H., M.Y., and T.N.),
Grant-in-Aid for Scientific Research (to S.T.), and Research Grant from
Sumitomo Chemicals Co (to S.T.).
The nucleotide sequence data reported in this report will appear in the
DDBJ/EMBL/GenBank nucleotide sequence databases with the accession
numbers AB011237 to AB011243.
Address reprint requests to Tadashi Nagai, MD, PhD, Department of
Biochemistry, Tohoku University School of Medicine, 2-1 Seiryo-machi,
Sendai, 980-77, Japan; e-mail: shintak{at}mail.cc.tohoku.ac.jp.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Drs H. Motohashi, N. Minegishi, K. Furuyama, S. Suzuki, and H. Ohtsu for helpful discussions.
| |
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Abstract
Introduction
Abstract