 |
INTRODUCTION |
Mitochondrial fatty acid
-oxidation is a major energy-producing
pathway, and at least 10 different enzymes are involved in this
function. A mitochondrial trifunctional protein
(TP)1 catalyzes the last
three steps of the
-oxidation of long-chain fatty acids (1-3). This
enzyme complex is composed of four
- and four
-subunits; the
-subunit harbors the enoyl-CoA hydratase and 3-hydroxyacyl-CoA
dehydrogenase domains, whereas the 3-ketoacyl-CoA thiolase domain is
located on the
-subunit (3). These two subunits are encoded in
independent genes, HADHA and HADHB (3, 4). In
humans, genomic organization of the
-subunit (HADHA) was
determined by Zhang and Baldwin (5), and that of the
-subunit (HADHB) was determined in our laboratories (6). Both
HADHA and HADHB are located on chromosome 2p23
(6, 7). Additionally, the distance between the two loci is short,
which suggests that the two genes exist side by side (8), as do the two
genes of the subunits of the bacterial fatty acid
-oxidation
multienzyme complex (9).
Deficiencies in this enzyme complex can cause sudden, unexplained death
in children, acute hepatic encephalopathy, skeletal myopathy, and
cardiomyopathy (10, 11). TP deficiency was classified into two
biochemical phenotypes. The most common phenotype is long-chain
3-hydroxyacyl-CoA dehydrogenase deficiency, with normal or near-normal
activities of long-chain enoyl-CoA hydratase and long-chain
3-ketoacyl-CoA thiolase (10, 11). The other phenotype is characterized
by decreased activity of all three enzyme activities (12, 13). These
analyses showed that formation of the enzyme complex is important for
related functions (14, 15). Formation of the enzyme complex apparently
requires the presence of a similar number of molecules of
- and
-subunits in mitochondria, which raises the possibility of a
synchronized regulation of the expression of the both subunits. We
isolated clones of the two genes, including their 5' flanking regions,
and characterized their structures and transcriptional regulation.
 |
EXPERIMENTAL PROCEDURES |
Materials--
A Sau3AI human leukocyte genomic
library containing 1 × 106
EMBL3 recombinant
phages was purchased from CLONTECH. The radioactive nucleotides [
-32P]dATP and [
-32P]dATP
were purchased from Amersham Pharmacia Biotech. The DNA-modifying enzymes were obtained from New England Biolabs (Beverly, MA), TaKaRa
(Tokyo, Japan), and Toyobo (Tokyo, Japan). Moloney murine leukemia
virus reverse transcriptase and culture medium were purchased from Life
Technologies, Inc. AmpliTaq DNA polymerase and DNA sequencing kits were
purchased from Applied Biosystems Japan (Tokyo, Japan). The LA PCR kit
was purchased from TaKaRa. HEK293 cells were obtained from Dr. S. Asano
(Department of Pharmaceutical Physiology, Toyama Medical & Pharmaceutical University). Drosophila Schneider S2 cells
were a gift of Dr. S. Suzuki (Department of Geriatrics, Shinshu
University School of Medicine). pCMV-Sp1 expression vector was a
generous gift from Dr. R. Tjian (Howard Hughes Medical Institute Investigator at University of California, Berkeley, CA). The CAT assay
kit was purchased from CLONTECH. The PicaGene
luciferase assay system was purchased from Wako (Osaka, Japan).
Isolation and Characterization of Genomic Clones--
The human
leukocyte genomic library was screened at a density of 40,000 plaques/200-mm plate using as probes
[
-32P]dATP-labeled cDNA fragments of human
HADHA (
23 to +428, 451 bp) or HADHB (
26 to
+204, 230 bp) (6). Approximately 2 × 106 plaques were
screened. Positive clones were purified by repeated screening, and
three different clones were obtained (Fig. 1). The phage DNAs from the
purified clones were isolated using standard methods (16).
EcoRI-BamHI fragments from clone 1 (5 kb) and clone 3 (5 kb) and a BamHI fragment from clone 2 (400 bp)
were subcloned into pBluescript KS(+) plasmid (Stratagene) and
sequenced with M13 primers and with primers derived from the human
HADHA and HADHB cDNA sequences (4).
Primer Extension--
The appropriate oligonucleotides (primers
1 and 2; Table I) were end labeled with
[
-32P]dATP, using Multiprime DNA labeling systems
(Amersham Pharmacia Biotech). The radioactive oligonucleotide (5 × 104 cpm) was hybridized to 50 µg of total RNA isolated
from HepG2, HeLa, and HEK293 cell lines for 12 h at 45 °C in 20 µl of hybridization buffer (20 mM Hepes-KOH, pH 7.5, 0.1 mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, and 30 units of RNase inhibitor) (16). Control reactions were run using the same amount of yeast RNA.
View this table:
[in this window]
[in a new window]
|
Table I
Oligonucleotides used
Regions of fragments A and B were used for DNase I footprint analysis.
A and B mutants are mutated fragments. These mutated positions are
represented by small characters and are underlined.
|
|
The hybridized oligonucleotide with RNA was dissolved with 20 µl of
reverse transcription buffer; 50 units of Moloney murine leukemia virus
reverse transcriptase was added, followed by incubation at 37 °C for
2 h. After RNase A treatment, the products were purified by
phenol-chloroform extraction, recovered by ethanol precipitation, and
then electrophoresed, using a 6% polyacrylamide gel containing 8 M urea. The gels were exposed to x-ray films for 18 h.
Size of the labeled products was determined by comparison with
DNA-sequencing reactions run using the same primer.
Ribonuclease Protection Assay--
Ribonuclease protection assay
was done using the Guardian RNase protection assay kit
(CLONTECH). To prepare the riboprobe, PCR was done
using primers 2 and 3 (Table I) and human leukocyte cDNA. An
[
-32P]ATP-labeled antisense RNA probe was synthesized
by in vitro transcription reaction, using the PCR product as
a template. Eighty micrograms of total RNA from HeLa cells were
precipitated with ~1 × 105 cpm of the probe and
resuspended in 20 µl of hybridization buffer. Samples were denatured
at 95 °C for 3 min and incubated at 42 °C for 12 h. After
hybridization, 50 µl of RNA digestion buffer containing RNase A (0.3 µg) and RNase T1 (5 units) were added, and the samples were incubated
at 37 °C for 30 min. RNA was subsequently precipitated, dissolved
with 5 µl of gel-loading buffer, and separated in a denaturing 8 M urea, 6% polyacrylamide gel. The gel was analyzed by
autoradiography using a BAS 1500 bioimaging analyzer (Fuji Film, Fuji, Japan).
Construction of Reporter Gene Plasmids--
The human
HADHA and HADHB reporter gene plasmids were
constructed using a pCAT-Basic Vector (Promega) and a PGV-B2 luciferase reporter gene plasmid (Wako). Human HADHA and
HADHB gene fragments were generated by digesting the DNA
with either restriction enzymes or Bal-31 exonuclease (TaKaRa) or PCR
reaction. The Bal-31-processed DNA fragments were blunt ended using the
Klenow fragment (TaKaRa) and were cloned upstream of the CAT gene in
pCAT-Basic vector. We amplified a 1.2-kb HADHA gene fragment
(nucleotide +25 to 1.2 kb) and a 2.2-kb HADHB gene fragment
(nucleotides
369 to
2200) by PCR, using primers 4 and 5 or primers
6 and 7, respectively (Table I), followed by subcloning into a
pCAT-Basic vector (pCAT-
1 and pCAT-
1). The PCR fragments were
sequenced to confirm that the sequences matched the original genomic
clones. To construct 5' deletion mutants of the HADHA and
HADHB 5' upstream region, the 1.2 kb of HADHA
gene fragment and 2.2 kb of HADHB gene fragment were
inserted in pBluescript II KS(+), in reverse orientation. These
plasmids were linearized with the EcoRI restriction enzyme, partially digested with exonuclease Bal-31 for various lengths of time,
treated with the Klenow fragment to form blunt ends, digested out from
the plasmid, using the EcoRV restriction enzyme, and ligated
into blunt-ended pCAT-Basic vector. The 5'-deleted ends were determined
by DNA sequencing.
To determine whether an intron promoter exists for transcripts
initiated at
-exon 2, a 3.2-kb fragment was amplified using primers
8-10 (Table I). The deletion mutants were inserted into pCAT-Basic
Vector as well as HADHA promoter constructs.
To analyze the overlapping 350-bp promoter region, we prepared serial
deletion mutants (pCAT-
3-9 and pCAT-
4-9) by PCR: pCAT-
3, +25
to
366; pCAT-
4, +25 to
285; pCAT-
5, +25 to
253; pCAT-
6, +25 to
223; pCAT-
7, +25 to
213; pCAT-
8, +25 to
195;
pCAT-
9, +25 to
170; pCAT-
4,
366 to +25; pCAT-
6,
366 to
107; pCAT-
7,
366 to
203; pCAT-
8,
366 to
223; pCAT-
9,
366 to
238; and pCAT-
10,
366 to
248. Deletion mutants of
luciferase construct were obtained by PCR, using primers 11-14
(pLUC-
3 and pLUC-
4). The internal deletion mutants (pCAT-
6 and
pCAT-
7) were constructed between Nae I and
ApaI sites (
106 to
271). To construct pCAT-
3 m1-3,
pCAT-
4 m1-3, pLUC-
m3, and pLUC-
m3, site-directed mutagenesis was performed by overlap extension, using primers 15 and 16. The positions of the upper strand sequences of mutations are indicated in
Fig. 1. The control vector of Wilms' tumor-1 gene were obtained by PCR
using primers 17 and 18. The plasmid vectors were prepared by CsCl
density gradient centrifugation (16).
Cell Culture Media and Conditions--
HepG2, HeLa, and HEK293
cells were grown in Dulbecco's modified Eagle's medium supplemented
with 10% fetal bovine serum (Life Technologies, Inc.).
Drosophila cell line Schneider S2 was maintained in
Schneider's Drosophila medium (Life Technologies, Inc.)
supplemented with 10% fetal bovine serum.
Transfections and CAT Assays--
At 20-24 h before
transfection, cells were replated into 60-mm dishes. Cells were
cotransfected with 5 µg of reporter construct and 1 µg of
CMV-
-galactosidase using the calcium phosphate precipitation technique (16). Duplicate tissue culture dishes for each construct were
transfected in each experiment. CAT activities were corrected for
-galactosidase activities. Aliquots of cell extracts were used for
the CAT and luciferase assays. The CAT assay was performed using Quanty
CAT assay kits (Amersham Pharmacia Biotech). CAT activity was counted
in a Beckman (Fullerton, CA) LS6000SC liquid scintillation counter. The
luciferase activity was measured in relative light units using a
Picagene luciferase assay kit and a Lumat LB 9507 (EG&G, Berthold, Germany).
Electrophoretic Mobility Shift and Antibody Supershift
Assays--
Nuclear extracts from HeLa cells were then prepared (17).
Two double-stranded synthetic oligonucleotides corresponding to nucleotides
210 to
182 and
239 to
210 of a common promoter of
HADHA and HADHB containing 5'-GC overhangs were
used as probes (Fig. 1, fragments A and B).
Electrophoretic mobility shift assays were done as described, but with
minor modification (18). 32P-End-labeled probes (5 fmol of
oligonucleotide, 15,000-20,000 cpm) were incubated with 5 µg of
nuclear extract from HeLa cells for 15 min at room temperature in 10 µl of reaction mixture containing 15 mM Hepes-KOH (pH
7.1) buffer, 0.3 mM EDTA, 60 mM KCl, 0.1 µg of poly(dI-dC), 1.2 mM dithiothreitol, 0.72 mM
MgCl2, and 10% glycerol. These reactions were
electrophoresed using 5% polyacrylamide gels in Tris-glycine buffer
(50 mM Tris, 380 mM glycine, and 2 mM EDTA) at 200 V for 90 min at 4 °C. The gel was
analyzed by autoradiography. Competition experiments were done using
oligonucleotides as follows (only one strand is shown): Sp1 consensus
oligonuceotide, 5'-ATTCGATCGGGGCGGGGCGCGAGC-3'; AP1 consensus
oligonucleotide, 5'-CGCTTGATGAGTCAGCCGCTAA-3'; and AP2 consensus
oligonucleotide, 5'-GATCGAACTGACCGCCCGCGGCCCGT-3'. The sequences of
mutated fragments A and B (A mutant and B mutant) are shown in Table I.
For some reactions we used 2 µl of commercially available
affinity-purified rabbit polyclonal antibody against human Sp1 (Santa
Cruz Biotechnology), and 10,000 cpm of 32P-labeled probe
(~30,000 cpm/ng) were added 5 min later. Next, binding for 20 min at
room temperature occurred, and the reactions were loaded on a 10%
glycerol, 5% polyacrylamide gel and run at 40 mA in 1 × Tris-borate EDTA buffer at 10 °C. The gel was analyzed by
autoradiography using a BAS 1500 bioimaging analyzer.
DNase I Footprinting--
The 276-bp-length probe was prepared
by PCR amplification using primers 19 and 20, including
BamHI and PstI sites, respectively. After
BamHI and PstI treatment, the probe was end
labeled by 32P. Footprinting reaction was performed in 50 µl of 20 mM Hepes-KOH (pH 7.5) buffer, 0.1 mM
EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 1 µg of poly(dI-dC), nuclear extract from HeLa cells (25 and 40 µg),
10% glycerol, and a 32P-end-labeled probe (5 × 104 cpm) at 4 °C for 15 min. DNase I (25 and 50 µg)
was then added followed by incubation for 40 s. The DNase I
reaction was terminated by adding stop solution, which included 100 mM Tris-HCl (pH 7.5), 0.375% SDS, 15 mM EDTA,
100 mM NaCl, and salmon sperm DNA (25 µg/ml). After
proteinase K treatment, phenol-chloroform extraction, and recovery by
ethanol precipitation, the DNA pellet was dissolved in 4 µl of
gel-loading buffer and separated on a denaturing 8 M urea,
6% polyacrylamide gel. The gels were dried and exposed to x-ray films
for 18 h. We used the Maxam-Gilbert sequence reaction to determine
accurate location of footprints.
 |
RESULTS |
Head-to-Head Arrangement of Human HADHA and HADHB
Genes--
Chromosomal localization of the human HADHA and
HADHB genes was previously identified in the same region,
2p23 (6, 7). Because the distance between the two loci was short (8),
we examined the structure of the region between the two loci. When
EMBL3 recombinant phages from a Sau3AI human leukocyte
genomic library were screened using as probe a 5' fragment of either
human HADHA (
23 to +428) or HADHB (
26 to
+204) cDNA, three different genomic DNA clones were isolated.
Sequencing revealed that these clones partially overlapped and covered
approximately a 15-kb region. Clones 2 and 3 carried both exon 1 of
HADHA and exon 1 of HADHB, on opposite strands in
approximately a 400-bp region, respectively (Fig.
1A), thereby indicating that
human HADHA and HADHB genes exist side by side in
a head-to-head arrangement.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 1.
Organization of HADHA and
HADHB genes in human genomic DNA. A, three
clones obtained from a EMBL3 human leukocyte genomic DNA library are
represented. Untranslated and coding regions of both genes are shown as
open and solid boxes, respectively.
Arrows indicate positions of oligonucleotides 1 and 2 used
for primer extension analyses. E and B are the
restriction enzyme sites of EcoRI and BamHI,
respectively. B, nucleotide sequence between exon 1 of
HADHA and exon 2 of HADHB. Exonic regions of both
genes are shown by capital letters, and translation
initiation codons of HADHA and HADHB are
boxed. Arrows and open arrowheads are
the major and minor transcription initiation sites, respectively, as
determined by primer extension analysis in Fig. 2. Nucleotide positions
are numbered for the sense strand of HADHA as the
major transcription initiation site to be +1, and
numbers in parentheses start from that of
HADHB. Sequences of Oligo 1 and Oligo
2, the regions protected in DNase I footprinting (I-V;
see Fig. 4) are also represented.
|
|
The sequence between exons 1 of the two genes is shown in Fig.
1B. The nucleotide sequence data reported in this paper will appear in the DNA Data Bank of Japan-EMBL-GenBank nucleotide sequence data bases under accession number AB020811. The translation initiation
codon ATG of HADHA was present in exon 1, whereas that of
HADHB was present in exon 2. The sequence of exon 1 of
HADHB and 5' flanking region (Fig. 1B,
369 to
333) corresponded to part of the 5' untranslated region of the
cDNA clone (Ref. 4;
45 to
9).
Determination of Transcription Initiation Sites--
To
determine the transcription initiation sites of the HADHA
and HADHB genes, primer extension experiments were done
using primers located in exon 1 of HADHA or in exon 2 of
HADHB (Fig. 1B, Oligo 1 and
Oligo 2, respectively). Using oligonucleotide 1 with total
RNAs from HeLa, HepG2, and HEK293 cells, respectively, three bands of
the extended cDNAs were detected at 86, 97, and 121 bp; the 86-bp
band is the major product in these cell lines (Fig.
2, left panel). The major
transcription initiation site of HADHA corresponded to the
5' end of the cDNA clone (4), as shown by arrow +1 in
Fig. 1B. Two other sites corresponded to positions
11 and
35, respectively (Fig. 1B).

View larger version (92K):
[in this window]
[in a new window]
|
Fig. 2.
Mapping of transcriptional initiation sites
for HADHA and HADHB. The transcription
initiation sites of human HADHA and HADHB were
determined by primer extension analyses using mRNA obtained from
HepG2, HEK293, and HeLa cells. All procedures are described under
"Experimental Procedures." Oligonucleotides 1 and 2 described in
Fig. 1 were used for HADHA and HADHB,
respectively. Lanes G, A, T, and C show a known
sequence ladder that we used as a molecular weight marker.
|
|
Six bands of extended cDNAs (60, 77, 86, 92, 100, and 107 bp)
appeared when using oligonucleotide 2 (Fig. 2, right panel); the positions of 5' ends of these fragments corresponded to nucleotide positions +20, +1,
9,
15,
23, and
30, respectively, in the HADHB cDNA clone (4). The main transcription initiation
site of HADHB seems to be position of the 77-bp band (Fig.
2, right panel), which is nucleotide position
351 relative
to the major transcription start site of the HADHA gene
(Fig. 1B). These findings was confirmed by reverse
transcription-PCR and ribonuclease protection assay (data not shown).
Determination of the Promoter Region(s) for HADHA and HADHB
Genes--
To examine promoter activities of the 5' flanking regions
of HADHA and HADHB genes, 1.2-kb (
1200 to +25,
pCAT-
1) and 2.2-kb (+1850 to
366, pCAT-
1) fragments were
connected to CAT cDNA, as shown in Fig.
3, and the constructs were transfected to
HeLa or HEK293 cells. The construct pCAT-
1 showed a 10- and 7-fold higher level of the reporter gene activity in HeLa and HEK293 cells,
respectively, when compared with the pCAT-Basic with no promoter.
pCAT-
1 exhibited a 12-fold higher expression of CAT in both cell
lines. Because promoter activity of the 5' flanking region of
HADHB exon 2 (pCAT-
8 and 9; Fig. 3) was low compared with
that of pCAT-
1 in both cell lines, the transcription band of
HADHB mRNA initiated at the 5' end of exon 2 in some
degree (Fig. 2 right panel) might be derived from a small
amount of nuclear precursor RNA, retaining intron 1, in the total RNA
sample.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 3.
Promoter activities of the 5' flanking
regions of HADHA and HADHB. Various CAT
vector constructs are shown on the left. The nucleotide
numbers indicated correspond with those in Fig.
1B, except pCAT- 12 and 13. Numbers in
parentheses indicate those against the major transcription
initiation site of HADHB in its exon 1 (pCAT- 1-11) or the 5'-end of HADHB
exon 2 (pCAT- 12 and 13).
Transfection of the constructs into HeLa (open bar) or
HEK293 (solid bar) cells was performed in triplicate, and
means of CAT activities were compared with that of pCAT-Basic.
|
|
Deletion analyses of the 5' flanking regions of the both genes were
done to define the active promoter regions. The promoter activity of
pCAT-
1 was at a similar level or increased until truncating between
positions
366 and
253 (pCAT-
5) in both cell lines. Furthermore,
significant decreases occurred when truncation to position
169 was
tested (pCAT-
9). On the other hand, the activity of pCAT-
1 was
completely retained by truncation to position +25 (pCAT-
4).
Truncation to position
238 led to a significant decrease in the
activity. Activities of internal deletion (
101 to
275) mutants
(pCAT-
10 and
11) were not above background in both cases. These
observations suggest that the region between positions +25 and
366,
being common to both genes, has promoter activity for both directions,
whereas the more limited region (
169 to
238) is important for
bidirectional promoter function.
Transcriptional Factors Binding to the Bidirectional Promoter
Region--
DNase I footprint analysis for the
169 to
238 region
was done using nuclear extract from HeLa cells (Fig.
4). Five protected regions appeared when
the 276-bp (
90 to
366) fragment was digested with DNase I in the
presence of the nuclear extract: region I, positions
185 to
190;
region II,
192 to
203; region III,
213 to
224; region IV,
230
to
240; and region V,
250 to
265. Protection in these regions was
enhanced by increasing the amount of nuclear extract.

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 4.
DNase I footprint analysis of the
HADHA and HADHB promoter. DNase I
footprint analysis of the region between nucleotide positions 169 and
238 was done as described under "Experimental Procedures."
Twenty-five or fifty micrograms of protein of nuclear extract
(NE) from HeLa cells were used. Protected segments are
diagrammatically shown to the right. Lane G
represents a chemical sequencing reaction of the fragment.
|
|
To confirm which protected regions have promoter activity for both
genes, we further analyzed several deletion mutants (pCAT-
4-9 and
pCAT-
6-10) (Fig. 5). Significant
decreases of promoter activities occurred when truncation to region II
for HADHA (pCAT-
8) and region IV for HADHB
(pCAT-
10) genes. These findings suggested that regions II and IV are
important for HADHA and HADHB, respectively.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 5.
Promoter activities of common 5' flanking
regions of HADHA and HADHB. Various
deletion mutants of CAT constructs are shown on the left.
The nucleotide numbers indicated correspond with those in
Fig. 1B. Transfection of the constructs into HEK293 cells
was done in triplicate, and means of CAT activities were compared with
that of pCAT-Basic. Protected regions I-V of DNase
footprint analysis are presented as solid boxes.
|
|
To confirm the binding of nuclear proteins to regions II and IV, gel
mobility shift assays were done using 32P-labeled synthetic
oligonucleotides, fragments A and B for regions II and IV,
respectively. The major complexes, C1 and C2, formed with fragments A
and B, respectively, disappeared in a dose-dependent manner
when unlabeled probes were added (Fig.
6). When the unlabeled oligonucleotides
containing mutations in regions II and IV were used as competitors, the
intensity of complexes C1 and C2 remained unchanged (Fig. 6).

View larger version (69K):
[in this window]
[in a new window]
|
Fig. 6.
Gel mobility shift assays of regions II and
IV. To confirm the binding of nuclear proteins to regions II and
IV, gel mobility shift assays were done using 32P-labeled
synthetic oligonucleotides, fragments A and B for regions II and IV,
respectively. Five micrograms of protein of nuclear extract from HeLa
cells were used. Sequences of oligonucleotides used as probes
(Normal) and competitors (Normal and
Mutant) are given at the bottom. The substituted
bases in mutants are shown by small letters. Amounts of
competitors used are 10-, 100-, and 400-fold molar excess,
respectively. The positions of major complexes C1 and C2 are indicated
on the right.
|
|
Both sites contain consensus binding sites for transcription factors
Sp1 and AP2 (Fig. 1B). When 400-fold molar excess
oligonucleotides of the Sp1, AP1, and AP2 consensus sites were used as
competitors, the Sp1 consensus oligonucleotide specifically competed in
formation of complexes C1 and C2. The AP1 consensus oligonucleotide did not compete, and the AP2 consensus oligonucleotide slightly inhibited the binding but only in fragment B (Fig.
7A). Binding of Sp1 to sites A
and B was confirmed in supershift experiments using a polyclonal
anti-Sp1 antibody. Electrophoretic mobilities of both complexes C1 and
C2 were slower in the presence of an antibody (Fig. 7B).

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 7.
Identification of transcription factors
binding to the regions II and IV. A, 400-fold molar excess
of unlabeled oligonucleotides of fragments A and B for regions II and
IV, respectively, and consensus binding sites of Sp1, Ap1, and Ap2 were
used as competitors. Regions II and IV bound with nuclear proteins. Its
major complexes, C1 and C2, respectively, disappeared by adding
unlabeled the Sp1-consensus oligonucleotide specifically. B,
supershift assay was performed using 1 µg of anti-human Sp1 antibody,
which recognizes Jun family proteins (c-Jun, JunB, and JunD). The
positions of complexes C1 and C2 are indicated on the right.
Open arrowheads indicate the complexes with antibody.
|
|
Regulative Function of Sp1 on the Bidirectional
Promoter--
Mutations were introduced at regions II and IV in
pCAT-
3 and pCAT-
4, respectively. Mutations at regions II and IV
were the same as for Fig. 6, to which Sp1 did not bind. Each of the
recombinants was transfected to HEK293 cells, and reporter gene
expression in the transfected cells was examined (Fig.
8). The promoter activity of pCAT-
3
decreased to 25, 40, and 25% in constructs mutated at regions II, IV,
and both, respectively. Similarly, the activity of pCAT-
4 was
reduced to 50, 40, and 40% by mutation at regions II, IV, and both,
respectively. Drosophila Schneider S2 cells lacking Sp1 were
used to examine the function of Sp1 on the bidirectional promoter (Fig.
9). Luciferase constructs were
transfected into the S2 cells with or without Sp1-expressing vector
(pCMV-Sp1). Without Sp1-expressing vector, luciferase constructs of
pLUC-
3,
4,
m3, and
m3 did not have promoter activities, as
seen in the control wild type Wilms' tumor-1 gene, the promoter
activity of which depends on Sp1 (19). Co-transfection of pCMV-Sp1 with normal constructs led to an 11- and 10-fold increase in expression of
pLUC-
3 and -
4, respectively, but to a <2-fold increase in the
expression of pLUC-
m3 and -
m3 (Fig. 9). These findings suggest that expressing of HADHA and HADHB genes depends
on Sp1, and that regions II and IV are "minimal" elements required
for expression.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 8.
Mutational evidence for the roles of regions
II and IV on the promoter. The 366 to +25 region was mutated at
regions II and IV using mutant oligonucleotides described in Fig. 6.
The CAT constructs represented on the left were
independently transfected into HEK293 cells, and CAT activity in the
transfected cells was assayed. Normal and mutant sequence within
regions II and IV are indicated by the open box and
X within the open box, respectively. Transfection
of constructs into HEK293 was performed in triplicate, and means of CAT
activities were compared with that of pCAT-Basic.
|
|

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 9.
Effect of Sp1 on promoter activity. The
constructs pLUC- 3, 4, Am3, and Bm3 were transfected into
Drosophila Schneider S2 cells without (open bar)
or with pCMV-Sp1 (solid bar). Transfection of the constructs
was triplicated, and means of luciferase activities were compared with
that of control vector pLUC-WT, Wilms' tumor-1 gene, regulated by
Sp1.
|
|
 |
DISCUSSION |
We obtained evidence that human HADHA and
HADHB genes encoding subunits of the multienzyme complex TP
exist side by side in a head-to-head arrangement on chromosome 2p23.
The multienzyme complex of fatty acid
oxidation in various
organisms has been identified. The complex consists of two subunits
with high amino acid sequence homology to human TP (3, 20, 21). In
prokaryotes, two genes for the enzyme complex fadA and B in
Escherichia coli and faoA and B in
Pseudomonas fragi, which are multienzyme complexes similar
to human TP, are tandemly arranged on the bacterial operon (9, 20, 21).
Head-to-head arrangement of human TP genes differs from these cases and
may have occurred by gene inversion with during processes of evolution.
The structure of the HADHA and HADHB locus is unique.
The intergenic region between exons 1 of HADHA and
HADHB has bidirectional promoter function regulated by
transcription factor Sp1. The two Sp1 sites are critical for promoter
expression of both genes, but they are not the minimal elements
required for expression. Thus, this region probably functions as a
common promoter of the two genes. In our foregoing reports, we stated
that the association of both subunits is required for stabilization in mitochondria (14, 15). The coordinated gene expression of HADHA and HADHB, regulated by the common
promoter, seems appropriate for the association and for subsequent
stabilization. However, levels of individual subunit mRNA in organs
are not always similar (3, 13). Promoter activities of HADHA
constructs are different in HeLa and HEK293 cell lines (Fig. 3).
Therefore, other regions and/or other transcription factors may be
involved in regulating the cell type-specific expression of the two genes.
Promoters having bidirectional functions have been identified in
surf-1 and surf-2 of the surfeit locus (22), the murine and human
collagen IV genes a1 and a2 (23, 24), histone H2A and
H2B genes (25), DHFR and Rep-1 genes
(26, 27), and the Wilms' tumor locus (28). These pairs of genes are
coordinately expressed in general, and their gene products have similar
functions. HADHA and HADHB genes code for
proteins different in structure and function, although the proteins
belong to the same metabolic system, fatty acid
oxidation, and form
an enzyme complex. This character differs from the gene pairs described
above; hence, the HADHA-HADHB gene pair is unique.
Expression of the mitochondrial medium-chain acyl-CoA dehydrogenase
gene is regulated by nuclear receptor response elements, which bind
with orphan members of the steroid-thyroid nuclear receptor
superfamily, and Sp1 (29). In addition, it is known that several genes
in fatty acid
oxidation systems, such as peroxisomal acyl-CoA
oxidase (30), peroxisomal bifunctional enzyme (31), and mitochondrial
acetoacyl-CoA thiolase (32), have GC boxes and lack typical TATA and/or
CAAT boxes in their putative promoter regions (29). Thus the expression
of many genes at
oxidation of fatty acids seems to relate to Sp1.
It was reported that induction of TP in rat liver by the administration
of di-(2-ethylhexyl) phthalate, one of the peroxisome proliferators,
was 10-fold (1). Using peroxisome proliferator-activated receptor
null mice, it has just been reported that expression of
HADHA and HADHB genes in the liver is enhanced
coordinately by administration of a potent hypolipidemic reagent,
Wy-14,643, and that these enhancements are mediated by peroxisome
proliferator-activated receptor
(33). In our numerous experiments
done to identify peroxisome proliferator responsive elements on both
the HADHA and HADHB genes, we found no active
element binding with peroxisome proliferator-activated receptor
, at
least in 15-kb regions sandwiching the common promoter region (data not
shown). Thus, the mechanism of peroxisome proliferator-activated
receptor
-dependent induction of HADHA and
HADHB expression remains to be determined.