From the
The human alcohol dehydrogenase 5 gene ( ADH5) differs
from all other human alcohol dehydrogenase genes in its ubiquitous
expression, although there are tissue-specific differences in the level
of expression. To understand the expression of ADH5, we
characterized the structure and function of its 5` region by DNase I
footprinting and transient transfection assays. The region from base
pair (bp) -34 to +61, flanking the major transcription start
site, had strong promoter activity in three different cell lines: HeLa,
H4IIE-C3, and CV-1, and could explain the ubiquitous expression. Two
Sp1 sites within that region are footprinted by nuclear extracts from
all tissues and cells tested. There are sites further upstream that
show cell- and tissue-specific differences in both their patterns of
occupancy and their effects on promoter activity. The region between bp
-34 and -64 strongly increases promoter activity in
H4IIE-C3 cells, weakly activates in CV-1 cells, but has no effect in
HeLa cells. The region between bp -127 and -163 is a
positive element in both HeLa cells and CV-1 cells, but is a negative
regulatory element in H4IIE-C3 cells. These differences in part explain
the levels of expression of ADH5 in various tissues. Two
regions (bp -64 to -127 and bp -163 to -365)
contain negative regulatory elements that reduce promoter activity in
all three cells. The 5`-nontranslated region of ADH5 contains
two upstream ATGs. Insertion of 12 bp within the putative coding region
of these upstream ATGs led to a 1.6-2.3-fold increase in
activity. This suggests that the 5`-nontranslated region has regulatory
significance.
The human ADH5 gene encodes
ADH5 was recently cloned in
our laboratory
(18) . The 5` nontranslated region of ADH5 is unusual; it contains two upstream ATG codons that are in frame
with each other but out of frame with the
pCAT-X-1342 contains the 1.4-kb FokI fragment of
ADH5 subcloned into pCAT Basic at a blunted XbaI site
in front of the CAT gene; it contains ADH5 sequences extending
from bp -1342 to +61 relative to the transcription
initiation site. The pCAT-X series of plasmids was derived from
pCAT-X-1342 by 5` nested deletions using exonuclease III or using
restriction endonucleases followed by Klenow treatment to create blunt
ends before subcloning. All pCAT-X plasmids extend to bp +61;
their 5` ends are at the positions designated in their names ( i.e. pCAT-X-163 contains from bp -163 to +61 of the ADH5
promoter; see Fig. 2). pCAT-AA was created by cloning the AvaI
restriction fragment (bp -726 to -61) into pCAT Basic at
the PstI site (blunted).
pCAT-H-1342 contains the same
1.4-kb FokI fragment of ADH5 as in pCAT-X-1342 (bp
-1342 to +61), except that it is subcloned into pCAT Basic
at a blunted HindIII site in front of the CAT gene. pCAT-H-253
was made from pCAT-H-1342 by 3` deletion as described above; it
contains ADH5 sequences from bp -1342 to -253
(Fig. 2).
DNase I
digestion and electrophoresis were as described previously
(23) . Nuclear extract (10-40 µg), probe (40,000 cpm)
and poly(dI
15 µg of pCAT-X-1342 (or the molar
equivalent of other plasmids), 2 µg of pCMV-Luc, and sufficient
pUC18 to bring the total DNA to 25 µg were transfected into the
cells by the CaCl
Luciferase activity was used as an internal control to normalize the
plate to plate variation in transfection efficiency. Luciferase
activity was assayed
(19) using 30 µl of each supernatant
in 370 µl of assay buffer (25 m
M Gly-Gly (free base), 15
m
M MgCl
CAT assays
(25) were
conducted by incubating cell extracts containing 20,000 relative light
units of luciferase activity in 0.25
M Tris-HCl (pH 7.8), 50
m
M acetyl-CoA, 75 nCi
[
There are consensus sequences for Sp1
(29, 30) and
Sp1-related factors
(31, 32, 33) and for AP-2
(34) clustered around the transcription start point
(Fig. 1); these factors create footprints in this region (see
below). There are consensus sequences
(35) for C/EBP and other
CCAAT-binding proteins, and for HNF5, which might be involved in the
higher expression of ADH5 in liver. Consensus sequences for
other nuclear transcription factors, e.g. E-boxes, AP1, SRE,
and XRE, are also found. It is interesting that there are five regions
that contain groups of heat shock elements. Normally three or more
clustered nGAAn sites are needed; the group of sites from -290 to
-258 is the most likely of these to be functional. There are as
yet no data about the induction of ADH5 by heat shock, but
since the protein is protective against formaldehyde and other
potential toxins, this might be physiologically relevant.
In H4IIE-C3 cells, pCAT-X-1342 produced high levels
of CAT activity, 42% of that obtained with the SV40 promoter plus
enhancer construct pCAT Control (Fig. 3 A). Deletions
extending down to bp -365 had little effect on CAT activity. The
next three deletions each increased CAT activity: pCAT-X-163 showed 57%
more CAT activity than pCAT-X-365, pCAT-X-127 showed 65% more activity
than pCAT-163, and pCAT-X-64 showed 39% more activity than pCAT-X-127.
pCAT-X-127 and pCAT-X-64 were more active than the SV40
promoter/enhancer combination in pCAT Control. Further deletion to
-34 bp (pCAT-X-34) reduced activity 4-fold. This smallest
promoter tested, pCAT-X-34, was nearly as active as the entire 1.4-kb
fragment contained in pCAT-X-1342. pCAT-H-253, a derivative of
pCAT-H-1342 in which the promoter is deleted from the 3` end to bp -253
was not active (data not shown).
In CV-1 cells, as in the other cells tested, pCAT-X-1342
produced high levels of CAT activity, 44% of that obtained with pCAT
Control (Fig. 3 C). The effects of the deletions differed from
those seen in either H4IIE-C3 or HeLa cells. Deletions extending to bp
-583 did not substantially affect the activity of the ADH5 promoter, and further deletions to bp -365 increased
activity slightly. In CV-1, as in the other two cell lines, deletion of
the region between bp -365 and -163 increased activity by
58%. Deletion of the region from bp -163 to -127 decreased
activity 2-fold. Deletion to bp -64 increased activity by 55%,
and further deletion to bp -34 decreased activity by 31%.
pCAT-H-253 was not active (Fig. 3 C).
Extracts from mouse liver, kidney, and spleen showed
footprints B, C, and D at bp +3 to +22, bp -38 to
-4 and bp -57 to -40, respectively (Fig. 5,
A and B). Liver showed a cluster of hypersensitive
sites from bp +24 to +27 (Fig. 5 A). The pattern in
mouse brain extract is more similar to that of the other cells when 20
µg of extract was used (data not shown; it did not footprint well
in Fig. 5 A, lane 7, where only 10 µg of brain
extract was used). Among these tissues, only kidney showed footprint A
(bp +32 to +61), and only on one strand (Fig. 5, A versus
B). Further upstream, additional DNase I footprints were detected
(Fig. 5 C). There were differences among the cell lines.
CV-1 showed a strong footprint J (bp -384 to -343) that was
not seen with other extracts (Fig. 5 C). Footprint I (bp
-318 to -284) just downstream of that is seen in liver
extracts and also in CV-1 and H4IIE-C3 extracts.
Fig. 7
summarizes the footprints. There are differences in the
boundaries of the footprints, e.g. the footprint in the region
from bp -57 to -40 is slightly shorter in brain and spleen
than in liver and kidney (Fig. 5 A, lanes 7-10).
Footprint F (bp -151 to -112) is seen in liver extract but
not seen with the other tissue extracts; a slightly smaller footprint F
(-151 to -118) is detected in CV-1 and H4IIE-C3 extracts
but not in HeLa cells.
Given the high G
+ C content and presence of Sp1 consensus sequences, we tested the
binding of Sp1 and AP-2 to this region of the ADH5 promoter.
Sp1 produced four footprints; two strong footprints, B and C,
correspond to footprints seen in the cell extracts. To further test
whether Sp1 was the protein responsible for the footprinting of sites B
and C, gel mobility shift assays were carried out using antibodies to
Sp1. Fig. 6 A shows that purified Sp1 can bind to site B
( lane 8) but not to a mutated site B ( lane 12).
Antibody to Sp1 shifts the retarded band to lower mobility. The
protein(s) in the HeLa extract also bind specifically to site B, as
shown in lane 4, and most of the retarded band is shifted to
lower mobility by the antibody to Sp1, indicating that Sp1 is indeed
the major protein binding to the site. The small residue of unshifted
band might be due to insufficient antibody or to the presence of a
second protein able to bind to the oligonucleotide. The mobility of the
shifted band in the HeLa extract is slightly greater than that due to
purified Sp1; this might be due to post-translational modifications in
the protein, since the Sp1 gene product can produce bands with
mobilities approximating 95 and 105 kDa. Fig. 6 B shows
that the predominant protein that binds to site C is also Sp1; again a
fraction of the band does not shift with antibody, and again the
mobility of the shifted band in the HeLa extract is slightly greater
than that due to purified Sp1, but similar to that which binds to site
B. These data demonstrate that the bulk of the protein binding to sites
B and C in the HeLa extract is Sp1.
The human ADH5 gene has characteristics of both a
housekeeping gene and a tissue-specific gene. It is expressed in all
tissues and in all stages of development, but at higher levels in liver
and kidney. The data presented here demonstrate that although the
proximal promoter of ADH5 has some characteristics of a
housekeeping gene, particularly a G + C-rich and TATA-less
promoter, it also contains several tissue-specific cis-acting
elements that affect expression differently in different cellular
contexts.
The region from bp
-163 to -128 is another cell-specific element; it acts as
positive element in CV-1 and HeLa cells, causing the 2-2.8-fold
higher CAT expression of pCAT-X-163 compared with pCAT-X-127
(Fig. 3, B and C). Surprisingly, this region
down-regulated gene expression in H4IIE-C3 cells by 39% (Fig.
3 A). Thus the sequences between bp -163 and -128
bp can act as either a positive or negative regulatory element in
different cellular contexts. The cell-specific function of this element
suggests that it is bound by different transcription factors in
different cells or that the factor(s) bound interact differently with
other tissue-specific factors also bound to the promoter. We observed a
footprint in the region from bp -to -130 bp in CV-1 and
H4IIE-C3 extracts and a larger footprint from bp -151 to
-112 in liver extracts (Fig. 5 C). This region
contains a consensus sequence for C/EBP or other CAAT-binding proteins
(35) .
The region between bp -163 and
-365 reduced CAT activity to the level of pCAT-X-34 in all three
cell lines tested (Fig. 3). This region contains several
footprinted sequences (Figs. 5 and 7). Regions further upstream did not
substantially change promoter activity in H4IIE-C3 or CV-1 cells
(Fig. 3, A and C), but in HeLa cells there was
a modest stimulation by sequences located between bp -464 to
-583 and between bp -837 to -1342
(Fig. 3 B).
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank/EMBL Data Bank with accession number(s) U10902.
We thank Ronald E. Jerome for excellent technical
assistance with tissue culture, Lu Zhang and Jinghua Zhao for
assistance with the antibody shift experiments, and Dr. Celeste Brown,
Dr. Mang Yu, and Lu Zhang for their helpful advice.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-alcohol
dehydrogenase (EC 1.1.1.1), which is expressed in all tissues tested
and at all stages of development. This makes
-alcohol
dehydrogenase unique among the mammalian alcohol dehydrogenases, since
the others are all are expressed in tissue-specific patterns
(1, 2, 3, 4, 5, 6, 7) .
The level of expression of
-alcohol dehydrogenase differs among
different tissues, however. Human liver and kidney show two to three
fold higher levels of
-alcohol dehydrogenase expression than the
other tissues. In rodents, baboons, and horses the difference in
expression is even greater
(1, 2, 3, 4, 5, 6) .
-Alcohol dehydrogenase also differs from other mammalian
alcohol dehydrogenases in its substrate specificity.
-ADH
(
)
most efficiently oxidizes long chain
alcohols and
-hydroxyfatty acids
(8, 9, 10, 11) . It is also the
NAD
and glutathione-dependent formaldehyde
dehydrogenase (EC 1.2.1.1), as shown by the identity in partial amino
acid sequence and in structural and enzymatic properties of the
homologous rat liver alcohol dehydrogenase and formaldehyde
dehydrogenase
(12) .
-Like alcohol dehydrogenases are
highly conserved during mammalian evolution
(13, 14, 15, 16) . Escherichia coli contains an alcohol/formaldehyde dehydrogenase with 60% amino acid
sequence identity
(17) , indicating much longer evolutionary
conservation. This suggests
-like alcohol dehydrogenases play
important physiological roles.
-ADH coding sequence
(18) . The high conservation and presumed physiological
importance of
-ADH, the quantitative differences in expression in
different tissues, and the unusual 5` nontranslated region all make
ADH5 an interesting gene to study. To analyze the molecular
mechanisms underlying the expression of ADH5 in different
tissues, we used DNase I footprinting to detect cis-acting
elements. We carried out functional assays of the promoter using
transient transfections of a reporter gene into three different cell
lines: H4IIE-C3 (a rat hepatoma line), HeLa (a human cervical carcinoma
line), and CV-1 (a monkey kidney fibroblast line). We found that a
small promoter region functions well in all three cell lines, and we
detected multiple positive and negative regulatory regions further
upstream. Several cis-acting elements act as positive
regulatory elements in one cell line but as negative regulatory
elements in another. We also showed that a minor alteration in the
5`-nontranslated region can have a significant effect on gene
expression.
Reagents
All enzymes were purchased from Life
Technologies, Inc., Promega (Madison, WI), and Boehringer Mannheim. The
buffers for restriction enzymes were supplied by the manufacturers.
Poly(dIdC) was purchased from Boehringer Mannheim and Pharmacia
Biotech Inc. The exonuclease III nested deletion kit and
phosphorothioate dNTP were purchased from Promega (Erase a Base System,
Promega). Other nucleotides were purchased from Pharmacia.
[
-
P]ATP and
[
-
P]dNTPs were purchased from DuPont.
Silica gel TLC plates were purchased from Eastman Kodak Co. or J. T.
Baker (Baker Flex; Phillipsburg, NJ).
Plasmids and DNA Sequencing
pCAT Control and pCAT
Basic were purchased from Promega. pCAT Control contains the SV40
promoter, enhancer, and CAT coding sequence. pCAT Basic contains the
CAT coding sequence but lacks a eukaryotic promoter and enhancer.
pCMV-Luc
(19) contains a firefly luciferase gene driven by a
Cytomegalovirus (CMV) promoter in a vector called pCDNAI (Invitrogen);
it was obtained from Dr. Y.-C. Yang (Indiana University School of
Medicine).
Figure 2:
Schematic diagram of the pCAT constructs,
cloned into pCAT Basic. A, the pCAT Basic vector (Promega),
showing the location of the cloning sites relative to the CAT coding
region and eukaryotic 3` splice and polyadenylation signals.
B, diagram of the inserts. The pCAT-X series contains the
inserts in the XbaI site (blunted), the pCAT-H series contains
the inserts in the HindIII site (blunted), and pCAT-AA
contains the AvaI fragment cloned at the PstI site
(blunted).
The deleted plasmids were sequenced from both
direction using primers flanking the insertion site (HE13,
CAGGAAACAGCTATGACC and HE66, CAACGGTGGTATATCCAGTG). Two additional
oligonucleotides were used to sequence regions originally obtained in
only one direction.
Nuclear Extracts and Transcription Factors
Nuclear
extracts were prepared from liver, kidney, spleen, and brain of
C57BL/6J mice according to Gorski et al. (20) . Nuclear
extracts from cultured cells (CV-1, H4IIE-C3) were made according to
Shapiro et al. (21) . HeLa cell nuclear extract, Sp1,
AP-2, and TFIID were purchased from Promega. Antibodies to Sp1
(Sp1(PEP2)) were obtained from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA).
DNase I Footprinting Assays
To examine the
proximal region, pCAT-H-1342 was digested with NarI (at bp
-128), dephosphorylated with calf intestine alkaline phosphatase,
and labeled with polynucleotide kinase and
[-
P]ATP. After removal of the free
nucleotides by Sephadex G25 spin column chromatography
(22) ,
the DNA was digested with XbaI (a restriction site in the
polylinker). For footprinting from the opposite end of the fragment,
the order of digestion was opposite, so the XbaI site was
labeled. The NarI to XbaI fragment was purified by
anion exchange HPLC column chromatography (Gen-Pak Fax column, Waters,
Milford, MA). For footprinting upstream of the NarI site, the
AvaI fragment from pCAT-X-1342 was dephosphorylated, kinased
with [
-
P]ATP, and digested with
HinfI; the 331-bp fragment (bp -402 bp to -61) was
isolated by HPLC as described above. For footprinting in opposite
direction, the AvaI fragment was digested with HinfI
and HaeIII, the resulting HinfI to AvaI
fragment was separated, dephosphorylated, kinased, and further digested
with EcoRII. The HinfI/ EcoRII fragment was
separated by HPLC and used for the DNase I footprint assays.
dC) (1-2 µg) were mixed in binding buffer (10
m
M HEPES (pH 7.9), 60 m
M KCl, 1 m
M EDTA, 7%
glycerol, 0.2 m
M dithiothreitol) and incubated at room
temperature for 15 min. 5 µl of DNase I (0.2-0.4 unit) was
added and incubated for 2 min. When extract was omitted, 0.04 unit of
DNase I were used. For purified transcription factors and their control
reactions, 60 ng of factors (Sp1, AP-2, TFIID), 0.004 unit of DNase I,
and no poly(dI
dC) were used. DNase I digestion was stopped by
adding 75 µl of DNase I stop solution (20 m
M Tris (pH
7.5), 20 m
M EDTA, 5 m
M EGTA, and 5 µg/ml yeast
tRNA). The mixture was extracted once with phenol/CHCl
and
precipitated by the addition of 0.5 volume of 7.5
M ammonium
acetate and 2.5 volume of ethanol. The pellets were resuspended in 5
µl of sequencing loading buffer. The digested DNA (10,000 cpm) was
electrophoresed in a 6% polyacrylamide/7
M urea sequencing gel
(Life Technologies, Inc.) for about 50 min. An autoradiogram was
obtained by exposing the dried gel to film (XAR-5, Eastman Kodak Co.)
overnight at -70 °C with a Quanta III image enhancing screen
(DuPont).
Gel Mobility Shift Assays
Gel mobility shift
assays were carried out as described previously
(23) . The site
B oligonucleotide was a double-stranded oligonucleotide with the
sequence (top strand) TAGGCGCTCGCCACGCCCATGCCTCCGTC. The mutant site B
oligonucleotide had the sequence TAGGCGCTCGCTCTAGATATGCCTCCGTC. The
site C oligonucleotide was a double-stranded oligonucleotide with the
sequence (top strand) CCCCCACGCCCCGCCCCCCTCGCTAGGCGC. The mutant site C
oligonucleotide had the sequence CCCCCCACGCAAGATCTCACTCGCTAGGC. One
strand of each oligonucleotide was labeled using polynucleotide kinase
as described above, and the complementary oligonucleotides were
annealed. Each assay contained (in 20 µl) 10 m
M HEPES (pH
7.9), 60 m
M KCl, 1 m
M dithiothreitol, 1 m
M EDTA, 7% glycerol and the appropriate extract (5.2 µg of HeLa
nuclear extract or 1 footprinting unit of purified Sp1. Where
indicated, 0.5 or 2.0 µl of unlabeled competitor oligonucleotide
(at 4 pmol/µl) or 1 µg of antibody against Sp1 was added to the
incubation. Electrophoresis was at 80 V for 4 h in 4% polyacrylamide
gel with 0.167 TAE buffer (1
TAE, 40 m
M Tris,
20 m
M acetic acid, 1 m
M EDTA).
Transient Expression Assays
CV-1 (African green
monkey kidney cells), H4IIE-C3 (rat hepatoma cells), and HeLa (human
cervical carcinoma cells) were grown on 10-cm dishes in minimal
essential medium (Life Technologies, Inc.) supplemented with 5% fetal
calf serum (CV-1 and HeLa) or in SWIM'S medium (Sigma)
supplemented with 10% fetal calf serum (H4IIE-C3). Medium was replaced
4 h before the transfections.
-DNA coprecipitation method
(24) .
The DNA was allowed to remain on the cells for 4 h (CV-1, HeLa cells)
or 18 h (H4IIE-C3 cells), then the medium was removed and replaced for
2 min with medium containing either 20% (CV-1, HeLa) or 15% (H4IIE-C3)
glycerol. The glycerol-containing medium was replaced with the
appropriate growth medium, and incubation was continued for a total of
48 h. The plates were gently washed four times with phosphate-buffered
saline, the cells were harvested, and cell pellets were resuspended in
150 µl of lysis buffer (100 m
M K
PO
(pH
7.8), 1 m
M dithiothreitol) and broken by sonication.
, 5 m
M ATP). 100 µl of
luciferin (6 mg/ml) was added automatically, and activity was measured
as relative light units for 10 s in a LB9501 Luminometer (Berthold
Analytical Instruments, Nashua, NH).
C]chloramphenicol, 5 m
M EDTA in 200
µl at 37 °C for 2.5 h. The acetylation of chloramphenicol was
analyzed by silica gel thin layer chromatography
(25) and
quantitated with the AMBIS Radioanalytic Imaging System (AMBIS, San
Diego, CA). CAT activity is expressed as percent chloramphenicol
acetylated per 20,000 luciferase units (average of three to four
independent experiments). When CAT activity was high, smaller aliquots
were reassayed for shorter times to keep the reactions in the linear
range, and the results were normalized to 2.5 h assays containing
20,000 relative light units of luciferase.
Sequence of the 5` Region of ADH5
The proximal
promoter region does not contain either a TATA box or a CCAAT box (Fig.
1). It is very high in G + C content (73% in the first 200 bp
upstream of the translation initiation codon and 61% in the 358 bp
upstream region). This region has the characteristics of a CpG island
(26, 27) : CpGs are found approximately as often as
expected from the base composition, and CpGs are about equal in
frequency to GpCs
(18) . These are characteristics of the
promoters of housekeeping genes. ADH5 is the only human
alcohol dehydrogenase gene with these characteristics
(28) .
Figure 1:
DNA sequence of the
5`-upstream region of ADH5, numbered at the right.
The main transcription start sites are at +1 (marked) and +3.
The boundaries of the deletion mutants are in uppercase
letters, underlined, and numbered above. Several
consensus sequences are in uppercase letters and indicated
above.
Promoter Activity in Three Cell Lines
We prepared
a series of plasmids in which various portions of the ADH5 promoter were placed in front of the cat gene in the
vector pCAT Basic (Fig. 2). We carried out transient expression
assays in three different cell lines (H4IIE-C3, HeLa, and CV-1) to
determine the extent to which each promoter fragment could direct
transcription.
Figure 3:
Promoter activity of ADH5 in
different cells. A, H4IIE-C3 cells (four independent
experiments). B, HeLa cells (four independent experiments).
C, CV-1 cells (three independent experiments). Black
columns indicate the CAT activity of ADH5 promoter
constructs as percent conversion (average percent acetylated
chloramphenicol per 20,000 relative light units of luciferase
activity); lines show the standard error of the mean. Smaller
aliquots were assayed for extracts with high CAT activity to keep the
results in the linear range, and the data were normalized to luciferase
activity, as detailed under ``Experimental Procedures.''
In HeLa cells, pCAT-X-1342 produced
high levels of CAT activity, 31% of that obtained with pCAT Control
(Fig. 3 B). The effects of deletions differed from those
seen in H4IIE-C3 cells. Deletion of the region between bp -1342
and -838 and between bp -583 and -465 reduced CAT
activity by 15 and 25%, respectively. Deletion of the region between bp
-365 and -163 increased activity 77%. Deletion of the
region from bp -163 to -127 decreased activity 3-fold, and
there was a 27% increase in activity upon further deletion to bp
-64. pCAT-H-253 was not active (Fig. 3 B and data
not shown).
Effect of 5`-Nontranslated Region on CAT
Activity
Both pCAT-X-1342 and pCAT-H-1342 contain the same
1.4-kb promoter fragment, cloned into different positions in the
polylinker of pCAT Basic (see ``Experimental Procedures'').
Both contain the two upstream ATGs that are found in the ADH5 gene itself
(18) , followed by in-frame (and also out of
frame) termination codons, as they are in ADH5. pCAT-H-1342
has an additional 12 nucleotides (from the polylinker) inserted within
the putative coding region of these upstream ATGs (Fig. 4). Both
upstream ATGs are out of frame with the ATG of the cat gene,
just as they are out of frame with the ATG of ADH5 (18) . In all three cell lines, pCAT-H-1342 directed from
1.6- to 2.3-fold more CAT activity than did pCAT-X-1342 (Fig. 4).
Figure 4:
Effects of
an altered 5`-nontranslated region on CAT activity. A,
sequences upstream of the cat gene. X =
pCAT-X-1342, H = pCAT-H-1342, which has an additional
12 nucleotides ( underlined). Both pCAT-X-1342 and pCAT-H-1342
contain two upstream ATGs ( bold) at +16 and +46 bp
relative to the major ADH5 transcription start site, followed
by an in-frame termination codon TAG (+79 bp; marked with
three asterisks). There are additional termination codons
( bold) at +88, +112, and +121 bp. B,
CAT activity of pCAT-X-1342 and pCAT-H-1342 in three cell lines,
normalized to the activity of pCAT Control (containing the SV40
promoter and enhancer) in each cell line. Solid boxes,
pCAT-H-1342; striped boxes, pCAT-X-1342; shaded box (near .00), pCAT Basic (no eukaryotic promoter);
lines, standard error of the mean.
Does ADH5 Have a Dual Promoter?
Primer extension
assays
(18) showed two major transcription start sites (bp
+1 and +3 in Fig. 1) and one minor transcription start
site (at bp -82). The minor start site suggested that ADH5 might have a second promoter upstream. To investigate this,
pCAT-AA was analyzed. pCAT-AA contains sequences from bp -726 to
-61 bp, which includes the putative upstream transcription start
site at bp -82 but not the major downstream sites at bp +1
and +3. pCAT-AA showed no promoter activity in any of the cell
lines tested (Fig. 3).
cis-Acting Elements in the Proximal ADH5
Promoter
To identify the location of cis-acting
elements in the proximal ADH5 promoter, we carried out DNase I
footprinting assays
(36) on fragments spanning the region from
bp -401 to +61 relative to the major transcription start
point of ADH5 (18) . Footprinting experiments were
carried out using nuclear extracts prepared from the cell lines used
for transfection studies and from various mouse tissues; we also
examined the binding of purified transcription factors Sp1, AP-2, and
TFIID.
Figure 5:
DNaseI footprinting of the proximal
promoter region of ADH5. A, NarI to
XbaI restriction fragment (from -127 bp to +61 bp
labeled at NarI site). B, NarI to
XbaI restriction fragment labeled at XbaI site.
C, HinfI to AvaI restriction fragment
(-401 bp to -61 bp) labeled at AvaI site.
Boxes at left indicate footprints in nuclear
extracts; boxes at the right indicate footprints of
purified Sp1 protein or AP-2 protein. Arrows indicate DNase I
hypersensitive sites.
Figure 7:
Summary of DNase I footprints and their
effects on gene expression. Boxes indicate footprints (see
Fig. 5); solid boxes are footprints seen in liver;
stippled boxes A and J are seen in other tissues. The
arrows beneath reflect the regions tested in successive
deletion mutants (Fig. 3); their effects on CAT activity are
represented as + for positive, 0 for no effect, and - for
negative effect.
Nuclear extracts prepared from H4IIE-C3,
HeLa, and CV-1 produced two strong footprints, B and C, immediately
flanking the transcription start point (Fig. 5, A lanes
4-6, and B, lanes 3-5). There are
hypersensitive sites right around the transcription start site
(+1) in all tissues except brain. There are two weaker footprints
flanking B and C: D from bp -57 to -40 and A from bp
+32 to +61 (Fig. 5, A and B). The
HeLa extracts produced the strongest footprints B and C and looked most
like the pattern seen with purified Sp1 (see below). Both H4IIE-C3 and
CV-1 produced several footprints upstream of bp -60, whereas HeLa
cells did not. As was seen for different tissues, the patterns of
protection differ slightly among the extracts from different cells
(Fig. 5). Footprint J was seen in CV-1 cells.
Figure 6:
The major protein binding to sites B and C
is Sp1. A, double-stranded oligonucleotide containing site B
was used in gel mobility shift assays with purified Sp1 and HeLa cell
extract. Lanes 1-9, site B oligonucleotide; lanes
10-12, mutant site B oligonucleotide. Lane 1, no
extract. Lane 2, HeLa extract plus 200-fold unlabeled
competitor site B oligonucleotide. Lane 3, HeLa extract plus
50-fold unlabeled competitor site B oligonucleotide. Lane 4,
HeLa extract. Lane 5, HeLa extract plus 1 µg of antibody
to Sp1. Lane 6, purified Sp1 plus 200-fold unlabeled
competitor site B oligonucleotide. Lane 7, Sp1 plus 100-fold
unlabeled competitor site B oligonucleotide. Lane 8, Sp1.
Lane 9, Sp1 plus 1 µg of antibody to Sp1. Lane
10, mutant site B oligonucleotide without extract. Lane
11, mutant site B oligonucleotide with HeLa extract. Lane
12, mutant site B oligonucleotide with Sp1. B,
double-stranded oligonucleotide containing site C was used in gel
mobility shift assays with purified Sp1 and HeLa cell extract. All
lanes are as in A except that the oligonucleotides are site C
and mutant site C. The rapidly migrating retarded band visible in
lanes 2-5 is nonspecific, as shown by the lack of
self-competition in lanes 2 and
3.
The pattern of AP-2 binding
differs from that seen in any of the cell lines. AP-2 protects both
strands at site A; this differs from the footprinting on only one
strand seen in the cell extracts. AP-2 also produces weak footprints in
sites extending from bp -66 to +27, partially overlapping
footprints produced by various extracts and Sp-1. TFIID did not produce
detectable footprints under our conditions.
A Minimal Promoter Containing Two Sp1 Sites May Direct
Ubiquitous Expression
A small region between bp -34 and
+61 (contained in pCAT-X-34) has surprisingly strong promoter
activity in all three cell lines tested. Its activity in these cells
ranges from 18 to 46% of the activity of the SV40 promoter +
enhancer combination. This small region contains two Sp1 sites flanking
the major transcription start site (sites B and C) that were
footprinted by all nuclear extracts (Fig. 5). These two sites are
bound strongly by purified Sp1 protein. The protein in HeLa extract
that binds to these sites is Sp1, as shown by antibody double-gel
shifts (Fig. 6). In the absence of a TATA box, Sp1 bound to this
unusual arrangement of sites might directly stimulate transcription by
actively recruiting the TFIID complex to the promoter
(37, 38) . Tazi and Bird
(39) showed that the G
+ C-rich promoters of housekeeping genes are depleted of histones
and actively transcribed. Croston et al. (40) suggested that Sp1 can act as an antirepressor by actively
displacing histone. Sp1 or related factors might also displace histones
from the ADH5 promoter region. Sp1 is expressed in all
tissues, but the mRNA level can vary as much as 100-fold
(30, 41) . Thus, Sp1
(30, 42) or
Sp1-related proteins
(31, 32, 33) may be
important in the widespread expression of -ADH.
Cell-specific Effects of cis-Acting Promoter
Elements
Upstream of this minimal promoter, there are cell- and
tissue-specific differences in both the binding of transcription
factors and the effects of the cis-acting sequences on gene
expression (Fig. 7). For example, the region containing site D
had different effects in different cells. Site D was footprinted by all
nuclear extracts and strongly by Sp1 (Fig. 5, A and
B). Its effect on promoter function, however, differed. In
H4IIE-C3 cells, it was a strong positive regulatory element,
stimulating CAT expression 5-fold ( cf. pCAT-X-64 and
pCAT-X-34, Fig. 3 A). pCAT-X-64 was a better promoter in
the hepatoma cells than the SV40 promoter and enhancer (by 1.5-fold).
In CV-1 cells, this sequence was a weak positive element that increased
CAT expression by 45% (Fig. 3 C). In contrast, the same
sequence did not have any effect in HeLa cells
(Fig. 3 B). Thus the differential expression of Sp1
(30, 41) and/or related proteins may be important in
the differential expression of this promoter.
Negative Elements in the ADH5 Promoter
The region
from bp -127 to -65 acted as a weak negative element in all
three cell lines tested, as evidenced by the lower expression of
pCAT-X-127 relative to pCAT-X-64 (Fig. 3). A reduction in CAT
activity by 21, 28, and 36% was seen in HeLa, H4IIE-C3, and CV-1 cells,
respectively. A very weak footprint was observed in region from bp
-100 to -61 only with CV-1 nuclear extract
(Fig. 5 C).
Effects of the 5`-Nontranslated Region on Gene
Expression
The 5` non-translated region of ADH5 is
unusual in having two upstream ATGs. pCAT-X-1342 and pCAT-H-1342 have
the same 1.4 kb ADH5 upstream sequence inserted at different
sites in the polylinker of pCAT Basic. This introduces an extra 12 bp
in front of the CAT coding sequence in pCAT-H-1342, relative to
pCAT-X-1342 (Fig. 4A). pCAT-H-1342 produced a 1.6-2.3-fold higher
CAT activity than pCAT-X-1342 in all cell lines tested
(Fig. 4 B). The higher CAT activity in pCAT-H-1342 may be
due to the slightly longer upstream coding sequences (4 amino acids
longer) in pCAT-H-1342, which may affect the translocation and
reinitiation of ribosomes
(43) . Alternatively, the enhancement
may be the result of an alteration in the secondary structure of the
5`-nontranslated region.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.