(Received for publication, August 17, 1996, and in revised form, January 30, 1997)
From the Departments of Pathology and
§ Cell Biology, Baylor College of Medicine,
Houston, Texas 77030
The mouse -glutamyl transpeptidase (GGT) gene
encodes seven distinct mRNAs that are transcribed from seven
separate promoters. Type II mRNA is the most abundant in kidney. We
have developed a cell line with features of renal proximal tubular
cells which expresses GGT mRNA types with a pattern similar to that
of mouse kidney. Because a 346-bp sequence from the type II promoter
directed the highest level of CAT activity in these cells, this region was used to drive the expression of a
-galactosidase reporter gene
in transgenic mice. Two transgenic mouse lines expressed
-galactosidase limited to the renal proximal tubules. Site-directed deletions within this 346-bp promoter region demonstrated that cis-elements containing the consensus binding sites for AP2, a glucocorticoid response element (GRE)-like element, and the initiator region were required for transcriptional activity and were not additive. Purified AP2 bound and footprinted the AP2 consensus region,
making it likely that transcription from the GGT type II promoter is
regulated in part by AP2. These data suggest that transcription of the
type II promoter requires multiple protein DNA interactions involving
at least an AP2 element, and probably a GRE-like element and the
initiator region.
-glutamyl transpeptidase (GGT)1 is
a key enzyme in glutathione metabolism (1-3). It is expressed in many
epithelial cells, but the highest levels are found in kidney, small
intestine, pancreas, fetal liver, and other organs, which have
secretory or absorptive function (1, 3). In kidney GGT expression is
restricted to proximal tubules, where the
-glutamyl cycle plays an
important role in the recycling of GSH (1, 3). Renal GGT is primarily associated with the apical surface of the proximal tubule with its
active site in the extracellular milieu. GGT activity in proximal tubules results in reabsorption of greater than 99.9% of the tubular glutathione (as the constituent amino acids) and thus
functions in cysteine reabsorption (1, 4).
We have previously identified and characterized the structure of six
different GGT mRNAs in mouse kidney (5). The GGT mRNA species
differ in their 5-untranslated sequences but share a common coding
region (5, 6). The different GGT mRNAs are expressed from separate
promoters that are present in the 10-kb 5
-flanking region of the GGT
gene (7). We have studied the relative abundance of the GGT mRNAs
in kidney and found that type II mRNA is the most abundant,
representing approximately 45% of the total, while the five remaining
GGT mRNAs are present at lower levels (7). Different GGT RNAs are
expressed in a tissue restricted-pattern, and in general one type is
present in only a few different tissues (3). For example, type III is
expressed only in fetal liver and type IV is also detected in
epididymis and in embryonic cells derived from the endoderm of the yolk
sac (3, 8).
Although GGT is expressed in a relatively ubiquitous manner, the
restricted pattern of expression of individual GGT mRNAs has led to
the hypothesis that the different promoters are tissue-restricted. In
contrast to other GGT mRNAs, significant expression of type II is
limited to the kidney where it is also the most abundant GGT mRNA
(7). Our previous studies also demonstrated that the type II GGT
promoter conferred significantly higher levels of CAT activity in
transient transfections in mouse proximal tubular cells than in
fibroblasts, indicating that those cis-acting elements were sufficient
to direct the expression of the reporter gene in a cell specific manner
(7). We therefore examined the type II promoter to determine if it
contains sufficient cis-acting elements to direct kidney restricted
expression of a -galactosidase reporter gene in vivo by
generating transgenic mice.
In addition we have performed deletion analyses of the 346-bp type II
promoter to determine the cis-acting binding sequences that are
responsible for transcriptional activity in mouse proximal tubular
(MPT) cells. We obtained a series of 5 truncations and short sequence
deletions by site-directed mutagenesis and tested them by transient
transfection assays of the CAT reporter gene. Selected sequences
containing critical elements required for promoter activity were tested
for their ability to activate a minimal GGT promoter region and
heterologous minimal promoters.
The ras-transformed MPT cell line
was established from the kidneys of p21ras-transgenic mice line
499 (9). The culture medium contained a 1:1 mixture of Dulbecco
modified Eagle's medium and Ham's F12 medium (Life Technologies,
Inc.) with 10 mM Hepes buffer, sodium bicarbonate at 1.1 mg/ml, 10 nM
Na2SeO35H2O (SFFD) and was
supplemented with insulin (5 mg/ml), PGE1 (25 ng/ml),
triiodothyronine (5 × 1011 M),
hydrocortisone (5 × 10
8 M), and
transferrin (5 mg/ml) (10). Whole kidneys were minced and suspended in
1 mg/ml collagenase in SFFD. Cells were harvested and plated in 25-mm
plates. The cells were maintained as mixed cell cultures in medium
supplemented with 7% fetal calf serum and passed every 3-4 days.
The antisense RNA probes used for the quantification of
the GGT mRNAs II and IV were obtained by in vitro
transcription of the GGT cDNAs previously reported (5), and their
structures are represented in Fig. 1A. The lengths of their
unique 5 sequences are 94 and 99 bp for probes II and IV,
respectively. The
-galactosidase ribonuclease probe was obtained
from Ambion, Inc. (Austin, TX). The plasmids were linearized and then
transcribed with either T3 or T7 polymerase (Stratagene Inc., La Jolla,
CA) in the presence of [
-32P]UTP to obtain the
uniformly labeled antisense strand.
Ribonuclease Protection Assays
Total RNA was isolated from the organs of transgenic mice using the acid-phenol guanidine procedure (11). Poly(A)+ RNA was obtained from MPT cells and adult Friend leukemia virus strain B mice (FVB) kidneys and selected by oligo(dT)-cellulose type III (Collaborative Research Inc., Bedford, MA).
Ribonuclease protection assays were performed with the RPA II
ribonuclease protection kit (Ambion, Inc.). Briefly,
poly(A)+ RNA from MPT cells or total RNA from FVB or
transgenic mouse tissues was hybridized for 18 h at 45 °C with
1 × 105 cpm of a [-32P]UTP labeled
RNA probe. Ribonuclease digestion of the hybridized probe and sample
RNA was performed at 30 °C for 30 min, with 0.1 unit of ribonuclease
A and 20 units of ribonuclease T1. The protected RNA fragments were
separated on 6% polyacrylamide, 7 M urea gels. For
quantitation of GGT mRNA types in MPT cells, the number of counts
in each band was determined with the AMBIS Radioanalytic Imaging System
and the AMBIS QuantProbe Software version 4.01 (AMBIS, Inc., San Diego,
CA).
The promoterless plasmid
pJFCAT1 (12) was used to subclone the 5-flanking regions of GGT in
front of CAT. PII-2.7 is a 2.7-kb fragment that results from
PstI digestion of the 6.0-kb XhoI clone (7); the
PstI ends were flushed with T4 polymerase, and the fragment
was cloned into the XhoI site of pJFCAT1. PII-346 (
346 to
+70 bp) and PII-746 (
746 to +70) have been described (7) and were
previously named PII-416 and PII-816, respectively. Since convenient
restriction sites were absent in some regions, constructs PII-230
(
230 to +70) and PII-95 (
95 to +70) were obtained by amplifying
sequences of appropriate size using PII-346 as a template. The PCR
amplifications were performed as described previously (13). PII-230 was
obtained by PCR with the oligonucleotides 5
(5
-CTCGAGAAGGGTTCACCGGTGGCCTCTGC) and 3
(5
-GCCGCCCTCGAGGCAAGAGGTCAGCTAA), and PII-95 was obtained by PCR with
the oligonucleotides 5
(5
-CTCGAGGTCACAAGCCTGACGCTGCGCC) and 3
(5
-GCCGCCCTCGAGGCAAGAGGTCAGCTAA). The PCR products were cloned into
the pT7Blue vector (Novagen, Madison, WI), digested with
XhoI, and subcloned into the XhoI site of
pJFCAT1.
The 116-bp fragment was obtained by PCR using PII-346 as a template,
and using the oligonucleotides 5 (5
-AAGGATCCGATCTAAGCTATGGTCTAGTG) and 3
(5
-AAGGATCCAGATCTTCCAGACAGCCCCTGCTAAG), subcloned into the
pT7Blue vector, excised with BamHI, and cloned upstream of PII-95 (7). The double-stranded oligonucleotides p6a
(5
-GATCACTCGAGCCCCTTAGAGGGAACCAAATCTGGAAAGTGGGGA) and p6b
(5
-GATCACTCGAGGGAACCAAATCTGGA) were cloned into the
BglII site of the enhancerless CAT reporter vector (pSV40
CAT) (Promega, Madison, WI), to obtain pSV-6a and pSV-6b, respectively.
Single copies of each double-stranded oligonucleotide upstream of
CAT were obtained after screening by restriction digestion with
XhoI and confirmation of the sequence by DNA sequencing. The
sense orientations were used.
The TATA-Luc minimal
promoter reporter containing 58 bp of the cardiac
-actin promoter
was kindly provided by R. Schwartz (Baylor College of Medicine) (14).
The double-stranded oligonucleotide p6a (see above) was cloned upstream
of the
-actin minimal promoter into a SacI site in the
polylinker of the vector. A construct containing seven copies of the
double strand oligonucleotide was used in transient transfection
assays.
A 416-bp DNA
fragment containing 346 bp of the GGT type II promoter region was
cloned into the vector PCRTMII (Invitrogen, Inc.) and released after
digestion with HindIII and NotI (7). This probe
was end-labeled with a T4 polynucleotide kinase (Promega) and
[-32P]ATP, followed by digestion with BamHI
to release the 3
end of the GGT promoter. Footprint assays were
performed with the Core Footprinting System (Promega) using 1 or 2 footprint units of purified human AP2 protein (Promega) and 1 × 105 cpm of labeled probe. The probe was incubated with the
protein extract for 10 min, followed by digestion with 0.15 unit of RQ1 RNase-free DNase (Promega). The products were resolved on 6%
polyacrylamide, 7 M urea sequencing gels.
Gel mobility shift analyses were performed with a double-stranded
oligonucleotide (5-ATGGTCTAGTGCCTGGGGTACCCC) containing the AP2
consensus binding site (GCCTGGGG) present at
326 to
319 bp in the
GGT type II promoter (7). Control oligonucleotides included the human
metallothionein AP2 binding site (5
-GATCGAACTGACCGCCCGCGGCCCGT) (58);
an oligonucleotide containing a mutated human metallothionein AP2
binding site (5
-GATCGAACTGAAATGTAGATGCCCGT); and an oligonucleotide containing the binding site for AP1 (5
-CGCTTGATGAGTCAGCCGGAA) (24). The double-stranded oligonucleotides were end-labeled with
T4 polynucleotide kinase (Promega) and [
-32P]ATP, and
1 × 105 cpm were incubated with 1 µg of purified
human AP2 (Promega), using the gel shift assay system (Promega). The
products were resolved in 4% nondenaturing acrylamide gels.
Quantitation of free and bound probe was performed with the AMBIS
Radioanalytic Imaging System and the AMBIS QuantProbe Software version
4.01 (AMBIS, Inc.).
Site-directed mutagenesis was
performed by oligonucleotide in vitro mutagenesis as
described by Kunkel, with the Muta-Gene Phagemid In Vitro
Mutagenesis Version 2 (Bio-Rad). The template for mutagenesis was the
single-stranded DNA pJFCAT-346 (PII-346), previously described (7). The
oligonucleotides used for mutagenesis were: P1rs (AP2)
(5-GCAATAAAGTAGGGGTATAGACCATAGCTTAGA) or P1
(5
-GCAATAAAGTAGGGGTATAGACCATAGCTTAGA), P2rs (GRE)
(5
-TCTGTGAATTTAGAGACTAGACCATAGCTTAGA) or P2
(5
-TCTGTGAATTTAGAGACTAGACCATAGCTTAGA), P3
(5
-CTAAGGGGCAGGCCACTTCCAGACAGCCCCTGCT), P4
(5
-TTGGTTCCCTCTAAGGGGCGGTGAACCCTTCCAGAC), P5
(5
-CTTTCCAGATTTGGTTCCCAGAGGCCACCGGTGAACC), P6
(5
-GGCTCCCCCACTTTCCAGCTCTAAGGGGCAGAGGC), P7
(5
-ACTAAGAGCCTGGGAAACATTTCCAGATTTGGT), and P8
(INR)(5
-CATCTGAAGGCTTCTGAGTCCTCTGTAAGA). The
underlined sequences correspond to the AatII (GACGTC) or
NotI (GCGGCCGC) restriction sites. The sequences deleted in
each construct were PII-P1 and PII-P1rs (
310 to
318), PII-P2 and
PII-P2rs (
283 to
273), PII-P3 (
229 to
220), PII-P4 (
219
to
210), PII-P5 (
209 to
200), PII-P6 (
199 to
190), PII-P7
(
179 to
170) and PII-P8 (
9 to +10). Positive clones were
screened by digestion with the restriction enzyme specific for the
sequence present within the mutated primers, AatII in
primers P1rs, P2rs, and P8 and NotI in P3, P4, P5, P6, and
P7. The oligonucleotides P1 and P2 have complete deletions of the base
pairs (
327 to
320) and (
294 to
284), and do not contain a
restriction enzyme recognition site; these clones were screened
by sequencing.
Sequence analyses of the type II promoter region were performed with the Quest program from Intelligenetics, using the release 6.0 of the Transcription Factors Data Base (15), and with the Transfac data base (16).
Cell TransfectionsApproximately 1 × 106
MPT cells in 100-mm plates were transfected using the calcium phosphate
co-precipitation method (17). Precipitates contained 20 µg of the
GGT-CAT construct and 500 ng of plasmid pCMV
(CLONTECH, Palo Alto, CA), which expresses
-galactosidase. The
-galactosidase activity was used for
correction of the transfection efficiency. Five hours after addition of
the DNA to MPT cells a glycerol shock was performed. The cells were harvested and extracts obtained at 48 h post-transfection by
freezing and thawing. CAT assays were performed from at least three
independent transfections, as described elsewhere (7).
Transfection of luciferase reporter genes was performed using LipofectAMINE (Life Technologies, Inc.) with a DNA/LipofectAMINE ratio of 1/5 (w/v). 0.5 × 106 cells were plated onto 60-mm plates and transfected with a total of 2 µg of DNA. After 48 h of post-transfection, cell extracts were obtained using 200 µl of lysis buffer solution (Promega). Thirty microliters were used to determine luciferase activity using a standard protocol (Promega).
Generation of Transgenic MiceTo obtain transgenic mice,
346 bp of the GGT type II promoter were cloned upstream of the
-galactosidase reporter gene of the plasmid pNASS
(CLONTECH). The type II promoter 416-bp fragment containing the 346-bp 5
-flanking region was excised with
XhoI from the pT7Blue vector (7) and subcloned into the
XhoI site of the
-galactosidase reporter vector pNASS
to yield pII-346/
-galactosidase. Clones containing the 416- bp
insert were sequenced, and a clone with the sense orientation was
selected for microinjection. A 4.3-kb fragment that resulted from
digestion of the pII-346/
-galactosidase with EcoRI and
HindIII was used for microinjection of fertilized eggs.
Injections and implantations were performed using standard protocols
(18).
The transgenes from founder animals and F1 progeny were analyzed by Southern blot of tail DNA. Briefly, 10 µg of genomic DNA were digested with SacI, electrophoresed through a 0.8% agarose gel, and transferred to Zeta-Probe (Bio-Rad) nylon membranes. The DNA probe used was the 4.3-kb EcoRI-HindIII fragment that was also used for microinjections. Labeling was performed using a random primer labeling kit (Boehringer Mannheim). Hybridization and washing conditions were used as recommended by the Zeta-Probe membrane manufacturer.
Reverse Transcription-PCR (RT-PCR)The oligonucleotides 5
(5
-GAACTGAAAAACCAGAAAGTTAACT), 3
a
(5
-TCCCAGTCACGAGTTGTAAAACGACG), and 3
b
(5
-CAATGCCTCCCAGACCGGCAAC) were designed to amplify the transgene
across the SV40 introns present in the
-galactosidase reporter
vector pNASS
. RT-PCR was performed with the Access RT-PCR System
(Promega). A sample of total RNA extracted from transgenic mouse
tissues was first digested with BamHI, which cleaves a site
that is present in the intronic region of SV40, to eliminate residual
genomic DNAs in the RNA preparations, and was followed by treatment of
the sample with 3 units of DNase RQ1 (Promega). One microgram of total
RNA was used in each RT-PCR reaction. The integrity of the RNAs was confirmed by amplification of the correct product of G3PDH
(CLONTECH). The reaction products were
electrophoresed in 2.5% agarose gels and visualized with ethidium
bromide staining.
Mouse tissues were
sampled, and 8-µm thick frozen sections were prepared. The tissues
were fixed in 1% glutaraldehyde for 5 min and stained at 30 °C
overnight to 24 h in a solution containing 100 mM
sodium phosphate, pH 7.3, 1.3 mM MgCl2, 3 mM K3Fe(CN)6, 3 mM
K4Fe(CN)6, and 1 mg/ml
5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-gal).
Tissue sections were counterstained with neutral red.
-Galactosidase
activity in tissues was identified by its ability to convert X-gal into
a water-insoluble blue product. To demonstrate GGT in tissue sections
histochemistry was performed on frozen sections fixed with methanol
(100%) for 5 min and stained as described previously (19).
We
have previously demonstrated that five separate 5-flanking regions of
the GGT gene have promoter activity in mouse kidney C1.1 cells (7, 20).
However, ribonuclease protection assays performed to determine the
steady state levels of the most abundant GGT mRNA types in kidney
(types II and IV) did not show significant expression of these messages
in C1.1 cells (Fig. 1 and data not shown). We therefore
developed a cell line that would reproduce the pattern of GGT
expression in vivo.
To establish a mouse proximal tubular cell line, we took advantage of the fact that a line of transgenic mice carrying a rat GGT(I)-rasVal-12 develops proximal tubular hyperplasia (9). We were able to establish an MPT cell line from the kidneys of GGT-ras transgenic mice. After 30 passages, the cells maintained a polygonal epithelial morphology, formed cell islands, and, when grown to confluency, formed occasional domes. Histochemical staining for GGT in the MPT cell population was positive and most accentuated in the domes after confluency was reached (data not shown). Northern blot analyses showed that they expressed GGT RNA at a level that is greater than 10% of that present in total kidney RNA (21). This level is approximately 3-fold higher than the level expressed by C1.1 cells. Since types II and IV are the most abundant GGT mRNA types in kidney, we used ribonuclease protection assays (RPA) to perform a quantitative analysis of these GGT mRNA types in established MPT cells (7).
The structural features of the GGT cDNAs, with a common 3 segment
and a unique 5
-flanking region with a DNA sequence that is
type-specific, allows a determination of the relative abundance of each
GGT transcript relative to total GGT RNA in a cell population by
ribonuclease protection (7). Using this approach we found that the type
II mRNA represents approximately 45% and the type IV approximately
25% of the total GGT mRNA in MPT cells (Fig. 1). These results
agree well with those from studies in which we quantified GGT mRNAs
in the kidney and found 45% of the GGT mRNA was type II and 33%
was type IV (7).
In previous
experiments we found that in transient transfection assays of CAT
reporter constructs a 346-bp 5 fragment directed higher CAT activity
than a 746-bp promoter region in C1.1 cells (7). In addition, no
significant CAT activity was found in NIH-3T3 cells with these
constructs, indicating that cis-elements present in the 346-bp fragment
were sufficient for kidney cell specificity. We have evaluated a series
of GGT (II) constructs for CAT activity in MPT cells. A construct
containing additional 5
-flanking sequences (PII-2.7) (Fig.
2) displayed lower level of CAT activity (~60%)
relative to PII-346. Sequential 5
truncations of the 346-bp region
were tested next. Removal of 50 bp (
346 to
296) (PII-296) resulted
in a decline in transcription to approximately 30% of PII-346. This
50-bp region contains an AP2 consensus site (GCCTGGGG) (
326 to
319)
(22) which is a candidate for an activator of GGT transcription (see
below). Removal of the 5
116 bp of PII-346 (PII-230) results in a
reduction in CAT activity to about 25-30% of PII-346. This finding
indicates that putative activating cis-elements are present within this
116-bp region. Also, within this region there is an imperfect
glucocorticoid receptor consensus binding site (GRE) (GACATCATGTC)
(
294 to
284) (23). The GRE-like site and the AP2 site do not appear
to be additive because deletion of the AP2 site alone (PII-296) results
in the same level of activity of PII-230, in which both the AP2
consensus and the GRE-like sequence are removed. Deletion of 249 bp
(PII-95) results in a marked decrease in transcription with essentially
no activity remaining with this construct (Fig. 2). Within the region
deleted in PII-95 there are several consensus binding sites, including
putative binding sites for AP1 (TTAGTCACC) (
105 to
97) (24), SP1
and AP2 (TCCCCCGCCCA) (
141 to
132) (22, 24), and cAMP response
element binding factor/activating transcription factor (TGACGTCA) (
62
to
55) (25) transcription factors.
Characterization of Transgenic Mice Carrying the pII-346/
Of the constructs we tested
PII-346 has the highest CAT activity in transient transfection assays
in MPT cells (Fig. 2). Thus we used this region to construct a fusion
reporter gene by cloning it upstream of the bacterial -galactosidase
gene (Fig. 3A). The pII-346/
-galactosidase
construct was transfected into the MPT cell line by transient
transfections. Transfected cells stained positively for
-galactosidase (data not shown). The same construct was used to
generate transgenic mice by microinjection (see "Experimental Procedures").
Putative transgenic mice were screened by Southern blotting of genomic
DNA digested with SacI (Fig. 3B). The hybridizing
probe was the same EcoRI-HindIII fragment that we
used for microinjection. Because this probe contains 416 bp of the type
II 5 region, both transgenic and nontransgenic genomic DNAs yield a
band of ~9 kb which corresponds to the endogenous GGT gene. Complete
SacI digestion of the integrated transgene results in a
2.4-kb SacI-SacI fragment which includes the type
II 5
region and part of the
-galactosidase cDNA and a fragment
greater than 1.8 kb in length, which includes part of the 3
end of the
-galactosidase cDNA and a segment of mouse genomic DNA of
varying size. Tandem integrations which are head to tail also result in
a 1.8-kb band (Fig. 3, A and B).
We obtained four female founder mice that were positive for the transgene (F0-17, F0-1, F0-52, and F0-46) (Fig. 3B). The transgenes integrated as multiple copies in a tandem head to tail orientation in the four animals, as demonstrated by the presence of a 1.8-kb band present in all lanes. The mouse F0-17 yielded F1 progeny, while the other females did not after multiple attempts.
Analyses of the Expression of the Transgene in Mouse TissuesWe stained sections of kidney, liver, small intestine,
spleen, uterus, adrenal gland, lung, heart, and brain for
-galactosidase. Only kidney sections stained positively for
-galactosidase. Expression of
-galactosidase was found only in a
population of renal cortical tubules and not in glomeruli, interstitium
or in the medullary portions of the kidney (Fig.
4A). Both founder F0-1 and mice of line 17 were positive while nontransgenic FVB mice were negative. We took
advantage of a histochemical stain for GGT which is specific for
proximal tubules to localize
-galactosidase expression (19, 26).
Serial sections were performed, and adjacent levels were stained for
GGT and
-galactosidase. No
-galactosidase-positive tubules were
GGT-negative, while
-galactosidase-positive tubules were also
GGT-positive (Fig. 4, B and C), demonstrating
that 346 bp of the type II promoter directed expression of the
transgene to the correct cell population in kidney.
To confirm the histochemistry results, we used RT-PCR and ribonuclease
protection to demonstrate the expression of -galactosidase in
transgenic mouse tissues. Total RNAs from kidney, spleen, liver, small
intestine, pancreas, lung, brain, skeletal muscle, and heart were
examined. RT-PCR was designed to identify the transgene by the presence
of the two spliced RNA variants that are originated through utilization
of two separate splice acceptor sites present in the vector pNASS
,
resulting in two bands of 262 and 188 bp in length (Fig.
5, A and B) (27). Only the kidneys
of F0-1 and mice of line 17 were positive (Fig. 5A). To
demonstrate the integrity of RNA samples used in the RT-PCR reactions,
parallel amplifications with oligonucleotide primers specific for G3PDH
were performed. The expected G3PDH reaction product was present in all
RNAs tested. In addition, ribonuclease protection using a
-galactosidase specific probe was performed with RNA from F0-1 and
line 17 kidneys. We were able to demonstrate a protected 300-bp band
with kidney RNA from mouse F0-1 but not with RNAs from negative control
FVB mice (Fig. 5C). We did not observe a specific
ribonuclease protected band in line 17, probably as a result of low
levels of expression.
Contribution of Cis-elements within the Type II 346-bp Promoter Region to Transcriptional Regulation
As an initial attempt to
identify key cis-acting elements of the type II promoter, we performed
site-directed mutagenesis of sequences that match or have high identity
with the binding sites for known transcription factors. Analysis of 5
truncations of the 346-bp region of promoter II demonstrated that
deletion of the 116-bp (
346 to
230) region (PII-230) resulted in a
70-75% decline in transcription of the CAT reporter gene in MPT cells (Fig. 2, A and B). Sequence analyses revealed
consensus binding sites for AP2 (
326 to
319) and a GRE-like
sequence (
284 to
271) within these 116 bp (Fig.
6A) (22, 28). Similarly, deletion of the 5
50-bp (PII-296) that includes the AP2 consensus binding site resulted
in a 75-80% decrease in transcription activity of the CAT reporter
construct (Fig. 2B). There are two possible explanations for
the results of the transfection data. First it is possible that the GRE
is not required for transcription activation. The second explanation is
that the GRE is important for transcription but it does not have an
additive effect with AP2. Because the GRE-like site is conserved in
mouse and rat, we decided to investigate the second possibility by
testing whether a mutation of the GRE-like site alone would affect
transcription of the GGT promoter. To demonstrate that the putative
binding sequences for AP2 and GRE are involved in the regulation of the
type II promoter, we performed site-directed mutagenesis and tested two
types of deletion mutations of the AP2 and GRE-like sites (Fig. 6,
A and B). In one type the consensus binding
sequence was simply removed, and in the other, the site was partially
replaced by a restriction enzyme site that could be used for screening
purposes. Both yielded similar results, indicating that utilization of
the restriction enzyme site did not affect the level of transcription.
Both deletions of the AP2 site (PII-P1rs and PII-P1) resulted in an
approximately 80% drop in transcription (Fig. 6B); this is
the same extent of transcriptional decrease that was observed when the
AP2 consensus site was removed in the 5
truncation PII-296 (Fig. 2).
Deletions of the GRE-like element (PII-P2rs and PII-P2) resulted in a
drop in CAT activity of 65 and 82%, respectively. Taken together,
these findings indicate that the AP2 and GRE-like elements do not
appear to act synergistically, although both appear to be required for
transcription activation, because their individual deletions affected
transcription to a similar extent.
To demonstrate the role of the 116-bp (346 to
230) region in
increasing transcription from the type II promoter, we tested its
ability to activate a minimal type II promoter (PII-95) by cloning this
116-bp sequence upstream of the
95-bp region (PII-95/116) (Fig.
7A). This construct corresponds to an
internal deletion of the region
230 to
95 (Fig. 7A).
Transient transfection experiments in MPT cells showed approximately a
7-fold increase in CAT activity with PII-95/116 relative to PII-95
(Fig. 7B); the overall activity, however, was only 18% of
the maximal promoter. These data suggest that, although the
cis-elements present in the 116-bp region can activate the
transcription of type II promoter, additional elements present within
the
230 to
95 region may be required for full promoter
activity.
AP2 Binds to the GGT AP2 Binding Site and Footprints a Region of the 5
To determine
whether the putative AP2 binding site actually binds AP2, we performed
gel shift assays. This analysis showed that purified AP2 is able to
bind an oligonucleotide that contains the AP2 consensus sequence
present in the (310 to
318) region of the GGT type II promoter
(Fig. 8). This binding ability is sequence-specific,
since it was abolished in the presence of a cold competitor
oligonucleotide containing the GGT AP2 consensus sequence but not by an
oligonucleotide with a mutation in the AP2 binding site nor by an
oligonucleotide containing the sequence of the human metallothionein
AP1 binding site (Fig. 8). The affinity of the purified AP2 protein for
the GGT AP2 sequence was 10-fold less than for the metallothionein AP2
consensus. This was determined by the ratio of bound to free counts
(Fig. 8).
Footprint analysis with an end-labeled DNA probe spanning the GGT AP2
binding sequence revealed that a 45-bp region (338 to
294) is
protected by human AP2 (Fig. 9). The protected 45-bp region includes the GGT AP2 binding site.
Putative Regulatory Cis-elements within the Type II Promoter
We performed the initial sequence analysis of the
type II promoter with the Transcription Factors Data base release 6.0 and searched for consensus sequence matches with a calculated intrinsic probability of random occurrence of <2.0 × 104.
This approach resulted in a relative paucity of putative binding sites
within the
230- to
95-bp region. We, therefore, performed site-directed deletion mutations of a series of individual random sequences to pinpoint possible cis-acting sequences. We obtained five
constructs: PII-P3, PII-P4, PII-P5, PII-P6, and PII-P7 (Fig. 6,
A and B). The results of transient transfections
into MPT cells revealed that two deletions (PII-P4 (
219 to
210) and
PII-P7 (
179 to
170)) resulted in approximately 5- and 3-fold
transcriptional increases, respectively (Fig. 6, A and
B). Two sequences do not appear to affect transcription
(PII-P3 (
229 to
220) and PII-P5 (
209 to
200)). One deletion
resulted in a 71% drop in CAT activity (PII-P6 (
199 to
190)).
Because the type II promoter is a TATA-less promoter that displays
partial sequence identity with the terminal deoxynucleotidyltransferase
initiator and matches a general consensus for initiators around the
transcription start site (29, 30), we performed site-directed
mutagenesis in the region that overlaps the major start site (
9 to
+10) (PII-P8) (Fig. 6A). Transient transfection assays
showed that these bases appear to be required for activity of the
promoter, because deletion of this region results in 80% drop in CAT
activity, relative to the promoter region PII-346 (Fig. 6B).
Other cis-elements that match the consensus binding sites for putative
transcription regulators are present within the
230- to
95-bp
region, including a CREB/ATF consensus sequence (TGACGTCA;
62 to
55) (31), and consensus binding sites for AP1 (
105 to
97) (24),
SP1 (
141 to
132) (32), and AP2 (
141 to
132) (22) (Fig.
6A).
Additional sequence analyses were performed in an attempt to identify
putative transcription factor binding sites that overlap the sequences
that were deleted in constructs PII-P6, PII-P4, and PII-P7. Using the
TESS Transfac data base we identified a putative serum response element
(SRE) overlapping PII-P6 (Fig. 6A) (33). The SRE is followed
immediately by a consensus binding sequence for Nkx-2.5, a murine
homeobox homologue of Drosophila tinman (Fig. 6A)
(186 to
180) (14). Because transcription activity and specificity
of transcription activators may be achieved through interactions
between a limited and specific set of proteins, these sites appeared to
represent good targets for transcription regulation. We therefore
tested if a region containing the SRE/Nkx binding site had the ability
to activate a minimal heterologous promoter. Single copies of
double-stranded oligonucleotides containing the sequences (p6a:
167
to
200) or (p6b:
175 to
192) of the type II promoter were cloned
upstream of the CAT reporter gene in the enhancerless pSV40-CAT vector.
Transfection studies revealed no significant transcriptional activation
with these constructs relative to the vector pSV40-CAT alone (data not
shown). To determine whether the lack of enhancing activity was related
to this particular vector, we cloned a fragment containing seven
multimerized copies of the double-stranded oligonucleotide p6a upstream
of a minimal promoter driving the luciferase reporter gene in the
TATA-Luc vector (14) and failed to observe increased expression (data not shown).
Sequences matching the consensus binding site for the ubiquitous
transcription factor NF1 (34) are present in a palindrome that extends
through a region that overlaps the sequences deleted in PII-P6 and
PII-P7. This finding suggests a complex regulation of the GGT promoter
through this region, since the PII-P7 deletion results in a
transcriptional activation (Fig. 6A). Interestingly, sequences matching a consensus binding site for 1-acid
glycoprotein/enhancer binding protein, a member of C/EBP family of
transcription factors (35), partially overlaps the NF1 sequences with a
similar palindromic organization. In addition, the sequence deleted in
PII-P4, which results in transcriptional increase and therefore
predicts a site for down-regulation, matches the binding site for the
liver-enriched transcription factor HNF4 (36) in 10 out of 11 bp
(Fig. 6A).
In this study we report the isolation and establishment of MPT cells in culture from the kidneys of GGT(I)-rasVal-12 transgenic mice. These cells are useful to study the regulation of the GGT gene in kidney because, unlike other kidney cell lines, they express GGT in a pattern that parallels the expression of GGT in vivo (Fig. 1). MPT cells make substantial GGT mRNA and, as in the kidney, GGT type II mRNA is the most abundant of the GGT mRNAs.
Characterization of promoters of genes that are expressed in kidney is
in its early stages (37). Even less is known about transcriptional
regulation in specific segments of the nephron in the differentiated
kidney. Examples of promoters of genes normally expressed in kidney
that have began to be analyzed include erythropoietin (38) and renin
(39, 40). Many genes have been shown to be expressed in proximal renal
tubules, including the enzymes of metabolic pathways such as
phosphoenolpyruvate carboxykinase (41, 42), argininosuccinate lyase
(43), and aldolase B (44); channel and channel-associated proteins such
as the - and
-isoforms of (Na+K+)-ATPase
(45, 46); and the angiotensin type II receptor, which is an important
regulator of proximal tubule salt water reabsorption and
angiotensinogen (47).
A major limitation to the study of mechanisms of kidney and in particular renal proximal tubule-specific gene transcription has been the lack of promoters that show expression restricted to the kidney. The GGT gene is expressed in many epithelial cells including those of the proximal renal tubules; however, of the six different GGT mRNAs that are expressed in kidney, type II is the most abundant. Further, type II mRNA is not found in other visceral organs (7). Thus the type II promoter region is a good candidate to study mechanisms of transcriptional regulation in kidney.
Our data demonstrate that a 346-bp region immediately 5 of the
transcription start of promoter II shows maximal promoter activity.
Inclusion of as much as 2.7-kb does not augment expression. Our
experiments with transgenic mice demonstrate that this region contains
sufficient information to direct transcription to the proximal
convoluted tubules and is not promiscuously expressed in other tissues.
Although we have established only one line of mice carrying our
-galactosidase construct, we do not believe the results are
explained by site-specific integration. First, the possibility is
extremely unlikely on a chance basis, and second, we have obtained
similar results in a second founder (Fig. 3).
To identify the cis-acting elements that are required for the
transcription of GGT type II in kidney cells (MPT) we performed a
series of 5 truncations and site-directed deletions. Deletion of 116 bp from PII-346 (construct PII-230), resulted in a drop in
transcription to 25-30%, which indicates that important regulatory elements are present in this region. A consensus binding site for the
transcription factor AP2 and a GRE-like site are present within this
region (22, 24). The PII-296 construct, in which the AP2 site was
removed, showed a significant drop in transcription to about 25% of
the activity seen with PII-346 (Fig. 2). Further, purified AP2 is able
to specifically bind an oligonucleotide containing the GGT AP2 binding
sequence. In addition, purified AP2 determines a footprint in the
region of the type II promoter that includes the AP2 binding site.
These findings strongly support a role of AP2 in the activation of the
GGT type II promoter. Construct PII-95, with only 95 base pairs
upstream of the major transcription start site, showed negligible
activity. Although addition of the 116 bp containing the AP2 and
GRE-like element to a minimal GGT promoter resulted in 6-fold
activation, the CAT activity of this construct was about 5-fold less
than that of the 346-bp promoter, indicating that other elements in the
promoter are necessary for full activity. In addition, these two
elements did not appear to have additive activity. These results
indicate that cis-elements present within
230 to
95 bp are required
for maximal activity of the type II promoter. An alternative
explanation is that the deletions affected the spacing of elements
relative to the transcription basal machinery. Changes in the relative
positions of factor binding sites often decreases enhancer function as
well as specificity. A suggested explanation for this result is that
there are architectural factors that have no transcriptional activity
on their own but can act synergistically with other transcriptional
factors, through the assembly of higher order nucleoprotein complexes.
A good example of the required three-dimensional enhancer complex is
the binding of four factors to the T cell receptor
enhancer
in vitro (48-51).
Transient transfections of site-directed mutations within the 230- to
95-bp region identified two sequences that do not appear to affect
transcription, one deletion, which resulted in a 71% drop in CAT
activity (PII-P6), and two deletions (PII-P4 and PII-P7), which
resulted in approximately 5- and 3-fold transcriptional increases,
respectively (Fig. 6). These findings show that GGT type II promoter,
like other promoters, contains both positive and negative cis-acting
elements (44, 52, 53).
Several reports indicate that the tissue specific expression of genes
is achieved through complex interactions that involve numerous
cis-acting regions that exert positive and negative effects on promoter
activity. This type of regulation is exemplified by the human
erythropoietin receptor gene (54) and the skeletal -actin gene
(55).
Regarding the mechanisms that direct kidney specificity of the GGT type II promoter, it is possible that non-DNA-binding factors, such as coactivators or adapters, can determine tissue specificity. Examples of this type of regulation include the B cell-specific coactivator OCA-B (56) and the tissue-specific coactivator DCoH (57). It is possible that negative regulatory elements function in tissues other than kidney. For example, the consensus binding sequence for the liver enriched factor HNF4 has a negative effect on the transcription of type II promoter and may mediate the repression of this promoter in liver cells. It is also possible that there are tissue-specific elements in the GGT promoter that were not revealed by the mutagenesis analysis.
The combination of transient transfection assays in the novel kidney
MPT cell line and expression of a pII-346/-galactosidase reporter
gene in transgenic mice demonstrates that 346 bp of the mouse type II
GGT promoter are sufficient to confer specific tissue expression of GGT
type II to proximal tubular renal cells. Future dissection of the
protein factors that bind to regulatory sites in the promoter and of
their cooperative interactions should help elucidate the regulation of
transcription in proximal renal tubules.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) L17331[GenBank] and L17336[GenBank].
We thank Karl Frindrich for help with cell cultures.