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(Received for publication, June 5,
1995; and in revised form, August 21, 1995) From the
Two sequence elements located at -111 to -100 base
pairs and -70 to -50 base pairs in the 5`-flanking region
of the bovine CYP11A gene and in closely related positions in CYP11A of
other species contain G-rich regions that are similar to the consensus
Sp1-binding site. These sequences bind the purified transcription
factor Sp1 as well as nuclear proteins from mouse Y1 adrenal cells that
interact with an antibody specific for Sp1. Both of these CYP11A
sequences support basal and cAMP-dependent transcription of reporter
gene plasmids transfected into Y1 cells, and mutations within the
G-rich -111/-100-base pair sequence that reduce or
eliminate the binding of Sp1-related Y1 nuclear proteins also markedly
reduce cAMP-induced transcription. cAMP-dependent transcription
supported by both CYP11A sequence elements is mediated by protein
kinase A at levels comparable to that promoted by different
cAMP-response sequences and transcription factors in other genes
involved in steroidogenesis. These results indicate that ACTH-dependent
regulation of cholesterol side chain cleavage cytochrome P450 levels in
the adrenal cortex which is mediated through cAMP involves the
ubiquitous transcription factor Sp1.
In the adrenal cortex of vertebrate species, synthesis of
glucocorticoids, mineralocorticoids, and precursors of sex hormones is
catalyzed by steroid hydroxylases which are members of the cytochrome
P450 gene superfamily (Simpson and Waterman, 1988). The initial and
rate-limiting step in steroid hormone biosynthesis is the conversion of
cholesterol to pregnenolone by the mitochondrial steroid hydroxylase
cholesterol side chain cleavage cytochrome P450 which is the product of
CYP11A gene (Nelson et al., 1993). The transcription of this
gene along with three other steroid hydroxylase genes, namely CYP11B1,
CYP17, and CYP21, is coordinately regulated in the bovine adrenal
cortex by the peptide hormone corticotropin (ACTH) via cAMP (John et al., 1986). Constitutive expression of CYP11A, in the
absence of ACTH, is also observed in the adult bovine adrenocortical
cells (John et al., 1984), fetal bovine adrenal cortex (Lund et al., 1988), and fetal human adrenal cells (John et
al., 1987). Functional analyses of the 5`-flanking regions of
CYP11A genes from human (Inoue et al., 1988; Moore et
al., 1990, 1992; Hum et al., 1993; Guo et al.,
1994), bovine (Ahlgren et al., 1990; Momoi et al.,
1992), rat (Oonk et al., 1990; Clemens et al., 1994),
and mouse (Rice et al., 1990) have revealed species-specific
variation in the composition of cAMP-responsive regions within this
gene as well as in their ability to support cAMP-induced transcription
in cell lines originating from different steroidogenic tissues. Serial
deletions of the proximal 896 base pairs (bp) ( Herein we demonstrate that 1)
one of the proteins binding to the sequence between -111 and
-100 bp within the bovine CYP11A promoter is Sp1 or a protein
antigenically related to it, 2) mutations in this region either
eliminate or markedly reduce both binding of Sp1 and cAMP-dependent
transcription mediated by this element in Y1 adrenocortical cells, 3)
there is at least one additional sequence between -70 and
-50 bp of the bovine CYP11A gene which also binds Sp1 and
supports cAMP-induced transcription, and 4) the cAMP-induced
transcription mediated by the Sp1-binding sequences of the bovine
CYP11A gene is dependent on the cAMP-dependent protein kinase (PKA)
catalytic subunit.
The
double-stranded bovine CYP11A wild type and mutant oligonucleotide
sequences with SacI and SalI ends were inserted into
the unique SacI and SalI sites of the OVEC vector to
generate various bovine CYP11A promoter-OVEC plasmids (Fig. 1C). The OVEC construct having multiple tandem
copies of -118/-100 bp from bovine CYP11A was made
according to the scheme described by Westin et al.(1987). The
OVEC plasmids with one and two tandem copies of the human
-126/-113 CYP21 sequence, one and two tandem copies of the
bovine -243/-225 CYP17 sequence, and one copy of the bovine
699/740 adrenodoxin gene sequence are designated as 1
Figure 1:
Schematic diagrams of
the bovine CYP11A proximal promoter region and its sequences used in
the reporter gene plasmids. A, the 5`-flanking bovine CYP11A
gene sequence from -118 to -32. The G-rich sequences are
boxed in rectangles while a known SF-1 binding site is encircled by an oval. The solid line above and the dashed line below mark sequences that show strong homology to the consensus
binding sites for ASP and AP-1, respectively. B, wild type and
mutant -118/-100 promoter sequences. The arrows indicate base changes in the wild type sequence. The wild-type
G-rich sequence is shown inside the rectangle. C,
OVEC-
Luciferase reporter gene
constructs (Fig. 1D) -186/+12LUC, and
-101/+12LUC were made by inserting XhoI/PstI fragments from -186CATSCC, and
-101CATSCC (Ahlgren et al., 1990) into XhoI/PstI sites of the pA
Figure 2:
Gel mobility shift analysis of binding of
Y1 nuclear proteins and purified Sp1 to wild type and mutant
-118/-100 sequences.
When both wild type and mutant
-118/-100 fragments were used as probes only the
-116/-114M showed a binding pattern similar to that
observed with -118/-100 in the presence of either Y1
nuclear extracts or purified Sp1 (Fig. 2B). Formation
of complexes I and II for which the consensus Sp1 oligonucleotide
competed (Fig. 2A) were either markedly reduced, as
observed with -103/-101M, or completely absent as seen in
the case of -105/-104M and -108/-107M. All
mutant fragments, however, still formed complex III with both Y1
nuclear extracts and purified Sp1. Also, the complexes formed by both
the -118/-100 sequence and the consensus Sp1
oligonucleotide using the Y1 nuclear extracts interacted with an
antibody raised against Sp1 transcription factor resulting in a
supershift complex (Fig. 2C). A similar supershift
complex is also formed when the consensus Sp1 sequence was incubated
with purified Sp1 in the presence of Sp1 antibody. The same
complexes, including the supershift complex formed in the presence of
Sp1 antibody, were observed when the -111/-100 sequence,
which lacked seven nucleotides present at the 5`-end of the
-118/100 sequence, was used as the probe (Fig. 2D). In contrast, these complexes were not
observed when four nucleotides were removed from the 3`-end of the
-118/-100 fragment (-118/-104). Together, the
observations in Fig. 2suggest that the nucleotides present
between positions -108 and -100 of the CYP11A promoter are
important for the formation of DNA-protein complexes using Y1 nuclear
extracts that contain Sp1 or a protein that is antigenically related to
it.
Figure 3:
Analysis of wild type and mutant bovine
CYP11A promoter activity by transient reporter gene expression. A, mouse Y1 adrenal tumor cells were cotransfected with CYP11A
promoter constructs and an internal reference plasmid using the
CaPO
Figure 4:
Y1
nuclear proteins associated with Sp1 bind to the CYP11A promoter region
between nucleotides -101 and -32. Y1 nuclear extracts were
incubated with labeled probes in A, -111/-100,
-101/-50, -70/-32; and B,
-118/-100, and -70/-50 as described in the
legend for Fig. 2. P, probe; E, Y1 nuclear
extract; SP1 OLIGO, consensus Sp1-binding oligonucleotide, SP1 AB or
Figure 5:
cAMP-dependent transcription supported by
-118/-100 and -70/-50 sequences is mediated
through the PKA signal transduction pathway. Mutant KIN8 (A)
and normal Y1 (B) cells were cotransfected with 15 µg of
test plasmid, 1 µg of an internal reference plasmid, and 4 µg
of either PKA catalytic or mutant regulatory expression vector. After
transfection, cells were grown in the presence of 100 µM ZnSO
These
findings were further corroborated by data obtained upon cotransfection
of the PKA subunit into Y1 cells which, unlike KIN8, express a
functional endogenous PKA enzyme. In these cells, constructs with
either -118/-100 or -70/-50 sequences from the
bovine CYP11A promoter respond to the exogenously supplied free
catalytic subunit by supporting a 4-5-fold increased
transcriptional activity compared to that observed in the control cells (Fig. 5B). The same effect could also be reproduced by
treating the cells with forskolin alone. Conversely, when a mutant form
of the PKA regulatory subunit was expressed in these cells, forskolin
treatment did not support the same level of increased expression as
observed when the cells were treated with forskolin alone. This
suggests that exogenous mutant PKA regulatory subunit binds a portion
of the catalytic subunit that is dissociated from the endogenous wild
type regulatory PKA subunit by cAMP action. However, since the mutant
regulatory subunit lacks the ability to interact with cAMP, the bound
catalytic subunit can no longer participate in the signal transduction
pathway responsible for the activation of cAMP-dependent transcription
(Rae et al., 1979). The plasmid -105/-104MOV,
which carries mutations in the G-rich region of the
-118/-100 sequence and cannot bind Sp1, did not support
transcriptional activity in response to either forskolin treatment or
exogenously supplied PKA catalytic subunit. In contrast, 2
Figure 6:
Comparison of transcriptional activity
supported by CRSs of different genes involved in steroidogenesis.
Transcriptional activity directed by promoter constructs containing one
(1
In previous studies the sequence -118/-100,
containing a putative binding site for the transcription factor Sp1,
has been shown to be involved in the cAMP-dependent transcription of
the bovine CYP11A gene in Y1 (Momoi et al., 1992) and bovine
ovarian luteal (Begeot et al., 1993) cells. We now report that
a minimal 12-bp G-rich sequence present between -111 and
-100 can indeed bind to Sp1 or an antigenically related protein
and this fragment is sufficient to support cAMP-dependent transcription
at the same level as that observed with the longer
-118/-100 fragment. Evidence is also presented for the
identification of an additional cAMP responsive sequence in the region
between -70 and -50. While mutagenesis of the putative
Sp1-binding site within -70/-50 has not been carried out,
all experimental features of this element including gel shift pattern
with Y1 nuclear extracts or purified Sp1, supershift by anti-Sp1, and
transcriptional responsiveness to both forskolin and PKA catalytic
subunit are similar to -118/-100. We infer from these
similarities that both -118/-100 and -70/-50
can bind Sp1 and function independently as cAMP-responsive sequences.
Thus, within the proximal 110 bp of the bovine CYP11A promoter
sequence, two CRS elements participate in cAMP-induced transcription in
Y1 cells. Consistent with these results, the -101/+12 and
-186/+12 bovine CYP11A fragments support cAMP-induced
transcription in Y1 cells at levels expected from one or both of the
identified Sp1-binding elements. Among the steroidogenic genes,
evidence for the existence of multiple CRS sequences has also been
documented for mouse (Rice et al., 1990) and human (Guo et
al., 1994) CYP11A and bovine CYP17 (Lund et al., 1990).
When the available sequences from the 5`-flanking regions of bovine
(Ahlgren et al., 1990), ovine (Pestell et al., 1993),
mouse (Rice et al., 1990), rat (Oonk et al., 1990),
and human (Morohashi et al., 1987) CYP11A genes are compared (Fig. 7), it is noticed that -111/-100 and
-70/-50 sequences are among the five upstream regions of
greatest sequence homology between the genes (boxed areas).
There is a 100% identity in the regions -111/-100 and
-70/-50 between bovine and ovine sequences, while putative
Sp1-binding sites are also present in the regions of the human, mouse,
and rat genes corresponding to bovine -110/-100. It is also
noteworthy that the corresponding sequences within the
-118/-100 and -70/-50 regions of homology have
been shown to be important for the cAMP-induced transcription of mouse
CYP11A in Y1 cells (Rice et al., 1990). Similarly, the human
-118/-100 sequence and the rat -73/-38 sequence
have also been shown to support cAMP-dependent transcription in Y1/JEG3
(Guo et al., 1994) and rat granulosa cells (Clemens et
al., 1994), respectively.
Figure 7:
Alignment of the sequences in the proximal
promoter regions of CYP11A from different species. The sequences
showing the greatest homology in the proximal promoter regions of
CYP11A genes from different species are shown in the boxes.
The bases that differ from the bovine sequence are underlined and shown in bold. Dots indicate the gaps introduced to
maximize the sequence homology. The numbers above the boxes
indicate the nucleotide positions relative to the transcriptional start
site of bovine CYP11A. The 5`-end of ovine is -109, mouse is
-118, rat is -119, human is
-117.
It has been suggested by Rice et
al.(1990) that a sequence similar to the consensus SF-1 binding
site (AGGTCA) is responsible for the cAMP-induced transcription
observed with the -77/-60 region of the mouse gene in Y1
cells. However, this sequence present at similar position in an
identical context in the rat gene is not required for cAMP-dependent
transcription in rat granulosa cells (Clemens et al., 1994).
In addition, when AGGAGC, present at nucleotide positions -106 to
-101 in the bovine CYP11A sequence, was changed to AGGTCA (Fig. 1B, -103/-101M), to make it identical
to the SF-1 binding sequence in the mouse gene cited above, cAMP
induced transcription was abolished (Fig. 3A). Whether
the highly conserved sequence -60/-50, which is nearly
identical in the CYP11A genes from different species (Fig. 7),
plays a more important part than the GC-rich -70/-60
sequence in conferring the cAMP responsiveness to the proximal bovine
CYP11A promoter (-70/-50) remains to be studied. There
are other examples of genes that contain Sp1-binding sites either close
to or within a cAMP-responsive element. These include genes that encode
human CYP21 (Kagawa and Waterman, 1992), type II In genes such as
CYP11A and adrenodoxin which are expressed constitutively, the former
being active exclusively or predominantly in steroidogenic tissues, the
general transcription factor Sp1 might play a very important role in
maintaining the basal transcription. Indeed, each cAMP-responsive
sequence of the bovine CYP11A gene (-118/-100 and
-70/-50) which has one Sp1-binding site and the bovine
adrenodoxin sequence from 699 to 740 which contains two Sp1 sites
confer basal expression to a Coexpression studies of PKA catalytic
and regulatory subunits in KIN8 and Y1 cells show that the bovine
CYP11A cAMP-responsive sequences -118/-100 and
-70/-50 function via a cAMP-PKA signal transduction
pathway. Since Sp1 does not have a PKA phosphorylation site and at
least in -111/-100 an Sp1-like protein is the only one
which binds directly to the DNA, regulation of transcription directed
by the G-rich elements at -111/-100 and -70/-50
sequences probably involves Sp1 or an Sp1-like factor in combination
with an as yet unidentified cell-specific protein. This unknown protein
would be predicted to interact directly with Sp1 and to not be a
DNA-binding protein. Perhaps it could function like CBP, the nuclear
protein which serves as a coactivator for the phosphorylated form of
CREB (Chrivia et al., 1993; Kwok et al., 1994).
However, in this case the unknown coactivator might indeed be a target
for PKA in execution of its role in coupling the ubiquitous
transcription factor Sp1 with cAMP-dependent activation of CYP11A
transcription.
Volume 270,
Number 43,
Issue of October 27, 1995 pp. 25402-25410
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
)of the
5`-flanking region of the bovine CYP11A gene identified a cAMP-response
sequence (CRS) between -183 and -83 bp (Ahlgren et
al., 1990). Subsequently, this element was found to reside within
-118 and -100 bp and was shown to be sufficient for
cAMP-dependent transcription of a reporter gene in mouse adrenocortical
Y1 tumor cells (Momoi et al., 1992) as well as in bovine
ovarian luteal cells (Begeot et al., 1993). This G-rich
sequence, conserved at similar positions in the CYP11A genes of
different species, has also been implicated in the negative regulation
of forskolin-induced bovine CYP11A gene transcription by phorbol esters
in bovine ovarian luteal cells (Begeot et al., 1993; Lauber et al., 1993). It does not share homology with the well
characterized cAMP-response element (CRE) (Montminy et al.,
1990) but contains a site that is similar to the consensus Sp1 binding
sequence (Kadonaga et al., 1986). The human CYP11A gene does
contain a sequence similar to the CRE between -1654 and
-1648 (Inoue et al., 1988; Moore et al., 1992;
Guo et al., 1994) which is far upstream from the G-rich region
sharing homology with bovine CYP11A.
Oligonucleotides
Complimentary single-stranded
oligonucleotides were synthesized and annealed to generate the
following double-stranded oligonucleotides representing both wild type
and mutant bovine CYP11A CRS fragments. The sequence for a consensus
GC-rich double-stranded oligonucleotide that binds transcription factor
Sp1 is also shown (k). The staggered sequences, in lower case
letters, at the 5`- and 3`-ends of the antisense strand were included
to enable the insertion of the oligonucleotides into SacI and SalI restriction enzyme sites of OVEC or luciferase reporter
plasmid DNA. The bold letters identify the positions where changes in
wild type sequence were made to create mutant oligonucleotides. (a) -118/-100, 5`-cgagACTGAGTCTGGGAGGAGCTg-3`,
3`-tcgagctcTGACTCAGACCCTCCTCGAcagct-5`; (b)
-116/-114M, 5`-cgagACATTGTCTGGGAGGAGCTg-3`,
3`-tcgagctcTGTAACAGACCCTCCTCGAcagct-5`; (c)
-108/-107M, 5`-cgagACTGAGTCTGCCAGGAGCTg-3`,
3`-tcgagctcTGACTCAGACGGTCCTCGAcagct-5`; (d)
-105/-104M, 5`-cgagACTGAGTCTGGGACCAGCTg-3`,
3`-tcgagctcTGACTCAGACCCTGGTCGAcagct-5`; (e)
-103/-101M, 5`-cgagACTGAGTCTGGGAGGTCATg-3`,
3`-tcgagctcTGACTCAGACCCTCCAGTAcagct-5`; (f)
-118/-104, 5`-cgagACTGAGTCTGGGAGGg-3`,
3`-tcgagctcTGACTCAGACCCTCCcagct-5`; (g) -111/-100,
5`-cgagCTGGGAGGAGCTg-3`, 3`-tcgagctcGACCCTCCTCGAcagct-5`; (h)
-101/-50,
5`-cgagCTGTGTGGGCTGGAGTCAGCCGGAGGAGGCTGACCGCCCTGTCAGCTTCTCAG-3`,
3`-tcgagctcGACACACCCGACCTCAGTCGGCCTCCTCCGACTGGCGGGACAGTCGAAGAGTCagct-5`; (i) -70/-32,
5`-cgagGACCGCCCTGTCAGCTTCTCACTTAGCCTTGAGCTGGTGG-3`,
3`-tcgagctcCTGGCGGGACAGTCGAAGAGTGAATCGGAACTCGACCACCagct-5`; (j) -70/-50, 5`-cgagGACCGCCCTGTCAGCTTCTCAg-3`,
3`-tcgagctcCTGGCGGGACAGTCGAAGAGTcagct-5`; (k) consensus Sp1
oligonucleotide, 5`-CCTCGAGATCGGGGCGGGGCGATG-3`,
3`-tcgaGGAGCTCTAGCCCCGCCCCGCTACagct-5`.Plasmid Construction
The 
-globin reporter
gene plasmids, OVEC, OVECREF, and SVOVEC (Westin et al.,
1987), were obtained from Drs. Thomas Gerster and Walter Schaffner,
University of Zurich, Switzerland. The primary vector OVEC contains a
rabbit -globin coding sequence and a minimal -globin promoter
and is used in the subcloning of promoter fragments. The luciferase
reporter gene plasmids, pA
LUC, a promoterless vector
(Maxwell et al., 1989; Wood et al., 1989) and
pRSV186LUC, which contains the enhancerless Rous sarcoma virus (RSV)
promoter, were provided by Dr. David Gordon (University of Colorado).
The vector pALLUC was constructed by inserting a 100-bp KpnI/HindIII polylinker fragment from the PHECATN
plasmid (5 Prime 3 Prime, Inc., Boulder, CO) into the unique KpnI/HindIII sites of pA
LUC.
and 2
-126/-113h21OV (Kagawa and Waterman, 1992), 1
and 2 -243/-225b17OV (Lund et al.,
1990), and 699/740ADXOV (Chen and Waterman, 1992), respectively. The
plasmid 4 CREOV (Lund et al., 1990) contains four
tandem copies of the CRE from the human chorionic gonadotropin-
gene (Deutsch et al., 1987). Plasmids OVECREF and SV40OVEC,
containing the 72-bp repeat SV40 enhancer elements, served as an
internal reference control and a positive -globin reporter gene
expression control, respectively.
-globin reporter constructs. The CYP11A promoter sequences
shown in B were inserted between SacI (ScI)
and SalI (SlI) sites in front of a minimal
-globin promoter. D, CYP11A-luciferase reporer plasmids.
CYP11A promoter fragments that contained homologous TATA sequences were
inserted into XhoI (XhI) and PstI (PsI) sites of the ALLUC vector 5` to the
luciferase coding region. Three tandemly repeated boxes represent SV40 polyadenylation sites preceding the promoter
insertion site.
LLUC vector. The
-186/+12LUC and -101/+12LUC contained the
homologous bovine CYP11A TATA element. All plasmid constructions were
confirmed by restriction digestion and dideoxy sequencing. The
metallothionein promoter-controlled expression vectors for catalytic
and mutant type I regulatory subunits of PKA, CEVNeo, and
Mt-REV(AB)-Neo, respectively, were kindly provided by Dr. Stanley
McKnight, University of Washington, Seattle.Preparation of Nuclear Extracts
Nuclear extracts
were prepared from mouse Y1 adrenocortical cells as described by Dignam et al.(1983) and simplified for small scale preparation as
described by Andrews and Faller(1991). Protein concentrations were
determined by the BCA assay (Pierce Chemical Co., Rockford, IL) using
bovine serum albumin as standard.Gel Shift Assay
The double-stranded
oligonucleotides were labeled by polynucleotide kinase and
[-
P]dATP (6,000 Ci/mmol, Amersham Corp.) or
by a fill-in reaction with [
-P]dCTP (3,000
Ci/mmol, Amersham Corp.) and DNA polymerase I, Klenow fragment.
Twenty-five µg of nuclear extract protein or 7.5 ng of purified Sp1
transcription factor (Promega Biotech Inc.) were mixed with 15 µl
of binding buffer (20 mM Hepes, pH 7.9, 80 mM KCl, 5
mM MgCl
, 2% Ficoll, 5% glycerol, 0.1 mM EDTA, 0.2 mM DTT), 2 µg of poly(dI-dC), and P-labeled probe (10,000 cpm) on ice. For competition
assay, 10 pmol of unlabeled competitor oligonucleotide was used along
with the labeled probe. In the case of antibody supershift experiments,
the incubation conditions were identical except 1-2 µg of
anti-Sp1 antibody (Santa Cruz Biotech., Santa Cruz, CA) was
preincubated with 25 µg of nuclear extract proteins from Y1 cells
in the presence of binding buffer for 10 min prior to the addition of
other components. The DNA-protein complexes were resolved by
electrophoresis on a 5% polyacrylamide, 0.5% Ficoll gel (Sambrook et al., 1989) and visualized by autoradiography.
Cell Culture and Transient DNA Transfections
The
Y1 and KIN8 cells were routinely maintained in Dulbecco's
modified Eagle's medium supplemented with 10% bovine calf serum
at 37 °C in a 5% CO incubator. Near confluent cell
monolayers were trypsinized and split (1:6) into 10-cm plates 24 h
before the addition of DNA precipitates. Fresh medium was added to the
cells 4 h before transfection. Nineteen µg of test plasmid plus 1
µg of internal reference vector, OVECREF (in the case of
-globin reporter assays), or 15 µg of test plasmid together
with 4 µg of CEVNeo/Mt-REV(AB)-Neo, and 1 µg of OVECREF
(in the case of PKA cotransfection experiments) were precipitated with
0.125 mM CaCl in the presence of 50 mM Hepes, 1.5 mM sodium phosphate, 280 mM NaCl, pH
7.12 (Graham and Van Der Eb, 1973; Gorman et al., 1982). One
ml of DNA-calcium phosphate precipitate was added to each plate of
cells. After 4 h of exposure to the DNA precipitates, the cells were
shocked with 15% glycerol for 3 min at room temperature. The medium was
changed the morning after transfection, and the cells were maintained
in the presence of either 25 µM forskolin or dimethyl
sulfoxide (vehicle) for 6-8 h. The medium used after PKA subunit
transfections always contained 100 µM ZnSO
to
activate metallothionein promoter-controlled PKA subunit expression.
For luciferase reporter gene assays the transfection procedure was the
same as described before except 20 µg of test plasmid were used,
and the cells were exposed to forskolin for 36 h after the treatment
with glycerol.RNA Isolation and S1 Nuclease Analysis
Two
alternate procedures were followed for the preparation of RNA from
transfected cells. In the first method, the RNA was prepared as
described by Lund et al.(1990). In the second, RNA was
isolated using a commercially available Ultraspec reagent (Biotecx
Laboratories, Inc., Houston, TX). After removal of medium, 2 ml of
Ultraspec reagent were added directly to the cells on the plate for
lysis. The lysate was mixed by vortexing, transferred into 15-ml tubes,
and kept on ice for 5 min. Following the addition of 400 µl of
chloroform to the lysate, the samples were mixed vigorously and
incubated on ice for an additional period of 5 min. After a 10-min
centrifugation at 10,000 rpm, the RNA in the supernatant was
precipitated with an equal volume of isopropanol. S1 nuclease analysis
on 10-50 µg of cytoplasmic RNA was performed as described
previously (Lund et al., 1990). After autoradiography, the
intensities of the bands corresponding to the correctly initiated
transcripts were quantitated using a PhosphorImager (Molecular
Dynamics, Inc., Sunnyvale, CA).Luciferase Assays
Thirty-six hours after the
treatment with glycerol, the cells were lysed with 1 ml of 1% Triton
X-100, 25 mM glycylglycine, pH 7.8, 15 mM MgSO, 4 mM EGTA, and 1 mM DTT. The
assays were performed essentially as described previously by Brasier et al.(1989) and Venepally et al.(1992). One hundred
µl of lysate were added to 360 µl of 25 mM glycylglycine, pH 7.8, 15 mM MgSO, 4 mM EGTA, 15 mM potassium phosphate, pH 7.8, 1 mM DTT, and 2 mM ATP. The luciferase reactions were
initiated by the injection of 100 µl of 25 mM
glycylglycine, pH 7.8, 15 mM MgSO, 4 mM EGTA, 2 mM DTT, and 0.2 mM luciferin into the
sample, and light output was measured for 20 s at 25 °C using a
monolight luminometer (Analytical Luminescence Laboratory, San Diego,
CA).
Mutations in the -118/-100 Sequence Affect
Formation of DNA-Sp1 Complexes
The proximal promoter region of
the bovine CYP11A gene (Fig. 1A) that supports
cAMP-induced transcription in Y1 adrenocortical cells (Ahlgren et
al., 1990) contains potential binding sites for the general
transcription factor Sp1 (Kadonaga et al., 1986),
steroidogenic factor, SF-1 (Morohashi et al., 1992; Lala et al., 1992), and adrenal specific factor, ASP (Kagawa and
Waterman, 1992). Deletion analyses of this region have previously shown
that the sequence between -118 and -100 is sufficient for
supporting cAMP-dependent transcription in both Y1 (Momoi et
al., 1992) and bovine ovarian luteal cells (Begeot et
al., 1993). Moreover, the same sequence with nuclear extracts from
bovine ovarian luteal cells (Begeot et al., 1993) and a
slightly larger fragment, extending from -118 to -83, with
nuclear extracts from Y1 cells show binding to proteins that also
interact with the consensus Sp1 binding sequence. To more precisely
identify the nucleotides important for the binding of these nuclear
proteins, several mutations were made within the -118/-100
sequence (Fig. 1B) and their effects on the DNA-protein
interactions were examined using Y1 nuclear extracts. As seen in Fig. 2A, both wild type -118/-100 and a
consensus Sp1 binding sequence produce similar complexes I, II, and
III, although the latter showed greater affinity. Only the
-116/-114M sequence and the consensus Sp1 oligonucleotide
competed for the formation of these complexes by the wild type
-118/-100 sequence.
P-Labeled double-stranded
oligonucleotides were incubated with 25 µg of Y1 nuclear proteins
or 7.5 ng of purified Sp1. In the competition assays, 10 pmol (100-fold
molar excess) of unlabeled oligonucleotides were added along with the
probe. DNA-protein complexes were analyzed by electrophoresis on a
native 4% polyacrylamide gel. A, DNA-protein complexes formed
by the labeled -118/-100 CYP11A and consensus Sp1
oligonucleotide sequences. Excess unlabeled consenus Sp1 (Sp1
OLIGO) and CYP11A mutant oligonucleotides -108/-107M,
-105/-104M, -116/-114M, -103/-101M
were used as competitors. P, probe; E, extract. B, labeled wild type -118/-100 and mutant
-103/-101M, -105/-104M, -108/-107M,
-116/-114M fragments were used as probes in the presence of
Y1 nuclear extracts and purified Sp1. The consensus Sp1 oligonucleotide (Sp1 OLIGO) was used as competitor. P, probe; EXT, extract. C, 2 µg of polyclonal anti-Sp1
(
-Sp1) was added to the -118/-100 CYP11A and
consensus Sp1 sequence probes in incubation mixtures containing either
Y1 nuclear extracts or purified Sp1. P, probe; EXT,
extract; SS, supershift. D, 2 µg of polyclonal
anti-Sp1 was added to Y1 nuclear extracts in the presence of
-118/-100, -111/-100, and -118/-104
CYP11A probes.
Transient Expression Analysis of Wild Type and Mutant
CYP11A Reporter Gene Plamids in Y1 Cells
To examine the effects
of mutations and deletions in the -118/-100 region on
cAMP-dependent transcription, the wild type and mutant
-118/-100 sequences, used in in DNA-protein interaction
analyses shown in Fig. 2, were inserted into OVEC--globin
reporter gene vectors (Fig. 1C) and transfected into Y1
adrenal tumor cells. Cytoplasmic RNA, isolated from cells treated with
and without (control) forskolin, was analyzed by S1-nuclease digestion
for expression of the -globin reporter mRNA. As seen in Fig. 3A, forskolin treatment resulted in a
5-6-fold increase over control levels of -globin RNA
expression with both the -118/-100OV wild type and
-116/-114MOV mutant plasmids. The fold-induction in
response to forskolin observed with these two constructs is similar to
that obtained with -896/-32OV, although, the latter
supported the highest level of activity. Promoter constructs with
mutations between -108 and -100 bp, namely
-103/-101MOV, -105/-103MOV, and
-108/-107MOV, showed a markedly decreased forskolininduced
transcription compared with either -118/-100OV or
-116/-114MOV. Both basal and forskolin-induced
transcription observed from these mutant constructs was comparable to
that seen with the empty vector (OVEC). Also, the
-111/-100OV plasmid was as efficient as the longer wild
type -118/-100 construct in supporting cAMP
(forskolin)-induced transcription in Y1 adrenocortical tumor cells (Fig. 3B). Thus, the mutations in the G-rich region of
the -118/-100 sequence that reduced or eliminated the
formation of DNA-protein complexes I and II in Y1 nuclear extracts (Fig. 2B) also markedly reduced cAMP-induced
transcription in Y1 cells.
method. After transfection, cytoplasmic RNA from cells
grown in the presence or absence of 25 µM forskolin was
analyzed by S1 nuclease digestion for the expression of -globin
reporter messenger RNA. The results were quantified using a
PhosphorImager. The values obtained for correctly initiated transcripts
were normalized to the corresponding internal signal. The results
obtained in different experiments for each construct (OVEC,
-118/-100OV, n = 4;
-103/-101MOV, -105/-104MOV,
-108/-107MOV, and -116/-114MOV, n = 3) except in the case of -896/-32OV (n = 2) are shown as mean + S.D., error bars. B,
transcriptional activity directed by -118/-100 and
-111/-100 CYP11A sequences compared as described in A. The average values from two independent experiments are
shown.
Analysis of the Sequences between -100 and
-32 bp
The data from the initial deletion analysis of the
5`-flanking region of bovine CYP11A (Ahlgren et al., 1990)
showed that, although the fragment from -186/-32 promoted
near-maximal cAMP-dependent transcription, a smaller sequence between
-101 and 32 retained the ability to promote a 9-fold increase in
cAMP-dependent transcription over control levels in Y1 cells. This
suggested the possibility that more than one cAMP-responsive sequence
might be present in the proximal 5`-flanking region of the bovine
CYP11A promoter. To look for elements in addition to
-111/-100, two overlapping fragments, -101/-50
and -70/-32 spanning the sequence -101 to -32
bp, were screened for their ability to bind to nuclear proteins from Y1
cells. As seen in Fig. 4A, both overlapping fragments
-101/-50 and -70/-32 formed a complex similar
to complex I observed with the -111/-100. Formation of this
complex disappeared when the unlabeled consensus Sp1 oligonucleotide
was used as a competitor. Complexes III and IV, observed in the case of
-101/-50 and -70/-50, respectively, were not
affected by such competition. Further, in each case, protein(s) present
in complex I interacted with the Sp1 antibody, resulting in a
supershift complex, suggesting that, as with -111/-100,
either Sp1 or proteins antigenically related to it bind to sequences
within -101/-50 and -70/-32. A sequence that
shares homology with the consensus Sp1-binding site is present between
-70 and -60 of the CYP11A promoter (Fig. 1A). Thus, the -70/-50 sequence,
which is common to fragments -101/-50 and
-70/-32, was compared with the -118/-100
sequence for both its ability to bind Sp1 and to support cAMP-induced
transcription in Y1 cells (Fig. 4, B and C).
In the presence of Y1 nuclear extracts, both fragments
-118/-100 and -70/-50 yielded complexes I and
II, although the former showed stronger complex formation (Fig. 4B). Also, from the appearance of a supershift
complex in the presence of Sp1 antibody, the complexes formed by
-70/-50 appear to contain Sp1 or a protein
antigenically related to it. Addition of antibody against CREB
transcription factor did not affect any of the complexes, revealing
that CREB is not involved in their formation. Moreover, when assayed by
transient expression analysis, the plasmids containing both
-118/-100 and -70/-50 fragments supported
similar levels of cAMP-induced transcription in Y1 cells (Fig. 4C). Thus, there is an additional cAMP-responsive
element in the bovine CYP11A promoter region which is located between
-70 and -50 bp, and this sequence like
-111/-100 has the ability to bind Sp1. Consistent with
these results, when -186/+12 and -101/+12 CYP11A
fragments, which contain both or only the proximal Sp1-binding site(s),
respectively, were analyzed for transcriptional activity in Y1 cells (Fig. 4D), the former showed a 5-fold greater activity
than the latter in both basal and cAMP-induced transcription. However,
as compared to the vector ALLUC, the -101/+12
fragment itself supported a 10- and 20-fold increase in basal and
cAMP-induced transcription, respectively. Thus, the transcriptional
elements present between -186 and -101 and between
-101 and +12 of the bovine CYP11A promoter each participate
in both basal and cAMP-induced transcription in Y1 cells.
-Sp1, anti-Sp1 antibody;
-CREB, anti-CREB antibody; SS, supershift
complex. C, comparative analysis of the bovine CYP11A promoter
activity observed with -118/-100 and -70/-50
sequences: error bars indicate S.D. of the mean for three
separate experiments, performed as described in Fig. 3A.
D, luciferase activity directed by constructs containing
homologous CYP11A TATA sequence in Y1 cells. The average values from
two separate experiments is presented.
cAMP-induced Transcription Supported by
-118/-100 and -70/-50 Sequences Is Mediated by
Protein Kinase A
The role of PKA in the forskolin-stimulated
transcription supported by CYP11A sequences was examined by the
analysis of reporter gene expression in KIN8 and Y1 cells (Fig. 5). When the activity of promoter constructs containing
one or two copies of the -118/-100 sequence was analyzed in
PKA-deficient KIN8 cells, little cAMP-induced transcription was
observed over the background levels (Fig. 5A). However,
the same plasmids showed a 5-6-fold increase in the reporter gene
transcription when the free catalytic subunit of PKA was overexpressed
in these cells. As expected, forskolin treatment did not enhance the
PKA catalytic subunit-dependent transcription, since the catalytic
subunit does not interact with cAMP (Rae et al., 1979). The
high level of transcriptional activity observed from the SV40OV was
unaffected by the absence or the presence of a functional PKA.
throughout the experiment to activate the
metallothionein promoter-driven expression of PKA subunits. The
cytoplasmic RNA, isolated from both untreated(-) and
forskolin-treated cells (+), was analyzed for -globin mRNA by
S1 nuclease digestion as described under methods and Fig. 3A. Because of the low level of transcriptional
activity in KIN8 cells, the average values from two experiments are
presented relative to the expression observed from the negative
controls OVEC (forskolin(-), and PKA CAT(-); not shown). In
the case of Y1 cells, which support a high level of cAMP-dependent
transcription, the data is presented as a percentage of activity
observed from 2 CREOV in the presence of forskolin. P,
probe; C.I., correctly initiated transcripts; REF,
internal control. PKA CAT and PKA REG represent PKA
catalytic and mutant regulatory subunit expression
vectors.
CREOV, the plasmid that contains two consensus CRE elements, showed a
very high level of transcriptional activity under these conditions.Comparison of cAMP Responsiveness of Various CRS Elements
Involved in Steroidogenesis
The levels of basal and
cAMP-dependent transcription supported by the -118/-100 and
-70/-50 fragments of the bovine CYP11A gene and CRSs
characterized in other genes involved in steroidogenesis were compared
to evaluate their relative strengths (Fig. 6). An approximate
3-5-fold increase in cAMP-dependent transcription was observed by
equivalent constructs containing one or two tandem copies of bovine
CYP11A CRSs (-118/-100 and -70/-50), bovine
CYP17 CRS1 (-243/-225) and human CYP21 CRS
(-126/-113) promoter sequences. The CRS from intron 1 of
the bovine adrenodoxin gene in ADXOV (699/740) (Chen and Waterman,
1992) that contains two GC-rich sequences supported the highest level
of basal and cAMP-induced transcription among the genes associated with
steroidogenic metabolism. The level of cAMP-induced expression directed
by 2 CREOV was similar to that observed with 1 ADXOV,
although the induction effect observed in the case of the former was
much higher because of its low basal activity. OVEC served as the
control for background expression.
) or two (2) copies of discrete CRS elements from
bovine CYP11A (-118/-100bSCCOV and
-70/-50bSCCOV), bovine CYP17
(-243/-225b17OV), human CYP21
(-126/-113h21OV), and bovine adrenodoxin (699/740ADXOV) genes were compared in Y1 cells alongside CREOV
plasmids which carry one or two copies of the CRE
sequence.
cAMP-dependent
protein kinase regulatory subunit (Kurten et al., 1992),
bovine adrenodoxin (Chen and Waterman, 1992), human ferredoxin (Chang et al., 1992), and human urokinase (Grimaldi et al.,
1993). Although, the importance of Sp1 in the constitutive expression
of genes, in general, is well understood, the evidence for its direct
role in cAMP-mediated transcription has not yet been clearly
established at a mechanistic level. In the case of the human CYP21
gene, Sp1 and the adrenal-specific protein (ASP) have overlapping
binding sites in the -129/-96 sequence, although ASP alone
can support cAMP-dependent transcription from a shorter
-126/-113 promoter fragment (Kagawa and Waterman, 1992).
Interestingly, ASP also binds to the bovine CYP11A sequence around
-101 to -89 (Momoi et al., 1992) but is not
involved in cAMP-dependent transcription of this gene. In another
example, a sequence similar to the consensus Sp1-binding site in the
cAMP-responsive element (-54/-42) of the human urokinase
gene interacts with cAMP-induced DNA-protein complexes in mouse Sertoli
cells (Grimaldi et al., 1993). In other genes, evidence for
the involvement of Sp1 is also documented in the case of retinoic
acid/cAMP-dependent and differentiation-specific transcription of the
tissue plasminogen activator gene (Darrow et al., 1990).
Acting through retinoblastoma control elements, Sp1 has also been shown
to activate transcription of early response genes such as c-fos (Udvadia et al., 1993), the expression of which is
induced by ACTH (Miyamoto et al., 1992).-globin reporter gene in Y1 cells (Fig. 6). However, it is also noticed that these same sequences
also support cAMP-dependent transcription (Fig. 6) and any
mutations that reduce Sp1 binding to the -118/-100 sequence
in bovine CYP11A gene reduce, in parallel, both basal and cAMP-induced
transcription (Fig. 2B and Fig. 3A).
Thus, these multiple dual role responsive sequences of bovine CYP11A,
just as in their murine counterparts (Rice et al., 1990),
might function independently or in association in supporting optimal
level of CYP11A transcription.
)
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