(Received for publication, August 10, 1994; and in revised form, December 19, 1994)
From the
Transcription of the luteinizing hormone receptor gene is
dependent on Sp1-induced promoter activation from two Sp1 binding
domains (Sp1 and Sp1
) within the 173-base pair
promoter. Of the two Sp1 binding domains, the canonical GC box (GGGCGG)
was determined by mutation to be the binding element for only the
Sp1
domain. The Sp1 binding element within the Sp1
domain was identified by mutation and immunological/competition
studies as the 5`-GGG GTG GGG that conforms to a Zif-268 like three
zinc finger binding domain, rather than the canonical 3` Sp1
GC box (GGGCGG). The guanines in the third trinucleotide (GGG GTG
GGG) were not required for Sp1 binding, although they
increased binding affinity. Non-Sp1 protein(s) bind the 3` Sp1
GC box, and by themselves exhibit transcriptional activity.
Tissue specific differences were localized to this non-Sp1 binding
domain, which functionally substituted for the downstream activating M1
regulatory domain in non-expressing but not in expressing cells.
Mutations of both non-Sp1 and M1 domains were required for inhibition
of promoter activity in constructs that retained the Sp1 binding
elements in non-expressing cells, indicating that together these
domains may play a role in regulation of luteinizing hormone receptor
gene expression.
The promoter region of the luteinizing hormone receptor (LHR) ()gene has been localized to the 173-nucleotide 5`-flanking
domain, which induces maximal basal transcriptional initiation of a
reporter gene in both expressing and non-expressing cell types.
Repression of this activity is governed by regions upstream of
-173 bp (1) that are of critical importance in silencing
gene expression in non-expressing cell types and regulating LHR gene
expression in the gonadal cell.
The structure of the isolated LHR
promoter domain that elicits constitutive promoter activity in both
cell types has been characterized previously as a TATA-less region that
carries multiple GC boxes(2) . Without an obvious TATA element,
our investigation has centered on a possible Sp1-driven promoter
activity(3, 4) . Two potential Sp1 binding domains,
Sp1 (-73 to -94 bp), and Sp1
(-135 to -154 bp), each contain a single GC box that
competes for trans-binding factors with standard Sp1 elements. These
LHR Sp1 domains were identified by deletion and competition studies to
be essential for basal promoter activity(1) . The Sp1 binding
element in Sp1
was previously localized through mutation to
the canonical GC box (GGGCGG) that conforms to those identified in the
promoter regions of GC-rich housekeeping genes(5) . Addition of
LHR 5`-flanking nucleotides (-138 to -173 bp) to the
construct that contains the Sp1
domain (+1 to
-137 bp) resulted in an additional 100% increase in promoter
activity(5) . The 20-nucleotide domain that was determined to
be responsible for this increase was defined as Sp1
and
contained a canonical GC box. These studies also indicated that unlike
the Sp1
domain, mutation of the entire 20-nucleotide G-rich
Sp1
domain was necessary to abolish competition for Sp1
trans-factors, and Sp1
-induced promoter
activity(5) . In addition, tissue-specific differences by the
downstream protein binding M1 domain (-24 to -42 bp), that
influenced Sp1
-induced promoter activation were
demonstrated(1, 5) . To further explore the mechanism
of Sp1 activation from the Sp1
domain, we analyzed the
potential Sp1 binding elements on Sp1
, and their individual
effect on transcriptional activation in the presence or absence of
viable Sp1
and M1 domains, in both expressing and
non-expressing cell types. Tandem Sp1/non-Sp1 binding elements were
identified on the Sp1
domain, and each element induced
transcript activation in a differential expressing versus non-expressing cell mode of action.
All mutants were generated by recombinant circle polymerase chain reaction (6) as described previously (5) and verified by sequence analysis using the dideoxy chain termination method.
IC values for competitive mobility shift assays (Fig. 3) (15) were determined in Kaleidograph (version 2.1, Abelbeck
Software, Synergy Software, Reading, PA) using the logistic function of
Delean et al.(16) .
Figure 3:
Competitive binding of WT and mutant
Sp1 Domains to the Sp1 factor. Gel retardation analysis of
labeled DNA binding to MLTC or CHO nuclear extracts in presence or
absence of unlabeled competitor(s) oligonucleotides b, b+2, or
Sp1
or Std Sp1. Semilog plots of % protein/labeled DNA
complex reflecting quantitative changes in the level of DNA/protein
complex with increasing doses of unlabeled competitor DNA. Labeled DNA: panelsA and C, b/aX*; panels B and D, b+2/(a-2)X*.
Figure 1:
Sp1
dependence of LHR promoter activity. Luciferase reporter gene activity
measured from Sp1-deficient SL cells (13) upon cotransfection
of p174GL or the mutant p174Sp1X/Sp1
XGL with
the Sp1 containing plasmid pPacSp1 (+). Sp1
X is bX/aX
LHR mutant (see Table 1). Mutant Sp1
domain:
(GGGGCGGG to atctgcaG). Controls are the plasmid without Sp1 cDNA
insert (pPac
)(-).
Sp1 was subdivided
into two domains: the 3`-subdomain (a) retaining the complete
Sp1 consensus element GGGCGG (14) , and the 5`-subdomain b (Table 1, upper panel). When the canonical Sp1 element within
subdomain a was mutated to ``atctgcag'' (b/aX), the mutant Sp1
oligonucleotide was still able to compete for LHR-expressing MLTC
and non-expressing CHO nuclear trans-factors that bound to the standard
Sp1 oligonucleotide in gel retardation studies (Fig. 2, lane
3 versuslane 5). Competition for Sp1 binding
trans-factors was, however, abolished with mutation of the 5`-end of
the Sp1
domain (subdomain b) (Fig. 2, lane2). Control autocompetition by unlabeled Std Sp1
is shown in lane6. These studies indicate that the
LHR Sp1 binding domain lies within the G-rich 5`-subdomain b of
Sp1
, rather than the GC box within subdomain a.
Figure 2:
Competition for Sp1 factors by wild type
and mutant Sp1 oligomers. Gel retardation analysis of CHO
and MLTC nuclear factors bound to labeled standard Sp1 ((cons)Sp1*) in the presence or absence(-) of 1000-fold
molar excess of designated unlabeled competitors in CHO or MLTC nuclear
extracts. X, mutations; minus, unchanged nucleotide. Asterisk is labeled oligonucleotide in this figure and
subsequent gel retardation studies (Fig. 3, Fig. 4, and Fig. 6).
Figure 4:
Supershift of Sp1 complexes binding to the
b and b+2 elements of the Sp1 domain. Gel retardation
analysis of MLTC and CHO nuclear factor Sp1 bound to labeled DNA probes (panelA, b/aX*; panelB,
(b+2)/(a-2)X*) identified by band supershift with Sp1
antibody. Nuclear extracts were preincubated for 30 min with rabbit
antiserum (Ab) raised against the human Sp1 protein prior to
the addition of labeled DNA. NRS, non-immune rabbit serum
control; -, no competitor.
Figure 6:
DNA/protein complexes formed from the
[a] element in the Sp1 domain. Gel retardation
analysis of CHO and mLTC nuclear factors bound to the a domain on the
mutant oligonucleotide (bx/a) (panels A and B), or
the mutant a domain (bx/a-) (panelB). Specific
complexes that are competed by the native Sp1
domain are
designated with arrows (1, 2 and 3, CHO; 1 and 3, MLTC).
Competing DNA (top) is present at 1000-fold molar excess.
-, no competitive DNA. *, labeled
DNA.
A
similarity was noted between subdomain b (GGG GTG GGG), and the
Sp1 three triplet nucleotide binding sequence GGG GCG GGG (15, 17, 18) with a substitution of the
pyrimidine C for T. The 5` first two trinucleotides and the first
nucleotide of the third trinucleotide (GGG GTG GGG; Table 1,
overlined) are within the Sp1 binding subdomain b, while the 3`
final two GG are within subdomain a (Table 1,
). To determine whether this element is the Sp1 binding
site, and the role of the 3` two guanines within domain a in
protein binding, mutant oligonucleotides containing either the entire b+2 element (GGG GTG GGG) or the b element (GGG GTG
Gat) were evaluated (see ``Materials and Methods''; mutants 3
and 2, respectively).
Competition mobility shift assays for labeled
mutant Sp1/nuclear protein complexes was used to determine
the IC
of the Sp1
b or b+2 elements for Sp1 (Fig. 3). The IC
for mutant
Sp1
DNA was similar to the IC
for the standard
consensus Sp1 oligonucleotide only when the two consecutive GG of the
putative third trinucleotide element were retained in the
oligonucleotide sequence (GGG GTG GGG; the b+2 element) (Fig. 3, panels A and BversusC and D) (mutant 2 versus mutant 3) (Table 2).
The labeled oligonucleotide b/aX, which does not
carry the final consecutive GG (GGG GTG Gat), exhibits a significant
increase in the IC for nuclear-binding proteins when
compared with the standard Sp1 (GGG GCG GGG) or the
Sp1
(b+2)/(a-2)X (GGG GTG GGG) DNA elements (Fig. 3, A and BversusC and D). In addition, a greater increase in the IC
was observed with competition by the native Sp1
(b/a)
sequence than the isolated b (mutant b/aX) or b+2 (mutant (b+2)/(a-2)X) element (Fig. 3). This
observation can be explained by the presence of non-Sp1 proteins on the
unlabeled native Sp1
domain a that interfere with
competition for the Sp1 trans-factor (see below). Mutation of the a-2 domain, leaving only the native Sp1
(b+2) element, converts the Sp1
domain
into an Sp1-binding oligonucleotide with similar IC
as
standard Sp1.
In subsequent studies, incubation of either the b or b+2/nuclear protein complexes with human
Sp1-directed antibody caused an electrophoretic band supershift,
indicating that the MLTC and CHO Sp1 protein does bind to both the b and b+2 Sp1 domains (Fig. 4, A and B). This finding substantiates that the protein that
binds b or b+2 is Sp1, and that the final two GG of
the third trinucleotide element are not essential for Sp1 binding to
the Sp1
domain. Individual mutation of nucleotides within
the putative LHR Sp1 binding element reveals that substitution of the
guanines in the 5` first trinucleotide element (GGG GTG GGG) abolishes
Sp1 binding to the LHR Sp1
domain (data not shown).
The b+2 element in the Sp1 domain is also a
consensus sequence for the binding domain of the AP-2
protein(19) . However, there is no indication that nuclear MLTC
or CHO AP-2 constitutively binds this element under basal conditions.
Gel retardation studies using the AP-2 consensus oligonucleotide as
labeled probe displayed a band that was not competed by either the b+2 ((b+2)/(a-2)X) or b (b/aX) element.
Incubation of labeled SP1
or the isolated b+2 ((b+2)/(a-2)X) with human AP-2 antibody did not result
in a decrease in the mobility of the DNA/protein complex, and similar
findings were observed with the b (b/aX)-labeled probe, although
a supershift was evident with the control AP-2 standard oligonucleotide
(data not shown).
Figure 5:
Transcriptional activity of Sp1 wild type and mutant variants of the luteinizing hormone receptor
promoter. Mean of relative luciferase activity of wild type and mutant
Sp1
domains in p174GL (A) or
p174Sp1
XGL (B) constructs transfected in
expressing cells (MLTC-1 and MLTC-2, upperpanels) or
non-expressing cells (CHO, Y1, and I10, lowerpanels). Wild type p174GL is normalized at 100% of
promoter activity. p174Sp1
XGL is also normalized at 100%,
although it represents approximately 50% of wild type p174GL
activity(5) . Mutated nucleotides are listed under
``Materials and Methods.''
The question of whether this non-expressing
cell specific reduction by the construct p174b/aXGL is due solely to a
reduced affinity for the Sp1 protein (Fig. 3) or to the absence
of activating non-Sp1 factors on the mutated Sp1a domain, and/or both was investigated. The subdomain a sequence (GGGGCGGAGA), by itself, was capable of inducing Sp1
activity at levels that are comparable to wild type p174b/aGL and
p174b/aSp1
XGL in the MLTC cell, and of inducing a partial
promoter activation in the non-expressing cell (Fig. 5, Table 1, mutant bX/a versus b/a). The requisite
nucleotide elements for this activation included the 5` two guanines
within the a subdomain GC box (GGGGCGG), since mutation of these
two nucleotides in the mutant sequence bX/a abolished Sp1
activation (Table 1,
, mutant bX/a versus (b+2)X/(a-2)). No significant difference was observed
between expressing and non-expressing cells with mutant
(b+2)X/(a-2).
Promoter activity induced by subdomain a does not appear to be related to the Sp1 protein. Gel retardation
studies show three nuclear protein complexes that bind to the labeled
bX/a oligonucleotide in the presence of CHO nuclear extract and two
nuclear protein complexes with MLTC nuclear extract, that are competed
by excess unlabeled Sp1 b/a and bX/a oligonucleotides (Fig. 6A, arrows). The observed competition by
both b/a and bX/a demonstrates protein binding specificity exclusively
to the a domain.
Mutation of the 5` second G in subdomain a to T (GGGGCGGAGA) to GtGGCGGAGA) results in a loss of subdomain a-induced promoter activity (Table 1, mutant bX/a versus bX/a-, Fig. 5A), and this mutation also results in the loss of protein/DNA complexes 1, 2, and 3 with CHO nuclear protein, and complex 3 with MLTC nuclear protein (Fig. 6, A and B). Thus, subdomain a-induced promoter activity can be attributed to CHO complexes 1, 2, and 3, and complex 3 from MLTC extract, and the second guanine of the GC box (GGGGCGG) in the subdomain a appears to be essential for the formation of these specific protein/DNA complexes. MLTC DNA/protein complex 1 was still visible on the transcriptionally inactive a- domain of bx/a- (Fig. 6B, lanes4, 5, and 7). The observation that bX/a- can form complex 1 but not 3 in MLTC is confirmed in lane11, where bX/a- was able to compete for complex 1, but not complex 3. Complex 1 was minimally competed by the GC box (GGGGCGGGG) in the standard Sp1 oligonucleotide (Fig. 6A, lanes 4 and 8), while protein/DNA complex 3 (MLTC and CHO) and complex 2 (CHO) showed no competition (Fig. 6A, lanes4 and 8). It can therefore be inferred that the underlined GGGGCGGAGA are requisite nucleotides in the complex 2 and 3 binding domain. The 5` second G requirement is demonstrated above with the mutant bX/a-, while the relevance of the 3`AGA can be deduced by a comparison of the nucleotide sequence of the non-competing GC box in the standard Sp1 oligonucleotide (GGGGCGGGG), and the nonmutated a (GGGGCGGAGA) domain of the competing oligonucleotides, bX/a and b/a.
Incubation of these Sp1 mutant oligonucleotide/protein complexes with anti human Sp1
antibody did not alter the mobility of DNA/protein complexes 1, 2, or 3 (Fig. 6B, lanes5 and 10),
confirming that protein(s) that bind to the a domain are not
Sp1, and that an Sp1 protein is not involved in the formation of
complex 1. Since this binding protein tolerates the substitution of
GtGGCGG for GGGGCGG in MLTC, but not CHO extract (Fig. 6B), complex 1 may involve different non-Sp1
proteins between the two cell types. However, only the CHO, not the
MLTC complex 1, may be of relevance to transcriptional activity since
it is not observed upon mutation to a transcriptionally inactive
promoter p174bX/a-GL (Fig. 6, B, lanes2 and 4, and Table 1).
Thus, in the absence of the LHR Sp1 binding domain (b), non-Sp1 proteins that exhibit different characteristics in MLTC and CHO nuclear extract, bind to domain a, and themselves induce transcript activation. The relevant putative functional proteins have been identified as CHO complexes 1, 2, and 3, and MLTC complex 3.
However, in all of the non-expressing cells, a potentiation by each of the a- and b-binding proteins of wild type activity is evident, since
individual mutation of either the a or b subdomains
resulted in statistically significant losses of promoter activity (Table 1, Fig. 5A, lowerpanel). Similar results were observed in the
p174Sp1XGL constructs (Fig. 5B, lower
panel). These data indicate that, unlike the expressing MLTC cell,
both the b and a domains in the non-expressing cell are
simultaneously functional.
Figure 7:
Effect
of simultaneous mutation of the M1 and Sp1 domain on LHR
promoter activity. Relative luciferase activity of wild type and mutant
p174GL (leftaxis) and p138GL (rightaxis) transfected in MLTC and CHO cells (openbars, wild type; shadedbars, mutant).
Different y axis scales reflect the 1-fold increase in
promoter activity from p138GL to p174GL. This figure contains data
reported in (5) for comparison as well as new experiments.
Statistically significant differences: MLTC (p174GL versus p174M1XGL) and CHO (p174M1XGL versus p174M1X/aXGL), p < 0.0001.
In non-expressing
cells, addition of the native Sp1 domain did overcome the
inhibitory effect of mutation of the M1 domain (Fig. 7, laneb). This activation was exclusively dependent on a viable
subdomain a since mutation of this domain prevented
Sp1
-induced activation (lane b versuslanec). However subdomain a could not overcome
inhibition by mutation of the M1 domain in MLTC cells (Fig. 7, lanesg, b, and c). This
observation is consistent with our Sp1
mutant studies in
the p174GL and p174Sp1
XGL constructs, where trans-factor(s)
binding to the a domain increase wild type activity only in the
non-expressing CHO, Y1, and I10 cells (Table 1, Fig. 5).
The LHR Sp1 domain is configured as adjacent
non-canonical (GGG GTG GGG) and canonical (14) (GGGCGG) Sp1 elements with a two-nucleotide
overlap. This domain binds the Sp1 protein exclusively on the
non-canonical 5`-element, and non-Sp1 trans-activators on the 3`-GC box
in both expressing and non-expressing cells. The two-nucleotide overlap
is essential for binding of the non-Sp1 protein to the a domain,
but not for binding of Sp1 to the b domain (Table 1, mutant 4 versus 5, and Fig. 3and Fig. 4). Thus either competition or simultaneous binding of the
two factors are feasible. The LHR Sp1 binding element, GGG GTG GGG, in
which the C of the Sp1 consensus element GGG GCG GGG (15) is
replaced by a T, potentially conforms to the Zif-268 DNA binding
pattern (20) since the zinc fingers contact the guanines of the
GCG trinucleotide, and not the central cytosine(15) . Sp1 does
not bind to the GC box in domain a even in the absence of the
non-canonical Sp1 binding element, and this may be attributed to a
higher affinity for the MLTC and CHO non-Sp1 proteins (Fig. 6).
The only nucleotide difference between the Sp1 binding element of
Sp1
(GGGGCGGGCAGA), and the non-Sp1 binding element of
Sp1
a (GGGGCGGAGA) is the 3`-AGA extension of the GC
box; therefore, these nucleotides play an important role in the binding
of specific MLTC and CHO non-Sp1 factors.
Our studies indicate that the non-Sp1 trans-activator that binds to domain a, may represent different proteins in the non-expressing and expressing cell type. In the presence of CHO nuclear extract, domain a forms three functional protein/DNA complexes, whereas in MLTC nuclear extract, domain a forms only one functional complex ( Fig. 5and Fig. 6). In addition, the MLTC a domain trans-activator is only functional when the b element is mutated, whereas the CHO trans-activator is constitutively functional (Fig. 8, Table 1). Thus, a distinction between basal LHR transcription of the wild type p174 promoter in the expressing or non-expressing cell is that domain a is functionally occupied only in the non-expressing cell, inferring that the expressing cell may contain an exclusive open domain for the binding of putative activated proteins (Fig. 8). Several non-Sp1 transcriptional factors such as LSF(21) , ETF(22) , GCF-1(23) , and BTEB (24, 25) have been reported to bind GC boxes. Most notably, the BTEB1 protein is a ubiquitous protein, whereas the BTEB2 protein is found predominantly in the placenta and testis. The binding specificity of BTEB1 and BTEB2 for the GC box are indistinguishable from that of the Sp1 protein. The proteins BTEB1 and BTEB2 exhibit a 72% and 59% similarity, respectively, with the Sp1 protein within the DNA binding zinc finger domains, but little or no similarity to the activation domains of the Sp1 protein(24, 25) . The Sp1 protein can only be distinguished from GC binding non-Sp1 proteins in this study with immunological supershifts of DNA/protein complexes, since these involve binding of antibody to regions other than the DNA binding domain. These studies indicate that only subdomain b binds Sp1.
Figure 8:
Summary model of Sp1 and M1
binding and activation in CHO and MLTC cells. Diagram depicting model
of binding of three zinc finger Sp1, non-Sp1 A, and downstream
activator M1 trans-factors to the b+2, a, and M1 domains,
respectively of the 173-bp LHR promoter. Alternate binding of Sp1 and A
to the b+2 and a domains, respectively, is shown in the expressing
cell to correlate with functional studies that indicate competition
between the two activators. Sp1 alone activates Sp1
-induced
promoter activity from the non-canonical Sp1 binding b domain.
Simultaneous binding of Sp1 and A is shown for the non-expressing cell,
based on independent contributions by each domain. Subdomain a-induced
activation compensates for mutation of the activating M1 domain, and
elimination of both M1 and A are necessary to inhibit promoter activity
in the non-expressing cell. Sp1 is related to the human Sp1 protein in
both cell types, but A and M1 proteins may represent different
trans-factors in the two cell types (A, A
, M1,
M1
). Crossed-out arrow represents loss of
Sp1
-induced promoter activity.
In vitro identification of the non-canonical
Sp1 binding element on the rat LHR promoter is suitable using the mouse
Sp1 nuclear protein and anti-human Sp1 antibody (Fig. 4), since
Sp1 is a ubiquitous protein that is expressed in immunologically
related forms in mouse and human cells (26) and CHO cells (Fig. 4), and its sequence is highly conserved in the
rat(24) . However, non-Sp1 proteins that compete against Sp1 or
bind simultaneously to the Sp1 domain may vary with species
or cell type. Our studies indicate common functional changes with
subdomain a mutation, between the LHR-expressing MLTC and LHR
non-expressing mouse Leydig cells (I10), CHO cell, and mouse adrenal
cells (Y1) (Table 1). Thus trans-factors that bind to the a domain in a non-expressing cell type are functionally similar
across species and tissue type, although they may represent different
proteins. In addition, expressing and non-expressing mouse Leydig tumor
cell lines (MLTC and I10) with a common genetic origin exhibit the same
differential subdomain a function as MLTC versus CHO.
Although both expressing and non-expressing cells exhibit
significant levels of p174 transcriptional activation, the presence or
absence of the a-binding protein may be linked to regulation of
promoter activity by inhibitory upstream domains or the activating
downstream M1 domain(5) . The MLTC-specific M1 protein by
itself can activate Sp1-induced LHR gene transcription in
the intact p174 promoter, and mutation of the Sp1
a domain does not further change this promoter activity ( Fig. 7and Fig. 8). However, in the non-expressing CHO
cell, the a domain in the p174 promoter can functionally
substitute for the activating M1 domain. These tissue-specific
differences in a/M1X promoter activation mirrors our observation
that the trans-binding A protein is constitutively functional in the
non-expressing cell, and only potentially functional in the expressing
cell (Fig. 8). Since addition of the Sp1
a domain inactivates M1 function in the non-expressing cell (Fig. 7, lanes a and b), and a competitive
interaction exists between the M1- and A-binding proteins (data not
shown), the A protein may be involved with removal of the CHO M1
trans-factor from the M1 domain. Deletion or mutation of the Sp1
a domain restores M1 function in the CHO cell (Fig. 7, lanes f and g and lanes b and c). In contrast, the Sp1
a domain
does not influence M1 function in the MLTC cell (Fig. 7, lanes b and c), and this can be correlated with the
absence of a function, or the A-binding protein(s), on the wild
type promoter in this cell type (Fig. 8).
The two
transcriptionally active Sp1 and non-Sp1 binding factors may support
unique interactions with distal domains, resulting in differential
regulation of gene expression. In the non-expressing cell, simultaneous
mutation of both the a and M1 subdomains is necessary to
effectively inhibit Sp1-induced p174 LHR promoter activity
( Fig. 7and Fig. 8) and removal of these trans-factors
from the a and M1 subdomain in the non-expressing cell may play
a role in the permanent silencing of gene expression.