©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of Diverse Functional Elements in the Upstream Sp1 Domain of the Rat Luteinizing Hormone Receptor Gene Promoter (*)

(Received for publication, August 10, 1994; and in revised form, December 19, 1994)

Chon-Hwa Tsai-Morris Yi Geng Ellen Buczko Maria L. Dufau (§)

From the Section on Molecular Endocrinology, Endocrinology and Reproduction Research Branch, NICHHD, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Transcription of the luteinizing hormone receptor gene is dependent on Sp1-induced promoter activation from two Sp1 binding domains (Sp1(2) and Sp1(4)) 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(2) domain. The Sp1 binding element within the Sp1(4) 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(4) 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(4) 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.


INTRODUCTION

The promoter region of the luteinizing hormone receptor (LHR) (^1)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(2) (-73 to -94 bp), and Sp1(4) (-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(2) 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(2) 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(4) and contained a canonical GC box. These studies also indicated that unlike the Sp1(2) domain, mutation of the entire 20-nucleotide G-rich Sp1(4) domain was necessary to abolish competition for Sp1 trans-factors, and Sp1(4)-induced promoter activity(5) . In addition, tissue-specific differences by the downstream protein binding M1 domain (-24 to -42 bp), that influenced Sp1(4)-induced promoter activation were demonstrated(1, 5) . To further explore the mechanism of Sp1 activation from the Sp1(4) domain, we analyzed the potential Sp1 binding elements on Sp1(4), and their individual effect on transcriptional activation in the presence or absence of viable Sp1(2) and M1 domains, in both expressing and non-expressing cell types. Tandem Sp1/non-Sp1 binding elements were identified on the Sp1(4) domain, and each element induced transcript activation in a differential expressing versus non-expressing cell mode of action.


MATERIALS AND METHODS

Construction of Sp1(4) Mutant Plasmids

Designated nucleotides of subdomains 5` b (-154 to -145 bp) and 3` a (-144 to -135 bp) in the Sp1(4) domain (-135 to -154 bp) were individually mutated within 20-bp oligonucleotides for gel retardation studies, and within LHR promoter domain of the LHR/luciferase fusion constructs (pLHRGL) for transient expression (see below and Table 1). Mutated domains in all constructs are designated with an X, and mutated nucleotides are in lower case. Transcriptional activity was measured for Sp1(4) wild type (b/a) and mutant (b/aX, bX/a, bX/aX) constructs in the context of three general forms of the LHR promoter: 1) the wild type 173-bp 5`-flanking domain (p174GL) (+1 to -173 bp), 2) the Sp1(2)X mutant form (p174Sp1(2)XGL) (Sp1(2) domain: -77 to -84 bp) (GGGGCGGG mutated to atctgcaG), and 3) the M1X mutant form (p174M1XGL) (M1 domain: -24 to -42 bp) (CTCA mutated to gcCc](5) . Sp1(4)X is defined as bX/aX. Mutant nucleotide substitutions are identical for oligonucleotides used in gel retardation and mutant plasmid constructs.



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.

Transient Expression

Transient expression studies of LHR promoter constructs were performed in two LHR-expressing mouse Leydig tumor cell lines possessing luteinizing hormone/human choriogonadotropin receptors, gonadotropin-responsive adenylate cyclase, and steroidogenic capacity (MLTC-1 and MLTC-2) (ATCC CRL 2065) (kindly provided by Dr. R. V. Rebois, NIH, Bethesda, MD; (7) ), and LHR non-expressing cell lines, Chinese hamster ovary (CHO) (ATCC CCL 61), I10 tumor cell line (mouse Leydig cell testicular tumor obtained from American Type Culture Collection, Rockville, MD; ATCC CCL 83)(8) , and Y1 (mouse adrenal tumor obtained from American Type Culture Collection, Rockville, MD; ATCC CCL 79)(9) . The DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate) method (10) was used for transfection (5) according to the manufacturer's protocol (Boehringer Mannheim) using 7:1 (w/w) DOTAP/DNA. Luciferase activity was determined in the cell extract by luminometry as described previously (11) and normalized with beta-galactosidase activity (12) or protein concentration. In experiments to determine the direct effect of the Sp1 protein on LHR promoter activity, transient expression was performed with Sp1-deficient Drosophila cells (13) where LHR promoter constructs (wild type p174GL or mutant p174Sp1(2)X/Sp1(4)XGL) were transfected in the presence or absence of the Sp1 DNA insert in the expressing plasmid (pPacSp1 or pPacSp1, respectively). These expression vectors, kindly provided by Dr. R. Tjian (Howard Hughes Medical Institute, Berkeley, CA), are driven by the actin 5C promoter(13) . Conditions for optimal promoter activity were attained with cotransfection of 10 ng of pPacSp1 and used in this study. Control pPac plasmid without Sp1 cDNA insert (pPac-) was used at the same concentration.

Gel Retardation Analysis

Sp1(4) mutant oligonucleotides (Midland Certified Regent Co, Midland, TX) were annealed and end-labeled with [- P]ATP. These were incubated at designated concentrations with nuclear protein extracted from MLTC and CHO cells. The Sp1 standard oligonucleotide (Promega) contains the Sp1 binding GC box sequence (ATTCGATCGGGGCGGGGCGAGC)(14) , and is termed either consensus Sp1 (cons. Sp1) or standard Sp1 (Std. Sp1) in figures. This oligonucleotide sequence was derived from the SV40 promoter, and specifically binds the Sp1 protein, in contrast to the Sp1(4) domain that appears to bind multiple proteins. The AP-2 consensus oligonucleotide was purchased from Promega. Electrophoresis of protein/DNA complexes were performed as described previously(5) , and individual bands were quantitated by PhosphorImager (Molecular Dynamics, Sunnyvale, CA). In antibody complex/supershift experiments, nuclear extracts prepared as described previously(5) , were preincubated with rabbit anti-human Sp1 or AP-2 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 min prior to the addition of labeled DNA and gel retardation analysis.

Analysis of Data

Transcriptional activity of mutants is normalized as percent of the wild type p174GL and p174Sp1(2)XGL luciferase fusion constructs. The former represents the Sp1(4) mutational effects in the presence of a viable Sp1(2) domain, where each contributes approximately 50% transcript activity(5) . The latter represents the mutational effects from the Sp1(4) domain exclusively. Individual data points from 2 to 10 separate experiments in triplicate were normalized with the mean of the wild type constructs. Differences between groups were sought using analysis of variance with groups representing Sp1(4) mutant type (bX/a, b/aX, (b+2)/(a-2)X, (b+2)X/(a-2), bX/a-, bX/aX), cell type (LHR expressing MLTC-1 and MLTC-2, and non-expressing CHO, Y1, I10), and general promoter type (p174 wild type, p174Sp1(2)X, and p174M1X). Post hoc analyses with Fisher LSD and Duncan tests were performed, and a 95% significance level was interpreted with the Bonferroni adjustment for multiple comparisons in the computer programs Statview (Abacus Concepts, Berkeley, CA) and Superanova (Abacus Concepts). Results are expressed as the mean ± S.E. Statistical significance was construed for p < 0.01.

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(4) 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(4) 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*.




RESULTS

Sp1 Dependence of LHR Promoter Activity

An Sp1 protein requirement for LHR transcription was initially established with transfection of LHR promoter/luciferase gene constructs (p174GL) in a Drosophila Sp1-deficient SL cell line (13) (Fig. 1). Cotransfection of a human Sp1-expressing plasmid (pPacSp1) with the wild type LHR promoter/luciferase construct elevated transcriptional activity over control levels (pPacSp1 without the Sp1 insert) by 600% (+ versus -) (lanes 1 and 2). Cotransfection of the construct with mutated Sp1(2) and Sp1(4) domains (p174Sp1(2)X/Sp1(4)XGL) resulted in a major decrease in LHR promoter activity to levels equivalent to that observed with the pPac vector insert. These data confirm our previous findings that the Sp1(2) and Sp1(4) domains are the only elements in the LHR promoter (+1 to -173 bp) involved in Sp1-induced transcriptional activation.


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 p174Sp1(2)X/Sp1(4)XGL with the Sp1 containing plasmid pPacSp1 (+). Sp1(4)X is bX/aX LHR mutant (see Table 1). Mutant Sp1(2) domain: (GGGGCGGG to atctgcaG). Controls are the plasmid without Sp1 cDNA insert (pPac)(-).



Identification of the Sp1 Binding Element in Sp1(4)

Previous mutational studies of the Sp1(2) domain have shown that the canonical GC box (GGGCGG) within Sp1(2) binds a nuclear factor that has the binding and immunological properties of the Sp1 protein(5) . However, the Sp1 binding element within the Sp1(4) domain could not be localized to its GC box (GGGCGG)(5) , and mutational studies to identify this element were performed.

Sp1(4) 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(4) 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(4) 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(4), rather than the GC box within subdomain a.


Figure 2: Competition for Sp1 factors by wild type and mutant Sp1(4) 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(4) 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(4) 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(4) 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, bulletbullet). 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(4)/nuclear protein complexes was used to determine the IC of the Sp1(4)b or b+2 elements for Sp1 (Fig. 3). The IC for mutant Sp1(4) 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(4)(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(4) (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(4) domain a that interfere with competition for the Sp1 trans-factor (see below). Mutation of the a-2 domain, leaving only the native Sp1(4) (b+2) element, converts the Sp1(4) 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(4) 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(4) 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(4) domain (data not shown).

The b+2 element in the Sp1(4) 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(4) 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).

Transcriptional Activation by Sp1(4) Mutant Constructs and Characterization of the [a]Binding Element

Promoter activity was studied in the corresponding p174 GL mutant constructs in the presence or absence of the viable Sp1(2) domain (Table 1, Fig. 5). These studies were performed in two individual LHR-expressing clonal cell lines (MLTC-1 and MLTC-2) and three different LHR non-expressing cells (CHO, Y1, and I10). Activation by the trans-factor that binds to the Sp1(4)b+2 element, in an Sp1(4) sequence that contains the mutated a domain [Sp1(4) mutant (b+2)/(a-2)X], is not statistically different from wild type in either the transfected LHR-expressing or non-expressing cell lines (Table 1, Fig. 5A). However, substitution of the 3` GG of the b+2 element GGG GTG GGG in the Sp1(4) mutant ((b+2)/(a-2)X) to ``AT'' (mutant b/aX) gives a significant reduction in transcriptional activation in the non-expressing cell lines (Table 1, compare mutants 2 and 3) (p range = 0.0001-0.01). This reduction is noted either in the presence or absence of the Sp1(2) domain (p174b/aXGL and p174b/aX/Sp1(2)XGL, respectively; Table 1, Fig. 5, A and B, lowerpanels) and it is more marked in the Sp1(2)XGL mutants (expressing versus non-expressing, p range = 0.0002-0.001). In contrast, this reduction is not observed in LHR-expressing MLTC-1 or MLTC-2 cells (Table 1, Fig. 5, A and B, upperpanels).


Figure 5: Transcriptional activity of Sp1(4) wild type and mutant variants of the luteinizing hormone receptor promoter. Mean of relative luciferase activity of wild type and mutant Sp1(4) domains in p174GL (A) or p174Sp1(2)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(2)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 Sp1(4)a domain, and/or both was investigated. The subdomain a sequence (GGGGCGGAGA), by itself, was capable of inducing Sp1(4) activity at levels that are comparable to wild type p174b/aGL and p174b/aSp1(2)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(4) activation (Table 1, bulletbullet, 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(4) 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(4) 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.

Functional Placement of Sp1 and Non-Sp1 Binding Trans-factors on the Wild Type Sp1(4) Domain

Differences in transcriptional activation of the b/aX and bX/a constructs between the expressing and non-expressing cell types also suggests a cell specific pattern of non-Sp1 and Sp1 contribution to Sp1(4)-induced activity (Table 1). In the expressing cell, there is no significant difference between the wild type p174GL, p174b/aXGL, or p174bX/aGL constructs, indicating that either subdomain a or b can support 100% of the basal p174 promoter activity. Similar results were obtained in constructs carrying a mutated Sp1(2) domain, where maximal basal transcriptional levels were approximately 50% that of the wild type(5) . No additive effect or potentiation was observed by the individual subdomain a or b elements in the wild type p174b/aGL construct, and a competition between the two binding factors is indicated.

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 p174Sp1(2)XGL 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.

Sp1(4) Functional Differences Involving Cooperation with the Tissue-specific M1 Domain

Since our previous studies indicated that Sp1(4) activation was influenced in a cell-specific mode by the downstream activating M1 domain(1, 5) , Sp1(4) transcriptional activity was measured in p174M1X mutant constructs (CTCA mutated to gcCc) to determine individual effects by the Sp1(4) Sp1/non-Sp1 binding domains. The M1 domain is an activating domain in both the expressing and non-expressing cell in the minimal Sp1(2) promoter p138GL(5) , and M1 mutation reduces transcript activity by 50% (Fig. 7, lanesf and g). Addition of the Sp1(4) domain to the wild type p138GL construct results in a 1-fold transcriptional activation in both mLTC and CHO cells, and this activation was inhibited upon mutation of the Sp1(4) domain (bX/aX, lanee). Thus, this effect can directly be attributed to the Sp1(4) domain (b/a) (5) (Fig. 7, lanesf, a, and e).


Figure 7: Effect of simultaneous mutation of the M1 and Sp1(4) 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(4) 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(4)-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(4) mutant studies in the p174GL and p174Sp1(2)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).


DISCUSSION

The LHR Sp1(4) 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(2) (GGGGCGGGCAGA), and the non-Sp1 binding element of Sp1(4)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(4) 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(4)-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(4)-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(4) 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(4)-induced LHR gene transcription in the intact p174 promoter, and mutation of the Sp1(4)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(4)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(4)a domain restores M1 function in the CHO cell (Fig. 7, lanes f and g and lanes b and c). In contrast, the Sp1(4)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(4)-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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 301-496-2021; Fax: 301-496-480-8010.

(^1)
The abbreviations used are: LHR, luteinizing hormone receptor; MLTC, mouse Leydig tumor cell; CHO, Chinese hamster ovarian cell; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium methylsulfate; SL, Drosophila Sp1-deficient cell line; bp, base pair(s).


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