A Novel Cyclic Adenosine 3',5'-Monophosphate-Responsive Element Involved In the Transcriptional Regulation of the Lutropin Receptor Gene in Granulosa Cells

Shiyou Chen1, Xuebo Liu1 and Deborah L. Segaloff

Department of Physiology and Biophysics The University of Iowa College of Medicine Iowa City, Iowa 52242


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The induction of the lutropin receptor (LHR) in granulosa cells by FSH is mediated, at least in part, by cAMP. However, the classic cAMP-responsive element (CRE) is not present in the 5'-flanking region of the rat LHR gene. Previous studies from our laboratory had shown that three Sp1 sites within the promoter region of the rat LHR (rLHR) bind Sp1 and Sp3 and are involved in the basal and cAMP-mediated transcription of the rLHR gene. In the present studies we show that the rLHR promoter region forms a complex (designated complex A) with nuclear extracts from rat granulosa cells, and the abundance of complex A is markedly increased when using cells that had been pretreated with 8-bromo (Br)-cAMP. We have localized the binding of the protein(s) in complex A to a DNA sequence immediately upstream and partially overlapping with the Sp1c binding site. The core site (designated SAS for Sp1c adjacent sequence) is localized to nucleotide (nt) -146 to -142 and contains the sequence GGGGG. The consensus sequence for the core portion of this element appears to be (G/T)GGGG. Mutations of the SAS site, but not SP1c site, abolish complex A formation. Experiments utilizing rat granulosa cells transfected with luciferase reporter genes driven by the 5'-flanking region of the rLHR gene demonstrate a functional role for the SAS site in the cAMP responsiveness of the rLHR gene.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The induction of the lutropin receptor (LHR) in granulosa cells during the growth of small antral follicles to preovulatory follicles is of physiological importance for LH-induced ovulation and corpus luteum formation (1, 2, 3, 4). Earlier studies have shown that the induction of the LHR in granulosa cells is mediated by the concerted actions of both FSH and estradiol (5, 6, 7, 8, 9, 10). Whereas the effects of FSH on LHR induction are apparently due to its ability to increase intracellular cAMP levels (11, 12, 13, 14), the role of estradiol in the induction of the LHR is still unclear.

It has since been shown that the FSH or 8-bromo (Br)-cAMP-mediated increase in the LHR and LHR mRNA in granulosa cells obtained from diethylstilbestrol (DES)-treated rats is associated with a marked increase in LHR gene transcription. (15). However, increases in LHR gene transcription are not apparent within the first 24 h of treatment with FSH or 8-Br-cAMP, consistent with there being a 24-h lag time before increases in LHR mRNA are apparent (15). Interestingly, the 5'-flanking region of the rat LHR (rLHR) gene does not contain any classical cAMP-responsive elements (16). However, granulosa cells transfected with a luciferase reporter gene driven by 2.1 kb of the 5'-flanking region of the rLHR gene respond to exogenously added 8-Br-cAMP (15). A further characterization of that region of the gene has revealed that a number of cis-regulatory elements and trans-acting factors are involved in the transcriptional regulation of the rLHR gene in rat granulosa cells (17). Among these are three Sp1 sites (Sp1a, Sp1b, and Sp1c) in the promoter region, which we have shown to be functionally important. Thus, each of these sites forms complexes with Sp1 and/or Sp3 as determined by mobility shift assays. Furthermore, mutation of each of these sites results in both diminished basal as well as cAMP-induced transcriptional activity of the rLHR gene as determined by reporter gene assays (17).

Mobility gel shift assays have shown the marked increase in the abundance of a complex formed between the promoter region of the rLHR gene and nuclear extracts from 8-Br-cAMP-induced rat granulosa cells as opposed to extracts from control cells (17). The current studies were undertaken to identify the DNA element involved in this complex formation. We report herein the identification of a unique cAMP-responsive element in the promoter region of the rLHR gene. The nature of this site and its role with the Sp1c site in regulating basal and cAMP responsiveness of the rLHR gene are presented.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Delineation of nt -151/-136 as Containing the Region Involved in Complex A Formation
Previous studies from our laboratory using electrophoretic mobility shift assays (EMSAs) had examined the ability of probes corresponding to overlapping regions of the nt -2056/-2 portion of the rLHR gene to form complexes with nuclear extracts from granulosa cells that had been untreated or treated with 8-Br-cAMP (17). All but one probe exhibited several DNA/protein complexes, the patterns of which were different with the various probes. In many cases, the intensity of a given complex was increased when the extracts were derived from 8-Br-cAMP-treated cells. In the present study we chose to focus on a cAMP-dependent complex that was observed using a probe corresponding to the promoter region of the rLHR. Figure 1Go shows the formation of this complex, which we have designated complex A, with the -187/-2 region of the rLHR gene. As seen in Fig. 1Go, its abundance is markedly increased by pretreatment of granulosa cells with 8-Br-cAMP.



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Figure 1. Analysis of the Involvement of Consensus Elements in the Formation of Complex A

Panel A, Mobility shift assays were performed using a 32P-labeled fragment corresponding to nt -187/-2 of the rLHR gene promoter. The labeled probe was incubated with 2 µg of nuclear extracts isolated from uninduced (C) or 8-Br-cAMP-induced (I) rat granulosa cells as described in Materials and Methods. Competitions between the labeled fragment and unlabeled fragments containing mutations in consensus sequences at molar excess ratios of 100 and 200, respectively, are shown for each element. Panel B, The sequences of the synthetic oligomer sequences used for competition are shown. The core binding residues for each corresponding factor are underlined.

 
To precisely define the DNA binding region contributing to complex A, we first examined its relationship with the consensus elements in the promoter region. The 5'-flanking region of rLHR gene contains three Sp1 sites (Sp1a, Sp1b, and Sp1c), two steroidogenic factor 1 (SF1) sites (SF1a and SF1b), and one AP2 site (17). Previous studies have shown the SF1 and AP2 sites are not required for basal or cAMP-induced expression of hLHR gene transcription. However, mutation of any of the three Sp1 sites decreases both basal and cAMP-induced rLHR gene transcription. EMSAs further showed that S1 and Sp3 proteins bind to each of the three Sp1 sites in the rLHR gene promoter region. However, it is important to note, for the purposes of the present studies, that the EMSA conditions required for the detection of the Sp1 and Sp3-containing complexes with the Sp1 sites were quite different than those required for the detection of complex A formation with the -187/-2 rLHR probe. As a result, experiments designed to detect Sp1/Sp3-containing complexes with the Sp1 sites cannot detect complex A. Conversely, those designed to detect complex A cannot detect the Sp1/Sp3-containing complexes with the Sp1 sites. The experiments shown in Fig. 1Go were performed under EMSA conditions chosen to optimize detection of complex A. Therefore, the uppermost bands on the gel (which are specific since all the complexes can be completed by the inclusion of an excess of an unlabeled -187/-2 fragment) do not correspond to Sp1/Sp3-containing complexes. Competing DNA fragments containing the SF1, Sp1a, Sp1b, Sp1c, or AP2 sites were synthesized and added to EMSAs examining complex A formation to the -187/-2 region of the rLHR gene. As shown in Fig. 1AGo, fragment D, which contains the Sp1c site (nt -151 to -130), completely inhibited the formation of complex A. Fragment B, which contains the Sp1a site, also inhibited complex A formation, but to a smaller extent. Fragments A, C, and E, which contain SF1a, Sp1b, and AP2 binding sites, respectively, exhibited no competition.

Additional EMSAs were performed using competing fragments of different lengths to narrow down the region of the rLHR gene contributing to complex A formation. From these experiments we were able to determine that the cis-element contributing to complex A formation lies within nt -151 to -136 of the rLHR gene (data not shown).

Identification of the GGGGG Sequence of nt -146/-142 as the Core Sequence Required for Complex A Formation
To more precisely define the DNA sequence in the -151 to -136 region of the rLHR gene involved in complex A formation, the following experiments were performed. Thirteen pairs of complementary oligonucleotides were synthesized based upon the wild-type sequence of fragment D in which a single nucleotide at a time spanning the region of nt -150 to -138 was substituted. These fragments were then used as competing oligonucleotides in EMSAs to determine whether any decreased the formation of complex A. The results shown in Fig. 2Go were obtained utilizing nuclear extracts that had been obtained from 8-Br-cAMP-treated granulosa cells. Each of the fragments containing single substitutions of the nucleotides GGGGG located between nt -146/-142 prevented the formation of complex A. In contrast, all the other singly substituted oligonucleotides did not inhibit complex A formation. These results suggest that the GGGGG sequence located between nt -146 and -142 is the core sequence necessary for complex A formation. The same results were obtained when nuclear extracts from untreated granulosa cells were used (data not shown).



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Figure 2. Identification of the Core DNA Element That Contributes to Complex A Formation

Shown are the results of a mobility shift assay using a fragment corresponding to nt -187/-2 of the rLHR gene as the labeled probe. The labeled probe was incubated with 2 µg of nuclear extracts isolated from 8-Br-cAMP-induced rat granulosa cells in the absence or the presence of an unlabeled competing oligonucleotide at a 100-fold molar excess. The unlabeled competing oligonucleotides corresponded to nt -151/-130 (fragment D of Fig. 1Go) with either the wild-type sequence (lane 1) or a sequence in which one residue between -150 and -138 was substituted (lanes 3–15). Lanes 1 and 2 show the results in the presence or absence, respectively, of a competing fragment corresponding to the wild-type sequence. Lanes 3–15 show the results using competing fragments containing a mutation. The five G’s whose substitution did not cause competition are underlined.

 
To further determine the consensus sequence for the element forming complex A, each of the five G’s was separately substituted by A, C, or T within the context of the -151/-130 fragment. Each of the substituted fragments or wild-type fragment was used in EMSAs at different concentrations to competitively inhibit the formation of complex A. The intensities of complex A before and after competition by wild-type or mutant fragments are shown in Fig. 3Go. The wild-type oligonucleotide corresponding to each G completely inhibited complex A formation even at a 50-fold molar excess. Therefore, the wild-type GGGGG sequence is the best candidate. When the G corresponding to nt -146 was substituted with T, complex A formation was also significantly inhibited. All other substitutions did not show significant inhibition of complex A formation. We conclude, therefore, that the consensus sequence for this core element is (G/T)GGGG.



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Figure 3. Determination of the SAS Element Consensus Sequence

Mobility shift assays were performed in which a labeled probe corresponding to nt -187/-2 of the rLHR gene was incubated with 2 µg of nuclear extracts isolated from 8-Br-cAMP-induced rat granulosa cells in the absence or the presence of an unlabeled competing oligonucleotide at molar excess ratios of 50, 100, and 200. The competing oligonucleotides corresponded to nt -151 to -130 of the rLHR gene in which each of the five consecutive G’s corresponding to nt -146/142 was left intact or mutated to either C, A, or T. The complexes were resolved on polyacrylamide gels, which were then exposed to a phosphor screen for 4 h at room temperature. The screen was scanned and the intensity of complex A (relative to background) was quantified.

 
As shown by the sequence in Fig. 4Go, the core sequence identified as the cis element forming complex A with granulosa cell nuclear protein(s) is upstream of and partially overlapping the Sp1c site. Therefore, we have termed this cis element SAS for Sp1 adjacent sequence.



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Figure 4. Schematic Representation of the SAS Site and Its Relationship to the Sp1c Site Shown is the sequence of nt -151/-130 of the rLHR gene. The consensus sequence for the Sp1/Sp3 binding site corresponding to Sp1c between nt -142/-137 is noted in bold. The core SAS sequence of nt -146/-142 is noted by the shaded box. Both sequences are also overlined. It can be appreciated that nt -142 and -143 are within both the core SAS site as well as the Sp1c site. Residues that were substituted in the creation of site-selective mutations are noted by the underlined sequences. For Sp1c(mt), the GCGG sequence of nt -141/-138 was replaced by AGTC. For SAS(mt1), the GG sequence of nt -146/-145 was replaced by AT. For SAS(mt2), the GGGGG sequence of -150/-145 was replaced by TATCAT.

 
The SAS Site Is Related to cis Elements Binding G String Factors
A search of the literature and databases for DNA cis elements revealed that the SAS core sequence shares the common feature of a G-rich sequence with some other cis-acting elements. These include the cis-elements that bind the transcription factors G-string (18), G-fer (19), BGP1 (20, 21), IF-1 (22, 23), and Zif268 (24). We, therefore, examined whether fragments corresponding to these G-string and related binding elements could compete for complex A formation. Figure 5Go shows the results of an EMSA using a labeled fragment corresponding to -187/-2 of the rLHR gene incubated with nuclear extracts from induced granulosa cells where unlabeled fragments corresponding to the wild-type or mutated sequences of G-string and related binding elements were added in excess. Fragments corresponding to the wild-type sequences that bind G-string, G-fer, BGP1, or IF-1 effectively competed for complex A formation, and mutations in the G-string, G-fer, and IF-1-binding fragments abolished their ability to compete for complex A formation. However, two different mutations of the BGP1-binding DNA fragment did not diminish the ability of the fragments to inhibit complex A formation. This could imply that the inhibition of complex A formation by the wild-type BGP1-binding fragment is mediated by residues other than those that we mutated. The latter is not an unlikely scenario given the large tracts of G-rich sequences in the BGP1-binding DNA fragment. It should also be noted that the wild-type BGP1-binding sequence, in addition to inhibiting complex A formation, also inhibited the formation of other complexes in the upper portion of the gel. This may reflect a nonspecific effect of the BGP1-binding DNA fragment. Alternatively, this fragment may be blocking the formation of multimerized complexes. As also shown in Fig. 5Go, not all G-rich DNA elements competed for complex A formation, as evidenced by the inability of the Zif268-binding fragment to inhibit complex A formation. These data suggest that the SAS site may be related to some (e.g. the G-string and G-fer binding sites), but not all, G-rich DNA binding cis-elements.



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Figure 5. DNA Binding Sites for G-String-Related Transcription Factors Compete for Complex A Formation

Shown are the results of a mobility shift assay using a fragment corresponding to nt -187/-2 of the rLHR gene as the labeled probe. The labeled probe was incubated with 2 µg of nuclear extracts isolated from 8-Br-cAMP-induced rat granulosa cells in the absence (NA) or the presence of an unlabeled competing oligonucleotide at a 100-fold molar excess. The unlabeled competing oligonucleotides corresponded to wild-type or mutated (mt) DNA binding sequences for G-string, G-fer, BGP1, IF-1, and Zif268. The residues in the wild-type sequence that were substituted are highlighted and underlined. The mutated sequences showing the residues that were inserted in place of the wild-type residues are highlighted and underlined.

 
The SAS Site, but Not Sp1c Site, Is Required for Complex A Formation
Because the SAS site partially overlaps the Sp1c site, it is important to determine whether complex A formation is dependent upon nuclear proteins binding to the SAS site only or the SAS and Sp1c sites. To address this question, we designed mutations of the SAS and Sp1c sites that would be site-specific (Fig. 6Go). A mutation of the Sp1c site was made by substituting the GCGG residues immediately downstream of the core SAS site. Two different mutations of the SAS site were examined. The SAS(mt1) mutation substituted the two G nucleotides at the 5'-end of the core SAS site. The SAS(mt2) mutation substituted these same two G nucleotides as well as additional four nucleotides immediately upstream of the core SAS sequence. Both the mt1 and mt2 mutations of the SAS sites left the Sp1c site intact. EMSAs were performed examining the ability of the wild-type or mutated -187/-2 fragments to compete with complex A formation to a labeled -187/-2 fragment. As shown in Fig. 6Go, the Sp1c(mt1) fragment completely inhibited complex A formation, demonstrating that an intact Sp1c site is not required for complex A formation. In contrast, the SAS(mt2) fragment caused no inhibition of complex A formation. Although a 20-fold excess of SAS(mt1) did not compete, a 50-fold excess of SAS(mt1) caused a slight inhibition of complex A formation. The ability of the SAS(mt1) fragment at high concentrations to partially compete for fragment A formation suggests that nucleotides in addition to the guanines at -146 and -145 are required for high affinity binding of complex A. One possibility, of course, is that the other three guanines in the core sequence are required. Unfortunately, we cannot readily test this because disruption of guanines -143 and -142 would affect the Sp1c site as well. Notably, the lack of ability of the SAS(mt2) fragment to compete for complex A formation implicates residues upstream of the core SAS site as being involved in complex A formation. Although the individual substitutions of nt -150 through -147 did not implicate these residues (see Fig. 2Go), the aggregate mutation of these residues suggests a role for them in the formation of complex A (Fig. 6Go). Importantly, these data show that mutation of the Sp1c site does not affect the formation of complex A to the SAS site. We further addressed the question of the potential role of Sp1 and Sp3 binding contributing to complex A formation by examining the effects of antibodies to Sp1 and Sp3 on complex A formation in EMSAs. As shown in Fig. 7Go, the addition of antibodies to Sp1 and/or Sp3 had no effect on the mobility or the intensity of complex A. Because the complexes binding to the Sp1c site cannot be visualized under the EMSA conditions used to detect complex A, controls showing the effects of antibodies to Sp1 and Sp3 on known Sp1/Sp3-containing complexes cannot be determined from the experiments shown in Fig. 7Go. However, as shown in Fig. 8Go, the concentrations of antibodies used are indeed effective in inhibiting the binding of Sp1 and Sp3 to the Sp1c site in the rLHR promoter. Therefore, the lack of effect of these antibodies on complex A formation (as seen in Fig. 7Go) suggests that complex A does not contain either Sp1 or Sp3.



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Figure 6. The SAS Site, but Not the Sp1c Site, Is Required for Complex A Formation

Shown are the results of a mobility shift assay using a fragment corresponding to nt -187/-2 of the rLHR gene as the labeled probe. The labeled probe was incubated with 2 µg of nuclear extracts isolated from 8-Br-cAMP-induced rat granulosa cells in the absence or the presence of unlabeled competing oligonucleotides at 20- or 50-fold molar excess. The competing oligonucleotides correspond to nt -164/-130 with the wild-type sequence or with Sp1c(mt1), SAS(mt1), or SAS(mt2) mutations as described in Fig. 4Go.

 


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Figure 7. Complex A Is Not Composed of Sp1-Binding Proteins

Shown are the results of a mobility shift assay using a fragment corresponding to nt -187/-2 of the rLHR gene as the labeled probe. The labeled probe was incubated with 2 µg of nuclear extracts isolated from 8-Br-cAMP-induced rat granulosa cells in the absence or the presence of antibodies to Sp1 and/or Sp3. In some cases, the antibodies were added to the reaction mixture with all the other components (lanes 2–4). In other cases (lanes 6–8), the antibodies were preincubated with the nuclear extracts for 30 min at 0 C before the addition of labeled probe.

 


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Figure 8. The SAS Site Is Not Required for Sp1 and Sp3 Binding to the Sp1c Site

Shown are the results of a mobility shift assay using a fragment corresponding to nt -165/-131 of the rLHR gene as the labeled probe. Results of the labeled probe incubated with 2 mg of nuclear extracts isolated from 8-Br-cAMP-induced rat granulosa cells are shown in lane 4. Lanes 1–3 show the results when antibodies to Sp1 binding proteins were added to the reaction mixture. Lanes 6–8 show the results when competing oligonucleotides were added at a 70-fold molar excess. The competing oligonucleotides correspond to nt -165/-131 corresponding either to the wild-type sequence or the Sp1c(mt1), SAS(mt1), or SAS(mt2) mutations as described in Fig. 4Go. Note that the optimal conditions for observing Sp1/Sp3-containing complexes are different than those for detecting complex A and are described in Materials and Methods.

 
Conversely, we also examined whether or not the SAS site is required for Sp1 and Sp3 binding to the Sp1c site. To optimally visualize the binding of Sp1 and Sp3 to the Sp1c site, it was necessary to perform EMSAs using a smaller labeled fragment, -156/-131. Also, as noted before, EMSA conditions different than those used to visualize complex A are required to detect Sp1 and Sp3 proteins binding to the Sp1 sites. Under these EMSA conditions, complex A is not detected. As we have previously shown (17) and is shown herein in Fig. 8Go, two complexes are discerned binding to the Sp1c site. The upper complex I is composed of both Sp1 and Sp3 as its intensity is decreased by the addition of either anti-Sp1 or anti-Sp3, and the lower complex II is composed of Sp3 as its intensity is decreased by anti-Sp3. The simultaneous addition of antibodies to Sp1 and Sp3 prevented the formation of both complexes I and II. We then examined the ability of unlabeled oligonucleotides corresponding to either the wild-type -156/-131 or those containing mutations of the Sp1c or SAS site (as shown in Fig. 4Go) to compete for complex I and II formation. Whereas the wild-type fragment, SAS(mt1), and SAS(mt2) competed for complexes I and II, the Sp1c(mt) fragment was ineffective in competing. These data demonstrate that the residues mutated in the Sp1c(mt) are sufficient to disrupt Sp1 and Sp3 binding to the Sp1c site. In contrast, mutations of the SAS site had no effect on competition for binding of Sp1 or Sp3 to the probe. These data demonstrate that the mt1 and mt2 mutations of the SAS site do not disrupt the Sp1c site. Taken altogether, our data support the conclusion that the mutations chosen to disrupt the SAS vs. the Sp1c sites are indeed site-selective. Furthermore, the cAMP-dependent complex A, which arises from the binding of nuclear proteins to the SAS site, does not require Sp1 or Sp3 binding. Thus, although the SAS and Sp1c sites overlap, they appear to behave independently.

The SAS Site Is Required for cAMP-Induced Transcription of the rLHR Gene
The data thus far have identified a cis-element in the 5'-flanking region of the rLHR, termed SAS, which binds a granulosa cell nuclear protein(s) giving rise to a complex termed complex A. The observation that the abundance of complex A increases when the granulosa cells have been exposed to 8-Br-cAMP suggests that the SAS site may be involved in the cAMP-mediated induction of the rLHR gene. To address this question, rat granulosa cells were transiently transfected with luciferase reported gene constructs driven by the wild-type 5'-flanking rLHR gene sequence or by sequences containing SAS(mt1), SAS(mt2), or Sp1c(mt1) mutations. As shown in Fig. 9Go, mutation of the Sp1c site inhibits both basal as well as cAMP-mediated transcription of the rLHR gene. Although the SAS(mt1) mutation inhibited basal transcription slightly, the larger SAS(mt2) mutation had no effect on basal activity. Both the mt1 and mt2 mutations of SAS, however, markedly reduced 8-Br-cAMP-induced transcription of the rLHR gene. These data clearly demonstrate that mutation of the SAS site significantly attenuates the cAMP-mediated induction of rLHR gene transcription. These results show that the SAS site is required, but not necessarily sufficient, for the cAMP-mediated increase in rLHR gene transcription.



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Figure 9. The Effects of the Sp1c and SAS Sites on Promoter Activity of the rLHR Gene in Rat Granulosa Cells

Rat granulosa cells were transfected with luciferase reporter gene constructs containing either nt -2056/-1 of the rLHR gene (2056(wt)), or that region of the rLHR gene containing mutations within the Sp1c site or the SAS site. The mutations corresponding to Sp1c(mt1), SAS(mt1), and SAS(mt2) are described in Fig. 4Go. The cells were incubated without (C) or with (I) 8-Br-cAMP and then assayed for luciferase activity. Results shown are the mean ± SEM of three independent experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Previous studies from this laboratory have suggested that numerous cis elements within the 5'-flanking region of the rLHR gene contribute to the cAMP responsiveness of the gene (17). Two lines of evidence support this conclusion. First, when granulosa cells were transfected with luciferase reporter genes driven by increasing lengths (up to 2.1 kb) of the 5'-flanking region of the rLHR gene, a gradual increase in cAMP responsiveness was observed (17). Second, in EMSAs utilizing labeled fragments corresponding to overlapping portions spanning nt -2 to -2056 of the rLHR gene, numerous complexes were of greater intensity when nuclear extracts from 8-Br-cAMP-induced granulosa cells were used as compared with extracts from noninduced cells (17). These observations suggest that there are likely to be numerous transcription factors binding to several cis-elements within the 5'-flanking region of the rLHR gene that act in a combinatorial manner to elicit the cAMP-mediated increase in rLHR gene transcription in granulosa cells.

To begin to dissect this potentially complex system, we focused in the present study on one particular EMSA complex whose formation was increased when using nuclear extracts from 8-Br-cAMP-treated granulosa cells.2 This complex (which we have designated complex A) was initially observed in EMSAs using a labeled probe corresponding to nt -187/-2 of the rLHR gene. As presented herein, the core sequence required for protein A complex is a string of five G’s at positions -146/-142. Thus, in EMSAs using nt -187/-2 as the labeled probe and competing with unlabeled fragments corresponding to the wild-type sequence of nt -149/-137 or mutations thereof containing only one substitution at a time it was found that mutations of any of these five guanine nucleotides abolished the ability of the competing fragment to inhibit complex A formation. Interestingly, nt -146/-142 are situated overlapping with and extending upstream of one of three Sp1 sites (designated Sp1c) in the 5'-flanking region of the rLHR gene (see Fig. 4Go). Accordingly, we have named the -146/-142 core sequence which forms complex A the SAS site (Sp1c adjacent site). By performing competitive EMSAs using competing oligonucleotides in which each of the five G’s in the core SAS site was mutated to A, T, or C, it was determined that the core consensus sequence for complex A formation is (G/T)GGGG. However, other experiments suggest that additional nucleotides upstream of the core SAS site may also be involved in complex A formation (cf. Figure 6Go). Presumably, they may be interacting with lower affinity as only disruption of the aggregate, rather than the individual, nucleotides suggests their role in complex A formation. Although the SAS site overlaps the Sp1c site, SAS- vs. Sp1c-specific mutations show that complex A formation on EMSAs is dependent only upon the integrity of the SAS site. Furthermore, antibodies to Sp1 and Sp3 do not affect complex A formation, further suggesting that Sp1c-binding proteins are not required for complex A formation (cf. Figs. 6Go and 7Go). It is not uncommon for Sp1 sites to overlap other cis-elements. Whereas in some cases a concerted action between the two sites is required for gene regulation (25, 26, 27), this is not always the case. For example, the binding of BGP1 to a G-rich DNA cis-element in the ß-globin gene promoter does not require Sp1 binding to the overlapping Sp1 cis-element (20).

Interestingly, there are several other transcription factor-binding elements which, like the SAS site, are known to be G-rich. These include the cis-elements which bind the G-string-related transcription factors G-string (18), G-fer (19), BGP1 (20, 21), and IF-1 (22, 23), as well as Zif268 (24). These transcription factors have been shown to regulate genes in different ways. For example, G-string positively regulates the LpS1ß gene in Lytechinus pictus embryos (18), G-fer inhibits the expression of human H-ferritin-encoding gene (19), BGP1 appears to destabilize or exclude a positioned nucleosome, allowing access of transcription factors to their cognate sites (20, 21), IF-1 negatively regulates the mouse {alpha}1(I) and {alpha}2(I) collagen genes (22, 23), and Zif268 is thought to modulate the transcription of genes regulated by growth factors (24). It was of interest, therefore, to determine whether the transcription factor binding the SAS site was related to any of these previously described factors. To address this, we asked whether oligonucleotides corresponding to the binding sequences of these transcription factors could compete for complex A formation in EMSAs. Whereas binding sequences for G-string, G-fer, and IF-1 appear to specifically inhibit complex A formation, Zif228 does not, and the results for BGP1 are ambiguous. Taken altogether, our data suggest that the SAS-binding protein may be related to the G-string family of transcription factors. A more definitive assessment cannot be made until the cDNA encoding the transcription factor binding to the SAS site is cloned.

Two of the most widely characterized cAMP-responsive elements are the cAMP-responsive element (CRE) (28) and AP2 (29). Recently, it has been shown that some other elements, for example SF1 (30, 31, 32) and Sp1/Sp3 (26, 27, 33, 34), can also mediate cAMP effects on gene expression. Although the consensus binding sequence for the classical CRE is not present in the 5'-flanking region of rLHR gene (16), reporter gene assays using 5'-deletion constructs of nt -2100/-1 have suggested numerous cAMP-responsive elements within this region (17). Recent studies from our laboratory have shown that the three Sp1/Sp3 binding sites in the 5'-flanking region of the rLHR gene contribute both to the basal and the cAMP-stimulated expression of this gene (17). It should be noted that the earlier studies were performed before the identification of the SAS site, which is also involved in the cAMP responsiveness of the rLHR gene. Unfortunately, the Sp1c site in the prior study was mutated in such a way that both the Sp1c and SAS sites would be predicted to be disrupted. However, as shown herein, a more selective mutation of the Sp1c site still results in decreased basal and cAMP-inducible transcriptional activity. Therefore, the conclusions regarding the functional role of the three Sp1 sites remain valid. Although the rLHR 5'-flanking sequence also contains an AP2 site and two SF1 sites, these do not appear to be involved in the cAMP-induced transcription of this gene in rat granulosa cells (17). The studies presented herein show that the novel SAS site is functionally important for the cAMP inducibility of the rLHR gene since disruption of this site attenuates the ability of 8-Br-cAMP to stimulate transcription of a reporter gene (cf. Fig. 9Go). Experiments performed thus far testing the ability of the SAS element to confer cAMP responsiveness to a heterologous promoter have been negative. These negative results may reflect that the experiment has not been performed optimally. However, they may also result if the SAS site is necessary, but not sufficient, for cAMP inducibility. Such a conclusion would be consistent with the observation that the three Sp1 sites in the 5'-flanking region also mediate cAMP induction of the rLHR gene (17) and the data suggestive of several sites throughout the 5'-flanking region acting in a combinatorial manner to allow for cAMP induction of the rLHR gene (17).

Using reporter gene assays, we have shown that the selective mutation of the SAS site has a marked inhibitory effect on the cAMP induction of rLHR gene transcription. Given that cAMP increases the abundance of complex A formation at the SAS site, it is reasonable to postulate that the increase in complex A formation plays a role in the cAMP-mediated transcription of the rLHR gene, most likely in concert with other factors bound to other cis-elements as well. The effects of complex A on rLHR gene transcription may be affected by cAMP by one or more of several mechanisms. Thus, it is possible that cAMP treatment increases the steady state levels of the transcription factor binding to the SAS site. Alternatively, or additionally, phosphorylation of the transcription factor by activation of protein kinase A may increase its association with the SAS element (35). These are clearly important issues that will need to be addressed in future experiments but will first require the cloning of the factor(s) binding to the SAS site in the rLHR gene.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reagents
DMEM, Ham’s F-12 nutrient mixture, Waymouth’s MB 752/1 medium, and oligonucleotides were obtained from Life Technologies, Inc. (Gaithersburg, MD). 8-Br-cAMP, DES, BSA, and corticosterone were purchased from Sigma (St. Louis, MO). ITS was purchased from Collaborative Research Inc. (Bedford, MA). Restriction endonucleases MscI, NcoI, BstNI, and BstXI, and T4 polynucleotide kinase were obtained from New England Biolabs, Inc. (Beverly, MA). [32P]ATP was purchased from NEN Life Science Products (Boston, MA). Poly(dI-dC) was obtained from Pharmacia Biotech (Piscataway, NJ). Luciferin was from Analytical Luminescence Laboratory (Ann Arbor, MI). Antibodies to Sp1 and Sp3 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Primary Cultures of Granulosa Cells
Immature Sprague Dawley female rats (20–21 days of age, from Harlan Sprague, Indianapolis, IN) were primed subcutaneously with Silastic capsules (18 mm) containing 25 mg DES (a nonsteroidal estrogen) for 5 days. The ovaries were then excised and the granulosa cells were released as previously described (15). The granulosa cells were plated on fibronectin-coated tissue culture plates and cultured in growth medium (DMEM/Ham’s F-12 (1:1) supplemented with 10 mM HEPES, pH 7.4, 50 mg/ml gentamycin, 0.1% BSA, 1 mg/ml ITS, and 18 ng/ml corticosterone) at 37 C in a 5% CO2 incubator.

Nuclear Extracts and Gel Mobility Shift Assays
Granulosa cells released from the ovaries of DES-pretreated rats were plated on 60 mm fibronectin-coated plates at a cell density of 4 x 106 cells per dish and incubated with or without 3 mM 8-Br-cAMP for 40 h. The nuclear extracts were then prepared as previously described (36). The protein concentrations were determined by Bradford assay (37). Typically, the yield was approximately 6–12 mg nuclear protein/dish.

For EMSAs examining the formation of complex A, the following conditions were used. Nuclear proteins (2 mg) were incubated 60 min on ice with 10 fmol (~10,000 cpm) of a 32P-labeled probe corresponding to nt -187/-2 of the rLHR in a total volume of 25 ml of binding buffer (10 mM Tris, pH 7.6, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 100 mM KCl) also containing 3–5 mg poly (dI-dC). For competition experiments, unlabeled competitor DNA was added simultaneously with the labeled fragments at molar excess ratios as indicated. The resulting DNA-protein complexes were resolved by electrophoresis on 5% polyacrylamide gels run in 20 mM Tris-acetate, 1 mM EDTA, pH 8.0, for approximately 2 h at 180 V. Under these conditions, complex A can be observed in the lower portion of the gel. These conditions, however, do not permit detection of Sp1 and/or Sp3 binding to the Sp1 sites.

For optimal visualization of Sp1 and Sp3 binding to the Sp1c site, different EMSA conditions were used. In this case the labeled probe corresponded to nt -156/-131. For competition experiments, unlabeled competitor DNA was added simultaneously with the labeled fragments at molar excess ratios as indicated. The resulting DNA-protein complexes were resolved by electrophoresis on 5% polyacrylamide gels run in 25 mM Tris, 190 mM glycine, 1 mM EDTA, pH 8.4 for approximately 4 h at 165 V. EMSAs run under these conditions permit detection of the complex formed between the Sp1c site with Sp1 and/or Sp3, but not complex A formation.

Gels were either developed by autoradiography or exposed to a Cyclone storage phosphor screen (Packard Instrument Co., Meriden, CT) for 4 h at room temperature. The screen was scanned by Scan control software and quantified using OptiQuant software (Packard Instrument Company, Meriden, CT).

DNA Reporter Gene Constructs and Mutagenesis
Because the rLHR gene contains multiple transcriptional start sites, the numbering of the 5'-flanking region of the gene is based relative to the translation initiation codon (16). The rLHR-2056-luc construct, which contains the luciferase reporter gene and the fragment from -2056 to -2 of the 5'-flanking region of rLHR gene was a gift from Dr. Mario Ascoli (University of Iowa, Iowa City, IA). The promoterless pLLV3-luciferase plasmid was originally provided by Dr. Richard Maurer (Oregon Health Science University, Portland, OR). Mutants were generated by the PCR using the overlap extension method (38, 39). The template (fragment -187/-2) for mutagenesis of the Sp1c site and the SAS site was created from the LHR-2056-luc construct by digestion with NcoI and MscI. The Sp1c and SAS mutations are shown in Fig. 4Go. The entire PCR region for each mutant was verified by sequence analysis (40).

Transient Cell Transfection and Luciferase Assays
Granulosa cells were plated in 60-mm dishes at 4 x 106 cells per dish for 16 h and then were transiently transfected by the calcium-phosphate precipitation method (41) using 24 µg of plasmid/60-mm dish. After 4 h of exposure to the DNA precipitation, the cells were washed three times with Waymouth’s MB752/1 medium supplemented with 0.1% BSA, refed with fresh growth medium, and cultured in the absence or presence of 3 mM 8-Br-cAMP. In earlier experiments it was determined that maximal cAMP-inducible luciferase activity was observed 40–48 h after transfection. For the experiments shown, cells were lysed 40 h after transfection with 250 µl of lysis buffer (25 mM glycylglycine hydrochloride, 15 mM MgSO4, 4 mM EGTA, 1 mM dithiothreitol, 0.5% NP-40) for 15 min at room temperature. The cell debris was removed by brief centrifugation, and the supernatant was transferred to a clean tube. The luciferase assay was performed as described (42). Protein concentrations of the lysates were determined by the Bradford assay (37). The data for luciferase assays were normalized and reported as light units per mg protein. Earlier studies had shown that similar results were obtained regardless of whether or not the data were further standardized to ß-galactosidase activity as determined by cotransfection with the cDNA for ß-galactosidase. Therefore, in the experiments shown this was not performed.

Data Presentation
The gel shift assays and competition assays were repeated at least three times with different preparations of nuclear extracts, and the representative experiments are included shown.


    FOOTNOTES
 
Address requests for reprints to: Deborah L. Segaloff, Ph.D., Department of Physiology and Biophysics, The University of Iowa College of Medicine, Iowa City, Iowa 52242. E-mail: deborah-segaloff{at}uiowa.edu

These studies were supported by NIH Grants HD-22196 and HD-33931 (to D.L.S.). While these studies were in progress, D.L.S. was a recipient of a Research Career Development Award HD-00968. The services and facilities of the University of Diabetes and Endocrinology Research Center, supported by DK-25295, are also acknowledged.

1 S.C. and X.L. contributed equally to this study. Back

2 The detection of complex A in uninduced granulosa cells may at first seem contradictory to the observations that under these conditions neither rLHR mRNA nor rLHR binding activity is observed (10 15 ). However, uninduced granulosa cells do exhibit a detectable level of basal rLHR gene transcription, as measured by nuclear run-on assays (15 ). Back

Received for publication September 1, 1999. Accepted for publication May 18, 2000.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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