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a Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Department of Surgery, Harvard Medical School, Boston, Massachusetts 02114
b Division of Immunology and Department of Pathology, Sackler School of Graduate Studies, Tufts University School of Medicine, Boston, Massachusetts 02111
c University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, gene regulation, granulosa cells, Müllerian ducts, Sertoli cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Most protein-coding genes contain a TATA box that is required for accurate transcription initiation. However, the finding that some promoters lack a TATA box yet accurately initiate transcription led to the discovery of initiator (Inr) elements spanning the start site [21, 22]. The human MIS (hMIS) promoter lacks a consensus TATA element; therefore, we examined whether it had a functional Inr element. The sequence encompassing the hMIS start site [17, 23, 24] does not fit previously published initiator consensus sequences; however, when basal transcription directed by the wild-type core promoter was compared with the promoter in which the initiator site was mutated, the start site was found to be functionally necessary. Similar analysis of the nonconsensus TATA site showed that it was not required.
Initiator elements have been demonstrated to direct accurate transcription from heterologous [25, 26] as well as natural promoters [27, 28], and their mutation or deletion can decrease or abolish transcription [29]. Initiator elements can also function synergistically with a TATA box element [28, 30, 31]. Several models have been advanced to describe how specific transcription is initiated through Inr elements. One model suggests that TATA-binding protein (TBP)-associated factors (TAFs) are capable of binding to Inr elements to direct transcription [32–34]. A second model suggests that Inr elements are directly recognized by RNA polymerase II [35]. A third proposes that initiator-dependent transcription can be mediated by initiator-specific binding proteins such as TFII-I [36, 37] or YY1 [30], which can substitute for TFIIA [38, 39] or TBP [40], respectively. Gel shift analyses demonstrated that the hMIS Inr is recognized by the initiator-binding protein TFII-I, suggesting that hMIS core transcription may employ the third model for initiator-dependent transcription. Analysis of the sequences of several different functional Inr elements led to the definition of Py Py A+1 N T/A Py Py as the consensus sequence [41]. However, several genes, including the human TGF
gene [42], human chorionic somatomammotropin gene [43], and now, the hMIS gene, have been identified that contain functional Inr elements that do not match the consensus initiator sequence. In fact, TFII-I has six helix-loop-helix (HLH) domains, which may allow it to recognize several independent DNA elements; thus, the hMIS Inr may be a novel TFII-I-binding element.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The plasmid p-269/+8,hMIS-luc was created by amplifying a region of the 5'-flanking sequence of the hMIS gene [17]. The fragment (from -269 to +8 relative to the previously reported start site) was made by the polymerase chain reaction (PCR) using the primers NMI and NM2 as the 5'- and 3'-primers, respectively (Table 1), which introduced HindIII sites 5' and 3' of the fragment. The fragment was subcloned into the HindIII site of the pA3 luciferase vector, pA3-luc [44]. Constructs containing mutations in the nonconsensus TATA region (from -28 to -23 relative to the start site) or the initiator region (from -22 to +10) of the MIS promoter (Fig. 1A), called p-269hMIS,TATAmut-luc and p-269hMIS,Inrmut-luc, respectively, were created by PCR using primers NM1 as the 5'-primer and TATA mut or INR mut as the 3'-primer, respectively. For the construction of reporters containing the hMIS initiator (phMIS-Inr-luc [also called hMIS,-22+10-luc]), the adenovirus major late (AdML) promoter initiator (pAdML-Inr-luc), and the human immunodeficiency virus (HIV)-1 initiator (pHIV-1-Inr-luc), the annealed oligonucleotides representing -22 to +10 of the MIS promoter, -18 to +33 of the AdML promoter, or -3 to +42 of the HIV-1 promoter were inserted into the HindIII site of the pA3-luciferase vector, respectively. The constructs, p5'mut,3'wt,-22/+10-luc (for 5' mutated, 3' wild type) and p5'wt,3'mut,-22/+10-luc were created by replacing the 5' or 3' 16 nucleotides of the hMIS initiator with unrelated 
phage sequence (5'-TCTATCACCGCAAGGG-3'), respectively. An expression construct for the human sex-determining region of the Y chromosome (SRY) was made by inserting the complete open reading frame of human SRY into the BamHI site of the pCMV8 expression vector [45]. The fidelity of all PCR-amplified sequences was verified by dideoxyribonucleotide sequencing.
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Cell Transfection and Luciferase Assays
The CH34 cells, cloned from the urogenital ridge cells of 14.5-day-old male rat embryos that had been immortalized by 
2-v-myc retrovirus infection, were cultured as described elsewhere [46]. For transient transfections, CH34 cells in 9-cm2 wells were transfected by calcium phosphate precipitation with 2 µg of the luciferase reporter vector and 0.5 µg of pXGH5 vector [47] directing human growth hormone expression as a control for transfection efficiency. In experiments where SRY is overexpressed, 1 µg of pCMV8-SRY expression vector or the parental pCMV8 vector was cotransfected with the luciferase and growth hormone vectors. After 40 to 48 h of incubation, the transfected cells were washed three times with 2 ml of Hanks balanced salt solution, scraped in extraction buffer (100 mM K2HPO4 [pH 7.8], 1 mM dithiothreitol [DTT]), and resuspended in 100 µl of extraction buffer. Cells were lysed by three freeze-thaw cycles. Following centrifugation at 15 500 x g for 5 min, luciferase activity was detected in the supernatant by chemiluminescence with luciferin substrate (Promega, Inc., Madison, WI) using a Nichols Institute luminometer (San Juan Capistrano, CA) and expressed as mean ± SD from three to six independent transfections after normalization to growth hormone activity.
Electrophoretic Mobility Shift Assays
The oligonucleotides used in the assays were hMIS initiator -22/+10, randomized hMIS initiator, terminal deoxynucleotidyl transferase (TdT) initiator [22], AdML initiator [37], and Sp1 consensus elements [48] (Table 1). The wild-type and mutant T cell receptor-derived variable region ß-chain gene (Vß) Inr-containing oligonucleotides have been described [49], as has the E box oligonucleotide [50]. Nuclear extracts from CH34 cells were prepared as described elsewhere [51]. A typical reaction mixture for experiments shown in Figure 2, A and B, included 5 fmol of 32P-labeled double-stranded oligonucleotide (
50 000 cpm), 1 µg of poly (dI·dC) or poly (dA·dT), 10% glycerol, 10 mM Hepes (pH 7.9), 60 mM KCl, 1 mM DTT, 0.5 µg of BSA, and 0.5 to 2 µg of nuclear extract in a volume of 20 µl. Labeling of the oligonucleotides was accomplished by using either the Klenow fragment of DNA polymerase with [-32P]dATP or T4 polynucleotide kinase with [
-32P]ATP. In competition experiments, unlabeled double-stranded oligonucleotides were added to the reaction mixtures at different concentrations before the addition of nuclear extract. After a 30-min incubation at 30°C, the samples were loaded onto a 6% polyacrylamide gel in a glycerol-tolerant buffer (90 mM Tris, 30 mM taurine, 0.5 mM EDTA). For the electrophoretic mobility shift assays (EMSAs) shown in Figures 2C and 3A, reactions were carried out at 4°C in volumes of 20 µl containing 20 mM Tris (pH 7.9), 0.2 mM EDTA, 5 mM DTT, 0.5 mM PMSF, 10% glycerol, 80 mM KCl, and 100 ng of poly (dA·dT) per reaction as a carrier. The hMIS Inr probe (-22, +10) had a specific activity of 174 000 cpm/pmol, and 1 pmol was used in Figure 3A, lanes 4–8. The Vß Inr probe (-28, +12) had a specific activity of 99 000 cpm/pmol, and 0.017 pmol was used in Figure 3A, lanes 9–14. As competitor probes, the hMIS Inr-containing oligonucleotide was used at 40 pmol per 20 µl in Figure 2C, lanes 3 and 4, and 144 pmol per 20 µl in Figure 2C, lane 11. When used as a competitor, the Vß Inr (wild-type and mutant) oligonucleotides were used at 0.689 pmol per 20 µl in Figures 2C and 3A, lanes 6, 7, 12, and 13. For reactions employing the anti-TFII-I antibody to block complex formation, the antibody was preincubated with the protein source for 10 min at 30°C before addition of the labeled probe. DNA-binding reactions proceeded for 20 min at 30°C. For reactions exhibiting supershifts, probe and extract were incubated 20 min at 30°C, and the antibody was added, followed by 10 min of incubation at 30°C. The reactions were electrophoresed through a 5% native gel containing 5% acrylamide and 5% glycerol in 0.5x TBE buffer (45 mM Tris base, 45 mM boric acid, 1 mM EDTA), for 3 h at 140 V. The gel was then dried and subjected to autoradiography.
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TFII-I Antisera
Production of the anti-TFII-I antisera has been described elsewhere [27]. For the assays reported in Figures 2C and 3A, the anti-TFII-I antisera was affinity purified [52].
Recombinant TFII-I
The TFII-I cDNA was cloned according to the method described by Roy et al. [53]. The TFII-I protein was overexpressed as a hexa-histidine fusion protein in bacteria, purified on a Ni2+-agarose column, and shown to have DNA-binding properties that mirrored those of native TFII-I [53].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because the hMIS promoter does not contain a consensus TATA box (CTTAA at -26 to -22; Fig. 1A), we investigated for potential initiator function. Luciferase reporter constructs containing the hMIS promoter (-269 to +10) with mutations throughout either the potential initiator (-22 to +10) or the nonconsensus TATA regions (-28 to -23; Fig. 1A) were transiently transfected into CH34 cells, clonally derived from v-myc transformed urogenital ridge cells from 14.5-day-old male embryonic rats and considered to be Sertoli derivatives, because they express MIS (unpublished results) and Sox9 (unpublished results) by Northern blot analysis and/or reverse transcription-PCR. The construct containing mutations in the putative initiator region exhibited only 25% of the transcriptional activity of the wild-type promoter, whereas the construct in which the putative TATA region was mutated exhibited transcriptional activity identical to the wild-type promoter, indicating that the initiator region (-22 to +10) is essential for the transcription of hMIS and that the nonconsensus TATA element is not functional (Fig. 1B).
To establish that the putative hMIS Inr element (-22 to +10) can function as a core promoter, basal transcription conferred by the hMIS initiator was compared to that seen with characterized Inr elements. The hMIS initiator, as well as the previously identified AdML initiator [37], conferred threefold greater transcriptional activity compared with the empty luciferase vector, suggesting that similar mechanisms operate via these initiation elements, whereas the transcriptional activity of the putative HIV-1 initiator failed to activate basal transcription above the level seen with the luciferase vector alone (Fig. 1C). The HIV-1 initiator appears to require Rel p50, which may explain its lack of activation in this context [50, 54].
3' 16 Nucleotides in the MIS Initiator Region (-6 to +10) Are Essential for Transcription of hMIS
To further define the sequences required for core promoter activity, the 5' 16 nucleotides (-22 to -7) or the 3' 16 nucleotides (-6 to +10) of the hMIS initiator were replaced by an unrelated DNA sequence of the same length from phage (Fig. 1A). The construct with intact sequence corresponding to nucleotides -6 to +10 conferred almost the same transcriptional activity as the wild-type hMIS initiator region, whereas the activity of the construct in which nucleotides -6 to +10 were mutated did not exceed that of the empty vector (Fig. 1D).
Because the empty luciferase vector contains three SRY-binding sites (5'-ATTGTT-3') upstream of the cloning site, we used SRY as a heterologous trans-acting factor to determine whether the hMIS Inr element could function as a core promoter capable of responding to upstream enhancer element activation. Transcription of the construct containing the intact hMIS -6/+10 sequence, as well as the -22/+10 hMIS construct, were induced more than 20-fold by SRY, whereas transactivation of the construct in which the 3' region was mutated was almost the same as that of the luciferase vector alone (Fig. 1E).
hMIS Initiator Forms a Specific DNA-Protein Complex
To elucidate the biochemical interactions occurring via the hMIS initiator, EMSAs done using 32P-labeled, annealed oligonucleotides corresponding to the hMIS initiator generated one major DNA-protein complex and another minor complex in the presence of nuclear extract from CH34 cells (Fig. 2A, lane 2). The major complex was subjected to competition by increasing concentrations of unlabeled oligonucleotides corresponding to the hMIS initiator (Fig. 2A, lanes 3–5) or the AdML initiator (Fig. 2A, lanes 6–8), but oligonucleotides corresponding to a mutated hMIS initiator sequence failed to compete for binding to the nuclear extracts (Fig. 2A, lanes 9–11) or to induce luciferase reporter expression (Fig. 1D).
Transcription Factor TFII-I Binds to the hMIS Initiator
Because the AdML initiator, which binds to TFII-I from HeLa cell nuclear extract, competed for the factor (or factors) from CH34 cell nuclear extract that bind the hMIS initiator, we studied whether TFII-I could contribute to the complex formed with the hMIS Inr. Because TFII-I was originally purified from HeLa cell nuclear extract and the complex formed between TFII-I and the TdT initiator has been well documented [37], we used the hMIS initiator in EMSA experiments to compete HeLa cell nuclear protein binding to 32P-labeled TdT initiator. The major DNA-protein complex formed between the TdT initiator and HeLa nuclear extract (Fig. 2B, lane 2) was competed by unlabeled oligonucleotides corresponding to the hMIS initiator (Fig. 2B, lanes 7 and 8) as well as the TdT (Fig. 2B, lanes 3 and 4) and the AdML initiators (Fig. 2B, lanes 5 and 6), but not by an unrelated guanine-cytosine-rich oligonucleotide corresponding to the Sp1 consensus element (Fig. 2B, lanes 9 and 10). The TdT initiator also formed a complex with nuclear extracts from CH34 cells at the same position as the complex formed with HeLa nuclear extracts (Fig. 2B, lane 12). This complex was also competed by unlabeled oligonucleotides of TdT, AdML, and hMIS initiators (Fig. 2B, lanes 13–18), but not by the unrelated Sp1 element (Fig. 2B, lanes 19 and 20). Thus, a major binding species exists in CH34 and HeLa cell nuclear extracts that recognizes the consensus TdT and AdML Inr elements as well as the hMIS Inr.
To investigate the involvement of the ubiquitously expressed initiator-binding protein TFII-I in the complex with hMIS Inr, EMSA employing highly specific anti-TFII-I antibodies (Fig. 2C) and competition with a newly described TFII-I-binding initiator from the T cell receptor-derived Vß Inr was performed [49]. Figure 2C shows that the principal binding activity detected with the hMIS Inr oligonucleotide in the CH34 nuclear extract (lane 2) can be competed with a wild-type Inr (Vß Inr, lane 3)-containing oligonucleotide, but not with mutant Inr (Vß Inr mut, lane 4)-containing oligonucleotide, and that it can be blocked with an anti-TFII-I antibody (lane 5) raised to the putative DNA-binding domain of TFII-I [27]. In addition, TFII-I was detected in the complex by supershifting a portion of the complex with the TFII-I antiserum. Antiserum was added after DNA-protein complex formation (1 µl of a 1:10 v/v dilution in lane 8, or 1 µl undiluted in lane 9), and a supershift of the DNA/protein complex was observed at the higher concentration of the antibody. In lane 10, 1 µl of preimmune serum does not shift the complex. Figure 2C is representative of two separate experiments.
To confirm that TFII-I binds to the hMIS probe, EMSA was performed with highly purified TFII-I [55]. Complex formation observed between purified TFII-I and the hMIS initiator region was observed (Fig. 3A, lanes 2 and 5), and it was competed with hMIS Inr (Fig. 3A, lane 3) and by a Vß Inr-containing oligonucleotide (Fig. 3A, lane 6), but not by a mutant Vß Inr-containing oligonucleotide (Fig. 3A, lane 7). The identity of TFII-I binding was further confirmed using an anti-TFII-I antibody (Fig. 3A, lane 8). The control reactions in the right panel of Figure 3A demonstrate TFII-I binding to the unrelated Vß Inr probe. In this case, the labeled Vß Inr-containing oligonucleotide was competed by hMIS Inr and by the wild-type Vß Inr-containing oligonucleotides, but not by the mutant Vß Inr-containing oligonucleotide, suggesting that TFII-I binding is directed through the Inr. Because the binding of TFII-I to the Vß Inr was much higher (100-fold) compared with that of the hMIS Inr, the unlabeled competitor hMIS (144 pmol)-containing oligonucleotide was present in greater than 200-fold molar excess compared with the unlabeled Vß (0.689 pmol) competitor in the competition analysis demonstrated in the right panel of Figure 3A.
Further confirmation that TFII-I is the protein binding to the hMIS Inr was demonstrated with bacterially expressed TFII-I (Fig. 3B, lane 2). The complex can be competed with unlabeled hMIS Inr, Vß Inr, and to a lesser degree, with an E box-containing oligonucleotide (Fig. 3B, lanes 3, 4, and 6, respectively). An oligonucleotide containing a mutant Vß Inr sequence did not compete, showing the specificity of the complex (Fig. 3B, lane 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The control region of a typical regulated eukaryotic gene is comprised of proximal (core) and distal (enhancer) promoter regions. The core promoter region usually consists of one or both of two elements: the TATA box, and the Inr element. In TATA-containing promoters, the initiation of functional transcription involves the ordered assembly of at least six basal transcription factors, including TFIIA, -B,-D, -E, -F, and -H, in addition to RNA polymerase II [29, 56]. TFIID is the only factor known to possess sequence-specific DNA-binding activity and to initiate preinitiation complex assembly through recognition of the TATA box. However, the mechanism of preinitiation complex formation on TATA-less promoters has remained unclear. One postulated model is that a specific initiator-binding protein, such as TFII-I [37], YY1 [30], HIP-1 [57], or USF [58, 59], with specificity for a certain type of Inr element, binds to that element, recruits TBP, and then recruits the other transcription factors to the preinitiation complex [37]. For example, TFII-I is required for transcription of the promoter containing the AdML initiator [37]. In addition, recent in vitro transcription experiments using the T-cell receptor variable region-derived (Vß) promoter also showed that TFII-I is essential for transcription of this TATA-less initiator-containing promoter, whereas it is dispensable for the TATA element-containing B-cell immunoglobulin heavy chain-derived (immunoglobulin H) promoter [27]. Other initiators that require a precise initiator consensus have been identified that bind a component of TFIID for their function [33]. Several other genes have core promoters that rely on nonconsensus Inr elements [42, 43, 60]. Therefore, it is conceivable that initiator-containing promoters can be divided into several subgroups, with each employing different mechanisms for initiator function; the hMIS promoter uses a TFII-I-binding initiator, suggesting that transcriptional activity of hMIS may be dependent on TFII-I.
Sequence analysis of the hMIS initiator indicated that the sequence -13 to -5 (5'-CCCAGCCCC-3') rather than the sequence encompassing the transcriptional start site -2 to +5 (5'-GC
+1GCAC-3') was more similar to the previously reported, pyrimidine-rich initiator consensus YY+1N(T/A)YY [41]. However, the sequence between -14 and +2 did not form a complex with CH34 nuclear protein, whereas the region between -6 and +10 could form a DNA-protein complex that contained TFII-I. Furthermore, transient transfection assays affirmed that the region between -6 and +10 was both essential and sufficient for basal transcription of hMIS. These findings are in agreement with those of previous reports that demonstrated the transcriptional start site of the hMIS gene [17, 23]. There are six genes—AdML [37], TdT [37], HIV-1 [37], Vß [27], mouse ribonucleotide reductase R1 [61], and human KDR/flk-1 [62]—whose promoters are reported to bind to TFII-I, and all fit the initiator consensus sequence, except for HIV-1 and human KDR/flk-1. Here, we have shown that the nonhomologous hMIS initiator can also bind to TFII-I, although TFII-I appears to bind with higher affinity to the TdT, AdML, and Vß initiators (Fig. 2, A and B). How TFII-I recognizes both consensus and nonconsensus Inr elements is unclear. Because the optimal binding consensus for TFII-I has not yet been determined experimentally, it remains possible that TFII-I-binding requirements are flexible. Moreover, deduction of the primary amino acid sequence of TFII-I indicated that it is composed of six repeats of putative HLH domains [39], suggesting the possibility that TFII-I can bind distinct DNA elements and that independent interactions may occur with other distinct HLH proteins [53, 55]. That TFII-I has been shown to bind to E box elements, which bind HLH proteins [50], and that TFII-I binding to the hMIS Inr was competed less well with an E box-containing oligonucleotide than with the Vß Inr-containing oligonucleotide (Fig. 3C) is consistent with the possibility that TFII-I has multiple distinct DNA-binding domains.
In EMSA experiments, the observed binding of the hMIS initiator by proteins in CH34 nuclear extracts was also seen using nuclear extracts from HeLa cells (Fig. 2B) and Jurkat T lymphocytes (data not shown). The mobility of the DNA-protein complex formed by the TdT initiator and the hMIS initiators with CH34 nuclear extracts appears to be identical (Fig. 2C), indicating that the binding proteins in the extracts were neither tissue nor hMIS specific. Consistent with this notion, our data clearly demonstrate binding of a ubiquitous Inr-interacting protein, TFII-I, to the hMIS Inr (Fig. 3). However, the TdT initiator can bind to TFIID directly [33], and TAFs play an important role in the basal transcription of TdT by recruiting TBP to the preinitiation complexes [25, 28]. Thus, the TFIID complex containing TAFs, in addition to TFII-I, probably may also be functionally involved in the transcription of hMIS. This is especially true because TFII-I appears to recognizes a component of TFIID for efficient initiator-dependent function [27].
In summary, the basal transcription of hMIS appears to occur via a nonconsensus novel initiator, and it involves a complex of nuclear proteins that are not tissue specific and that include TFII-I, suggesting that the previously reported empirical initiator consensus sequence is not essential for recognition by TFII-I. Other Inr elements, which are dissimilar from the reported initiator consensus, have been described as well [42, 43, 60], raising the possibility that TFII-I has a broad specificity of interaction and may recognize multiple Inr elements.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH grant HD30812 to P.K.D. and in part by ACS grant RPG-98-104-01-TBE and NIH grant AI41147 to A.L.R., NIH grant HD08177 and a fellowship from ACS to T.R.C., NIH grant HD33462 to M.A.W., the Gerald Austen Surgical Research and Surdna GAR funds to N.M., and the M.G.H. Medical Discovery Award to C.M.H. 
2 Correspondence: Patricia K. Donahoe, Pediatric Surgical Research Laboratories, Warren 10, Massachusetts General Hospital, 32 Fruit St., Boston, MA 02114. FAX: 617 726 5057;donahoe.patricia{at}mgh.harvard.edu
Accepted: May 17, 2000.
Received: February 10, 2000.
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B homodimer binding within the HIV-1 initiator region and interactions with TFII-I. Proc Natl Acad Sci U S A 1996; 93:12376–12381This article has been cited by other articles:
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