©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A TATA-less Promoter Containing Binding Sites for Ubiquitous Transcription Factors Mediates Cell Type-specific Regulation of the Gene for Transcription Enhancer Factor-1 (TEF-1) (*)

(Received for publication, October 5, 1994; and in revised form, April 19, 1995)

David S. W. Boam (§) Irwin Davidson Pierre Chambon

From the Institut de Génétique et de Biologie Moléculaire et Cellulaire, Parc d'Innovations, 1 rue Laurent Freus, BP 163, 67404 Illkirch, Strasbourg, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

TEF-1 is a tissue-specific human transcription factor which binds to and activates transcription from the SV40 early promoter and the HPV-16 E6/E7 promoter and may be involved in regulation of muscle-specific and placenta-specific gene expression. To investigate the mechanism of its tissue-specific expression, we have isolated up to 3 kilobase pairs of 5`-flanking DNA and characterized the promoter of the gene for TEF-1. Multiple transcription start sites centering on a motif similar to the initiator element (Inr) were identified. A minimal promoter, which contains no recognizable TATA element but contains an Inr, delimited at -137 base pairs had full transcriptional activity both in vivo in HeLa cells and in vitro in HeLa cell extracts. This promoter is also highly active in vitro in lymphoid cell extracts, but not in vivo in lymphoid cell lines, which do not express the endogenous TEF-1 gene. The minimal promoter, which is sufficient to direct tissue-specific expression of the TEF-1 gene in vivo, contains multiple sites which bind the ubiquitous transcription factors Sp1 and ATF-1. Mutation of the Inr completely abolished transcription from the major start site while transcription from the minor sites was slightly augmented. Inactivation of the proximal Sp1 site abolished transcription from the principle start site and increased transcription from a 5` minor start site. Insertion of a TATA box element did not qualitatively alter the pattern of start site usage which seemed to be dependent upon integrity of the upstream Sp1 site. These observations suggest a ``cross-talk'' between the Inr and a proximal element to fix transcription start sites, which is independent of spacing and the presence of a TATA element.


INTRODUCTION

Transcriptional enhancers comprise multiple enhansons which bind transcriptional activator proteins. These factors ultimately determine the tissue-specific and developmental expression pattern of a given gene as well as its ability to be directly induced or repressed by various stimuli (reviewed in (1) and (2) ).

Mutational analysis of the SV40 enhancer has allowed the identification of multiple overlapping enhansons which function in a cell-specific manner(3, 4) . In HeLa cells, activity of the SV40 enhancer depends upon synergistic action of proteins bound to the TC-II, SPH-I+II, GT-I, and GT-IIC enhansons(3, 5, 6) . Transcriptional enhancer factor-1 (TEF-1) (^1)is a human transcriptional activator which binds to both the GT-IIC and SPH-I+II enhansons(6, 38) . A cDNA for TEF-1 has been isolated and its mRNA shown to be in HeLa and F9 EC cells, but not in lymphoid (BJA-B and MPC-11) cells where an alternate set of factors are responsible for the activity of the SV40 enhancer(7) . Furthermore TEF-1 cannot function in lymphoid cells possibly due to the presence of a factor which inhibits its stimulatory activity(39) .

TEF-1 is the prototypic member of a new family of transcription factors delineated by a homologous DNA-binding domain, the TEA/ATTS domain (8) which is highly conserved through evolution. A homolog of TEF-1, Scalloped, has been identified in Drosophila. Inactivation of this gene leads to multiple defects in sensory cells and certain subsets of neuroblasts(9) . Genes for TEF-1, and TEF-1-related factors, have now been isolated from mouse (10, 11) and chick(12) . The tissue-specific expression pattern of these genes gives no clue as to their function. TEF-1 has been shown to be involved in regulation of the chick cardiac tropomyosin gene (12) and it may also be involved in regulation of the hCS-B gene(41) . Moreover, TEF-1 has been implicated as a possible zygotic transcription factor, present in embryonic cells at the earliest developmental stages(13) . Inactivation of the mouse TEF-1 gene results in fetal death due to defects in cardiac maturation(16) . Nevertheless a coherent picture of the role of TEF-1 has not emerged.

In order to understand how spacial expression of the TEF-1 gene is controlled, we have cloned and begun to characterize the TEF-1 gene promoter. Here we delineate sequences important for high level expression of the gene and the sites of interaction with transcription factors within a defined minimal promoter region. The minimal promoter regions necessary for high activity both in vivo, in transfected HeLa cells, and in vitro are 80 and 137 bp, respectively, immediately 5` to the transcription start site. The TEF-1 promoter contains binding sites for the ubiquitous factors Sp1 and ATF1, and functions with high efficiency in both HeLa and lymphoid cells in vitro, but retains very little activity when introduced into lymphoid cells. The promoter region contains no recognizable TATA element and high level transcription is dependent upon an intact initiator element (Inr) (19) and upstream proximal Sp1 site. We demonstrate how transcriptional start site selection in this gene is controlled to a large extent by Sp1 and not influenced by the presence of a TATA element that was introduced between the Sp1 site and the Inr.


MATERIALS AND METHODS

Isolation of a TEF-1 5` Region Genomic Clone

A genomic DNA library derived from human lymphocyte DNA cloned into the vector EMBL3 was screened with a 40-bp oligonucleotide representing the 5` extremity of the TEF-1 cDNA 5`-UTR(7) . One positively hybridizing clone with an insert of approximately 13 kilobase pairs was isolated and subjected to restriction analysis. The digest was hybridized to the TEF-1 5`-UTR oligo which revealed a 3-kilobase pair EcoRI/NotI fragment, which was inserted into EcoRI and NotI sites in pBluescribe II SK and SK. This clone was sequenced by the chain termination technique (46) using a Sequenase kit (U. S. Biochemical Corp.).

Plasmid Constructs and Templates

All DNA manipulations and cloning steps were carried out essentially as according to Sambrook et al.(17) . A HindIII site was introduced by site-directed mutagenesis at +89 bp relative to a putative Inr and the resulting 2-kilobase pair KpnI/HindIII fragment was excised and cloned into the promoterless expression vector pAL4 (18) , containing the human beta-globin gene truncated to -9, to give pTDelta-1874. Further deletions of this construct were made using a combination of restriction sites and the polymerase chain reaction. The constructs pTDelta-530, pTDelta-455, pTDelta-365, pTDelta-178, pTDelta-156, and pTDelta-137 were constructed by cutting pTDelta-1874 with XhoI, HgiAI, HaeII, PvuII, BglI, and RsrII, respectively, followed by blunt-ending using T4 polymerase and restriction with HindIII (Fig. 1). pTDelta-80 and pTDelta-30 were constructed using polymerase chain reaction with relevant 5` primers annealing at -80 and -30, respectively, in the TEF-1 promoter and a 3` primer spanning the HindIII site at +89 T89 (see Table 1). The resulting fragments were subcloned into pAL4 cut with HindIII and SmaI. The polymerase chain reaction-generated fragments were sequenced after subcloning to verify absence of errors. All constructs were purified through 2 rounds of CsCl gradients and used for both in vivo and in vitro analysis.


Figure 1: Structure of reporter genes used for analysis of in vivo and in vitro of the TEF-1 promoter. Length of S1 probe and expected product after digestion is shown above the TEF-1 promoter-beta-globin reporter gene. Abbreviations to restriction sites are: K, KpnI; X, XhoI; Hg, HgiAI; Ha, HaeII; P, PvuII; B, BglI; R, RsrII.





Site-directed Mutagenesis

The template for site-directed mutagenesis, the template pTDelta-530, was constructed by inserting a XhoI/EcoRI fragment from pTDelta-1874 (see above) into pBluescribe II SK. Mutagenesis of the TEF-1 promoter was performed on antisense single-stranded DNA template derived from pTDelta-530 using the technique of Kunkel et al.(21) . The mutated inserts were sequenced then re-introduced as RsrII blunt-ended/HindIII fragments into pAL4 cut with SmaI and HindIII. Sequences of oligonucleotides used in mutagenesis are detailed in Table 1.

In Vitro Transcription Assay

Invitro transcriptions (25 µl) contained 100 µg of nuclear extract from HeLa or MPC-11 cells(6) , 50 ng of TEF-1 promoter template, 25 ng of internal control plasmid TATA-pAL7(18) , 50 mM Tris/HCl (pH 7.9), 50 mM KCl, 6 mM MgCl(2), 0.1 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol, and 0.4 mM of ATP, CTP, GTP, and UTP. Reactions were incubated for 45 min at 24 °C, then 150 µl of Stop buffer (0.1% SDS, 150 mM NaCl, 10 mM Tris, pH 7.9, 5 µg of yeast RNA) was added. Reactions were extracted with phenol/chloroform and RNA was precipitated in ethanol. Transcripts were quantified using S1 nuclease analysis as described below.

Transfections and RNA Analysis

Growth of cells (HeLa and MPC-11) and transfections were carried essentially as described(3, 5) . Briefly, HeLa cells and the murine plasmacytoma line MPC-11 were grown in 9-cm dishes to 30% confluence then transfected with 5 µg of reporter template, 1 µg of internal control template pG1B(3) , and carrier DNA (pBSII-SK) to 20 µg, using the calcium phosphate technique(45) . RNA was prepared 40 h post-transfection, and quantified by S1 nuclease analysis as described in (18) , except 150 units of S1 nuclease (Appligene) were used in digestion reactions.

For S1 nuclease mapping, an end-labeled single-stranded DNA probe from +69 in the beta-globin gene to -178 in the TEF-1 promoter was prepared as described in (18) with the following modifications. Single-stranded DNA template was prepared by helper phage-mediated rescue from the vector pTDelta-530. P-Labeled oligo M6 (18) was annealed to this template and extended using T7 DNA polymerase. Double-stranded DNA was cut with PvuII and the single-stranded probe was separated on a strand-separating gel(17) . For analysis of the Inr mutant promoter, a single-stranded DNA probe was generated from a template containing the Inr mutation (see Table 1).

Primer extension analysis was also carried out on transcripts isolated from HeLa cells transfected with pTDelta-1874. 10 µg of total RNA was hybridized to 20,000 cpm of end-labeled oligo M6 for 2 h at 35 °C then elongated with avian myeloblastosis virus reverse transcriptase at 42 °C for 1 h. Incubation conditions, buffer composition, and enzyme concentrations were as described(17) . All S1 nuclease-digested transcripts derived from transfections and invitro transcription reactions and products of primer extension analysis were analyzed on 8% sequencing gels alongside sequences generated from pTDelta-530 primed with oligo M6 as well as P-labeled pBR322/HpaII molecular weight markers.

DNase I Footprinting

30 µg of HeLa, BJA-B, or MPC-11 cell nuclear extract(6) , in a final volume of 20 µl of in vitro transcription buffer was incubated with 20,000 cpm of an end-labeled fragment of the TEF-1 promoter (-137 to +89), excised from pTDelta-137 with HindIII and EcoRI, for 30 min at 24 °C followed by digestion with DNase I (Boehringer) for 2 min. DNase I concentrations were determined empirically but were generally in the range 0.01-0.1 mg/ml. Incubations were terminated by addition of 150 µl of in vitro transcription stop buffer. Samples were phenol/chloroform extracted and DNA was ethanol precipitated and separated on a 8% sequencing gel alongside molecular weight and Maxam-Gilbert chemical sequencing markers(20) .

Gel Retardation Assays

Gel retardation assays containing 5 µg of nuclear extract in 10 mM Tris/HCl (pH 7.5), 50 mM NaCl, 5% (v/v) glycerol, 0.1 mM EDTA, 0.5 mM dithiothreitol, and 2 µg of poly(dI)bulletpoly(dC) (Pharmacia) as well as cold competitor oligonucleotide or antisera where indicated, were incubated on ice for 10 min then equilibrated to room temperature. 15,000 cpm (approximately 10-15 fmol) of end-labeled double-stranded oligonucleotide probe was added and incubations were continued for a further 20 min. Samples were run on 6% acrylamide (29:1 cross-linking ratio) gels in 0.5 TBE(17) , then dried and autoradiographed. Antisera to Sp1 and ATF factors were generous gifts from Dr. Stephen Jackson and Dr. Michael Green, respectively. Sequences of oligonucleotides used as probes and competitors are detailed in Table 1.


RESULTS

Organization of the TEF-1 5`-Region

We identified a sequence with 9 out of 11 identity with the Inr element previously identified by Smale et al.(19) (see Fig. 2E) 1874 bp downstream of the KpnI site identified in the TEF-1 5`-region. A HindIII site was introduced approximately 90 bp downstream of this element and the KpnI/HindIII fragment was subcloned into pAL4 to give pTDelta-1874. S1 nuclease and primer extension mapping of RNA isolated from HeLa cells transfected with pTDelta-1874 (Fig. 2, A and B) shows multiple transcription start sites centered around a putative Inr element (summarized in Fig. 2D). The 1-2 bp offset of start sites seen between S1 nuclease and primer extension analysis is artifactual due to the action of S1 nuclease. A genuine representation of start site positioning is given by the primer extension analysis, however, because the GC-rich 5`-UTR caused frequent pausing of the reverse transcriptase, quantization using reverse transcription was less reproducible. We therefore used S1 nuclease analysis for the rest of this study and the major start site seen using S1 nuclease mapping was designated +1.


Figure 2: Analysis of transcription initiation sites in the TEF-1 promoter. A, S1 mapping of initiation sites in the TEF-1 promoter. 10 µg of total RNA from HeLa cells transfected with 10 µg of pTDelta-1874 was analyzed by S1 mapping as described. Products were run alongside of a dideoxy sequencing ladder primed from the same template used to synthesize the S1 probe using oligo M6 as a primer. B, primer extension analysis of transcription initiation sites in the TEF-1 promoter. 10 µg of total RNA from HeLa cells transfected with 5 and 10 µg (left and right of sequence, respectively) of pTDelta-1874. Primer extension products were run alongside a sequence ladder generated as above. C, effect of mutation within the putative Inr of the TEF-1 promoter on transcription start site usage. HeLa cells were transfected with 10 µg of TDelta-1874 (left panel) or pTDelta-1874 (right panel). RNA was analyzed by S1 mapping. Products were run alongside sequences generated from corresponding templates (see results). D, summary of transcription start site positions in the TEF-1 promoter determined using primer extension and S1 mapping. E, comparison of sequences at the TEF-1 promoter start site with the TdT Inr. Identical sequences are shaded and an aligned sequence containing the TEF-1 mutant Inr is included for comparison.



Mutation of the Inr (Fig. 2E and Table 1) abolished transcription from the site at +1 but transcription from the other sites was relatively unaffected, although some augmentation of transcription from the minor site at -11 was observed (Fig. 2, C and D). The sequence from -400 to +174 is highly GC-rich (76% GC). Strikingly there is no apparent TATA element near the major start site nor downstream of it. The proportion of GC nucleotides both 5` and 3` to the start site would also rule out the possibility of a cryptic TATA box such as that identified in the adenovirus IVa2 promoter(22) . The sequences immediately 5` to the start site up to -156 contain multiple putative consensus binding sites for the ubiquitous transcription factors Sp1 and ATF/CREB. The sequences immediately surrounding the TEF-1 Inr (-40 to +30) have been previously described(47) . The complete sequence and chromosomal location of the TEF-1 5`-flanking region will be published elsewhere.

A Minimal Functional Promoter Requires 80 bp of 5`-Flanking DNA

In order to define minimal sequences necessary to support high level transcription, the TEF-1 5`-flanking sequence was deleted from -1874 to -30 (see ``Materials and Methods'' and Fig. 1). These deleted constructs were transfected into HeLa cells, which express the TEF-1 gene, and MPC-11 cells which do not. When transfected into HeLa cells, TEF-1 promoter constructs bearing progressive deletions of 5` DNA from -1874 to -137 had similar transcriptional activity, although the -365 and -455 deletions had an approximately 2-fold higher activity than other deletions (Fig. 3, A and E), indicating the possible presence of alternate positive and negative regulatory motifs. The -80 deletion still retained appreciable promoter activity (Fig. 3, A, lane 8, and E) when transcription levels were normalized against the beta-globin internal control but deletion to -30 almost completely abolished transcriptional activity (Fig. 3A, lane 9). In vitro analysis of these constructs using HeLa cell nuclear extract, revealed marked differences in activity (Fig. 3B, compare lanes 3 and 4) in comparison to that seen by in vivo analysis. These differences may be artifactual in nature because variation in relative levels of transcription was seen when the quantity of template DNA in the in vitro reaction was altered (results not shown). Also it should be noted that the mutant TEF-1 promoter deleted to -80 is almost inactive in vitro in comparison to its activity in vivo (compare Fig. 3E and Fig. 3, A, lane 8, with Fig. 3B, lane 8).


Figure 3: Deletion analysis of the TEF-1 promoter. In vivo analysis of TEF-1 promoter deletion mutants in (A) HeLa cells and (C) MPC-11 cells. 5 µg of reporter gene and 1 µg of internal control pG1B were transfected. beta-Globin mRNA was quantified by S1 mapping and normalized against RNA from the internal control. In vitro analysis of 100 ng of TEF-1 promoter deletion mutants by in vitro transcription in nuclear extracts from (B) HeLa cells and (D) MPC-11 cells. RNA levels were normalized against an internal control TATApAL7. E, relative in vivo transcription levels of TEF-1 deletion constructs in HeLa (black bars) and MPC-11 cells (shaded bars). Results were normalized against relative transcription levels of the internal control pG1B and represent mean ± S.E. from two to three separate experiments. The ratio of transcription between HeLa and MPC-11 cells is shown above pairs of bars for each deletion.



To analyze activity of TEF-1 promoter deletion mutants in cells which do not express the endogenous gene, the deletion constructs were transfected into MPC-11 cells (Fig. 3C) and analyzed in vitro using MPC-11 cell nuclear extract (Fig. 3D). After normalization against levels of beta-globin internal control transcripts, the level of activity of all constructs when transfected into MPC-11 cells ranged from between 3.3 and 18.4-fold less than corresponding levels in HeLa cells (Fig. 3, C and E), indicating that when introduced as a transgene, the promoter was able to direct cell specific expression, albeit at a lower level of selectivity than the endogenous gene. In contrast, all of these deletions up to -137 had high activity in vitro in MPC-11 cell nuclear extracts (Fig. 3D) and also in BJA-B cell extracts (results not shown).

Protein Binding Sites in the TEF-1 Promoter

The above results define a minimal promoter element extending to -80 which functions in a tissue-specific manner in vivo, -137 but not in vitro. To further investigate this we wished to identify trans-acting factors which bind to the TEF-1 proximal promoter. DNase I footprinting of the proximal region of the TEF-1 promoter revealed 4 major protected motifs (see Fig. 4and Fig. 7for summary) between -37 and -55, -58 and -73, -83 and -100, and a footprint at -109, the 5` end of which was undetermined because of its proximity to the labeled end of the probe possibly due to proteins which bind nonspecifically to the ends of DNA in such experiments. The protected sequences are summarized in Fig. 4B. DNase I-hypersensitive sites were found associated with footprint A at -36 and -33 and with footprint C at -82 and -81 (Fig. 4A, lane 2). Both footprints A and C with their associated hypersensitive sites were abolished when double-stranded oligonucleotide competitor A (Table 1) spanning footprint A was included in the footprint reaction (Fig. 4A, lanes 3-5). Oligo A, where A(49) was changed to G to create a canonical Sp1 site (23) was also an efficient competitor of protein binding to footprints A and C (Fig. 4A, lanes 8-10). In contrast, oligo A, where the putative Sp1 binding site was replaced with a BamHI restriction site (see Table 1), failed to compete out footprints A and C (Fig. 4A, lanes 13-15).


Figure 4: DNA protein interactions in the TEF-1 promoter region. A, DNA footprint analysis of the proximal TEF-1 promoter region. A probe spanning nucleotides -137 to +89 in the TEF-1 gene was end labeled at -137 then digested with DNase I. Lanes 1, 6, and 11, no protein; lanes 2,7, and 12, 30 µg of HeLa cell nuclear extract. Footprints were competed out with increasing quantities (10-, 50-, and 200-fold molar excess of probe in consecutive lanes) of oligos A (lanes 3-5), A (lanes 8-10), and A (lanes 13-15). B, sequences and positions of protected motifs in the TEF-1 promoter.




Figure 7: Sequence of the 5`-regulatory region of the TEF-1 promoter from -160 to +89. DNA-binding motifs are double underlined and the S1 nuclease-mapped major transcription start site, +1, is shown in bold underlined.



Footprints B and D were not abolished by competition with any of the oligonucleotides described above. Regions B and D contain exact consensus sites for members of the cAMP-inducible CREB/ATF factor families (CREs) (24) (Fig. 4B and 7). Box D also contains a good consensus Sp1 binding site close to the putative CRE (Figs. 4B and 7), but no competition with any of the A oligos was seen. This may be due to the fact that this motif was too close to the end of the probe or that proteins binding elsewhere within region D prevented protein binding to the putative Sp1 recognition site.

ATF1 and Sp1 Bind to the TEF-1 Promoter Region

To identify proteins binding within protected regions of the TEF-1 promoter we employed the gel retardation assay in combination with antibodies against known transcription factors to ``supershift'' or abolish retarded bands. Incubation of labeled oligo A with HeLa, BJA-B, or MPC-11 nuclear extracts gave rise to 2 major retarded bands of identical mobility (Fig. 5A, lanes 1-3). Identical retarded complexes were formed when oligo C, spanning footprint C (-77 to -106) was used as a probe (Fig. 5A, lanes 4-6). Footprint A spans a motif known to bind Sp1 and related factors(23, 25) . An anti-Sp1 antibody inhibited the formation of DNA-protein complexes on oligo A after incubation with HeLa cell nuclear extract (Fig. 5B, lanes 1 and 2). This was also seen with complexes formed on oligo C (Fig. 5B, lanes 3 and 4). Additionally recombinant Sp1 formed a complex of identical mobility to those formed by proteins in HeLa nuclear extract with both oligos A and C (Fig. 5B, lane 5, and results not shown). We therefore conclude that Sp1 is one of the proteins which binds to motifs within regions A and C in the TEF-1 promoter. Interestingly, the sequence within region C, although GC-rich does not contain a site previously shown to be a binding motif for Sp1. The identity of the faster migrating minor complex formed by nuclear proteins with oligos A and C (see Fig. 5A) has not been identified. It may be a fragment generated by proteolytic degradation of the nuclear extracts, which would also explain why the minor band was abolished by the anti-Sp1 antibody. Alternatively the lower band seen in Fig. 5A may represent the Sp1-related protein Sp2 (25) .


Figure 5: Sp1 and ATF-1 bind to the TEF-1 promoter. A, tissue specificity of proteins binding to oligos A (lanes 1-3) and C (lanes 1-3) incubated with HeLa (lanes 1 and 4), MPC-11 (lanes 2 and 5), and BJA-B (lanes 3 and 6) nuclear extracts. B, Sp1 binds to both oligo A and C: HeLa cell nuclear extract was incubated with oligo A (lanes 1, 2, and 5) or C (lanes 3 and 4) either in the presence of 1 µl of one-half dilution of preimmune serum (lanes 2 and 4) or anti-Sp1 serum 2892-E. 0.1 ng of recombinant Sp1 was incubated with oligo A (lane 6). C, tissue specificity of proteins binding to oligo B incubated with 5 µg of HeLa, MPC-11, and BJA-B nuclear extracts (lanes 1, 2, and 3, respectively). D, ATF-1 binds to oligos B and D. HeLa cell nuclear extract was incubated with oligo B (lanes 1-4) or oligo D (lanes 5-8) in the presence of 1 µl of a one-half dilution of preimmune serum (lanes 2 and 6), anti-ATF-1 (lanes 3 and 7), or anti-ATF-2 (lanes 4 and 8).



Gel retardation analysis of proteins binding within footprints B and D using oligonucleotides spanning these regions revealed binding of retarded complexes of identical mobility to oligos B (-56/-73) and D (-125/-127) (see Table 1). Both of these regions contain exact consensus sites for transcription factors belonging to the ATF/CREB family. Incubation of labeled oligo B with HeLa, BJA-B, or MPC-11 nuclear extracts gave rise to retarded bands of identical mobility (Fig. 5C, lanes 1-3). Identical retarded complexes were formed when oligo D was used as a probe (results not shown). Inclusion of anti-ATF1 resulted in supershifting of DNA protein complexes formed by both oligos B and D with HeLa nuclear extract (Fig. 5D, lanes 1, 2, 4, and 5), but not with anti-ATF2 (CRE-BP1) (Fig. 5D, lanes 1, 3, 4, and 6).

Effect of Mutations in the Proximal Promoter Region: Cross-talk between the Initiator and Proximal Upstream Element

To determine the role of the proximal upstream element in determining the activity of the TEF-1 promoter, a point mutation creating a BamHI site was introduced by substituting residues between -45 and -54 (see Table 1and Fig. 6A) to generate pTDelta-138. Both in vivo and in vitro, this mutation abolished transcription from the major site at +1 but augmented transcription 2- and 3.6-fold, respectively, from a minor site at -11 (Fig. 6, B, and C, lanes1 and 2) .


Figure 6: Mutational analysis of the TEF-1 promoter. A, TEF-1 proximal promoter mutants: 1, pTDelta-137 (wt); 2, pTDelta-137; 3, pTDelta-137; 4, pTDelta-137. B, in vivo analysis of TEF-1 proximal promoter mutants. 5 µg of reporter gene and 1 µg of internal control pG1B were transfected into HeLa cells. beta-Globin mRNA was quantified by S1 mapping and normalized against RNA from the internal control. C, in vitro analysis of TEF-1proximal promoter mutants. 100 ng of reporter gene and 50 ng of internal control TATApAL7 were incubated in the presence of HeLa nuclear extract. RNA was quantified as above.



The effect of introducing a TATA element into the proximal promoter of TEF-1 was examined. The TATA element and flanking sequences from the adenovirus major late promoter was inserted between the Inr and proximal upstream element to create pTDelta-138 (see Fig. 6A). This mutation also altered relative spacing between the Sp1 site, inserted TATA element, and Inr such that each was equidistant from the other by 30 bp and that the Sp1 site and Inr were 60 bp apart instead of 45 bp. In vitro, this mutation had the effect of dramatically increasing transcription from +1 and the downstream sites, but abolished transcription from the upstream sites (Fig. 6C, lane 3). In vivo transcription from +1 remained unchanged and transcription from all the minor sites was abolished (Fig. 6B, lane 3).

When the proximal Sp1 site was deleted from pTDelta-138, a shift in transcription start sites similar to that seen with the upstream mutation alone was observed. In vivo, the TATA insertion had only a marginal effect on the pattern and level of transcription when compared to that seen with the upstream element mutation alone (Fig. 6B, lane 4). In vitro, increased transcription associated with the TATA box insertion was seen, but transcription was initiated only at the -11 site (Fig. 6C, lane 4). Taken together these results suggest that the proximal Sp1 site is dominant over a TATA element in determining the transcription start site, even when a high affinity TATA sequence is used. Furthermore, these observations indicate that a TATA element may be redundant for maintaining high levels of transcription from a promoter which can function without one.


DISCUSSION

We have isolated 3 kilobase pairs of 5`-flanking DNA of the human TEF-1 gene and have delineated cis-acting sequences necessary for high level transcription of this gene. The minimal promoter consists of 80 bp of 5`-flanking DNA in vivo although 137 bp is required in vitro. This region contains multiple recognition motifs for the transcription factors Sp1 and ATF1. The upstream Sp1 site between -83 and -100 is atypical of any so far identified. The TEF-1 promoter resembles that of many housekeeping genes (28, 29, and references therein) in structure, but is expressed in a tissue-specific manner, and therefore belongs to the group of tissue-specific GC-rich and TATA-less promoters exemplified by the nerve growth factor receptor gene (35) and rat malic enzyme gene(36) .

TEF-1 mRNA is not present in BJA-B and MPC-11 cells(7) . We show here that the TEF-1 promoter functions less efficiently in MPC-11 cells despite the observation that it contains binding sites for factors present both in HeLa and lymphoid cells. Surprisingly, however, the TEF-1 promoter is equally active in lymphoid and HeLa cells in vitro. Several reasons may account for the apparent ability of the TEF-1 promoter to function in a tissue-specific manner in vivo, but not in vitro. First, the TEF-1 gene may be active in vivo in lymphoid cells but its mRNA may be selectively degraded. Xiao et al.(7) have noted the presence of multiple AUUUA motifs within the 3`-UTR which are potential signals for mRNA degradation(31) . Our constructs do not include the 3`-UTR and contain only 90 bp of 5`-UTR, and it is conceivable that this region contains motifs which selectively target TEF-1 mRNA for tissue-specific degradation. Further experiments need to be performed using TEF-1 promoter constructs with intact and fully truncated 5`-UTRs to verify this.

A more probable explanation for tissue specificity of this promoter is that it is subject to selective down-regulation by CpG methylation. The 5`-region of the TEF-1 gene contains numerous CpG dinucleotide motifs (Fig. 7). Although it has been shown that Sp1 can bind to de novo methylated DNA and activate transcription(37) , methylated DNA in vivo is complexed with specific methyl-CpG-binding proteins which would prevent access of Sp1 to the DNA ( (32) and references therein) and inhibit transcription. It has also been shown that ATF-1 cannot bind to its recognition site when it is methylated(33) . It is therefore likely that the TEF-1 promoter would be inactive when methylated.

The structure of the TEF-1 promoter and the nature of mechanisms controlling its tissue-specific function may be related to the function of TEF-1. Several studies have indicated that TEF-1 may be a zygotic transcription factor which is active at the earliest stages of development and is subsequently down-regulated during later stages(3, 13) . Activity of the TEF-1 promoter is highly dependent on the presence of Sp1 which is present at early developmental stages, including the 2 cell embryo (40) where there is evidence that TEF-1 is present(13) . Down-regulation of the TEF-1 promoter may occur later in specific developing lineages by a mechanism invoking a bimodal CpG methylation event where global CpG methylation occurs at gastrulation followed by selective demethylation of tissue-specific genes later during development ( (43) and references therein). The observation that the TEF-1 gene is almost ubiquitously active except in lymphoid tissue (16) may point to a mechanism favoring general activity but specific repression in certain tissues.

Transcription factor genes are commonly autoregulated (14, 30) but in this study we have not addressed whether the TEF-1 promoter is regulated in this manner. There are 3 sequence motifs flanking the Inr with partial identity to the GT-IIC enhanson in the SV40 enhancer(3, 6, 38) , but DNA-protein interactions in this region were undetected in this study. Nevertheless, preliminary work indicates that overexpression of TEF-1 in HeLa cells down-regulates the TEF-1 promoter. (^2)Elevated levels of TEF-1 may be able to bind GT-IIC sites near the Inr and interfere with transcription, alternatively the negative effect may result from transcriptional interference/squelching (7) between TEF-1 and/or the Sp1 and ATF-1 factors.

Mutation of the Inr inhibited transcription from the major start site and did not affect levels of transcription from the 3` start sites. However, transcription from the 5`-flanking site at -11 was augmented. These sites may function in an Inr-independent fashion. However, the sequences surrounding the minor initiation sites at -11 and +10 have partial identity to the consensus Inr (see below). These sequences may function as partial Inrs and become more important if the major Inr is mutated. This may explain our observation that the strength of flanking start sites increases if the Inr is mutated (see Fig. 2C).

When the proximal Sp1 site was mutated, transcription from the Inr-dependent site was markedly reduced both in vivo and in vitro, although transcription from the upstream start site at -11 increased dramatically. This site at -11 matches exactly the 3`-half of the TdT Inr but in the wild type promoter is very weak because T-16 is at a critical position normally occupied by C in a fully functional Inr(19) . These data suggest that Sp1 bound at the proximal GT box interacts specifically with factors bound at the Inr and plays a major role in fixing start site usage. In this case deletion of the proximal Sp1 site probably allows transcription to initiate from a weaker Inr which is able to interact with the transcription factors remaining bound upstream of the deleted Sp1 site. Numerous other studies have shown start site usage is dependent upon the integrity and position of the proximal Sp1 site where upstream Sp1 sites are juxtaposed to an Inr element(19, 29, 42, 44) . A mutant TEF-1 promoter containing a deletion of the proximal Sp1 site, and also into which a TATA element had been inserted, still initiated transcription from a start site(-11) upstream from the Inr, associated with deletion of the Sp1 site from the wild type promoter described above. We infer that a TATA element cannot direct correct initiation in place of the proximal Sp1 site in the TEF-1 promoter. The function of an Sp1 site and that of an introduced TATA box element are therefore distinct in their ability to direct initiation.

In a ``wild type'' TATA-less promoter, distance of the proximal Sp1 site from the Inr is critical to efficiency of initiation as well as positioning of start sites(42) . In this study, insertion of the adenovirus major late promoter TATA box and flanking elements spaced the Inr element and Sp1 site to 30 bp either side of the TATA box. The major start site in this promoter was identical to that in the wild type promoter. Our findings are at variance with an earlier study which proposed that positioning of transcription start sites is dominantly dependent upon positioning of a TATA element(44) . In these experiments an optimal spacing of 25 bp between a TATA element and Inr was required for high level transcription from the Inr. In the present study the TATA element was inserted 30 bp upstream from the Inr but did not influence the position of the transcription start site. This may be due to the influence of GC-rich regions, flanking the adenovirus major late promoter TATA element, which were also introduced with it, or other variations in experimental design between our work and that of O'Shea-Greenfield and Smale(44) .

It is noteworthy that introduction of a TATA element into a TATA-less promoter increased levels of transcription in vitro, but not in vivo in agreement with previous observations(49) . Inroduction of a TATA box in vivo also suppressed initiation from minor sites characteristic of a TATA-less promoter. The positions of the minor transcription start sites coincided with sequences with partial identity to the Inr which are probably capable of nucleating preinitiation complexes. Both TATA-less and TATA-containing promoters have a requirement for TFIID for efficient initiation of transcription (14, 48) and there is evidence that distinct subpopulations of TFIID exist which are specialized in function according to the nature of transactivation domains with which the TFIID-associated factors interact and the type of promoter(39, 47) . In the case of a TATA-less promoter, it is probable that a distinct species of TFIID interacts with the Inr, as opposed to that which interacts directly with a TATA element(47) . Both species may be recruited to a promoter containing a TATA element and Inr and interact to preferentially initiate transcription from a single site while repressing initiation from weaker sites. However, their ability to interact with transcriptional stimulators may be mutually exclusive, explaining why transcription levels from promoters containing either TATA element, Inr or both are similar in vivo.


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.

The full sequence for the TEF-1 gene 5`-region has been submitted to the EMBL nucleotide sequence data base with accession number X84839[GenBank].

§
Supported by a MRC French Exchange Fellowship and a grant from the Association pour la Récherche contre le Cancer. To whom correspondence should be addressed. Present address: School of Biological Sciences, 2.205 Stopford Building, Oxford Road, University of Manchester, Manchester M13 9PT, UK. Tel.: 61-275-5105; Fax: 61-275-5082; D.Boam{at}man.ac.uk.

(^1)
The abbreviations used are: TEF-1, transcriptional enhancer factor-1; bp, base pair(s); Inr, initiator element, UTR, untranslated region.

(^2)
D. S. W. Boam, I. Davidson, and P. Chambon, unpublished results.


ACKNOWLEDGEMENTS

Our thanks to Dr. Stephen Jackson for gifts of recombinant Sp1 and anti-Sp1 antiserum and to Dr. Michael Green for gifts of anti-ATF-1 and anti-ATF-2 antisera.


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