©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Functional Initiator Element in the Human -Globin Promoter (*)

(Received for publication, September 15, 1995)

Brian A. Lewis (§) Stuart H. Orkin (¶)

From the Division of Hematology/Oncology, The Children's Hospital Medical Center, the Dana Farber Cancer Institute, the Department of Pediatrics, Harvard Medical School, and the Howard Hughes Medical Institute, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Core promoters are defined by the presence of either a TATA box at approximately 30 base pairs upstream of the transcriptional start site (+1) and/or an initiator element centered around the +1 site. The prevalence, function, and significance of the various combinations of core promoter elements are as yet unclear. We describe here the identification and characterization of an initiator element in the TATA-containing human betaglobin promoter. Mutagenesis of the beta-globin initiator element at positions +2/+3 and +4/+5 abrogates transcription in a heterologous construct. Interestingly, we have found a beta-globin initiator binding activity in nuclear extracts whose presence or absence correlates with function of the beta-globin initiator. Accordingly, this binding activity may be part of the machinery required for beta-globin initiator-dependent transcription. Our analysis further describes a previously uncharacterized beta-thalassemia mutation at the +1 site as a mutation that decreases beta-globin initiator activity. Finally, consistent with other initiator elements, the beta-globin initiator requires a TFIID-containing fraction for in vitro activity. Thus, the human beta-globin promoter contains an initiator element whose function, as revealed by a betathalassemia mutation, is of physiological relevance.


INTRODUCTION

Study of RNA polymerase II transcription necessitates characterization of both the cis and trans elements involved in promoter function. Viral and minimal promoters, as well as those promoters directing highly regulated tissue-specific expression, contain a variety of upstream elements and exhibit heterogeneity in their core promoter elements. In this context, it is important to examine the relative roles of both the upstream elements and the core promoter, and their mutual interactions, in order to understand the regulation of complex tissue-specific promoters.

Accurate transcriptional initiation has been classically thought to require a TATA box. However, the finding of numerous promoters that do not contain a TATA box and yet accurately initiate transcription led to the discovery of elements centered around the start site as components of the core promoter(1, 2) . These initiator (Inr) (^1)elements direct accurate transcription from artificial constructs containing only upstream Sp1 sites(1, 2) . Mutation of Inr elements in several promoters decreased or abolished transcription (see (3) for review), and in heterologous constructs Inr elements stimulated transcription in the presence of a TATA box(4, 5, 6) . Experiments performed with promoters containing both a TATA box and Inr suggest that the TATA box is the predominant selector of the site of initiation and that the Inr contributes to the magnitude of the initiation(2) .

Several models have been put forward to describe how specific proteins initiate transcription through Inr elements. One suggests that factors binding to the Inr, and necessary for its function, are present in the TFIID complex(4, 5, 7, 8, 9, 10, 11) . A second model suggests that Inr-dependent transcription is mediated by initiator-binding proteins such as YY1 and TFII-I(6, 12, 13) , as both can substitute for members of the basal machinery in reconstituted systems in vitro (TBP and TFIIA, respectively)(14, 15) . In a third model, recognition of the Inr by RNA polymerase II serves as the nucleation event, analogous to the role of TBP in TATA-containing promoters(3, 16) . Finally, an alternate model suggests that TBP provides a nucleation function through its ability to recognize the -30 regions of TATA-less promoters(17) .

These additional complexities have prompted us to reevaluate the role of a core promoter in the expression of the human beta-globin gene, a paradigm for developmentally regulated genes. Early studies defined several sequences contributing to the activity of the beta-globin promoter. Internal deletion/substitution and point mutation analysis assigned the TATA box, a CCAAT box at approximately -75, and a CACC box at approximately -90 as the major determinants of transcriptional regulation(18, 19) . Mutations around the +1 site decreased transcription by approximately 50%(18, 19) . In a more recent study a C T mutation at -1 was shown to reduce beta-globin promoter expression to about 80% of wild-type activity in MEL cells(20) .

Human beta-thalassemia disease is a disorder characterized by reduced or absent beta-globin expression. The resulting globin chain imbalance due to unimpeded alpha-globin expression leads to precipitation of globin polypeptide chains in developing erythroid cells, and the ensuing anemia. Study of these naturally occurring beta-thalassemia mutations has proven useful in revealing the in vivo relevance of specific cis elements in the beta-globin promoter(21) . Wong et al.(22) reported a patient with mild asymptomatic beta-thalassemia whose DNA was homozygous for an A C transversion at +1 of the beta-globin promoter. In this report we describe the characterization of the +1 region of the human beta-globin promoter as a functional initiator element and demonstrate that the +1 beta-thalassemia mutation is a mutation in the beta-globin Inr element (betaInr). Furthermore, we show that in vitro transcription from the betaInr is dependent on partially purified TFIID and that a betaInr DNA binding activity exists whose binding correlates with betaInr functional activity.


MATERIALS AND METHODS

Cloning

pSp1 and pSp1/TdT plasmids were kindly provided by Steve Smale. Sp1/beta+1, Sp1/beta+1R, and the Sp1/Inr mutants (see Fig. 1and Fig. 2) were constructed by insertion of a double-stranded oligonucleotide into the SmaI site of pSp1. The oligonucleotide sequences for each construct are listed below. Sp1/beta+1 and Sp1/beta+1R, TATTGCTTACATTTGCTTCTG; Sp1/-1,-2, TATTGCAAACATTTGCTTCTG; Sp1/2,3, TATTGCTTAGGTTTGCTTCTG; Sp1/4,5, TATTGCTTACAGGTGCTTCTG; Sp1/7,8, TATTGCTTACATTTAGTTCTG; Sp1/9,10, TATTGCTTACATTTGCCCCTG. These same oligonucleotides were used for gel shift assays. The MLP template was kindly provided by Chris Parks and Tom Shenk. The beta-globin promoter constructs (betaGH and derivatives) contained the beta-globin promoter from -815 to +18, cloned into the XbaI site of pOGH (Nichols Institute).The betaGH derivatives were constructed by PCR amplification/mutagenesis. All Sp1 and betaGH constructs were sequenced to confirm their identity and orientation.


Figure 1: The human beta-globin promoter contains a functional initiator element. A, in vitro transcription with templates containing Sp1 sites either by themselves (Sp1; lane 1), or upstream of the TdT Inr (Sp1/TdT; lane 2), the beta-globin +1 region (from -8 to +13) (Sp1/beta+1; lanes 3 and 5), or the beta-globin +1 region in the reverse orientation (Sp1/beta+1R; lane 4). Lane 6 is a transcription reaction treated with 2 µg/ml alpha-amanitin (+alpha-aman). Heat-inactivated nuclear extract (HINE, 47 °C for 15 min) was used in the transcription reaction in lane 7(26) . Lanes 8-10 are identical to lanes 5-7 except that the adenovirus MLP was used as template. Arrows indicate the primer extension product representing the correctly initiated transcript. B, the primer extension product resulting from an in vitro transcription reaction using the Sp1/beta+1 template was electrophoresed next to the sequence of the Sp1/beta+1 template. Both primer extension and sequencing reactions used the same primer.




Figure 2: Mutational analysis of the beta-globin initiator element. In vitro transcriptions of the wild-type beta+1 region (lane 1) and double point mutations in the beta+1 region (lanes 2-6). The numbers above the lanes indicate the position of the mutations relative to the +1 transcriptional start site. The mutations in the Inr element are listed below the lanes. All constructs contained Sp1 sites upstream(1) .



TFIID Purification

MEL cells (7 l; approximately 10 cells) were harvested, and a nuclear extract was prepared as described by Dignam et al.(23, 24) and modified by Briggs et al.(25) . The pellet from the ammonium sulfate precipitation was suspended in H.1 (20 mM Hepes-KOH, pH 7.9, 1 mM EDTA, 1 mM DTT, 20% glycerol, 100 mM KCl). The extract was run over a P-11 column (Whatman), essentially as described by Roeder and colleagues(23, 24, 26) . 0.1, 0.3, 0.5, and 0.85 M fractions were assayed for TFIID activity by testing for rescue of transcription from heat-inactivated MEL crude nuclear extracts using an adenovirus major late promoter template(26) .

In Vitro Transcriptions

MEL crude nuclear extracts and in vitro transcriptions for all templates were prepared as described(1, 5) . 0.3 µg of the Sp1 templates (1) and 0.1 µg of the betaGH and MLP templates were using in 50 µl reactions. After incubation at 30 °C for 1 h, the transcription reactions were stopped by the addition of 225 µl of stop buffer (TES: 350 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1% SDS, 1 mM EDTA), 1 µg of glycogen, and 10 µg of proteinase K. The reactions were incubated at 37 °C for 30 min, extracted with phenol-chloroform, and ethanol-precipitated. The pellet was suspended in 10 µl of H(2)O, 50 µl of DNase buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl(2), 1 mM CaCl(2)), 5 mM DTT, 2 µl of RQ DNase (Promega), and 0.2 µl of RNasin (Promega), incubated at 37 °C for 15 min; reactions were terminated by the addition of 150 µl of TES, extracted with phenol-chloroform, and ethanol-precipitated. To the ethanol precipitation, approximately 50,000 cpm of the appropriate P-labeled single-stranded primer was added for the subsequent primer extension. Ethanol pellets were suspended in 8 µl of H(2)O and 2 µl of Buffer A (0.25 M Tris-HCl, pH 8.7, 2.7 M KCl, 5 mM EDTA), and primer annealing was done at 42 °C for 1 h. Primer extensions were done at 42 °C for 1 h after addition of 30 µl of extension buffer (2.4 µl of 10 mM dNTP, 6 µl of Buffer B, 1.5 µl of 0.1 M DTT, 1 µl of avian myeloblastosis virus-reverse transcriptase (Promega), 0.3 µl of RNasin (Promega), 18 µl of H(2)O) to the annealing reaction. Buffer B is 0.25 M Tris-HCl, pH 8.7, 0.065 M MgCl(2). Reactions were terminated by ethanol precipitation. Pellets were suspended in formamide loading buffer and run on 8% polyacrylamide/8 M urea, dried, and exposed at -70 °C with Kodak XAR-5 film. Quantitation was done by PhosporImager analysis (Molecular Dynamics). Heat-inactivation of MEL crude nuclear extracts was done as described(26) .

Gel Shift Assays

Gel shift binding reaction conditions were identical to the in vitro transcription conditions (10 mM Hepes-OH, pH 7.9, 50 mM KCl, 0.5 mM DTT, 10% glycerol, 6.25 mM MgCl(2)) plus the addition of 1 µg of poly(dI-dC) (Pharmacia Biotech Inc.), 50,000 cpm of the appropriate P-labeled probe, and 1 µl of crude MEL nuclear extract (5-10 mg/ml). Reactions were incubated at room temperature for 20 min and run on a 4% polyacrylamide (30:1) gel in 0.25 times TBE. Gels were dried and autoradiographed.


RESULTS

The Human beta-Globin +1 Region Contains an Inr Element

We employed an in vitro transcription assay to determine whether the +1 region of the beta-globin promoter can correctly initiate transcription from an artificial construct. In vitro transcriptions using MEL nuclear extracts and the parent template construct containing only Sp1 sites showed only minor, low level initiation (Fig. 1A, lane 1) (1) . Sp1/TdT, containing the TdT Inr downstream of the Sp1 sites, initiated high levels of transcription (Fig. 1A, lane 2)(1) . A similar construct, Sp1/beta+1, containing the beta-globin +1 region resulted in transcription from two regions (lane 3). Mapping of the initiation sites indicated that the site indicated by the arrow in Fig. 1B correctly maps to the previously observed +1 site for the betaglobin promoter(27) . The second site (bracket in Fig. 1B) maps to the junction of the vector and insert. Consistent with previous in vitro transcriptions using the TdT Inr(1) , an Sp1 construct containing the beta+1 region in the reverse orientation (Sp1/beta+1R) did not initiate transcription (lane 4). Finally, transcription from the Sp1/beta+1 is polymerase II-dependent, as shown by sensitivity to 2 µg/ml alpha-amanitin (Fig. 1A, lane 6); moreover, transcription is TFIID-dependent, as revealed by heat inactivation of a MEL nuclear extract (Fig. 1A, lane 7)(26) . As a control, we showed that transcription of a MLP template is sensitive to both alpha-amanitin and a 47 °C heat treatment (Fig. 1A, lanes 8-10). These data show that the +1 region of the beta-globin promoter is able to function as an initiator element in vitro.

Point Mutations in the beta-Globin Inr Element Abolish Transcription in Vitro

To further delineate the sequences necessary for the function of the beta-globin Inr element (betaInr), we introduced double point mutations in the beta-globin +1 region and assayed these mutants in the Sp1 construct using in vitro transcriptions. With the wild-type betaInr, transcription initiated predominantly at the A at +1; a minor transcript initiated at the C at +2 (Fig. 2, lane 1; Fig. 1B). The -1,-2 double mutant showed reduced transcription from the major initiation site, and additional downstream initiation sites (Fig. 2, lane 2). Conversion of the CA at 2/3 and the TT at 4/5 to GG abolished transcription from the betaInr element (Fig. 2, lanes 3 and 4). Mutation of positions 7/8 did not affect initiation from the major site (Fig. 2, lane 5), although one downstream initiation site was observed, similar to that seen with the -1,-2 double mutant. The 9/10 mutant displayed correct initiation (Fig. 2, lane 6). These data indicate that mutations in the +1 region specifically abolish betaInr-dependent transcription and define the boundaries of the betaInr element as approximately nucleotides -2 to +5 (TTACATT).

Protein Binding to the betaInr Element Correlates with betaInr Functional Activity

Our finding that the human beta-globin promoter contains a functional Inr element, and the apparent complexities of Inr-dependent transcription (see Introduction), led us to assay MEL cell nuclear extracts for a specific betaInr binding activity. We performed gel shift assays using P-labeled double-stranded oligonucleotides containing the wild-type betaInr sequence or double point mutants. As shown in Fig. 3(indicated by the arrow), a gel shift complex formed with the wild-type betaInr and the 7,8 and 9,10 double mutant probes. Binding was not detected with the 2,3 or 4,5 mutant probes, which were inactive as initiator sequences in vitro. Interestingly, the -1,-2 mutant template, which revealed reduced initiation from the +1 site, showed an intermediate level of complex formation. Thus, protein binding and transcription initiation were strictly correlated in this series of mutants. Competitions using the various cold wild-type or mutant binding sites against a labeled wild-type betaInr probe yielded similar results (data not shown). The betaInr binding activity does not appear to be restricted to MEL cells, as a comigrating activity was detected in HeLa nuclear extracts, nor does it comigrate with the YY1 initiator protein in gel shift assays (data not shown).


Figure 3: Nuclear extract contains a betaInr binding activity whose presence correlates with betaInr functional activity. P-Labeled double-stranded wild-type (lane 6) and mutant (lanes 1-5) oligomers were used in binding assays and run on a 4% polyacrylamide gel. The arrow indicates an activity whose presence correlates with the functional analysis of the betaInr mutations (Fig. 2). The mutant designations are identical to those in Fig. 2.



A Naturally Occurring beta-Thalassemia Is a Mutation in the beta-Globin Initiator Element

Wong et al.(22) described an Asian-Indian with a mild asymptomatic beta-thalassemia in which the only base substitution detected was an A C transversion at the +1 site of the beta-globin promoter. We surmised that this observation might suggest the presence of an initiator element in the +1 region. Although the +1 region appeared to function as an Inr element in an artificial promoter, we next sought to examine this region in the context of the beta-globin promoter. Constructs were assembled in which mutations were introduced into the beta-globin promoter (Fig. 4A).


Figure 4: A naturally occurring beta-thalassemia mutation is a mutation in the beta-globin Inr element. (A) Representative in vitro transcriptions of templates containing the beta-globin promoter from -815 to +18 upstream of a growth hormone (GH) reporter. Template designations are indicated above each lane and depicted below. Mutations in each template are underlined. B, graphical representation of the data in A. Experiments for each template were performed at least three times. Error bars indicate standard deviations from the mean transcription level relative to betaGH (all betaGH templates) or betaMLPGH (all templates with the MLP designation). Note that the error bars all show 10-15% deviation, which represents the intrinsic error in the assay.



Representative in vitro transcriptions from these constructs are shown in Fig. 4A. Fig. 4B provides quantitation of the results of three to four experiments. The incorporation of the A C transversion at the +1 site into the betaGH (wild-type) template resulted in transcriptional activity 75% of wild-type (Fig. 4A, lane 2). We also observed a slight shift in the pattern of initiation from three predominant sites to two major and two minor sites (compare lanes 1 and 2 in Fig. 4). Introduction of two known beta-thalassemia mutations into the beta-globin TATA box (-30betaGH: CATA to CACA; -31betaGH: CATA to CGTA) (28, 29) resulted in transcription levels 40% of wild-type (Fig. 4, A and B), consistent with transient expression data of TATA box beta-thalassemia mutations and mutagenesis studies(19, 20, 30, 31) . The TATA box mutations, however, did not alter the pattern of initiation (Fig. 4A, lanes 2 and 3). The double mutant (-30betaTHALGH), which contained both the -30 TATA box T C transition and the +1 A C transversion, further reduced transcription to 20% of wild-type activity.

Replacement of the beta-globin TATA box sequence (CATA) with the adenovirus MLP TATA box (TATA) provided a second promoter background into which we incorporated the +1 A C transversion and the 2,3 mutant. In the context of a stronger TATA box (compare lanes 1 and 8 in Fig. 4, A and B) (32) the A C +1 mutation reduced expression to 40% of the parent betaMLPGH template, compared to 75% in the wild-type background (compare betaMLPGH and betaMLPTHALGH to betaGH and betaTHALGH in Fig. 4, A and B). Here again the initiation pattern was altered (compare lanes 10, 11, and 12). Interestingly, the 2,3 mutant, which abolished transcription in the Sp1 assay (Fig. 2, lane 3), reduced transcription in the betaMLP2,3GH template to approximately 20% of betaMLPGH levels (Fig. 4, panel A, lane 12 and panel B). This similar reduction in initiation by the 2,3 and the +1 beta-thalassemia mutations in the betaMLPGH construct suggests that both mutations affect Inr-dependent transcription and that results using the Sp1-based templates can be reproduced in the context of a natural promoter.

Transcription from the betaInr Is Dependent on a Fraction Containing TFIID Activity

To begin to address the possible mechanisms of transcription from the betaInr element, we asked whether transcription of the Sp1/beta+1 template could be rescued by addition of a fraction containing TFIID to heat-inactivated nuclear extracts. This fraction was isolated from a MEL cell nuclear extract by passage over a phosphocellulose P-11 column and elution with 0.85 M KCl (see ``Materials and Methods''). Addition of the pooled peak 0.85 M fraction to MEL nuclear extracts that were heat-inactivated by incubation at 47 °C for 15 min restored transcriptional activity to both a control MLP template and the Sp1/beta+1 template (Fig. 5)(26) . Thus, consistent with previous data on other initiator elements the betaInr element requires an activity that copurifies with TFIID(4, 5, 10, 11, 33) .


Figure 5: A TFIID-containing phosphocellulose fraction rescues betaInr-dependent transcription in heat-inactivated nuclear extracts. In vitro transcriptions employed the adenovirus MLP (lanes 1-3) or the Sp1/beta+1 template (lanes 4-6). Lanes 1 and 4 are transcriptions using a MEL nuclear extract. Lanes 2 and 5 are transcriptions using heat-inactivated nuclear extracts (47 °C, 15 min). Lanes 3 and 6 are in vitro transcriptions using heat-inactivated nuclear extract and 2 µl of a 0.85 M P-11 fraction.




DISCUSSION

In this report we describe the identification and characterization of an initiator element in the TATA-containing human beta-globin promoter. In so doing, we provide evidence that a protein fraction containing TFIID is required for Inr-dependent transcription and detected a DNA binding activity whose binding to the betaInr correlates with its functional activity. Finally, we demonstrate that a base substitution at the beta-globin +1 site, found in association with a human beta-thalassemia, impairs the activity of the initiator element, thereby implicating the beta-globin Inr as a functional element in vivo.

Consistent with studies of other initiator elements(1, 2, 6) , the beta-globin Inr functions in a heterologous context and in an orientation-dependent manner (Fig. 1). Comparison of transcription from the Sp1/TdT Inr and Sp1/betaInr templates indicates that within these contexts the betaInr is weaker than the TdT Inr, a finding consistent with observations suggesting that deviations from the loose Inr consensus sequence element (YYANT/AYY) decrease Inr activity(7) . Accordingly, mutation of the beta-globin Inr with double point mutations replacing nucleotides -2 through +8 with purines reveals that positions -1,-2 (YY), 2,3 (NT/A), and 4,5 are necessary for betaInr activity (Fig. 2).

Gel shift analysis of MEL cell nuclear extracts with the betaInr sequence reveals a protein binding activity (Fig. 3) that strictly correlates with transcriptional activity in vitro (Fig. 2). Previous reports proposed TFII-I and YY1 transcription factors as candidates for mediating initiator activity(6, 12, 14, 15) . However, others have reported that the functional activities of YY1 mutant binding sites do not correlate precisely with YY1 binding activities over the same mutant sites(7) . This discrepancy is complicated by the assays used to define the activities of YY1. The mutational analysis performed by Javahery et al. (7) employed Sp1 templates containing a YY1 site and used crude nuclear extracts for in vitro transcriptions, whereas reconstituted in vitro systems were used to define YY1 as a functional initiator protein(15) . It is formally possible that these two functional assays do not assay similar activities. Further experiments are required to ascertain whether results obtained with systems using reconstituted factors are in accord with those using crude nuclear extracts. A second caveat is the possibility that the context of the Inr may influence the functional assays(7) . Nonetheless, our analysis of a panel of betaInr mutants provides an example of a correlation between Inr functional activity and Inr DNA binding activity.

A report by Wong et al.(22) described an Asian-Indian with a mild, asymptomatic beta-thalassemia. Their analysis of the patient's beta-globin promoter indicated that he was homozygous for an A C transversion at +1. Our analysis (Fig. 4) indicated that this transversion is a mutation in the initiator element, as levels were reduced to 75% of wild-type levels (Fig. 4, panel A, lane 2 and panel B). Previously described TATA box beta-thalassemia mutants express at approximately 25% of the wild-type levels in transient assays in HeLa cells(31) . Consistent with these results, templates containing a -30 beta-thalassemia mutation (T C) (-30betaGH) and a -31 beta-thalassemia mutation (A G) (-31betaGH) were expressed in vitro at 30% of wild-type levels (Fig. 4, A and B). These results indicate that the in vitro system data can accurately reflect the in vivo environment. Incorporation of the +1 A C beta-thalassemia into the -30betaGH template resulted in a further reduction in transcription, again indicating that the +1 mutation affects the function of the initiator element. Finally, the conversion of the beta-globin TATA box (CATA) to the adenovirus major late promoter TATA box (TATA) supplied a second template (betaMLPGH) with which to study the effects of the +1 beta-thalassemia mutation. Curiously, the incorporation of the +1 mutation into the betaMLPGH background resulted in transcription levels that were 35% of wild-type (Fig. 4B, compare betaMLPGH and betaMLPTHALGH). This effect is 2-fold greater than that seen with the wild-type betaglobin promoter background (Fig. 4B, compare betaGH with betaTHALGH). This difference may be due simply to the higher expression from the betaMLPGH template or may indicate a cooperativity between the TATA box and Inr element(34) . We observe that, despite the lack of transcription from the Sp1/2,3 double mutant (Fig. 2) the same mutation in the betaMLP2,3GH template results in transcription 20% of wild-type (Fig. 4). From these data we conclude that other elements are able to compensate for the mutation within the betaTHALGH, betaMLPTHALGH, and betaMLP2,3GH templates. Although other mechanisms are possible, these data are compatible with the requirement for TFIID, whose footprint on the adenovirus major late promoter extends from approximately -45 to +35(26) . If so, the mutations in the betaInr and TATA box may result in destabilization, or partial disruption, of the interaction of TFIID with the core promoter, and thereby lead to a decrease in transcription. Mutation of the Inr results in a complete loss of transcription (Fig. 2), as the template only contains one site for TFIID, whereas the partial loss of transcription in the beta-globin templates through either TATA box or Inr mutations (Fig. 4) is due to the destabilization/weakening of TFIID binding in the core promoter. Our results, therefore, are not mutually exclusive but may reflect the ability of TFIID to bind to both the TATA box and the Inr.

Consistent with this interpretation, our data reveal that a phosphocellulose fraction containing TFIID is required for betaInr activity (Fig. 5). These observations are consistent with other data and models proposing that the machinery for Inr-dependent transcription is contained in the TFIID complex(5, 8, 9, 10, 35) . Validation of this hypothesis will require further experiments to demonstrate a correlation between TFIID binding and the functional activity of the betaInr mutants(8) . If this is the case, the Inr binding activity we detect may reside within the TFIID complex. Alternatively, the existence of an independent DNA binding activity might suggest that Inr-dependent transcription requires distinct mechanisms which differ from those used by other promoters (see Introduction). Finally, the rescue of Inr-dependent transcription from heat-inactivated nuclear extracts by the addition of partially purified TFIID is incomplete, despite the complete rescue of adenovirus major late promoter transcription (Fig. 5). These data suggest the existence of an additional heat-sensitive factor that is required for optimal betaInr activity. These findings resemble those described for the TdT Inr(4, 10, 33) .

Several groups have produced data that suggest there are distinct mechanisms of initiation mediated by Inr elements. For example, Zenzie-Gregory et al.(36) have reported that Inr-dependent transcription in vitro does not show the lag period found using TATA-dependent templates. Moreover, increasing amounts of nuclear extract reduced activity of a TATA-dependent template, but not that of an Inr-dependent template. In addition, transient assays suggest that overexpression of TBP inhibits expression from TATA-containing, but not TATA-less, promoters(37) . With an upstream activator site, the combination of a TATA box and an Inr significantly increased promoter activity, suggesting that the Inr element may cooperate with a TATA box and also significantly enhance a promoter's response to activators(34) . Additional evidence of a lack of a TBP rate-limiting step in Inr-containing promoters is provided by Ham et al.(38) , who showed that overexpression of TBP can relieve a block on expression of minimal promoters containing a papillomavirus E2 activator binding site and a TATA box. However, the same promoter containing a TATA box and Inr is activated by E2 without the requirement for TBP overexpression. Consistent with this model are data suggesting that the interaction of c-Fos and TBP is required for TATA box-mediated, but not for Inr-dependent, transcription (39) . Finally, Lescure et al.(40) provide data that part of the N terminus of TBP is required for the assembly of preinitiation complexes in TATA-containing, but not TATA-less, promoters. Collectively, these data argue for distinct mechanisms of transcriptional initiation mediated by Inr elements and suggest that Inrs contribute to the response of promoters to upstream activators. The study of Inr-dependent promoters in TATA-containing and TATA-less contexts will thus further our understanding of the mechanisms of transcriptional initiation.


FOOTNOTES

(^1)
The abbreviations used are: Inr, initiator element; betaInr, beta-globin initiator element; TFIID, -A, and -I, transcription factors IID, -A, and -I, respectively; MEL, murine erythroleukemia; TdT, terminal deoxytransferase; MLP, major late promoter; TBP, TATA-binding protein; DTT, dithiothreitol; betaGH, beta-globin promoter upstream of a growth hormone reporter.

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

§
To whom correspondence should be addressed: Division of Hematology/Oncology, Enders, Rm. 750, The Children's Hospital, 300 Longwood Ave., Boston, MA 02115. Tel.: 617-355-8404; Fax: 617-355-7262; lewis@rascal.med.harvard.edu.

§
Fellow of the Howard Hughes Medical Institute.

Investigator of the Howard Hughes Medical Institute.


ACKNOWLEDGEMENTS

We thank the members of the Orkin laboratory for helpful discussions and Steve Smale, Chris Parks, and Tom Shenk for helpful advice and plasmids.


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