Proc. Natl. Acad. Sci. USAVol. 93, pp. 4336-4341, April 1996Biochemistry

Transcription of the lymphocyte-specific terminaldeoxynucleotidyltransferase gene requires a

specific core promoter structureISLA P. GARRAWAY, KATHLEEN SEMPLE, AND STEPHEN T. SMALE*Howard Hughes Medical Institute, Molecular Biology Institute and Department of Microbiology and Immunology, University of California, Los Angeles School ofMedicine, Los Angeles, CA 90095-1662

Communicated by Robert G. Roeder, The Rockefeller University, New York, NY, January 4, 1996 (received for review October 27, 1995)

ABSTRACT The terminal deoxynucleotidyltransferase(TdT) gene encodes a template-independent DNA polymerasethat is expressed exclusively in immature lymphocytes. TheTdT promoter lacks a TATA box, but an initiator element (Inr)overlaps the transcription start site. The Inr directs basaltranscription and also mediates activated transcription inconjunction with an upstream element called D'. We havebegun to address the fundamental question of why the TdTpromoter contains an Inr rather than a TATA box. First, wetested the possibility that the TdT promoter lacks a TATA boxbecause the -30 region is needed for the binding of anessential regulator. Mutations were introduced into the -30region, and the mutants were tested in transient transfectionand in vitro transcription assays. The mutations had onlyminor effects on promoter strength, suggesting that this firsthypothesis is incorrect. Next, the effect of inserting a TATAbox within the -30 region was tested. Although the TATA boxenhanced promoter strength, appropriate regulation ap-peared to be maintained, as transcription in lymphocytesremained dependent on the D' element. Finally, a promotervariant containing a TATA box at -30, but a mutant Inr, wastested. Surprisingly, transcription from this variant, both invitro and in vivo, was dramatically reduced. These resultssuggest that the TdT promoter, and possibly other naturalpromoters, contain an Inr element because one or moreactivator proteins that interact with surrounding controlelements preferentially function in its presence.

The development of B and T lymphocytes from a multipoten-tial stem cell is a complex process involving a network ofgenetic control loci that modulate gene expression in responseto extracellular signals and stochastic events (1). As lympho-cyte precursors mature, they express distinct sets of genes in acell-type and stage-specific manner. One of the earliest genesexpressed in both lymphoid lineages is the terminal de-oxynucleotidyltransferase (TdT) gene (2, 3). The protein en-coded by this gene contributes to diversity within the immu-noglobulin and T-cell receptor repertoires by adding randomnucleotides to coding junctions (4,5). Because of its expressionpattern, the TdT gene provides a paradigm for studying thecis-acting control elements and trans-acting factors that reg-ulate the earliest stages of lymphopoiesis.One control region that contributes to the regulated tran-

scription of the murine TdT gene is its promoter (6, 7), whichcontains at least two critical DNA sequence elements. The D'element, located 60 bp upstream of the transcription start site,is required for activated transcription in lymphocytes (6, 8).Multiple proteins bind to this element with high affinity,including several Ets family members (8) and a lymphocyte-restricted protein called Ikaros (LyF-1) (9, 10). It is not yet

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

4336

known whether an Ets protein, Ikaros, or a combination ofproteins is required for D' function.The second control element identified within the TdT

promoter is the initiator element (Inr) (7, 11). This elementoverlaps the start site and directs accurate, basal transcriptionin both lymphoid and nonlymphoid cells. In most eukaryoticpromoters, aTATA box located 30 bp upstream of the start siteis responsible for basal transcription and start-site localization(11-13). Because the TdT promoter lacks a TATA box, the Inrappears to carry out its functions. The sequence requirementsfor Inr activity in mammalian cells have been defined, reveal-ing a loose consensus sequence of PyPyA+iNT/APyPy (14,15). The start-site sequences of hundreds of promoters matchthis consensus (16), suggesting that functional Inr elements arequite common.The mechanisms of Inr-mediated transcription have been

explored by studying synthetic and natural promoters. Thesestudies have suggested that Inr-mediated transcription is sim-ilar to TATA-mediated transcription in several respects. Forexample, similar sets of general transcription factors arerequired for the function of both types of promoters (17-20).In fact, recognition of both TATA and Inr elements appearsto be carried out by the TFIID complex, which contains theTATA-binding protein (TBP) and several TBP-associatedfactors (TAFs) (15, 20-22). Following the association ofTFIID with either a TATA-containing or an Inr-containingcore promoter, it is reasonable to speculate that the remainingsteps leading to the formation of a preinitiation complex andtranscription initiation may be similar. This speculation issupported by comparisons of biochemical parameters of tran-scription initiation (20, 23, 24), although these comparisonssuggest that TATA- and Inr-mediated transcription may de-pend on distinct rate-limiting steps.

Because of the apparent similarities between TATA- andInr-mediated transcription, a complete understanding of TdTregulation depends on an understanding of why the TdTpromoter evolved to contain an Inr rather than a TATA box.This issue is important, not only for elucidating the mecha-nisms of TdT regulation, but also, more generally, for anunderstanding of why both TATA and Inr elements exist.There are four possible explanations for the presence of an Inrwithin the TdT promoter: (i) there may be no specific need forthe Inr, that is, a TATA box inserted at the -30 region mayfunction just as well; (ii) a TATA box may be detrimental forappropriate regulation by proteins bound to surroundingcontrol elements; (iii) an Inr may be required for appropriateregulation; and (iv) the -30 sequence may be used for anotherpurpose (e.g., for the binding of a critical activator or repres-

Abbreviations: TdT, terminal deoxynucleotidyltransferase; Inr, initi-ator element; TBP, TATA-binding protein; TAF, TBP-associatedfactor; HSV-TK, herpes simplex virus thymidine kinase.*To whom reprint requests should be addressed at: Howard HughesMedical Institute, 5-748 MRL, University of California, Los Angeles,675 Circle Drive South, Los Angeles, CA 90095-1662.

Dow

nloa

ded

by g

uest

on

Janu

ary

3, 2

021

Proc. Natl. Acad. Sci. USA 93 (1996) 4337

sor), preventing it from containing a TATA sequence. Thisfinal possibility is supported by significant sequence homologythat has been observed among the -30 sequences of severallymphocyte-specific, TATA-less genes (6).As a first step toward addressing this issue, we employed

transient transfection and in vitro transcription assays to ex-amine the roles of the Inr and the -30 region in TdTtranscription. Our results revealed no evidence of a bindingsite for an essential activator or repressor at the natural -30region of the TdT promoter. In addition, introduction of aTATA box at -30 enhanced promoter strength, but did notappear to influence the lymphocyte specificity of transcription.Surprisingly, however, transcription from a TdT promotervariant containing a TATA box at -30 instead of the Inrelement at the start site was dramatically reduced. Theseresults suggest that one or more essential activators within theTdT promoter preferentially stimulate transcription throughthe Inr.

MATERIALS AND METHODSPlasmid DNAs. Plasmids were constructed by first synthe-

sizing oligonucleotides containing the desired mutations foruse in PCR. The oligonucleotides containing the mutationsshown in the figures spanned the Kpn site at nt -41 in the TdTpromoter. The second primer used in all of the PCRs spanneda BamHI site at +58. Amplified products were cleaved withBamHI and Kpn I and inserted into the BamHI and Kpn I sitesofpIG (8), which contains TdT sequences from -1.7 kb to +58bp. For mutants pd'TI and pd'Ti, the PCR products wereinserted into plasmid 1060 (8), a variant of pIG containing amutated D' element (m7, ref. 8). The resulting plasmids werecleaved with BglII and BamHI, which excised the 1.7-kbmutant promoters. The promoter fragments then were in-serted into a BglII site upstream of the HSV-TK reporter genein the vector, pSVPyTK (7). Plasmids containing correctlyoriented inserts were sequenced to confirm the presence of thedesired mutations and the lack of other mutations. PlasmidsSpl-TI and Spl-I correspond to plasmids VI-a and VI-g asdescribed (25). Plasmid Spl-Ti was constructed by inserting an

A TDT PROMOTER

D) Activltior -30 Region

B RLm11T CellsIV

1,illm..-2

1 2 3 4 5

oligonucleotide containing an Inr mutant (-1G, +3G) intoplasmid III-a, using the cloning strategy described (25).

Transfections. RLm11 cells were grown to a density of 0.5-1x 106/ml in RPMI 1640 medium supplemented with 10%newborn calf serum. The cells were pelleted, washed once withphosphate-buffered saline (PBS), and resuspended in 1.2xRPMI 1640 medium with 24% fetal bovine serum at a densityof 2 x 107/250 ,l. Plasmid (60 ,tg) was added to 250 .ld of cells.Following a 10-min incubation on ice, cells were subjected toelectroporation at 260 V, 960 ,uF (Bio-Rad Gene Pulser).Following another 10-min incubation on ice, cells were washedin PBS and resuspended in 30 ml of growth medium. Cyto-plasmic RNA was isolated after 48 hr using an Nonidet P-40lysis method (25). 293 cells were grown in Dulbecco's modifiedEagle's medium supplemented with 10% HyClone bovine calfserum. Transfections were performed with 60 ,ug of plasmidDNA by a calcium phosphate method (25). RNA was isolated48 hr after transfection as described above. Cytoplasmic RNAs(30 ,ug) were analyzed via primer extension using 32P-labeledoligonucleotides complementary to HSV-TK sequences (7).

In Vitro Transcription Experiments. Nuclear extracts fromHeLa cells were prepared as described (7). Nuclear extractsfrom Jurkat T cells were prepared by the same method from10-20 liters of cells grown to a density of 1 X 106/ml. Nuclearproteins from both extracts were precipitated with ammoniumsulfate (0.35 mg/ml) and pelleted by ultracentrifugation. Thepellet was resuspended in one packed cell volume of HGED.1buffer (20 mM Hepes, pH 7.9/20% glycerol/0.1 M KCI/0.2mM EDTA/1 mM dithiothreitol) containing 12.5 mM MgCl2.Following dialysis, in vitro transcription and primer extensionreactions were performed as described (7).

RESULTS AND DISCUSSIONThe Conserved -30 Region of the TdT Core Promoter Does

Not Contribute to Promoter Activity in a Transient Transfec-tion Assay. The -30 regions of the murine and human TdTpromoters contain an identical 9-bp sequence (ref. 6; CTGCT-GGTG, between -28 and -20 in the murine promoter).Several other lymphocyte-specific, TATA-less genes contain a

PROMOTER MUTANTS

-28 -24o- \\WI1' ('T (;T (;C1 (; T (;

AInr'AI!nr

mil.2 (; C A ( (;(;G

ml.3 (' (; AC(;(;

m2.2 .X A T(l(' T(;

m2.3 ('T( ('T .\('

293 Embryonic Kidney

6 7 8 9 10

FIG. 1. Transient transfection analysis of TdT promoter mutants. (A) Schematic representation of the TdT promoter, indicating the D' activatorelement, the conserved -30 region, and the Inr. The sequences of the four mutants in the -30 region (-28 to -20) are shown. ml.2 and ml.3do not alter the A+T content, but m2.2 and m2.3 increase the AT content. (B) Results of a typical transient transfection experiment in whichplasmids containing wild-type or mutant promoters fused to the herpes simplex virus thymidine kinase (HSV-TK) reporter gene were transfectedinto RLmll T cells or 293 kidney cells. RNA was isolated 48 hr after transfection, and a 30-,ug sample was analyzed by primer extension usingan oligonucleotide complementary to an HSV-TK sequence. Arrows indicate the location of an 84-nt band corresponding to the expected size ofcDNAs resulting from correctly initiated transcripts. Similar results were obtained from three different experiments.

Biochemistry: Garraway et aL.

Dow

nloa

ded

by g

uest

on

Janu

ary

3, 2

021

4338 Biochemistry: Garraway et al.

B Jurkat

- N, V 'V* V,

1 2 3 4 5

HeLa

.Z .. "I 'V.Vl

6 7 8 9 10

FIG. 2. In vitro transcription analysis of TdT promoter mutants. In vitro reactions were carried out with 300 ng of template and 100 /ig of HeLaor Jurkat extracts. Transcripts were analyzed by primer extension with the HSV-TK primer. (A) Jurkat (lanes 1 and 2) and HeLa (lanes 3 and 4)extracts were used for analysis of plasmids containing the wild-type TdT promoter or a promoter (m7) containing a mutant D' element. (B) Invitro reactions were performed in Jurkat (lanes 1-5) and HeLa (lanes 6-10) extracts with the promoters described in Fig. 1.

related sequence at a similar location relative to their tran-scription start sites (6). This sequence does not bind with highaffinity to TBP (25) but may function as a binding site for an

essential activator or repressor. Therefore, the TdT promotermay contain an Inr rather than a TATA box because the -30region may be needed for this alternative purpose.To investigate a possible role for the conserved -30 se-

quence in directing transcriptional activation or repression,transient transfection experiments were performed with a

series of plasmids containing mutant TdT promoters withsequence alterations spanning nt -28 to -24 or nt -23 to -20(Fig. 1A). When designing the mutations, base pair substitu-tions were carefully monitored because previous experimentshave demonstrated that the strengths of TATA-less promotersare, in some cases, extremely sensitive to alteration of the -30sequence (25, 26). This sensitivity appears to result from an

interaction between TBP and the -30 sequence that may berequired for transcription initiation. Thus, a mutation thatalters the -30 sequence could change promoter strengthsimply by altering the TBP/DNA affinity, instead of disruptingthe binding of an activator or repressor. Unfortunately, al-though previous functional studies suggested that TBP iscapable of binding with low affinity to many sequences, we

have been unable to directly measure the affinities of TBP forthe suspected low-affinity interaction sites (25).The first two mutants, ml.2 and ml.3, contain base pair

changes that do not alter the A+T content of the -30 regionof the TdT promoter (Fig. 1A). Because the affinity ofTBP forDNA is thought to roughly correlate with A+T content (25),these mutations may prevent the binding of a postulatedregulatory protein without significantly altering TBP affinity.The next two mutants, m2.2 and m2.3 (Fig. 1A), contain basepair substitutions in which the cytosines and guanines are

changed to adenines or thymines. These mutants allow us tomeasure the sensitivity of promoter strength to increases inA+T content (and presumably to increases in the affinity ofTBP for the promoter). By comparing the relative promoterstrengths of the four mutants, we hoped to determine whetherthe -30 sequence is important for the binding of an activatoror repressor, or merely for TBP binding. If an activator or

repressor binds to the -30 region, promoter strength mightdecrease or increase in a manner that is largely independent ofthe A+T content of the mutant sequence. If, however, themutations only alter TBP affinity, then m2.2 and/or m2.3 mayexhibit increased promoter strengths, whereas ml.2 and ml.3might exhibit lesser effects.The TdT -30 mutants were inserted upstream of the

HSV-TK coding sequence in a vector that contains the poly-oma virus early region, which stimulates plasmid replication tohigh copy number in mouse cells. The resulting plasmids were

transiently transfected into the RLmll T-cell line, whichexpresses the endogenous TdT gene. After 48 hr, total cyto-plasmic RNA was isolated and analyzed for TdT promoteractivity via primer extension. The representative autoradio-graph in Fig. 1B shows that none of the mutations had a

reproducible effect on promoter strength (lanes 1-5). Similarresults were found after transfection into a nonlymphoid cellline, 293, which supports basal transcription from the TdTpromoter (lanes 6-10). (Although additional bands are visiblein lanes 6-10, the intensities of these bands vary from exper-iment to experiment and, therefore, are thought to be back-ground bands that arise during the primer extension experi-ments because of the exceedingly weak activity obtained withthe TdT promoter in 293 cells.) These results show that theconserved -30 sequence of the murine TdT promoter does notcontribute to the promoter activity detected in the transienttransfection assay. Additionally, with this assay, promoterstrength was not sensitive to the A+T content of the -30region.An in Vitro Transcription Assay Confirms the in Vivo

Results. To confirm and extend the above results, an in vitrotranscription assay was employed. This assay provides an

independent method for measuring TdT promoter activity.The in vitro assay also provides greater sensitivity than thetransient transfection assay, which yields extremely weakprimer extension signals because of the poor transfectionefficiencies obtained with TdT-positive cell lines. In vitroreactions were performed with nuclear extracts from eitherJurkat (Fig. 24) or RLmll (data not shown) lymphoid celllines, or the HeLa nonlymphoid cell line (Fig. 24). Transcriptsgenerated in the in vitro reactions were analyzed by primerextension.To first determine the relationship between the in vitro and

in vivo assays, the effect of a mutation in the upstream D'element of the TdT promoter was measured (Fig. 24, lanes1-4). Previously, it had been observed that, in the transienttransfection assay, D' mutations strongly reduced transcriptionin lymphoid cell lines but not in nonlymphoid cell lines (ref. 6).Analysis of the D' mutant promoter in Jurkat extracts revealeddiminished transcription (13-fold average) relative to thewild-type promoter (Fig. 2A, lanes 1 and 2). In contrast, a muchsmaller decrease (2-fold average) was found in HeLa extracts(Fig. 2A, lanes 3 and 4). Thus, Jurkat extracts appear to supportconsiderable activated transcription when the D' element isintact and weak basal transcription when the D' element ismutated. HeLa extracts, however, appear to support only basaltranscription, as the strengths of both the wild-type- andD'-mutant promoters were similar.Because the basal activity supported by the HeLa extracts

was considerably stronger than that supported by the Jurkat

HeLaA Jurkat

12. <

1 2 3 4

Proc. Natl. Acad. Sci. USA 93 (1996)

-N

.!;;z" lb-.ZP

1.14D

Dow

nloa

ded

by g

uest

on

Janu

ary

3, 2

021

Proc. Natl. Acad. Sci. USA 93 (1996) 4339

extracts (Fig. 2A4, compare lanes 2 and 4), template and extracttitrations were performed with the HeLa extracts to testwhether the strong basal activity might mask D' activation.With reduced concentrations of extract or template, the effectof the D' mutant was consistently less than 2-fold (data notshown), confirming the lymphocyte specificity of D' activity.Both the HeLa and Jurkat extracts were used to analyze the

strengths of the -30 mutant promoters described above.Unlike the transient transfection assay, the in vitro assayrevealed sensitivity to the sequence between -28 and -20,with the effect of each promoter mutant consistent betweenthe two extracts (Fig. 2B). The most striking variation inpromoter strength was observed with m2.2, which contains thesequence ATTAT from nt -28 to -24. This mutant exhibiteda 5-fold enhancement of promoter strength (relative to wildtype) in both extracts. In contrast, ml.2, ml.3, and m2.3exhibited moderate reductions in promoter strength.The variable promoter strengths observed in vitro most likely

reflect the variable affinities of TBP for the -30 sequences.This interpretation is supported by a previous study (25), whichshowed that variations in the affinity of TBP for the -30sequence of a synthetic promoter influenced promoter activitymore strongly in vitro than in vivo. In addition, this explanationis supported by the fact that the only mutation that enhancedpromoter strength, m2.2, introduces an A+T-rich sequenceinto the -30 region (-28 to -24). Enhanced promoterstrength was not seen with m2.3, which also contains anA+T-rich sequence, possibly because the A+T-rich sequence(-23 to -20) is positioned too close to the Inr for functionalcooperation.We favor a model in which TBP affinity dictates the relative

strengths of the mutants, but it certainly remains possible thatthe reduced promoter strengths exhibited by three of themutants result from the disruption of a binding site for a weak

A

pD'l (WT)~ TdT-30

pD'TI TATA

pD'Ti TTA

activator protein, whose activity cannot be detected with thetransient transfection assay. Regardless of the explanation,however, the in vitro and in vivo assays clearly demonstrate thatthe conserved sequence at the -30 region does not contain acontrol element that is essential for the promoter to directefficient transcription.A TATA Box Enhances the Strength of the TdT Promoter.

Because the above experiments failed to provide a clearexplanation for the presence of an Inr and lack of a TATA boxin the TdT promoter, the three alternative explanations pro-posed earlier must be considered. (i) A TATA box may bedetrimental to promoter function; (ii) an Inr may be essential;and (iii) the TATA and Inr may be interchangeable. Toinvestigate the first of these possibilities, we tested the activityof a TdT promoter variant containing a strong TATA box at-30, in addition to the Inr at the start site. The goal of thisexperiment was to determine if the TATA would influencetranscriptional regulation, as determined by the ability of theD' element to stimulate transcription in lymphoid cell lines andextracts.Mutants were constructed that contain a consensus TATA

box derived from the adenovirus major late promoter at the-30 region of the TdT promoter, downstream of either awild-type or mutant D' element (Fig. 3A, pD'TI and pd'TI,respectively). The transient and in vitro assays were then usedto determine the effects of the TATA box on promoterstrength and D' responsiveness. In both assays, the TATA boxenhanced promoter strength, but the promoter remainedresponsive to the D' element in lymphoid cells (i.e., the D'mutation reduced promoter strength). The specific degree ofenhancement by the TATA box varied among the assays. In thelymphoid cell lines (RLmll and Jurkat), the TATA boxincreased promoter strength (relative to the wild-type TdTpromoter) by 4-fold in the transient assay (Fig. 3B, lane 2) and

pd'l (WT) TdT-30

pd'TI

pd'Ti

B 4' Qp 9t6 94 9..1. 2 3

1 2 3 4 5 6

-l\s AS'.\ \ '\ s'< <\'Ql<? <? <P<6 9 '

r,<O'

1 2 3 4 5 6 7 8 9

.\' >A4\..\ z-<'\ \N > \91D9>D90<? 9 c,c.,^ ,z

10 11 12 13 14 15 16 17 18

FIG. 3. Transient transfection and in vitro transcription assays with TdT promoter variants containing a strong TATA box in addition to, orinstead of, the Inr. (A) Schematic diagram of the TdT promoter constructs. (B) Primer extension results (HSV-TK primer) are shown from atransient transfection experiment performed in RLmll T cells (lanes 1-6). Plasmids are indicated at the top and correspond to those diagrammed.(C) In vitro transcription reactions were performed in Jurkat (lanes 1-6) or HeLa extracts (lanes 10-15) with the plasmids described above. In vitroreactions were also performed in Jurkat (lanes 7-9) or HeLa (lanes 16-18) extracts with plasmids containing Spl binding sites upstream of awild-type TdT core promoter (-33 to +11; Spl-I), a core promoter containing a TATA box and the TdT Inr (SP1-TI), or a TATA box upstreamof the mutant Inr (-1G, -3G; SP1-Ti). An Sp6 primer (12) was employed for these experiments.

Biochemistry: Garraway et al.

-s. ;.. W.

.1 ..:'.. ..,4.,,a& -Mi.sus:- 10.00

99

Dow

nloa

ded

by g

uest

on

Janu

ary

3, 2

021

4340 Biochemistry: Garraway et al.

by 24-fold in the in vitro assay (Fig. 3C, lane 2). In nonlymphoidcell lines (293 and HeLa), the TATA box increased promoterstrength by 2-fold in the transient assay (not shown) and20-fold in the in vitro assay (Fig. 3C, lane 11). The effect of theD' mutation in lymphoid cells also varied among the assays,but with both assays, its effect appeared to be comparable bothin the presence and absence of a TATA box (Fig. 3B and 3C,compare lanes 1 and 2 with lanes 4 and 5). Importantly, inHeLa extracts, the D' mutant had little effect in either thepresence or the absence of the TATA box (Fig. 3C, comparelanes 10 and 11 with lanes 13 and 14).The above results demonstrate that the TATA box increases

the strength of the TdT promoter, but does not alter the abilityof the D' element to stimulate transcription. Although theseresults suggest that the TATA box is not detrimental topromoter function, increased basal and activated transcriptionof the endogenous TdT gene may indeed be detrimental to ananimal. Even low levels ofTdT enzymatic activity, for example,may be undesirable in nonlymphoid cells. Similarly, exceed-ingly high levels of enzymatic activity may be undesirable indeveloping lymphocytes. Therefore, one possible explanationfor the lack of both TATA and Inr elements within thepromoter is that an animal may require TdT expression at aprecise level in immature lymphocytes, with essentially noexpression in other cell types.TATA and Inr Elements Are Not Interchangeable in the TdT

Promoter. Although the above results may explain why theTdT promoter does not contain both TATA and Inr elements,we have not yet determined why the promoter contains an Inrinstead of a TATA box. The two remaining possibilities arethat (i) the Inr may be essential for promoter function and (ii)the TATA and Inr elements may be interchangeable, with nospecific preference. The Inr was previously shown to beessential for TdT promoter function (ref. 7; unpublishedresults), but it had not been determined whether promoteractivity could be supported by a TATA box in the absence ofthe Inr. Thus, to distinguish between these two remainingpossibilities, we examined whether a TATA box can supportTdT promoter activity in a promoter variant containing an Inrmutant. For this analysis, substitution mutations were intro-duced into the Inr elements of plasmids pD'TI and pd'TI,yielding plasmids pD'Ti and pd'Ti, respectively. Plasmid pD'Ticontains a wild-type D' element, a TATA box, and a mutantInr, and pd'Ti contains a mutant D' element, a TATA box, anda mutant Inr. The specific nucleotides mutated within the Inr,at -1 (cytidine to guanosine) and +3 (thymidine toguanosine), previously were shown to be critical for Inrfunction (ref. 14; K. Lo and S.T.S., unpublished data).

Surprisingly, with both the transient transfection and in vitrotranscription assays, transcription from the pD'Ti promoterwas undetectable or barely detectable (Fig. 3B, lane 3 and 3C,lanes 3 and 12). Interestingly, this promoter was extremelyweak, not only in lymphoid cells and extracts, but also in HeLaextracts, in which the D' element does not function.To provide a control for the above experiments, the natural

TdT core promoter (-33 to +11), the TATA/Inr core pro-moter, and the TATA/Inr mutant core promoter, were placeddownstream of multiple binding sites for the Spl transcriptionfactor. In HeLa and Jurkat extracts, the relative strengths ofthese promoters were very different from the strengths ob-served in the context of the TdT promoter; the Spl-TIpromoter was -2-fold stronger than the Spl-Ti promoter and-10-fold stronger than the Spl-I promoter (Fig. 3C, lanes 7-9and 16-18). These relative activities are consistent with theactivities of similar synthetic promoters reported previously(14, 17, 25). In addition, it is worth noting that the specific Inrmutant (-1G, +3G) used for these experiments has nodetrimental effect on the basal TATA-mediated transcriptiondetected with a more sensitive in vitro assay (J. Kaufmann and

S.T.S., unpublished work), confirming that the mutant se-quence does not interact with a specific repressor.One possible explanation for the Inr requirement within the

TdT promoter may be that the start-site region contains, inaddition to the basal Inr element, a binding site for a specificactivator protein that is needed for promoter function. In otherwords, the Inr mutant may disrupt a basal Inr element as wellas an essential activator element. To test this possibility, twodifferent functional Inr sequences identified in a randomscreen (14) were introduced into the TdT promoter in place ofthe TdT Inr. Both of these Inr elements supported TdTpromoter activity (data not shown), suggesting that any con-sensus Inr sequence would be adequate.Taken together, the results in Fig. 3 demonstrate that both

basal and activated transcription from the TdT promoterdepend on the presence of the Inr element, which cannot befunctionally replaced by a strong TATA box. The Inr prefer-ence is specific to the TdT promoter, as the preference was notmaintained when the same three core promoters were testedin the context of Spl binding sites. This observation stronglysuggests that the Inr preference is due to the specific nature ofthe activator proteins that interact with control elementssurrounding the core promoter.The control elements responsible for the Inr preference of

the TdT promoter remain to be established. Because thepreference was observed in HeLa extracts and with promoterscontaining a mutant D' element, the D' element is notprimarily responsible. It also is worth noting that a weak bandis detectable with the pD'Ti plasmid in Jurkat extracts and notwith the pd'Ti plasmid (Fig. 3C, lanes 3 and 6). This signal mayresult from a D'-binding protein acting through the TATA box,providing further support for the idea that the Inr preferenceis independent of the D' element. More likely to be responsiblefor the Inr preference is the downstream basal element locatedbetween +33 and +58, which we previously found to stimulatepromoter activity in extracts from nonlymphoid cell lines (7).Alternatively, other control elements that have yet to bediscovered may be responsible for the Inr preference.Concluding Remarks. We have demonstrated that the TdT

Inr is essential for activity of the natural TdT promoter. Thisfinding suggests that one or more activators that interact withcontrol elements surrounding the core promoter preferentiallyactivate transcription through the Inr. As discussed above, acontrol element that is active in both HeLa and lymphoid cellsmust be primarily responsible for the Inr preference. Theidentity of the critical activator proteins remain to be eluci-dated, as does the biochemical mechanism underlying the Inrpreference. Possibly, a general factor that is rate-limiting onlyduring Inr-mediated transcription is the target of the criticalactivator. An alternative possibility is that Inr-mediated tran-scription requires a novel factor that is not required forTATA-mediated transcription; this factor may be the target ofthe relevant activator. To distinguish between these possibil-ities, the Inr-dependent activators must first be defined andtheir mechanism of action dissected.Although an Inr preference within a natural TATA-less

promoter has not been described previously, recent studieshave revealed that the Spl activator contains a domain thatpreferentially activates transcription from core promoterscontaining either an Inr or an snRNA proximal sequenceelement, but not from core promoters containing only aTATAbox (27-29). Furthermore, preferences for specific TATAsequences have been reported within numerous TATA-containing promoters (11, 30). A well-characterized exampleis found in the yeast his3 gene, which contains two TATAboxes, TR and Tc, that direct transcription from distinct startsites (30). Interestingly, the TR element preferentially respondsto specific activators of the his3 gene. Insight into the mech-anism underlying this TATA preference has recently been

Proc. Natl. Acad. Sci. USA 93 (1996)

Dow

nloa

ded

by g

uest

on

Janu

ary

3, 2

021

Proc. Natl. Acad. Sci. USA 93 (1996) 4341

provided by a genetic screen that resulted in the identificationof several contributing proteins (31).The results presented in this study suggest that the sequence

of the -30 region within the TdT promoter is not importantfor promoter function. However, a closer examination of thisregion is needed. The -30 region of the TdT promoter is highlyconserved from mouse to man and has significant homologywith the -30 regions of promoters for other genes expressedspecifically in lymphocyte progenitors, including the AS5 andVpreB genes (6). Although this homologous sequence does notappear to be important in TdT-expressing cells, it may con-tribute to the inactivation of TdT transcription that occursduring lymphopoiesis. Indeed, given the possibility that se-quence-nonspecific TBP interactions may be required at the-30 region before transcription initiation (25, 26), this regionrepresents an ideal location for repressor binding.

This work was supported by Public Health Service Grant DK43726.I.P.G. was supported by Medical Scientist Training Program GrantGM08042 and by Public Health Service National Research ServiceAward HG00117. S.T.S. is an Assistant Investigator with the HowardHughes Medical Institute.

1. Ikuta, K., Uchida, N., Friedman, J. & Weissman, I. L. (1992)Annu. Rev. Immunol. 10, 759-783.

2. Bogue, M., Gilfillan, S., Benoist, C & Mathis, D. (1992) Proc.Natl. Acad. Sci. USA 89, 11011-11015.

3. Li, Y. S., Hayakawa, K. & Hardy, R. R. (1993) J. Exp. Med. 178,951-960.

4. Gilfillan, S., Dierich, A., Lemeur, M., Benoist, C. & Mathis, D.(1993) Science 261, 1175-1178.

5. Komori, T., Okada, A., Stewart, V. & Alt, F. W. (1993) Science261, 1171-1175.

6. Lo, K., Landau, N. R. & Smale, S. T. (1991) Mol. Cell. Biol. 10,5229-5243.

7. Smale, S. T. & Baltimore, D. (1989) Cell 57, 103-113.8. Ernst, P., Hahm, K. & Smale, S. T. (1993) Mol. Cell. Biol. 13,

2982-2992.

9. Georgopoulos, K., Moore, D. D. & Derfler, B. (1992) Science258, 808-812.

10. Hahm, K., Ernst, P., Lo, K., Kim, G. S., Turck, C. & Smale, S. T.(1994) Mol. Cell. Biol. 14, 7111-7123.

11. Smale, S. T. (1994) in Transcription: Mechanisms and Regulation,eds. Conaway, R. C. & Conaway, J. W. (Raven, New York), pp.63-81.

12. Grosschedl, R. & Birnstiel, M. L. (1980) Proc. Natl. Acad. Sci.USA 77, 1432-1436.

13. Breathnach, R. & Chambon, P. (1981) Annu. Rev. Biochem. 50,349-383.

14. Javahery, R., Khachi, A., Lo, K., Zenzie-Gregory, B. & Smale,S. T. (1994) Mol. Cell. Biol. 14, 116-127.

15. Kaufmann, J. & Smale, S. T. (1994) Genes Dev. 8, 821-829.16. Bucher, P. (1990) J. Mol. Biol. 212, 563-578.17. Smale, S. T., Schmidt, M. C., Berk, A. J. & Baltimore, D. (1990)

Proc. Natl. Acad. Sci. USA 87, 4509-4513.18. Pugh, B. F. & Tjian, R. (1990) Cell 61, 1187-1197.19. Aso, T., Conaway, J. W. & Conaway, R. C. (1994) J. Biol. Chem.

269, 26575-26583.20. Martinez, E., Chiang-Ming, C., Hui, G. & Roeder, R. G. (1994)

EMBO J. 13, 3115-3126.21. Purnell, B. A., Emanuel, P. A. & Gilmour, D. S. (1994) Genes

Dev. 8, 830-842.22. Hernandez, N. (1993) Genes Dev. 7, 1291-1308.23. Zenzie-Gregory, B., O'Shea-Greenfield, A. & Smale, S. T. (1992)

J. Biol. Chem. 267, 2823-2830.24. Colgan, J. & Manley, J. L. (1992) Genes Dev. 5, 304-315.25. Wiley, S. R., Kraus, R. J. & Mertz, J. E. (1992) Proc. Natl. Acad.

Sci. USA 89, 5814-5818.26. Zenzie-Gregory, B., Khachi, A., Garraway, I. P. & Smale, S. T.

(1993) Mol. Cell. Biol. 13, 3871-3879.27. Das, G., Hinkley, C. S. & Herr, W. (1995) Nature (London) 374,

657-660.28. Emami, K. H., Navarre, W. W. & Smale, S. T. (1995) Mol. Cell.

Biol. 15, 5906-5916.29. Colgan, J. & Manley, J. L. (1995) Proc. Natl. Acad. Sci. USA 92,

1955-1959.30. Struhl, K. (1989) Annu. Rev. Biochem. 58, 1051-1077.31. Collart, M. A. & Struhl, K. (1994) Genes Dev. 8, 525-537.

Biochemistry: Garraway et aL.

Dow

nloa

ded

by g

uest

on

Janu

ary

3, 2

021