Immunology 1998 93 213–220

Distal Vb

promoters transcribe novel T-cell receptor-b transcripts in earlydevelopment

K. A. SUTTON, M. N. VU & M. F. WILKINSONDepartment of Immunology, University of Texas M. D. Anderson Cancer Centre, Houston, TX, USA

SUMMARY

The transcriptional activation of germline T-cell receptor (TCR) and immunoglobulin (Ig) geneshas been proposed to promote the rearrangement of these genes. Here we report the identificationof distal TCR promoters (PDs), located upstream of the previously characterized promoters inthe mouse V

b5.1 and Vb8.1 gene segments, that are active in germline TCR genes in fetal thymus

and liver in vivo. We also identified an immature T-cell clone, SL12.4, that expresses bothendogenous and transfected PDs in a regulated manner in vitro. We propose that the transcriptionof these distal promoters in germline TCR genes may be important for inducing TCR generearrangements during T-cell development. Northern blot, RNase protection and reverse transcrip-tion–polymerase chain reaction (RT–PCR) analyses demonstrated that PDs are also transcribedfrom fully rearranged TCR genes in adult thymus, lymph node, and spleen. Although thefunctional significance of this expression is not known, our sequence analysis of the 5∞ leader inPD-derived V

b5.1 and Vb8.1 transcripts revealed the presence of several open reading frames

(ORFs) that may encode novel polypeptides or regulate the efficiency of TCRb translation.

INTRODUCTION tissues, such as fetal thymus and bone marrow, but not innon-lymphoid tissues.5

The differentiation of B and T lymphocytes depends upon the Although there have been several reports describing Vbsuccessful rearrangement and expression of T-cell receptor germline transcripts, to our knowledge no studies have identified

(TCR) and immunoglobulin (Ig) genes containing complete the promoters used to transcribe these germline transcripts. Theopen reading frames (ORFs). Expression of such rearranged only known V

bpromoters are located in the leader exon

TCR genes is driven by the activation of both promoter and upstream of each Vb

element. The transcription initiation sitesenhancer elements.1 Germline transcripts have also been for these promoters are located typically less than 60 nucleotidesdetected from unrearranged copies of TCR and immunoglob- from the initiator ATG that defines the TCRb reading frame.6ulin genes.2–5 It has been proposed that activation of the These promoters drive transcription of fully rearranged genespromoters that transcribe these ‘germline mRNAs’, favour the in both immature thymocytes and mature T lymphocytes.1 Inopening of chromatin structures at TCR and immunoglobulin the case of non-rearranged germline genes, it is not clear howloci, thus increasing their accessibility to recombination such enhancer-dependent V

bpromoters could be activated by

enzymes.2 Germline promoters that activate gene rearrange- the TCRb enhancer, as this positive regulatory element is 3∞ ofment are predicted to exhibit transcriptional activity early in C

b2, far downstream of most Vb

elements prior to genelymphocyte ontogeny and display cell-lineage specificity. rearrangement. Because germline V

btranscripts are still

expressed when the TCRb enhancer is inactivated by targetedConsistent with the first prediction, germline transcripts havedeletion,7 it is possible that other promoters are important forbeen detected by reverse transcription–polymerase chain reac-driving the expression of germline transcripts.tion (RT–PCR) at the earliest stages of T-lymphocyte develop-

Here we report the identification and characterization ofment, before the onset of TCR gene rearrangement.5 The cell-distal promoters (PDs) upstream of the previously describedlineage specificity of germline V

bexpression is evident from

promoters. Because these PDs are enhancer independent andthe observation that transcripts are detected in lymphoidare active early in thymocyte ontogeny, they may be respon-sible for the transcription of germline V

bgenes prior to the

Received 19 August 1997; revised 13 October 1997; accepted 13activation of the TCRb enhancer.October 1997.

Abbreviations: ORF, open reading frame; p.c., post coitum; PD,MATERIALS AND METHODSdistal promoter; PP, proximal promoter; RPA, RNase protection

analysis; RT, reverse transcriptase. RNA preparation and analysisRNA was prepared from tissues of BALB/C and SJL/J miceCorrespondence: Dr M. F. Wilkinson, Department of Immu-

nology, Box 180, 1515 Holcombe Blvd., Houston, TX 77 030 USA. obtained from Jackson Laboratories (Bar Harbor, ME).

© 1998 Blackwell Science Ltd 213

K. A. Sutton et al.214

Fetuses were harvested at 14·5, 16·5 and 17·5 days of gestation. the wells of a 24-well plate and G418 (800 mg/ml ) was addedto the cultures the following day. The cells were selected forAdult mice were killed at 8 weeks of age. Splenocytes from

adult mice were incubated with 10 mg/ml concanavalin A G418 resistance for 2–3 weeks, and Northern blot analysiswas performed to determine which cell lines expressed the(Con-A) in RPMI-1640 media with 10% fetal calf serum

(FCS) for 3 days.8 Total cellular RNA was isolated from transfected DNA.tissues and cell lines either by guanidinium isothiocyanate lysis

DNA sequencingand centrifugation over a CsCl cushion9 or by a guanidin-Vb5.1 and V

b8.1 genomic DNA was sequenced on both strandsium thiocyanate/phenol/chloroform extraction procedure.10with Sequenase according to the manufacturer’s instructionsNorthern blot hybridization and RNase protection analysis(USB, Cleveland, OH). The sequence was assembled and(RPA) were performed as described previously.11,12 Probes foranalysed with the GCG Wisconsin package (GCG, Madison,Northern blot analysis and RPA were generated from mouseWI ).V

b5.1 and Vb8.1 cDNAs, C

b1 genomic DNA (provided by E.Palmer, Basel Institute for Immunology, Basel, Switzerland),and V

b5.1 and Vb8.1 genomic clones (provided by D. Loh, RESULTS

Washington University, St. Louis, MO). The Cb1-specific (3∞

Distal Vb

promoters active in fetal and adult lymphoid tissuesuntranslated region), V

b8.1-specific (coding region), and Vb5.1

PD-specific (genomic region upstream of the PP) probes are Northern blot analysis of TCRb mRNAs expressed in fetalthymus revealed the presence of 1·7 kb transcripts, in addition0·4 kb EcoRI/HindIII, 0·29 kb EcoRI/PvuII, and 0·4 kb

SacI/ScaI fragments, respectively. For RPA, the following to the previously described 1·0 kb and 1·3 kb mRNAs tran-scribed from partially and fully rearranged TCRb genes,probes were used: a 0·2 kb SacI/AccI fragment from the V

b5.1PD, and a 0·7 kb NdeI/StyI fragment from the V

b8.1 PD. respectively.13–19 The 1·7 kb transcripts hybridized with bothCb1- and C

b2-specific probes (Fig. 1a and data not shown).They were expressed at least as early as day 14·5 post coitumRT-PCR analyses

To remove residual genomic DNA contaminants from the (p.c.) and remained at nearly constant levels throughoutthymic ontogeny. In contrast, the 1·0 kb and 1·3 kb transcriptsRNA preparations, all RNA samples were treated with 1 U

of DNase I for 30 min at 37° in 10 m Tris-HCl (pH 8·3), rose sharply in levels late in fetal development (Fig. 1a), inagreement with our previous observations and those of1·5 m MgCl2, and 50 m KCl. cDNA was prepared from

2 mg RNA by using an oligo-dT primer and Superscript II others.8,17–19 To determine whether the 1·7 kb transcripts werederived from rearranged V

bCb

genes, we assessed whether theyreverse transcriptase according to the manufacturer’s instruc-tions (GIBCO BRL, Grand Island, NY ). All PCR reactions hybridized with a V

b8.1 probe known to hybridize with 1·3 kbtranscripts from fully rearranged V

b8 genes but not 1·0 kbwere performed with 1 U Amplitaq polymerase (Perkin Elmer,Foster City, CA), 10 m Tris-HCl (pH 8·3), 1·5 m MgCl2, transcripts from partially rearranged genes that have only

undergone D-J rearrangement. The Vb8.1 probe hybridized50 m KCl, 0·2 m deoxynucleotides, and 10 pmol of each

primer. The cycling conditions for the Vb5.1 primers were with the 1·7 kb transcripts (Fig. 1b), suggesting that at least

some of these mRNAs were transcribed from fully30 seconds at 95°, 30 seconds at 60° and 30 seconds at 72°for 30–40 cycles. The primers used were 5∞-TGAAGA- rearranged genes.

Our subsequent analysis showed that the 1·7 kb mRNAsGGACTTTCCCACAC-3∞ (primer A, forward primer withinthe PD transcript 5∞ untranslated region), 5∞-TACA- were derived from distal promoters (PDs) upstream of those

that we now define as proximal promoters (PPs) that transcribeTCACCAGCTGGAACAGA-3∞ (primer B, reverse primer 30nt. downstream of V

b5.1); and 5∞-GAGAGCTCAACA- the well-characterized 1·3-kb TCR mRNAs. This was shownfirst using a probe from a region upstream of the previouslyAGGAGACCTT-3∞ (primer C, reverse primer in exon 1 of

Cb1). The PCR products were separated on 1% agarose gels described V

b5.1 PP promoter. This upstream probe hybridizedwith the 1·7 kb transcripts in both fetal thymus and ConA-and blotted onto Nytran membranes (Schleicher & Schuell,

Keene, NH). Southern blot analysis was performed with a stimulated splenocytes (Fig. 1c).We used RNase protection analysis (RPA) as an indepen-0·20 kb EcoRI/RsaI fragment corresponding to the V

b5.1coding region. dent assay of the existence of PDs and to map their transcrip-

tion initiation sites. Analysis with a Vb5.1 genomic probe that

spans the previously characterized PP and sequences 0·6 kbCell culture and transfectionThe cell clone SL12.4 and its culturing requirements have been upstream (a 0·7 kb BglII/EcoRI fragment) revealed two sets

of bands; one corresponding to the PP and the other to a PDdescribed.11 The cells were incubated with 100 mg/ml cyclohexi-mide or 1 mg/ml pactamycin for 6 hr, unless otherwise noted. (data not shown). The size of the bands corresponded to

transcription start sites of #−40 and ≥−400 for the PP andThe Vb8 enhancer− and V

b8 enhancer+ constructs transfectedinto SL12.4 cells contained V

b8.2Cb1 sequences [provided by PD, respectively, relative to the initiator ATG that defines theknown TCR reading frame. To more precisely assess theJ. Kaye (The Scripps Research Institute, La Jolla, CA)] and

Vb8.1Cb2 [provided by M. Blackman, P. Marrack and transcriptional initiation sites for the PD, we used a smaller

probe for RPA and found multiple bands that correspond toJ. Kappler (Howard Hughes Medical Institute, Denver, CO)],respectively. Stable transfection was performed by electropor- several transcriptional initiation sites in the PD (Fig. 2a). The

lengths of the major protected bands in thymus and spleenating 20–40 mg of DNA in 1×Hepes-buffered saline (140 m

NaCl, 1.5 m Na2HPO4, and 50 m Hepes, pH 7·005) with a correspond to PD transcription start sites between approxi-mately -473 and -403 (Fig. 2a). Inspection of the sequenceMoonlight Cat Door electroporator (Seattle, WA) at 2900–

3200 V. After transfection, the cell suspension was split among upstream of these initiation sites revealed no TATA box (see

© 1998 Blackwell Science Ltd, Immunology, 93, 213–220

Distal Vb

promoters transcribe novel T-cell receptor-b transcripts in early development 215

(a) (b) (c)

d 14

·5 th

ymus

d 15

·5 th

ymus

d 17

·5 th

ymus

Adu

lt th

ymus

d 14

·5 th

ymus

Adu

lt th

ymus

d 16

·5 th

ymus

Con

A -

spl

enoc

ytes

1·7kb1·3kb1·0kb

1·7kb

CHO-Aprobe

Cb1 probe

Vb8.1 probe Vb5.1 Pd probe

Figure 1. Developmental expression of mRNAs transcribed from proximal and distal Vb

promoters. Northern blot analysis oftotal cellular RNA from thymic tissue (the day of ontogeny for fetal thymus is shown). (a) The 1·0-kb mRNAs are transcriptsfrom D

band J

bpromoters; the 1·3-kb mRNAs are PP V

b-derived transcripts; and the 1·7-kb mRNAs are PD V

b-derived transcripts.

Hybridization with the CHO-A probe29 shows the amount of RNA loaded. All lanes contained 10 mg of RNA except for the adultthymus lane, which contained only 2 mg to permit evaluation of the transcripts at the exposure time shown. (b) The V

b8-specificprobe hybridized with both 1·3 kb and 1·7 kb transcripts because it corresponds to the V

b8.1 coding region. (c) The Vb5.1 PD-

specific probe (probe B from Fig. 5) hybridized with only 1·7 kb transcripts. All blots were stained with methylene blue to showintegrity of RNA and amount of RNA loaded (as described in ref. 30; data not shown).

(a) (b) (c)

tRN

AS

L12

.4/–

CH

X

SJL

/J th

ymus

tRN

AB

alb/

c th

ymus

tRN

A

1·7kb1·3kb

Vb8.1Vb5.1

Bal

b/c

sple

nocy

tes

SL

12.4

/+C

HX

Ladd

er

Bal

b/c

sple

nocy

tes

Vb8.

1 tr

ansf

ecte

d(e

nhan

cer+

)

Bal

b/c

thym

us

Vb8 transfected

Enhancer+ –

200

100

Figure 2. Expression and 5∞ mapping of Vb5.1 and V

b8.1 mRNAs transcribed from distal promoters. Total cellular RNA (10 mg)from the cell lines and tissues shown was analysed by RPA (a–b) and Northern blot analysis (c). (a) RPA using a V

b5.1-specificprobe. −CHX, without cycloheximide; +CHX, with cycloheximide. (b) RPA using a V

b8.1-specific probe. Shown is the expressionof the enhancer+ V

b8 construct transfected stably into SL12.4 cells. Non-transfected SL12.4 cells did not express the Vb8 sequences

(data not shown). The integrity and amount of RNA used for RPA in panels (a)-(b) was assessed by Northern blot analysisfollowed by methylene blue staining of 18S and 28S rRNA (performed as described in ref. 30; data not shown). (c) Northern blotanalysis of SL12.4 T cells stably transfected with enhancer+ and enhancer− V

b8 constructs (see the Materials and Methods fordetails). The V

b8.1 coding region probe hybridized with both PP- (1·3 kb) and PD- (1·7 kb) derived transcripts.

© 1998 Blackwell Science Ltd, Immunology, 93, 213–220

K. A. Sutton et al.216

later). Promoters that lack TATA boxes typically transcribemRNAs from multiple start sites.20 As a control to show thespecificity of the V

b5.1 PD probe, we used the same probe toanalyse expression from the SL12·4 T-lymphoma cell clone,which upregulates the levels of V

b5.1Cb1 transcripts after incu-bation with the protein synthesis inhibitor cycloheximide.21SL12.4 cells used the same multiple transcription start sites aslymphoid tissues, and that expression of transcripts from themultiple initiation sites was increased by cycloheximide. Thespecificity of the V

b5.1 PD probe was further assessed using amouse strain that lacks the V

b5.1 gene.22 As expected, theprobe failed to detected transcripts in SJL/J mice (Fig. 2a).

Because we had also detected 1·7 kb Vb8 transcripts by

Northern blot analysis, we assessed whether these were alsotranscribed from a PD. We performed RPA using a probecorresponding to the region upstream of the known PP regionin V

b8.1. This analysis revealed that BALB/c thymus andspleen used a PD that contain a transcription initiation site at#−513 nt (Fig. 2b). No signal was seen from SJL/J micethymus (data not shown), as expected since this strain of micelacks the V

b8.1 gene segment.22Because 1·7 kb transcripts are expressed early in fetal

ontogeny, before transcripts derived from enhancer-dependentpromoters, we considered the possibility that PDs may differfrom PPs in their requirement for the known enhancer element3∞ of C

b2. To test this, enhancer+ and enhancer− constructswere stably transfected into SL12.4 T-lymphoma cells andtheir expression was assessed. Figure 2 shows representativedata from two cell lines. The enhancer+ construct expressedhigher levels of 1·3 kb transcripts (PP-driven) than 1·7 kb(PD-driven) transcripts, as expected (Fig. 2c). RPA confirmedthat the enhancer+ construct was expressing PD-derived tran-scripts (Fig. 2b). The enhancer+ construct contained #9 kb

(a)

(b)

(c)

Spl

LN Ad

Thy

Test

is

+ – + – + – + –RT

d16.

5 T

hy

Ad

Thy

d16.

5 T

hy

Ad

Thy

Germline Rearranged

+RT –RTd1

6.5

Thy

Ad

Thy

LN d17.

5 F

L

Test

is

Germline

(d)

Rearranged

+RT –RT

Cb Exon 1

CBVb5.1

PdPp

A

d16.

5 T

hy

Ad

Thy

LN d17.

5 F

L

Test

is

Spl

LN Ad

Thy

Test

is

Figure 3. Distal promoter-driven Vb

mRNAs transcribed from germ-upstream of the Vb8.1 element and #8 kb downstream of the

line and rearranged genes. RT–PCR was performed to amplifyCb

element, demonstrating that such sequences are sufficientPD-derived V

b5.1 transcripts. After gel electrophoresis, the PCR prod-to direct both PD and PP transcription. The enhancer−ucts were blotted and hybridized with a V

b5.1-specific probe. (a) Theconstruct expressed similar levels of 1·7 kb and 1·3 kb tran-relative positions of the primers used for the analysis. The position ofscripts (Fig. 2c), demonstrating that neither the PD nor theprimer A is shown in Fig. 4. Primer C is equally complementary with

PP was absolutely dependent on the TCRb enhancer for Cb1 and C

b2. (b) Primers A and C were used to selectively amplifyexpression. transcripts from fully rearranged TCRb genes. (c) Amplification of

transcripts from both germline (primers A and B) and rearrangedgenes from day 16·5 p.c. fetal and adult thymus. (d) Primers A andV

b5.1distal promoter active in germline genes

B were used to selectively amplify transcripts from germline TCRb

genes. LN, lymph node; Thy, Thymus (fetal and adult); FL, fetalTo further examine the expression of transcripts from PDs weliver; Spl, spleen. All cDNAs generated in the presence of RTemployed RT–PCR analysis. To detect only those transcripts(including those from testis) were shown to be capable of beingderived from a PD we designed a sense primer (primer A)efficiently amplified by PCR, based on amplification with primersthat corresponds to the unique 5∞ untranslated region of thespecific for mouse b-actin (data not shown).V

b5.1 PD-derived transcripts (Fig. 3a). This primer was usedin combination with primer C (specific for exon 1 of both C

b1and C

b2) to amplify mRNA transcribed from TCRb genes in the testis (Fig. 3c and d). The failure to observe germlinetranscripts derived from PD in the testis suggests this promoterwhich had rearranged V

b5.1. This analysis showed that PDtranscripts from fully rearranged genes were present in spleen possesses lymphoid specificity. The observation that germline

transcripts derived from the PD can be detected in the fetal(Spl ), lymph node (LN ), fetal thymus (d16.5 Thy), and adultthymus (Ad Thy) (Figs 3b and 3c). As expected, such tran- thymus and fetal liver provides evidence that this promoter is

activated in the earliest T-lymphocyte progenitors. The pres-scripts were not detected in fetal liver or testis (Fig. 3b anddata not shown). Since the Northern blot data suggested that ence of germline transcripts in secondary lymphoid tissues is

in agreement with a study that described the expression ofthe PD may be transcribed early in lymphocyte development,prior to the activation of the PP, we examined the use of the germline V

btranscripts by mature T cells.3 Earlier studies on

germline transcripts have assumed that such transcripts areVb5.1 PD in germline genes using primers A and B (Fig. 3a).

We found that germline transcripts were expressed in both derived from the PP. Our observations suggest that germlinetranscripts are also derived from the PD.adult and fetal thymus, lymph node, and fetal liver, but not

© 1998 Blackwell Science Ltd, Immunology, 93, 213–220

Distal Vb

promoters transcribe novel T-cell receptor-b transcripts in early development 217

Upstream ORFs DISCUSSION

The generation of germline transcripts from TCR and immuno-To assess the potential consequences of the extended 5∞ leaderglobulin variable region gene segments has been proposed tosequences in PD-transcribed mRNAs we determined the nucle-be a consequence of critical events necessary to initiate TCRotide sequences of V

b5.1 and Vb8.1 genomic DNA that corre-

and immunoglobulin gene rearrangements. The act of tran-sponds to these regions (Fig. 4). The approximate sites of thescription may generate a more open chromatin structure toPD and PP are shown in Fig. 4, along with the conservedpermit access by the recombination machinery. It has generallydecamer sequence known to be important for PP-derivedbeen assumed that these germline transcripts arise from classi-transcription.23,24 Also shown are significant ORFs defined bycal enhancer-dependent TCR and immunoglobulin V pro-ATG initiator codons that closely match the Kozak consensusmoters. However, germline TCRb transcripts can still besequence; i.e. contain the conserved −3 purine and/or +4 G.25detected at the earliest stages of thymocyte ontogeny when theAccording to this analysis, two ORFs (encoding 36 and 39TCRb enhancer may be too distant in germline-configuredamino acids) are present in the 5∞ leader of PD-derived V

b8.1 genes to activate most known Vb

promoters. In this study wetranscripts. In contrast, Vb5.1 PD-derived transcripts contain

identified novel TCRb transcripts derived from a second classonly a single short ORF (encoding 7 amino acids) in the 5∞of promoters, termed PDs, located upstream of the previouslyterminus. The significance of these upstream ORFs is notdescribed V

bpromoters. PDs are able to drive the transcriptionknown; they may generate biologically active polypeptides,

of both germline and rearranged Vb

genes in a TCRb enhancer-down-regulate translation from the downstream TCRb readingindependent manner. Germline transcripts derived from PDsframe, or both. Interestingly, V

b5.1 PD-derived transcriptsare found in fetal liver, prior to the activation of the TCRbuniquely possess an upstream ATG in-frame with the knownenhancer and the onset of TCRb gene rearrangement events.V

b5.1 reading frame (Fig. 4). If translation initiates at thisOur sequence analysis revealed that at least four classes ofATG it would produce a TCRb protein with 23 additional

proteins could be translated from PD-derived transcripts: (i)amino acids at the amino terminus. This altered amino ter-full-length TCRb proteins; (ii) amino terminal-extended TCRbminus would thus have an unusual signal peptide that couldproteins translated from in-frame start codons in theinfluence the intracellular trafficking of this TCRb chain.PD-specific 5∞ leader; (iii) novel polypeptides encoded byWe hypothesized that the upstream ORFs in the 5∞ leadersORFs in the PD-specific 5∞ leader, and (iv) truncated V

bof PD-derived transcripts might trigger the rapid degradationproteins translated from germline transcripts. With regard toof these transcripts as a result of nonsense-mediated down-this last category, a germline transcript has been identifiedregulation. Several investigators have shown that prematurethat encodes a V

b8.2 polypeptide expressed on the cell surface.4nonsense codons cause a decrease in mRNA steady-state levelsInterestingly, this germline transcript contains V

b8.2 codingby a post-transcriptional mechanism (for reviews, refs 26 and

exons spliced to a Vb5.1 leader exon, and thus it may be

27). To investigate the role of premature termination codonstranscribed from the V

b5.1 PD described here. The function ofin regulating TCR mRNA levels, we used the SL12.4 cell clone

such Vb

polypeptides is not known; they may act as surrogatethat possesses a non-productively rearranged V

b5.1Jb1.6Cb1 receptors for immature thymocytes or they may participate in

gene.11,21 By virtue of the frameshift in the Vb5.1Jb1.6C

b1 gene, the establishment of tolerance by exposing developing thymo-it contains a premature termination codon in C

b1 exon 1, and cytes to multiple self Vb

gene products.thus V

b5.1 transcripts do not accumulate in SL12.4 cells.11,14,21 Another consequence of the upstream ORFs in PD-derivedBecause we have shown previously that protein synthesis transcripts is that these ORFs could inhibit the translation ofinhibitors reverse nonsense-mediated down-regulation,21 we TCR proteins. Several reports have shown that upstreamexamined the effect of the protein synthesis inhibitors cyclohex- ORFs depress the rate of translation from downstream readingimide and pactamycin on the TCRb transcripts expressed by frames in mammalian transcripts (reviewed, ref. 28). Thus, itthe SL12.4 cell clone. Northern blot analysis revealed that is possible that some classes of PD-derived transcripts areincubation with these protein synthesis inhibitors up-regulated infrequently translated and therefore they may represent aboth PP-and PD-derived transcripts (1·3 kb and 1·7 kb, new class of ‘sterile’ transcripts. Other examples of sterilerespectively) (Fig. 5a). To determine if the nonsense codons in transcripts include those from immunoglobulin genes andthe upstream ORFs of PD-derived transcripts were sufficient the 1·0-kb (D)J

bmRNAs transcribed from germline or

to trigger nonsense-mediated down-regulation we stably D-J rearranged TCRb genes.2,8,16–19 Therefore, many PDtransfected SL12.4 cells with a productively rearranged TCRb transcripts may not encode functional proteins, but insteadgene ( lacking premature nonsense codons in the coding their expression is a consequence of the activation of distalregion). This gene construct expressed both PP-derived 1·3 kb promoters necessary to trigger programmed DNA rearrange-transcripts and PD-derived 1·7 kb transcripts; the levels of ment events. It could be advantageous that PD-derived tran-these two transcripts were not significantly influenced by scripts are not efficiently translated, particularly in the case ofcycloheximide treatment. The inability of cycloheximide to germline PD transcripts, since these would encode potentiallyup-regulate the 1·7 kb mRNA indicates that the upstream deleterious V

bpolypeptides lacking D

b, J

b, and C

bsequences.

ORFs present in this PD-derived RNA do not trigger the Although the consequences of the expression of Vb

polypep-nonsense-mediated regulatory pathway. We conclude that at tides is not known, it is reasonable to suspect that theirleast some PD transcripts are not subject to down-regulation expression might interfere with the normal physiology ofas a result of their upstream ORFs. Therefore, these novel immature haematopoietic cells or mature T lymphocytes.transcripts may be expressed at sufficiently high levels to play The discovery of distal promoters that drive transcription

of germline TCR genes in early development raises at leastan important functional role in lymphocyte development.

© 1998 Blackwell Science Ltd, Immunology, 93, 213–220

K. A. Sutton et al.218

(a)

(b)

Figure 4. Sequence analysis of the Vb5.1 (a) and V

b8.1 (b) distal promoter regions. The sequences are numbered from the initiatorATG defining the known precursor TCRb protein. PD transcription start site regions (as mapped by RPA) are shown in boldlower case letters. The transcription start site of the V

b5.1 PP6 is shown by an arrow; the PP start site for Vb8.1 is not known. The

conserved decanucleotide motif which is important for PP transcription activity is also shown.1,23,24 The amino acids correspondingto ORFs defined by ATGs (in bold) containing surrounding sequences similar with the Kozak consensus sequence are shown. Theposition of the sense primer used for detecting V

b5.1 PD-derived transcripts by RT–PCR (see Fig. 5) is boxed. These sequence dataare available from EMBL under accession numbers U82703 and U82704.

© 1998 Blackwell Science Ltd, Immunology, 93, 213–220

Distal Vb

promoters transcribe novel T-cell receptor-b transcripts in early development 219

(a) (b)

–

Cb1

Vb5.1

PdPp

Probe B

Probe A

6 1 3 6Hr

Probe B

Probe A

Pac

tom

ycin

No

trea

tmen

t

Cycloheximide

1·7kb1·3kb

1·7kb

1·7kb1·3kb

28s

18s

No

trea

tmen

t

Cyc

lohe

xim

ide

Figure 5. The SL12.4 cell clone expresses both proximal and distal Vb5.1 promoters. (a) Northern blot analysis of total cellular

RNA (10 mg) from SL12.4 cells incubated with media alone or media with the translational inhibitors pactomycin or cycloheximide.The same blot was sequentially hybridized with a C

b1-specific fragment (probe A) and a Vb5.1 PD-specific fragment (probe B). All

lanes were shown to contain equal amounts of RNA by methylene blue staining of the 18S and 28S rRNA on the blot (as describedin ref. 30; data not shown). (b) Northern blot analysis of SL12.4 cells transfected stably with the enhancer+ V

b8 construct. Theblot was hybridized with the V

b8-specific probe described in Fig. 1. The lower panel shows methylene blue staining of rRNA todemonstrate equal loading.30

3. C S., H R.R., L Y.S. & S U.T. (1994) T cellthree important questions. First, what cis elements drivereceptor V

b8.2 gene germ-line transcription: an early event oftranscription from PDs? The identification of an immaturelymphocyte differentiation. Eur J Immunol 24, 3073.T-lymphoma cell clone, SL12.4, that transcribes a PD will be

4. J C.J. & O’N H.C. (1995) Expression of a TCR Vb8.2useful for elucidation of such cis elements. Transient transfec-

polypeptide from the unrearranged gene in a murine lymphoidtion experiments have revealed that sequences upstream of the

precursor cell line. Int Immunol 7, 1147.Vb8.1 PD are sufficient to drive modest luciferase expression in 5. S R.S., W T.-G., L L., D D. &

SL12.4 cells, but not in non-lymphoid COS-7 cells (K. A. S. C R. (1995) Transcription of the TCR-b locus initiates& M. F. W., unpublished observations). Second, does tran- in adult murine bone marrow. J Immunol 154, 3888.scription from PDs induce TCRb gene rearrangements? This 6. A S.J., C H.S. & L D.Y. (1988) A conserved

sequence in the T-cell receptor b-chain promoter region. Proccould be addressed by deletion of PD cis elements, followedNatl Acad Sci USA 85, 3551.by examination of the functional consequences in stably

7. B G., W F., N M., V C., N P.transfected T-cell lines or transgenic mice. Third, do the& F P. (1996) Deletion of the mouse T-cell receptor b geneproteins uniquely encoded by PD-derived transcripts possessenhancer blocks ab T-cell development. Proc Natl Acad Sci USAa functional role? Mutational analysis of the ORFs present in93, 7877.

PD transcripts will permit this issue to be addressed. We 8. D J. & W M.F. (1992) CD3-c, -d, -e,-j, T-cellconclude that the distal promoters described in this report receptor-a and -b transcripts are independently regulated duringhave the potential to contribute an additional level of com- thymocyte ontogeny and T-cell activation. Immunol 77, 465.plexity to the regulation of a set of genes that dictate the 9. W M. (1991) Purification of RNA. In: Essential

Molecular Biology: A Practical Approach (ed. T. A. Brown), p. 69.development and function of T cells.Oxford University Press, Oxford, UK.

10. C P. & S N. (1987) Single-step method of RNAACKNOWLEDGMENTS isolation by acid guanidinium thiocynate-phenol-chloroform

extraction. Anal Biochem 162, 156.This work was supported by National Institutes of Health grant

11. Q L., T L., C M., V M., S A.W. &GM39586. K. A. Sutton was the recipient of a Wellcome travel

W M.F. (1993) T cell receptor-b mRNA splicing: regu-fellowship. We would like to thank Hector Martinez-Valdez and David

lation of unusual splicing intermediates. Mol Cell Biol 13, 1686.McConkey for providing helpful comments on the manuscript.

12. M S., D J., L S., N R.P., L J.S. & W

M.F. (1996) The Pem Homeobox Gene. J Biol Chem 271, 17 536.13. S G., K M., S E., H R., M T.W. &REFERENCES

H L. (1984) The structure, rearrangement and expression of1. L J.M. (1993) Transcriptional regulation of T cell receptor D b gene segments of the murine T-cell antigen receptor. Nature

genes. Ann Rev Immunol 11, 539. 311, 344.2. S B.P., G J.R. & A F.W. (1996) Accessibility 14. W M.F. & ML C.L. (1988) Induction of T-cell

control of antigen-receptor variable-region gene assembly: role of receptor-a and -b mRNA in SL12 cell can occur by transcriptionaland post-transcriptional mechanisms. EMBO J 7, 101.cis-acting elements. Ann Rev Immunol 14, 459.

© 1998 Blackwell Science Ltd, Immunology, 93, 213–220

K. A. Sutton et al.220

15. D M.M. (1985) Molecular genetics of the T-cell-receptor b 24. A S.J., M S. & L D.Y. (1989) Transcription froma murine T-cell receptor V

bpromoter depends on a conservedchain. Ann Rev Immunol 3, 537.

16. C A.F. & P B.M. (1986) Expression of T cell decamer motif similar to the cyclic AMP response element. MolCell Biol 9, 4835.receptor genes in human B cells. J Exp Med 164, 1940.

17. S H.R., K P., K M., S M. & V 25. K M. (1989) The scanning model for translation: an update.J Cell Biol 108, 229.B H. (1985) Ontogeny of the T-cell antigen receptor within

the thymus. Nature 313, 592. 26. P S.W., H F., W E. & J A. (1994) Nonsense-mediated mRNA decay in yeast. Prog Nucl Acids Res Mol Biol18. R D.H., G R.D., S H. & T S. (1985)

Developmental regulation of T-cell receptor gene expression. 47, 271.27. M L.E. (1995) When cells stop making sense: effects ofNature 314, 103.

19. S J.L. (1989) Developmental biology of T cell recep- nonsense codons on RNA metabolism in vertebrate cells. RNA1, 453.tors. Science 244, 943.

20. Y G.Y., G W. & H N.B. (1994) Heterogeneity in the 5∞ 28. G A. (1996) Translational control mediated by upstreamAUG codons. In: Translational Control (eds J. W. B. Hershey, M.untranslated region of mouse cytochrome CT mRNAs leads to

altered translational status of the mRNAs. Nucleic Acids Res B. Mathews & N. Sonenberg), p. 173. Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY.22, 4599.

21. C M.S., D J., M P. et al. (1995) A regulatory 29. H M.M., E R.M., S-G M. & D

J.E. (1979) Production of mRNA in Chinese hamster cells:mechanism that detects premature nonsense codons in T-cellreceptor transcripts in vivo is reversed by protein synthesis inhibi- relationship of the rate of synthesis to the cytoplasmic concen-

tration of nine specific mRNA sequences. Cell 17, 1025.tors in vitro. J Biol Chem 270, 28 995.22. B M.A., C H.S., H K. & L D.Y. (1986) Murine 30. W M., D J. & L S. (1990) RNA blots:

staining procedures and optimization of conditions. Nucleic AcidsT-cell receptor mutants with deletions of b-chain variable regiongenes. Proc Natl Acad Sci USA 83, 767. Res 19, 679.

23. L M.-R., C C.-S., L M.-L. et al. (1992) Isolation andcharacterization of nuclear proteins that bind to T cell receptorVb

decamer motif. J Immunol 148, 1906.

© 1998 Blackwell Science Ltd, Immunology, 93, 213–220