Functional role of TTF-1 binding sites in bovine thyroglobulin promoter Fmnp&s Ja~up_

Fabien Batauxa. Alena Donda”, Helen Francis-Langb, Gilbert V~SSZXP, Roberto DXaurob and i)anieI Christophe”

Wehav~?stt&d the bindingofpurifkd~-1 on thebotine thyroglobulin gencpromotcr. DNasc I foolprintingcxperiments revealed three binding siw which auresponded in location to the A. B and C sites found in the rat thyroglobulin promoter. hlutants in the A and C regions showing r&u& binding of IXF-1, also exhibited largely decrtzzd promoter activity in transient exprcssiop experiments in primary-cultured dog thyrocyts TWY mutants in the B site that exhibited a reduced capaaty to hind ITF-I also displayed a drastically affcctcd transcriptional activity in tmnsient assays As in the rat sites A and C only are critical for promoter activity. these results suggest :hat fuli occupa~lcyof the T. site is required for thyroglobulin ptcrmntn activity in the cow only.

1. INTR0DUCTiON The expression of t floglobulin (Tg) gene is restricted to the follicular cells of the thyroid gland. The expression of the gene is under the positive control of thyrotropin which acts mainly via the intracellular messenger cAMP [I]. This control occurs. at least partly, at the transcriptional level [2,3]. It has been shown that the specific expression of a reporter-gene, the chloramphenicol acetyltran&rase (CAT) gcnc. rcquircd only the first 250 bp of the bovine Tg promoter when transfccted in thyroid cells in primary culture [4]. Likewise. 170 bp of the rat Tg promoter wcrc suficicnt lo direct the cell-type-specific expression of the same reporter gent when transfmed in FRTL-5 cells [SJ The contra! of gene expression depends largely on the binding of transcription factors on promoter sequences. A DNA sequence repeated three times in the rat T’g promoter has been sho&m to interact in footprinting experiments with a thyroid-specific nuclear factor called TTF-1 (s]. The lTF-1 protein hzs bccu purified and Cbdracterized by cDNA cloning i7]_ In this study. we compare the binding of TTF-1 on both rat and bovine promoters and its functional role in ?hc control of the cxpmsion of ihe corresponding gcna. Although ITF-l WCO@ZCS ilrra s~qu~na in bolh promotcrz in DIQ.sc I footprinting assays, o&y two caqmccs appear functionally rclcvaot in the rat promoter while occupancy of the three sites seems n_

scntial to sustain normal activity of the bovine promoter. 2. MATERIALS AND METWDS DNase I protection cxpcrilncnts wcrc performed on Saul znd-lab&d probes with purified TTF. I in the prcsencc of SOOflg/ml bovine serum albumin. 0.1% NP-40 and 2% polyvinylalcohol, accotiing to Uusti et al. 151.The DNasc I digestion was stopped with 1% SDS. S mM EDTA and 50 pg/nJ prouinasc K. The DNA was then ethanol prccipitalcd and analyzed on a 6% scqucncing gel.

Mutatidns wcrc introduccd in Ihe bovine Tg promoter in plasmid pbTgCAT 14 ([4] and Oonda ct al., ill preparation) using the PCR tcchniquc [S]. The description of :hc diffcrcnt substitutions is given Fig. 1. The rcsu$ing construct> :vcrc partially szqucnccd to vclify the prcscncc of the mutations and tu cxcludc the occurrcncc of undcsircd base changes in the PCR-amplified fraf!mcnls.

IJ?g thyrocytc primary crllturcs wcrc mtablished and maintained as dcscribcd m 191.‘li~ndCSti0 1 or primap cuhrd dog thyrocytcs was done as dcscribcd in [4]. After t: ansrmion.cells wcrc maintained thrcz days cithcr in conlrnl medium (control condilions) or in control mcxlium suppkmcnt~~ with IOpllI fonkolin (Foconditions). Analysis of CAT actwity ws alTccted according to !5ccd and Shccu IlO].

3. RESULn

XND DlSCUSSlON

Footprints were rcalizcd witi, a 250 bp fragment from the bnv;nc Tg promoter and \vith puriticd J-TF-;a Figure LA shows the rcsu!ts obtained with the wild-type promrtaea xqucnce. PpnrifieJTCF-I gcncratcd three

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Fig. I, interaction O~TTF-I with bovme Ifg promoter sequences. DNase 1 foolprin~s using (A) the wild-type pbTgCAT 14 or the mutated (89 pbTgCAT A, (Cl pbTgCAT C, 0) pb%CAT BI. IE9 pbT&AT D2. !F9 pbTgCAT B3 bovine TX promoler sequelus are shown. The positions of +f’fF-I binding sites A, D alid C are boxed. The mlltated Tl’F.1 recogniliou sequences are hatched and f,&tionr of mtz!slions are indica:cd by asterisks ( * 9. First lane: G + A sequenciog reaction; semod lane: naked DNA; third lane: probe preincubarcd with 0.5 pl of purified TTF.1.

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RR&S of ~bc transfcaion experiments with wild-type and mutated promoters obtained in three separate qcrimcnts an: shown. Cells were maintained cithcr in control medium (01) or in medium supplcmcntcd with 10 JJMfonkolin (Fo) after trattsfzction (rclativc activities of mutated promoters A. C and 83 wzsus 14 wild-type appear in brackets in Fo conditions only. 7hc background value of the assays WO0cpm and900cpmTorpancls(A)and(B1.~~ivcly) havebccn dCdUCCd.

footprints that correlated in positions, although less in DNA scquenzcs, to the regions A. B and C of the rat Tg promoter. So, in vitro, TTF-I has three similar binding sites on the rat and the bovine Tg promoters.

3.2. Binding of TTF-1 on mutated A and C regions Mutations introduced in, the A and C consensus sequenccs in the ‘rat ?jj promoter cIrasticaIIy decrease the transcription of a mpcuter-gene in hakfmioti e&n+ ments [I 11.Thii effect parallels the lack of binding to l-IF-1 ‘in vim. In order to correlate the biding of TfF-I with promoter activity in the beef, two mutations were introduced in the A and C m&ions of the bovine promoter at the -153 to -148 and at the -73 to -68 positions, respectively {Fig. 2). Weak or no binding of ‘ITF-I was observed respectively on the mutated A region (Fig. 1B) or on the mutated C region (Fig. 1C) in footprinting assays. Moreover, transfections of primary-cultured dog thyrocytes with the pbTgCAT A or pbTgCAT C constructs, that contained the mutated bovine Tg promoters, in the regions A and C respectively, linked to the CAT gene showed that the two substitutions greatly affected promoter activity (Table IA and II). As initially observed in the rat promoter, reduced or lack of binding of TfF-1 to the A and C mutants thus correlates with a dramatic decrease in transcriptional activity. 3.3. Binding of TTF-2 on mutated B region By contrast to what is observed for the TTF-I A and C sites, mutations introduced in the rat TfF-1 B binding site do not impair promoter activity in transfection experiments. Moreover, the rat B region has a much weaker affinity for TTF-1 than the A and C sites [12]. Three bovine promoters containing mutations in the B region were assayed for their capacity to bind TTF-1 in DNase I protection experiments (Fig. 2). As shown in Fig. ID. the GTG+TCT substitution of the 131mutation [13] did not prevent the binding of +M’F-1on the B site. On the other hand, the B2 (GGA+TTC substitution) (Donda et al., in preparation) and the B3 tTCC+GTT substitution) (this study) mutated sequences revealed a weaker affinity for TTF-t and displayed a narrower footprint of the protein 0~1this region of the promoter that correlated with the positions of the mutated residues (Fig. 1E and F). I-Iowcvcr, none of Table 11 CAT activity ofmutatcd prgmotcrs normitiizcd to the value obtained with the wild-type rg promoter (pbTgCAT 14 construct) in the prcsMCCof forskolin Construct pbTgCAT 14 pbTgCAT A pbTgCAT 81 pbT&ZAT 32 pbTISC‘ATB3 pbl+~CA-f C ---,I--.-. ll- ~“__-___

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Volume 300, number 3

FEBS LETTERS

April 1992

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of the isolated homeodomain of TTF-I produced in 17. coli with bovine Tg gene promoter sequences. DNase I footprints using pbTgCAT 14 or the mutated 03) pbTgCAT 92 or (C) pbTgCAT 83 bovine rg promoter scqucnccs arc shown. The positions sites A and B arc boxed. The mutated TTF-I recognition sequcnccs arc hatched and positions of mutations are indicated by (*)_ First lane: naked DNA; second lane: probe prcincubatcd with I pl of TTF-1 homeodomain produced in E. coli.

these two mutations completely abolished TTF-1 binding in vitro. Transcriptional activities of the 61 and B2 mutated promoters are displayed elsewhere ([13] and Donda et al. in preparation) and summarized in Table II. Results obtained with the B3 mutated promoter in gene-transfer cxpcriments are shown in Table IB. To exclude the intervention of other proteins that could have been co-purified with TTF-I, the isolated homeodomain of the TTF-1 protein produced in E. coli and which is sufficient to allow binding to DNA in vitro was also used in footprinting experiments. The observed footprints were, as expected, smaller than the ones displayed by the complete protein but the affinity of the homeodomain for the different B mutants paralleled that of the purified protein and, especially, was similarly distorted in the B2 and B3 mutants (Fig. 3). Altered binding of the TTF- 1 protein alone could thus account for the observed changes in the B footprints. Thus, from the three mutants in the bovine B site that were used here, one (Bl) displayed an apparently unatTected ability to bind to TTF-I and performed barely less than the wild-type promoter in a transient assay, and the two others (B2 and B3) showed reduced and distorted TTF-I footprints and were severely less aciivc than the natural scqucncc in the functional assay. 3.4. Punsienr sfrtfcf.s

c~xpres.siun of rat Tg prurrrrmr-C'A T LYJIIin pritrwry-wltimd dqq I/~_srw~yW.s to verify that the discrepancy obscrvcd bc-

in order tween the resulu of gcnc-transfer cxperimcnts with rat and bovine Tg promoters mutated in the B region did

not result from differences in the systems of cell culture used so far (FRTL-5 cell line for the rat promoter [I l] and dog thyrocytes in primary culture for the bovine

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REFERENCES

Tkresultsobtai&withtbemutatedsequenasWere similar to those obt+ed in WTL-5 transfixted cells [II]. in that the C~mutant (pTACAT14) exhibited reduced promoter activity- while both B mutants @core atrd~AcAIp)didnotTh~thefuactioaaldiff~~ ob6ervedbiztWznmtandbo*Tgpromotecsmutated is their B regions in ttatisf~~ experiments retkcts a real fimctional disparity between these promoters. On the otk bat& trim&&on of bovine promoter-conin FRTL-5 cells allowed the same tainingcu~ conclusions to be dmwtt (data not shown). Paradoti&y. although in the tat the three TTF-1 sites are much mote conserved in terms of DNA sequences, they disPhY better consenntiott of function in the cow. Ak-tsz Thr:amtinW sqp0rt and critical intcrcst of IIds work was -r&d Dr*J~dDumlmtarcdc&actaotulodgbd by grants from the Minis&c dc la Po1itiquc Scitmtifique (sciences de la vk and PAi), FRSM and ARBD a&L FJ. is the rccipicnt of a fclkw&ip from the Foundation Rose et Jean Hoguet. A.D. is the recipient of a long-tctm EMBQ felIou%hip. and D.C. is a Research Assaciatc of the National Fund for Scientific Research Wgium).

111 Vau Hcwas#)p &. Lcri&c. A_, Van Sande, J., Dumont, J.E. and”aaart, G. (198% FERs m 1~8.19%1%. m Van’H===yx B, Strqdb. C., Bwas, H.,‘RcfctolT’, S, Dumortt. J-E. and Va~ran. G. (1984) Pmc Natl. Aad Sci. USA 81. !wl-5945. [31 G&at& CM, Lcfort. A.. Christophc, D, Wcrt, F., Van Sand% J, DuPont J.E. and Vassar& G. (1989) Mol. Endocrinol. 3, 2110-2118. [4] Ctuistophe. D.. Gkard, C, Juvcna& G , Bacolla. A., Tcugels, E, Lcdcnt, C., christophe-Hobcrtus, CT..Dumont. LE. and Vassart, G. (1989) Mot CclL Endocrinol. 64, S-18. [q Musti, AM, Ursini. V-M, Avvcditncnto, EV.,Zimarino, V. and DiLauro. R. (1987) Nucleic Acids Rcs. IS, 8149-8166. [6J Civitarealc, D., Lonigro. R.. Sinclair, AJ. and DiLauro, R. (1989) EMw3 5. & 2537~2542. p1 Guatzi S, Prkzc, M, De F&q M, Dzunante,G., Matte& M.-G. and DiLautu. R (I%% EMEO J. 9.3631-3639. [S] HiguchL R.. Knunmcl, B. and Saiki. R.K. (1988) Nucfdc Acids Rcs. 16.7351-7367. p] Roger. P.P., Van Hcuvcrwyn, I& Lambert C., Reuse, S., Vassaxt. G. and Dumont. 3-E. (1985) Eur. J. Bicchcm. 152,239-245. Seed, 0. and Sheen, J.-Y. (1988) Gcnc 67,271-277. Sinclair, AJ., Lonipo, R., Civitareale, D., Ghibclli. L. and DiLauso, R (1990) Eur. J. BioEhcrn. 193,311318. Civitarcahz, D., Ghibclli, L., DiLauro, R. (1987) Hormone and Metabolic Research (supplement scrics) 17,73-77. Jauaux, F., Donda, A., Vassart, G. and Christophe, D. (1991) Nudcic Acids Rcs. 9.1121-I 127.

Functional role of TTF-1 binding sites in bovine thyroglobulin promoter.

We have studied the binding of purified TTF-1 on the bovine thyroglobulin gene promoter. DNase I footprinting experiments revealed three binding sites...
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