Biochimica el Biophysica Acta. 1090 ( 1991 ) 235- 237

235

© 1991 Elsevier Sc!ence Publishers B.V. All righls reserved 0167-4781/91/$03.50 ADONIS 016747819100226P

BBAEXP 00261

Short Sequence-Paper

Isolation and characterization of the canine thyroglobulin gene promoter region A l e n a D o n d a t, G i l b e r t V a s s a r t L2 a n d D a n i e l C h r i s t o p h e I Institute of Interdisciplinary Research and 2 Department of Medical Genetics, Unicersitd Libre de Bruxelles, Bnt~'eL~ (Belgium) (Received 2 April 1991)

Key words: Thryroglobulin gene; DNAa~e I footprinting; G e n e expression; (Canine)

The 5' flanking sequences from the canine thyroglolmlin gene were isolated by homology screening with the evolutionary conserved sequence from the bovine thyrogiobulin promoter and sequenced. Transient expression in primary cultured dog thyrocytes demonstrated that the canine clone contains a functional promo~r inducible by cAMP. DNAse I footprinting assays showed that the tbyroid-specific transcription factor TIT-l, purified from bovine thyroid, also recognizes the canine thyroglobulin promoter. Similar footprints were obtained with crude nuclear extracts from primary cultured dog thyrocytes. The sequence of the promoter region from the thyrogiobulin (Tg) gene is known for different mammals namely human, bovine and rat [1-3]. The proximal regions described as sufficient for proper promoter activity in vitro exhibit extensive sequence homology (about 70%) between - 140 and - 80 bp relative to the transcription start site [4]. However, transient expression experiments in primary cultured dog thyrocytes involving human or bovine Tg promoters mutated at homologous positions indicated that despite the overall DNA sequence conservation, promoter-specific mechanisms were involved in the control of transcriptional activity (A. Donda et al., unpublished data). To eliminate the possibility that the observed differences were due to the use of a heterologous transfection system, the promoter region of the canine Tg gene was isolated and sequenced. A 0.6 kb cDNA probe covering the first five exons of the bovine Tg gcne was used to screen 106 individual clones from a A DASH canine genomic library (Stratagene) according to standard procedures [5]. Hybridization was performed at 42 °C overnight in 40% formamide and 6 x SSC (1 × SSC = 0.15 M NaCI, 0.015 M sodium citrate (pH 7.0)); the use of a heterologous probe was taken into account by lowering the stringency of the washings (42°C, 0.1 × SSC for the last wash). Four positively hybridizing clones were scored.

D. Christophe, Institute of Interdisciplinary Research (IRIBHN), Campus Erasme, Bat. C, 808 Route de Lennik,

Correspondence:

13-1070Brussels, Belgium.

One of them, containing the promoter region, was selected at the second round of screening by hybridization with a 60-mer synthetic oligonucleotide probe corresponding to the bovine evolutionary conserved sequence ( - 1 3 6 to - 7 7 bp, see also Fig. 1). Briefly, the single-stranded bovine oligonucleotide probe was labelled at its 5' terminus, hybridization was performed at 30 °C overnight and stringency of the last -213 C A C C T ..TTC.T...~ ....... -162 ~ C / ~ . J ~

..........

-112 C ,,

-62 ~

~ ........

~ C C..~.~

T.......... ~

G

& ................

C C ~ - C . . . . . C . . . . . . . T,TCA . . . . A.

C

C G...A.T

+40

C.T

~ .....

C .........

A G C . A G C ~ a ' e

+lr ~ G A G C ~ ...... AA ......... Ca ..... T.A ...... TC..TG-

CAAA ~TG GCC CTG A.G ............. llet A l a L e u

T

CA

..... TG .... CCT. A..A.. ,&C ..... TGC ............

-ix

C

-C.G . . . . A . . . ,C . . . . . . . . . . . .

T ......

...... ..... CT,CC

GCC Ala

Fig. i. Sequence of the canine TE gene promoter region. TATA-box and translation initiator codon are boxed. The Iocalisations of the restriction sites H / n d l l l ( - 1 7 5 ) and B a m H I ( + 8 ) introduced for subsequent cloning are shown a n d the nneleotides that were substituted are underlined. A putative transcription start site ( + 1 ) is proposed on the basis of the homology with the bo~in~ Tg promoter sequence which is represented below the canine sequence. Dolts specify identical nucleotide between dog and bovine sequence a n d dashes indicate an additional nucleotide in the sequence of o r e o f the two species. The sequence o f the bovine oligonucleotide p r o b e u s e d f o r the screening is underlined.

236 washing step was further lowered (55°C, 2 × SSC, 0.1% SDS). Southern blot analysis of phage DNA using the same oligonucleotide probe identified a 2.0 kb BamHI fragment that was subcloned in both M13 mpl8 and mpl9 vectors and partially sequenced on both strands by the dideorynucleotide chain-termination method [6] using a 380A automated sequencer (Applied Biosystems). Conservation of sequence with the bovine Tg gene identified 213 bp of the canine Tg gene 5' flanking sequences with a putative transcription start site and the beginning of exon 1 (Fig. 1). To test the promoter activity, 180 bp of 5' flanking sequence were inserted either in the vector pBLCAT3 [7] in front of the CAT gene or in the vector p0GH [8] in front of the growth hormone gene (GH). Briefly, the DNA fragment to be subcloned was amplified by PCR using two primers, both of them containing two substituted nucleotides in order to introduce the appropriate cloning sites at each extremity of the amplification product, respectively, a H/ndlll site at - 1 7 5 and a BamHl site at + 8 bp (relative to the putative CAP site; see Fig. 1). These DNA constructs were transfected in primary cultures of dog thyrocytes according to an established protocol [4] and cells were kept in the presence or absence of 10 /zM forskolin (Fo) after transfection in order to assay cAMP-dependent transcriptional activity. Promoter activity was tested 3 days later by measuring either CAT activity in cellular extracts [9] or by quantifying the accumulation of GH in the cell culture media by immunoradiometric assay (IRMA) according to the manufacturer's protocol (RlAgnostics). The equivalent region of the bovine Tg promoter cloned in pBLCAT3 (pbTgCAT14M construct; A. Donda et al., unpublished data) was tested in the same experiments (Table

TABLE I 180 bp of 5' flanking sequence from the canine Tg gene were inserted in front of either the GH or the CAT gene and promoter activity was assayed by transient expression in primary cultured dog thyrucyles, cAMP stimulation of transcriptional activity was tested by the addition of 10 ~tM Fo to the culture medium ( - / + ) . 3 days after transfection GH accumulation was quantified in the cell culture medium and CAT activity (expressed as the amount, in cpm, of butyrylated [31-1]chloramphenicol)was measured in cellular extracts. The bovine Tg-CAT fusion gene (pbTgCATI4M; A. Donda et al., unpublished data) was transfected in parallel as a reference. Values between brackets denote the number of experiments. The limit of detection of the GH assay was 0.25 ng/ml and the background level of CAT assay was about 700 cpm Dog

Bovine

GH (ng/ml) --

CAT (cpm) +

L0

55.3

(3)

CAT (cpm)

-

-e

--

1990

35234

1466

(I)

+

48529

(3)

I

!

,

I

2

3

,

-156 -156

-128

-1

gllilF~ ' ' ~ - -78

C -60

3'

~

-60

Fig. 2. DNAse 1 protection on a 32p-labelled DNA fragment of the canine T8 promoter spanning from +8 to -175 bp, by purified TTF-I protein (left) or by crude nuclear extract of dog thyrocytes (right). Lane 1: Maxam and Gilbert purine ladder, G + A . Lane 2: no protein added to the reaction. Lane 3: addition of pure TI'F ! (left) or of crude nuclear extract of dog thyrocytes (right). The protected regions are specified by their boundaries and are termed A, B and C by homology with the three TIF-I binding sites initially found in the rat Tg promoter [12].

I). The results confirmed that the cloned canine sequence is a functional Tg promoter inducible by cAMP. The thyroid-specific transcription factor TFF-I, purified from bovine tissue [10] has been shown to bind specifically to the rat Tg promoter [12], and a similar pattern of binding was recently obtained for the bovine Tg promoter (F. Javaux et al., unpublished data). The characterization of the canine sequence prompted us to investigate TFF-1 binding by using DNAse I footprinting [11] on the Hindlll.BamHl DNA fragment

237

(see Fig. 1; purified TI'F-1 protein was kindly given to us by Dr. R. Di Lauro, EMBL Heidelberg, F.R.G.). TTF-1 footprints were detected on the canine Tg promoter (Fig. 2, leR panel) at three locations homologous to the regions termed A, B and C initially found in the rat [12]. Assays performed with crude nuclear extracts from dog thyrocytes, performed as described in Ref. 12, generated similar footprints (but not exactly identical on A and B sites; Fig. 2, right panel) suggesting that bovine TTF-1 has its homologue in dog thyroid and confirming its major role as transcription factor of Tg promoter in mammals. The availability of the canine Tg gene promoter will allow studying transcriptional regulation of the gene in the homologous system of primary cultured dog thyrocytes, which displays tight transcriptional control by cAMP-dependent mechanisms [13] in contrast to most systems available. The continuous support of Prof. J.-E. Dumont is deeply acknowledged. We are also grateful to Dr. R. Di Lauro for kindly providing purified TI'F-1 and to Dr. F. Javaux for helping in footprinting experiments. This work was supported by grants from the Minist~re de la Politique Scientifique (PAl) and from the FRSM. A.D. is a recipient of a long-term EMBO fellowship and D.C. is a Research Associate of FNRS (Belgium).

References I Christophe, D., Cabrer, B., Bacolla, A., Targovnik, H., Pohl, V. and Vassalt, G. (1985) Nucleic Acids Res. 13. 5127-5144. 2 De Martynoff, G., Pohl, V., Mcrcken, L, Van Ommen, G.L and Vassart, G. (1987) Eur. J. Biochem. 164, 591-599. 3 Musti, A.M., Ursini, V.M., Awedimento, E.V., Zimarino, V. and Di Lauro, R. (1987) Nucleic Acids Res. 15, 8149-8166. 4 Christoph¢, D., G~rard, C., Juvenal, G , Bucolla, A., Teugels, E., Ledent, C., Christophe-Hobertus, C., Damont, J.E. and Vassan, G. (1989) Mol. Cell. Endocrinol. 64, 5-18. 5 Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, Cotd Spring Harbor. 6 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 7 Luckow, B. and Schlitz, G. (1987) Nucleic Acids Res. 15, 5490. 8 Selden, R.F., Burke Howie, K., Rowe, M.E., Goodman, H.M. and Moore, D.D. (1986) Mol. Cell. Biol. 6, 3173-3179. 9 Ledent, C., Parmentier, M. and Vassart, G. (1990) Proc. Natl. Acad. Sci. USA 87, 6176-6180. 10 Guazzi, S., Price, M., De Felice, M., Damante, G., Mattei, M.G. and Di Lauro, R. (1990) EMBO J. 11, 3631-3639. !1 Musti, A.M., UrsinL V.M. Awedimento, E.V., Zimarino, V. And Di Lauro, R. (1987) Nucleic Acids Res. 15, 8149-8166. 12 Civitareale, D.. Lonigro, R., Sinclair, AJ. and Di Lauro, R. (1989) EMBO J. 9, 2537-2542. 13 G~rard, C.M., Lefort, A., Christophe, D., l.ibert, F., Van Sande, J., Dumont, J.E. and Vassart, G. (1989) Mot. Endocrinol. 3, 2110-2118.