induction of nuclear protein factors specific for hormone

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 13, Issue of May 5, pp. 7523-7530, 1989 Printed in U. S. A.

Induction of Nuclear Protein Factors Specific for Hormone-responsive Region during Activation of Thyroglobulin Gene by Thyrotropin in Rat Thyroid FRTL-5 Cells*

(Received for publication, November 7, 1988)

Nam-Taek Lee, Shihadeh N. Nayfeh, and Chi-Bom Chae From the Department of Biochemistry, University of North Carolina, Chapel Hill, North Carolina 27599-7260

We have investigated the mechanism of stimulation of thyroglobulin gene expression by thyrotropin (TSH) and cAMP in rat thyroid FRTL-5 cells. In contrast to the c-fos gene, induction of the thyroglobulin gene by TSH or cAMP is slow (10 h) and sensitive to cycloheximide treatment. We have identified a TSH and CAMP- responsive region of thyroglobulin gene between -171 and -140 base pairs from the transcription initiation site. The hormone-responsive region contains DNA sequence elements similar to the consensus CAMP-responsive element as well as the transcription factor AP-1-binding site but with opposite sequence polarity. Three DNA-protein complexes are formed when the hormone-responsive region is incubated with nuclear extracts of FRTL-5 cells. Formation of these complexes is dependent on TSH or cAMP stimulation, thus suggesting that the factors involved in binding to the hormone-responsive region may be induced by TSH. Although the identity of these factors is not yet known, they do not appear to be related to either CAMP-responsive element-binding protein or AP-1. These results suggest that thyroglobulin gene expression in FRTL-5 cells may be mediated by nuclear factors that are induced by cAMP in contrast to other genes (e.g. c-fos) whose activation involves post-translational modification of the pre-existing proteins specific for CAMP-responsive element.

TSH’ plays a key role in the maintenance of the differentiated function of the thyroid gland (Salvatore et al., 1980). It is generally accepted that most, if not all, of TSH actions are mediated by cAMP which is formed following interaction of TSH with specific thyroid cell surface receptors (for review, see Powell-Jones et al., 1980) and subsequent activation of adenylate cyclase (Field, 1975; Van Herle et al., 1979). One of the major thyroid proteins induced by TSH is thyroglobulin (TG), the precursor of thyroid hormones (Refetoff et al., 1983). The TG gene in the rat is 200 kb (Musti et al., 1986) while in humans it is 300 kb (Baas et al., 1986). The rat gene contains 42 exons with a combined length of about 9000 nucleotides (Musti et al., 1986). Both TSH (Van Heuverswyn et al., 1984; Santisteban et al., 1987) and cAMP (Bone et al.,

* This work was supported in part by National Institutes of Health Grants DK39019 and DK23080. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: TSH, thyrotropin; TG, thyroglobulin; kb, kilobases; CRE, CAMP-responsive element; IBMX, 3-isobutyl-1- methylxanthine; CAM, chloramphenicol; HRR, hormone-responsive region; bp, base pairs.

1986) activate TG gene transcription. Recent studies of several genes regulated by CAMP, e.g.

somatostatin (Montminy et al., 1986), the human chorionic gonadotropin a subunit (Darnel1 and Boime, 1985; Delegeane et al., 1987; Jameson et al., 1987; Silver et al., 1987), c-fos (Bravo et al., 1987; Colletta et al., 1987; Kruijer et al., 1985; Tramontano et al., 1986; Van Beveren et al., 19831, phos- phoenolpyruvate carboxykinase (Wynshaw-Boris et al., 1984), and plasminogen activator (Nagamine and Reich, 1985), indicate that they all have similar CAMP-responsive elements (CRE), and activation by cAMP is prompt and unaffected by inhibitors of protein synthesis (Nagamine et al., 1983; Colleta et al., 1987; Hofstetter et al., 1987; Hall et al., 1988). These results suggest that the CAMP-dependent activation of these genes may involve modification of pre-existing proteins by CAMP-dependent phosphorylation. This conclusion was supported by the observation that the somatostatin gene does not respond to cAMP in a mutant PC-12 cell line which lacks CAMP-dependent protein kinase type I1 activity (Montminy and Bilezikjian, 1987). Moreover, a 43,000 protein that selec- tively binds to CRE has recently been purified from nuclear extracts of PC-12 cells and rat brain (Montiminy and Bilezik- jian, 1987; Yamamoto et al., 1988). Phosphorylation of this 43,000 protein by CAMP-dependent protein kinase stimulates gene transcription in vitro (Yamamoto et al., 1988). However, neither the binding of this protein to CRE nor its concentration appears to be affected by pretreatment of PC-12 cells with CAMP. Similar results have been obtained for the human choriogonadotropin a-subunit CRE (Delegeane et al., 1987).

Although the minimum length of rat TG gene upstream sequence sufficient for tissue-specific transcription has recently been determined to be 170 bases from the transcription initiation site (Musti et al., 1987), thus far, neither the TSH- responsive DNA sequence nor the nuclear factors interacting with such a sequence have been identified. In this report, we have identified a 30-bp DNA region which is involved in the TSH and CAMP-dependent transcription of the TG gene in a normal rat thyroid (FRTL-5) cell line (Ambesi-Impiombato et al., 1980). The region contains DNA sequence elements which are similar to the consensus CRE (Nagamine and Reich, 1985; Montminy et al., 1986; Roesler et al., 1988) as well as the transcription factor AP-1-binding sites (Lee et al., 1987a, 1987b); Bohmann et al., 1987), albeit with opposite sequence polarity. The nuclear proteins which bind the hormone-responsive region (HRR) are induced by TSH and cAMP but apparently are not related to the known CRE- binding proteins or the AP-1 proteins. Furthermore, the rate of stimulation of TG gene expression by TSH and cAMP is sensitive to cycloheximide and is much slower than that of other CAMP-dependent genes, such as c-fos. Thus, the mechanism of CAMP-mediated activation of the TG gene by TSH

7523

7524 Thyroglobulin Gene Activation by TSH

appears to be quite different from that of other CAMP- responsive gene systems.

EXPERIMENTAL PROCEDURES

Materials-A TG cDNA was a generous gift from Dr. Gilbert Vassart and was prepared as described by Brocas et al. (1980). Bovine TSH, 8-bromo-CAMP, IBMX, and all other tissue culture medium and growth factors were purchased from Sigma.

Growth of Cells-FRTL-5 cells, a continuous line of functional epithelial cells from normal rat thyroid (Ambesi-Impiombato et al., 1982), were the generous gift of Dr. Kohn. The cells were grown in Coon’s modified Ham’s F-12 medium (Ambesi-Impiombato et al., 1980) containing 5% heat-inactivated calf serum, plus 6 hormones (6H), insulin (10 pg/ml), hydrocortisone (10 nM), transferrin (5 pg/

ng/ml), and TSH (1 X lo-’ M). This medium will be referred to as ml), glycyl-L-histidyl-L-lysine acetate (10 ng/ml), somatostatin (10

6H medium in this report. Three to 4 days before an experiment, the serum concentration was reduced from 5 to 0.2%, and TSH and insulin were removed (4H medium). Under this condition, the level of TG mRNA reaches a minimum (Santisteban et al., 1987). TSH and 8-bromo-CAMP were added to the 4H cells a t 1 X lo-* M and 1 mM, respectively. IBMX was always added with 8-bromo-CAMP at a concentration of 0.5 mM to prevent degradation CAMP.

Blot Hybridization-For extraction of RNA, the cells were lysed in 6 M guanidium isothiocyanate, and the samples were centrifuged through a 5.7 M CsCl cushion as described by Chirgwin et al. (1979). RNA was denatured in formamide and formaldehyde and fractionated by 1% agarose gel in formaldehyde (Lehrach et al., 1977). After blot transfer of the RNA onto a nitrocellulose filter, the filter was hybridized with 32P-labeled DNA probes as described (Lee et al., 1986; Kim et al., 1987). TG cDNA was labeled by random primer extension (Feinberg and Vogelstein, 1983).

Molecular Cloning of the DNA Containing the 5 “Flanking Sequence of Rat TG Gene-For molecular cloning of the DNA containing the 5”flanking sequence of rat TG gene, a 30-base oligonucleotide corresponding to the sequence upstream of the translation initiation codon (Musti et al., 1986) was synthesized and labeled with [32P]a- dATP in the presence of terminal transferase (Collins and Husanker, 1985). A rat genomic library established in Charon 4A (Sargent et al., 1979) was screened with the 32P-labeled oligonucleotide probe. A clone containing a 10-kb insert was obtained, and the 2.1-kb HindIII- EcoRI fragment containing the 5”flanking sequence was subcloned into M13mpll. The partial sequence of the 5”flanking region was determined by Sanger’s dideoxy sequencing protocol (Sanger et al., 1977) using the oligonucleotide probe as a primer. The sequence agreed with the published sequence of the partial TG gene 5”flanking DNA (Musti et al., 1986, 1987).

Fusion of the TG 5 “Flanking Sequence with Chloramphenicol Ace- tyltransferase Gene (pTGCAT)-A DNA fragment containing the TG sequence from +37 bp to -1262 bp was fused with chloramphenicol acetyltransferase gene present in the plasmid pLCAT. This plasmid was originally derived from pSV2CAT (Gorman et al., 1982) and contains multicloning sites upstream of the chloramphenicol acetyltransferase gene (Hwang and Chae, 1989). The fusion gene is referred to here as pTGCAT.

the 5’ end of TG DNA in pTGCAT were achieved by controlled Construction of Deletion Mutants-Unidirectional deletions from

digestion with exonuclease 111. The end points of deletions were confirmed by DNA sequencing.

Transfection and Assay of Chloramphenicol Acetyltransferase Activ-

DNA was transfected into FRTL-5 cells in a 100-mm dish (5 X lo6 ity-For transient gene expression, 4 pg of closed circular plasmid

cells) by the DEAE-dextran method (Sompayrac and Danna, 1981; Lopata et al., 1984). Transfected cells were grown in 4H medium for 50 h, followed by incubation with TSH or 8-bromo-CAMP + IBMX for 20 h. Cell extracts were prepared by freezing and thawing (Gorman et al., 1982) and chloramphenicol acetyltransferase activity was determined as described previously (Gorman et al., 1982; Mitsialis et al., 1987). [“CIAcetoxy-CAM was separated from the unmodified CAM by thin layer chromatography. Following autoradiography, the acetoxy CAM spots were scraped and the radioactivity was determined in a liquid scintillation counter. In all transfection experi-

plated in each culture dish, and different DNAs were transfected ments, an equal number of cells from the same stock culture was

under the same conditions. Variability in transfection efficiency among different dishes with the same DNA was 10-15%. We could not use other reporter genes such as @-galactosidase gene fused with

Rous sarcoma virus promoter for normalization of transfection efficiency due to the presence of galactosidase in FRTL-5 cells.

Gel Mobility Shift Assay-Nuclear extracts were prepared as described previously by Dignam et al. (1983). For gel mobility shift assay (Singh et al., 1986), nuclear proteins (4 pg) were preincubated in a final volume of 20 pl containing 10 mM Tris-HC1, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, 3-4 pg of poly(d1-dC). After 15 min of incubation on ice, 32P-labeled DNA probe (0.5-1.0 ng) with or without competitor DNA was added and incubated for 30 min at room temperature. The reaction mixture was loaded directly onto a 4% polyacrylamide gel (30:0.8, acrylamide/ bisacrylamide) in 25 mM Tris base, 25 mM boric acid, 1 mM EDTA, and electrophoresed at 120 V at room temperature. The gels were dried and analyzed by autoradiography. 3ZP-Labeled DNA probes were prepared by extension of the primers hybridized to template oligonucleotides (See Fig. 4) in the presence of 32P-nucleotides and Klenow fragment of Escherichia coli DNA polymerase I. When nec- essary, the double-stranded labeled DNAs were purified by polyacrylamide gel electrophoresis and elution.

RESULTS

Stimulation of TG Gene Transcription by TSH and CAMP- In this study, rat thyroid FRTL-5 cells which show the normal differentiated thyroid functions (Ambesi-Impiombato et al., 1980; Ambesi-Impiombato et al., 1982) were used. When the cells were nearly confluent, the serum concentration was reduced from 5 to 0.2%, and TSH and insulin were removed from the growth medium (4H medium, Santisteban et al., 1987). After 3 days, TG and c-fos mRNA accumulation was measured at various intervals of culture following readdition of either TSH or cAMP (Fig. 1). As can be seen in Fig. lA, removal of TSH, insulin, and most of the serum decreased the level of TG RNA to a barely detectable level. TSH at a concentration of 1 X lo-’ M stimulated TG mRNA synthesis after 10 h of readdition of the hormone, reaching maximal levels within 24 h. Similar effects were observed when cells grown in 4H medium were stimulated with 1 mM 8-bromo- cAMP plus 0.5 mM IBMX (an inhibitor of cAMP phospho- diesterase). In contrast, c-fos mRNA appeared within 30 min after the readdition of either TSH or cAMP to FRTL-5 cells in 4H medium (Fig. 1B). However, cAMP has no apparent effect on the synthesis of actin RNA (Fig. IC), thus indicating that the increase in TG and c-fos mRNA in response to cAMP is not due to an overall increase in the synthesis of cellular mRNA. Similar results were obtained with TSH (not shown here). The apparent difference between the level of actin mRNA in 4H cells compared with 6H cells is most likely due to the slower growth rate of the cells in the absence of TSH (Ambesi-Impiombato et al., 1980). It has been shown that expression of actin gene is related to cell growth rate (Farmer et al., 1983; Riddle et al., 1979).

Hormone-dependent Transcription of TG Gene Requires Protein Synthesk-The slow appearance of TG mRNA after addition of TSH and cAMP suggests that the stimulation of TG gene transcription by TSH and cAMP may require synthesis of new proteins. To investigate this possibility, cycloheximide was added to FRTL-5 cells treated with either TSH or cAMP and the level of TG mRNA was examined by blot hybridization. As shown in Fig. 2A, addition of cycloheximide 17 and 20 h after readdition of cAMP to 4H cells inhibited the accumulation of total cellular TG mRNA at 24 h by 80 and 50%, respectively. Similar results were obtained with the cells treated with TSH (data not shown). The inhibition appears to be not due to the possible turnover of transcription factors such as RNA polymerase and others. The same cells showed a severalfold increase in the accumulation of actin mRNA (Fig. 2A). Although the mode of action of cycloheximide on actin mRNA in FRTL-5 cells is not fully understood, it is possible that the actin gene is activated by cycloheximide

Thyroglobulin Gene Activation by TSH 7525

A. TSH

cAMP

TSH

cAMP

B. TSH

cAMP

C. cAMP

0 15' 30' 1 3 12 24 48h 8 H

."

0 10 13 18 20 2 4 h

I 0 16' 30' 1 3 12 24 48h 8 H

0 15' 30' 1 3 1 2 24 48h 6H

FIG. 1. Time-dependent stimulation of thyroglobulin and c- fos genes by TSH or CAMP. Confluent FRTL-5 cells, maintained in 4H medium for 3 days, were grown in the presence of 1 X lo-' M TSH or 1 mM 8-bromo-CAMP + 0.5 mM IBMX for various time intervals. Total RNA was extracted and analyzed by blot hybridization as described under "Experimental Procedures." Hybridization with TG cDNA ( A ) , c-10s gene ( B ) , and actin gene (C).

in a manner similar to that of quiescent mouse embryo cells (Elder et al., 1984). Furthermore, addition of cycloheximide together with cAMP or TSH to 4H cells at zero time inhibits the stimulation of TG transcription completely (data not shown). This is in contrast to the TSH-dependent stimulation of c-fos gene which is not affected by the addition of cycloheximide to FRTL-5 cells (Fig. 2B; Colleta et al., 1987). In fact, c-fos mRNA is stabilized by cycloheximide treatment, as reported by others (Hall et al., 1988). Similar effects on c-fos gene induction were observed following incubation of CAMP- treated cells with cycloheximide (data not shown).

Hormone-responsive DNA Region-To identify the TSH and CAMP-responsive DNA regions within the TG gene, we fused 1262 bases of the TG 5"flanking region to the bacterial chloramphenicol acetyltransferase gene (pTGCAT). Follow- ing sequential deletions from the 5' end of the TG DNA, the deletion mutants were introduced into FRTL-5 cells by the DEAE-dextran method (Sompayrac and Danna, 1981; Lopata et al., 1984). After DNA transfer, the cells were incubated in 4H medium for 50 h, and the production of chloramphenicol acetyltransferase from TG-chloramphenicol acetyltransferase fusion genes was examined following culture of the transfected cells with 1 X lo-* M TSH or 1 mM 8-bromo-CAMP plus 0.5 mM IBMX for 21 h. As shown in Fig. 3, TSH and cAMP caused 3- and 7-fold increases in chloramphenicol acetyltransferase activity, respectively, when the transfected constructs contained 1262 bp of TG 5'-flanking DNA. When the trans-

A.

TG rnRNA+

t;$A *

a b c d

no cAMP RNAext.

a- (43:) ' t 24hr

b. ' cAMP f 24hr

cAMP cnx 17hr c. ' 4 t 2 4 h r

d. ' cAMP CH X

20hr 4 f 2 4 h r

B. +CHX - CHX

-TIS 15' 30' l h 3h 12h24h -TIS 15' 30' l h 3h 12h 24h

C 4 O S mRNA

FIG. 2. Inhibition of CAMP-dependent stimulation of TG gene transcription by cycloheximide (CHX). A , FRTL-5 cells, maintained in 4H medium for 3 days, were incubated with 1 mM 8- bromo-CAMP + 0.5 mM IBMX for 24 h. At 17 and 20 h after the addition of CAMP, cycloheximide (20 pg/ml) was added. RNA was extracted at the end of 24 h of incubation, and TG and actin mRNA were analyzed by blot hybridization. The schedules of addition of cAMP and cycloheximide are shown in the diagram. B, FRTL-5 cells, maintained in 4H medium for 3 days (-TIS), were incubated with 1 X lo-' M TSH in the presence or absence of cycloheximide for various time intervals. RNA was extracted and the level of c-fos mRNA was determined by blot hybridization.

fected DNA contained deletions up to -171 bp, the induction of chloramphenicol acetyltransferase activity by TSH and cAMP was 2- and 3-fold, respectively. Further deletion to -140 bp abolished the TSH and CAMP-dependent production of chloramphenicol acetyltransferase. These results suggest that there is a region between -140 and -171 bp that is involved in stimulation by TSH and CAMP. The gradual decrease in stimulatory activity by deletions from -1262 to -171 bp suggests that the regions upstream of -171 bp are also involved in the TSH and CAMP-dependent stimulation of TG transcription. However, it is difficult to determine the boundary of the upstream regions based on the present results. Thus, in this report we mainly focused on the identity as well as the role of the 30-bp region (located between -140 and -170 bp) in TSH regulation of TG gene expression. This 30- bp segment will be referred to as the hormone-responsive region (HRR).

Interaction of Nuclear Proteins with HRR of the TG Gene- Close examination of the upstream sequence of the TG gene shows that there are at least three DNA elements with similar sequence (Fig. 4A). These elements are located at -68, -124, and -149 bp from the transcription initiation site. Sequences comprising -54 to -83 bp, -108 to -137 bp, and -130 to 163 bp are referred to herein as TG-I, TG-11, and TG-I11 sequence. The element at -149 bp is located within TSH and CAMP- responsive region (see Fig. 4). In addition, HRR contains a sequence motif (ATTACTCAA between -151 and -159 bp) somewhat similar to the consensus transcription factor AP-


1-binding sites (ATGACTCAT) (Lee et al., 1987a, 1987b; Bohmann et al., 1987).

To investigate whether nuclear proteins could bind to any of these sequences (TG-I, TG-11, and TG-111) and whether this binding is regulated by TSH and CAMP, protein-DNA interaction was examined by the gel DNA mobility shift assay. As a DNA probe, we first used a 32P-labeled double-stranded oligodeoxynucleotide corresponding to TG-I11 sequence (-130

A G T C A h G T I

re,a%,"* 10 -715 CAT aCt,r,,S

-171 m e 0.8 2.6 3.5

-140 m e 0.7 1.0 1.0

FIG. 3. The effect of TSH and cAMP on the expression of deletion mutants of TG-chloramphenicol acetyltransferase (CAT) fusion gene in FRTL-5 cells. TG gene upstream region (from +37 to -1262 bp) was fused with chloramphenicol acetyltransferase gene (pTGCAT) and sequential deletions were made from the 5' end of TG DNA. The deletion mutants were introduced into FRTL- 5 cells and production of chloramphenicol acetyltransferase in response to 1 X lo-' M TSH or 1 mM 8-bromo-CAMP was determined as described under "Experimental Procedures." Cells grown in 4H medium (-TIS) were used as control. Shown on the left are the deletion mutants of TGCAT gene with the end points of deletions. The heauy lines indicate the positions of the CRE-like sequences and TATAA box. In the -TIS column (a ) , the amount of ["Clacetoxy- CAM is expressed as a percent of the total radioactivity of [14C]CAM added. The values ( b ) in the +TSH and +CAMP columns represent the ratios of chloramphenicol acetyltransferase activity in cells treated with TSH or cAMP to that in cells grown in 4H medium (-TIS).

FIG. 4. The sequence of the region which control the expression of TG gene and the DNA probes used. A, the 5' upstream sequence of TG gene is shown. The similar sequence elements present in different regions are indicated by bold letters and indicated by Z, ZZ, and 111. The end points of deletion mutants are also shown. B, the sequence of oligonucleotide probes used in protein- binding assays are shown. The sequences of interest are shown by bold letters. The primers used in the labeling of DNA are underlined.

and -163 bp of the TG upstream sequence, Fig. 4B). The 3zP- labeled DNA probe was incubated with nuclear extracts prepared from FRTL-5 cells grown in either 4H medium or in the 4H cells containing TSH or CAMP. A large amount of poly(d1-dC) was also included to reduce the nonspecific binding of nuclear proteins to the labeled probe (Singh et al., 1986). The formation of DNA-protein complexes was examined by retardation of the labeled DNA probes during electrophoresis in nondenaturing polyacrylamide gels followed by autoradiography (Fried and Crothers, 1981). Autoradi- ographic analysis of the gels revealed the formation of four labeled bands (a-d) in nuclear extracts prepared from CAMP- treated cells (lane 2, Fig. 5). Similar bands were observed with nuclear proteins extracted from TSH-treated cells (data not shown). Band d, a mixture of two closely migrating complexes, was considered to be nonspecific since it was formed in the presence of a large excess of either poly(d1-dC) or unlabeled TG-I11 DNA probe. On the other hand, the formation of a, b, and c complexes was specific for TG-I11 since the formation of these three complexes with the labeled probe was prevented by the inclusion of an excess of unlabeled TG-I11 DNA in the reaction mixture (lanes 3-5, Fig. 5). Furthermore, labeling of the three bands a, b, and c was not affected by the presence of excess of unrelated DNA sequences, as will be discussed later. Similar bands (a-d) were observed when an oligodeoxynucleotide corresponding to HRR (-140 to -170 bp), instead of TG-I11 (-130 to -163 bp), was used as the 32P-labeled DNA probe (data not shown). However, because nonspecific labeling of complex d was extremely high with the probe corresponding to -140 to -170 bp, only TG-I11 sequence (-130 to -163) was used as a probe in all of the experiments described below.

The formation of these three complexes appears to be dependent on either TSH or CAMP, since more intense bands are observed on the gel when nuclear extracts are prepared from TSH and CAMP-treated cells ( l a n e 2, Fig. 5) rather than from 4H cells (lane 1 , Fig. 5). This is in contrast to proteins interacting with the consensus CRE whose binding activities are unaffected by treatment of the target cells with CAMP (Montiminy and Bilezikjian, 1987; Delegeane et al., 1987).

( A ) . T G g e n e u p s t r e a m s e q u e n c e :

180 e171 -170 -160 -150 -140 -130 -120 -110 -100

C C T G G A G T G G T C A C C C T A C T G A T T A C T C A A G T A T T C T T A G C G G G A G C A G A C T C A A G T A G A G G G A G T T C C T G T G A C T A G C A G A G A A A A C A A <140 <lo2

I 1 1 I 1

-90 -!O -!O -:0 -?O -?O -20 -20 -10

A G T G A G C C A C T G C C C A G T C A A G T G T T C T T G A A C A G T A G A G C A C T G C T T G C C A C T G T G ~ T A T A A A G G C T T C C T G A T A A G G G G A C T C A G A T G <36

I T A T A A

( 6 ) . Oligonucleotides used in protein-binding assays:

Somatostatin C R E S ' C T C T C T C T C T G A C G T C A G C C A A G G A G G C G G A G A G A G A G A C T G C A G T C G G T T C C T C C G C S '

Primer

-a3 T G I S ' C A C T G C C C A g T c a A G T G T T C T T G A A C A G T A

- 5 4

G T G A C G G G T c A g t T C A C A A G A A C T T G T C A T 5 ' Primer

-137 -108

C T C G T C T G A g t T C A T C T C C C T C A A G G A C A C S ' T G 1 1 S ' G A G C A G A C T c a A G T A G A G G G A G T T C C T G T G

Primer

T G 111 S ' A C T G A T T A C T c a A G T A T T C T T A G C G G G A G C A G A C -163 -130

T G A C T A A T G A g t T C A T A A G A A T C G C C C T C G T C T G S ' Primer

H u m a n c o l l a g e n a s e -79 A T A A A G C A T G A G T C A g A C A C C -59 A P - 1 b i n d i n g s i t e T A T T T C G T A C T C A G T c T G T G G

Thyroglobulin Gene Activation by TSH 7527

3%"TG I11 32

P - T G I11

comDetitor - TG -111 Competitor + 111 I I I SM

". .. .~ . . - + 10 50 2OOX

. " i".

a-

a +

b -

c " ,

d 4

".)

1 2 3 4 5 FIG. 5. Effect of CAMP on the formation of TG-111-protein

complexes. Nuclear extracts prepared from CAMP-treated and un- treated FRTL-5 cells were first incubated with a large amount of poly(d1-dC) (3-4 pg) to minimize nonspecific binding. Nuclear extracts were then incubated with 32P-TG-III probe (1 ng) in the presence of varying amounts of unlabeled TG-I11 as a competitor. DNA-protein complexes were analyzed by the mobility shift assay as described under "Experimental Procedures." The four DNA-protein complexes formed were designated as a-d. Lane 1, nuclear extracts prepared from the cells grown in 4H medium for 50 h; lune 2, nuclear extracts prepared from the cells treated with 1 mM 8-br-CAMP + 0.5 mM IBMX for 24 h. Competitor (unlabeled TG-111): No (lane 2); 10- fold excess (lane 3) ; 50-fold excess (lane 4 ) ; and 200-fold excess (lane 5 ) .

The Nature of TG HRR-To investigate the nature of sequence within TG HRR that interacts with the three nuclear proteins described above, we carried out DNA competition experiments using labeled TG-I11 probe and excess unlabeled DNA fragments corresponding to TG-I, TG-11, TG- 111, and somatostatin CRE (SM) as competitors. As can be seen in Fig, 6, the formation of these three complexes (a-c) was specific for TG-I11 sequence, since it was prevented when binding was performed in the presence of excess unlabeled TG-111, but only mildly with a thousand-fold excess of DNA fragments corresponding to TG-I, TG-11, and somatostatin CRE (SM). These results suggest that the binding to TG-I11 does not involve CRE-like sequences nor does it involve any of the sequences commonly shared by TG-I, TG-11, and TG- I11 (HRR).

b-

C-

FIG. 6. Lack of competition between TG-I11 and other DNA sequences for binding to FRTL-5 nuclear proteins. The formation of TG-111-protein complexes was assayed as described in the legend of Fig. 5. Lane 1, nuclear extract of the cells grown in 4H medium; lanes 2, nuclear extract from the cells treated with 1 mM 8- bromo-CAMP plus 0.5 mM IBMX. Competitors (all 1000-fold excess): TG-I11 (lane 3 ) , TG-I1 (lane 4 ) , TG-I (lane 5 ) , and somatostatin CRE (lane 6 ) .

As discussed before, HRR also contains a sequence motif very similar to the consensus transcription factor AP-l-binding site (see Fig. 4). To determine whether any of the nuclear proteins interacting with TG-I11 resembles the transcription factor AP-1, we next carried out competition studies using labeled TG-I11 probe, and the 20-bp DNA fragment containing the consensus AP-1-binding site in the human collagenase gene as competitor. As can be seen in Fig. 7, the formation of the DNA-protein complexes a, b, and c, was not prevented by the addition of 200-fold excess of unlabeled AP-1 DNA (lanes 5, Fig. 7), but there was some decrease in the formation of complexes a and c. This partial competition does not appear to be specific, since none of these complexes (a or c) was formed when the 20-bp labeled AP-1 DNA was incubated with FRTL-5 nuclear extracts (data not shown). In fact, only one complex is formed between FRTL-5 nuclear proteins and AP-1 DNA, with a mobility different from that of complexes a, b, and c (data not shown).

Comparison of HRR-Protein Complexes Formed with Nu- clear Extracts of Different Cell Types-To determine whether the proteins forming the a, b, and c complexes are present in


A P - 1

10 50 200X

a-

l 2 3 4 6 FIG. 7. Competition between TG-I11 and AP-1-binding site

for binding to FRTL-5 nuclear proteins. TG-111-protein complexes were assayed as described in the legend of Fig. 5. Lane 1, nuclear extract of cells grown in 4H medium; lanes 2, nuclear extracts from cells treated with cAMP as in Fig. 5. AP-1 DNA as competitor: 10- (lane 3 ) , 50- (lane 4 ) , and 200- (lane 5 ) fold excess over 3ZP-TG- 111.

all CAMP-responsive cells or are specific for thyroid tissues, labeled TG-I11 probe was next interacted with nuclear proteins extracted from HeLa, PC-12 pheochromocytoma or choriocarcinoma JEG-3 cells, and from rat liver or testis. The formation of complex d, which appears to be a nonspecific complex, will not be discussed here. Although three of the nuclear extracts (PC-12 cells, liver and testis) produced some DNA-protein complexes with mobilities similar to those obtained with thyroid nuclear extracts (Fig. 8A, lanes 2-5), none of the nuclear proteins forming complexes in PC-12 cells was induced by cAMP (Fig. 8A, lanes 2 and 3) . In contrast, JEG- 3 nuclear extracts produced only one complex with a mobility different from those of a, b, and c. The amount of the complex formed was also not altered by treatment of the cells with cAMP (Fig. 8B).

DISCUSSION

Although the expression of both the c-fos (Tramontano et al., 1986; Colleta et al., 1987) and TG (Van Heuverswyn et al., 1984; Santisteban et al., 1987; Bone et al., 1986) genes is regulated by TSH and cAMP in FRTL-5 cells, the present studies demonstrate that the rate of induction of these two

genes is markedly different. Like all of the known CAMP- dependent genes, c-fos gene is induced by post-translational modification of the CRE-binding protein (for review, see Roesler et al., 1988). The induction is prompt (within 30 min) and independent of protein synthesis (Nagamine et al., 1983; Colletta et al., 1986; Tramontano et al., 1986; Hofstetter et al., 1987; Hall et al., 1988). In contrast, the rate of synthesis of TG RNA in response to TSH or cAMP is much slower (10 h) than that of c-fos suggesting that the activation of the TG gene by TSH or cAMP may likely require synthesis of new protein factors that interact with the regulatory regions of TG gene. This suggestion is compatible with the demonstra- tion that cycloheximide inhibits the accumulation of TG, but not c-fos, mRNA in response to cAMP or TSH in FRTL-5 cells and that the activity of the nuclear factors interacting with the TG HRR appears to increase following incubation of control cells (4H) with TSH or CAMP.

The deletion-mutation data (Fig. 3) suggest that both TSH- and CAMP-responsive elements are present within a 30-bp region (HRR) located -140 bp upstream of the TG gene transcription initiation site. Although it is now generally accepted that most of TSH action is mediated by cAMP (Field, 1975; Van Herle et al., 1979), it is still not known whether the TSH- and CAMP-responsive elements are in fact the same or the two are different elements overlapping the 30-bp region. It is unlikely that TSH action on TG gene expression occurs through mechanisms not involving cAMP (Piot and Jacquemin, 1980; Dumont et al., 1981), since both TSH and cAMP stimulate TG gene expression with similar kinetics and both increase the levels of the same nuclear proteins forming DNA-protein complexes with HRR (see below). However, resolution of this question requires further studies including mapping of the protein contact sites within HRR.

Other elements that might participate in hormonal regulation of TG gene are located upstream of -170 bp and appear to be involved in the extent of induction of TG gene. Although the exact role of these latter sequences in hormonal respon- siveness remains to be determined, it is possible that more than one region act cooperatively to stimulate TG gene transcription maximally by TSH. Delegeane et al. (1987) have previously identified on the gonadotropin a-gene a sequence element upstream of the CRE that has no independent activity of its own, but in combination with CRE, it markedly increases tissue-specific expression of both the a-gene and a heterologous promoter.

Gel-mobility shift assays revealed the presence of three specific DNA-protein complexes resulting from incubation of labeled HRR with FRTL-5 nuclear extracts (Fig. 5). The levels of the three complexes are increased by TSH and CAMP, thus suggesting that they may be involved in TG gene regulation. The thyroid HRR-binding proteins appear to be related neither to CRE-binding proteins ngr to the transcription factor AP-1. This conclusion is based on the finding that binding of labeled HRR to FRTL-5 nuclear extracts is not prevented by the addition of excess of unlabeled consensus CRE. Furthermore, incubation of labeled AP-1 DNA probe with FRTL-5 nuclear extracts results in the formation of only one DNA-protein complex instead of three with mobility different from those obtained with labeled HRR.

Musti et al. (1987) reported that the DNA element which controls tissue-specific transcription of the rat TG gene are also contained within the same region which are involved in the TSH-dependent stimulation of TG gene expression. How- ever, the role of this region in the TSH- and CAMP-dependent activation of TG gene has not been investigated. It is possible

Thyroglobulin Gene Activation by T S H 7529

FIG. 8. Formation of DNA-protein complexes with labeled TG-I11 and nuclear extracts from different cell types and tissues. A, FRTL-5 cells were grown in the presence of cAMP (lane 1 ); PC-12 pheochromocytoma cells: absence (lane 3 ) and presence (lune 4 ) of CAMP; rat testis (lane 4 ) ; liver (lane 5 ) : and HeLa cells (lane 6). B, FRTL-5 cells: absence (lane 1) and presence (lane 2) of cAMP JEG-3 choriocarcinoma cells: absence (lane 3 ) and presence (lane 4 ) of CAMP.

FRTL-5 P C - 12 TESTIS LIVER HELA CAMP - + - +

a -

b -

c -

- d -

1 2 3

that HRR controls both hormone-dependent and tissue-specific expression of TG gene and that thyroid-specific expression of TG gene is controlled by thyroid-specific proteins that bind HRR. This suggestion will be consistent with the finding that TG gene is not expressed in other tissues lacking the HRR-binding proteins. To investigate this possibility, we next examined the mobilities of HRR-protein complexes formed in nuclear extracts prepared from different cell types and tissues. Of all tissues or cells examined, only nuclear extracts of pheochromocytoma PC-12 cells, and of liver and testis tissues resulted in HRR DNA-protein complexes with mobilities similar to those of thyroid extracts. However, in contrast to FRTL-5 cells, nuclear proteins interacting with HRR are not induced by cAMP in PC-12 cells. Furthermore, the PC- 12 proteins do not seem to play any role in gene regulation since pTGCAT is not expressed in PC-12 cells (data not shown). Whether or not some other cells, which contain DNA- binding proteins similar to those in FRTL-5 cells (e.g. liver), could be induced by cAMP and be involved in the CAMP- dependent activation of other genes containing similar sequence elements as TG HRR remains to be examined. Tissue- specific expression of TG gene and other similarly regulated genes could be achieved by cooperation of HRR and other sequence elements within such a gene. Recent experiments in our laboratory showed that mutations in the HRR abolish TSH-dependent expression of pTGCAT. However, HRR does not appear to be an enhancer and does not activate heterologous promoters in a TSH-dependent manner.’ The results suggest that the action of HRR may require the downstream sequence elements for the TSH-dependent and tissue-specific expression of TG gene. In this regard, it is interesting that both rat (Musti et al., 1986, 1987) and human (Christophe et al., 1985) TG upstream sequence share considerable smilari- ties in sequence in the HRR and also downstream region.

* N.-T. Lee, S. N. Nayfeh, and C.-B. Chae, unpublished results.

’’ 32 P - ‘I’ (; I l l

FRTL-5 JEG-3 CAMP - - + - +

a -

b -

C -

d -

4 5 6 1 2 3 4

The results described in this report suggest that the TSH- dependent activation of T G gene expression in FRTL-5 cells occurs through stimulation of synthesis of TG gene regulatory proteins by CAMP. Although the mechanism of this regulation is still not fully understood, it may involve activation of the genes encoding the regulatory factors by post-translational modification of CRE-binding proteins as other known CAMP- dependent genes including c-fos or through the action of c- fos/AP-1 complex which are also induced by cAMP (Rauscher et al., 1988; Sassone-Corsi et al., 1988).

Acknowledgment-We thank Inhwan Hwang for pLCAT and discussion, Gilbert Vassart for the rat thyroid cDNA, Leonard D. Kohn for FRTL-5 cells, and Dana Fawlkes for synthesis of oligonucleotides. The rat genomic X phage library constructed by T. D. Sargent was obtained through the Reproductive Biology Laboratory of the Uni- versity of North Carolina.

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