The EMBO Journal vol.9 no.3 pp.887-896, 1990

A major thyroid hormone response element in the third intron of the rat growth hormone gene

Jan Sap', Luisa de Magistris, Henk Stunnenberg and Bjorn Vennstrom2 EMBL, Differentiation and Gene Expression Programmes, Meyerhofstrasse 1, D-6900 Heidelberg, FRG 'Present address: Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot-76100, Israel 2Department of Molecular Biology, Karolinska Institute, Box 60400, S-10401 Stockholm, Sweden Communicated by H.Beng

The rat growth hormone (RGH) gene constitutes a welldocumented model system for the direct regulation of transcription by thyroid hormones. In order to analyse its interaction with sequences in the RGH gene, we have overproduced the thyroid hormone receptor-a (c-erbA) protein using a vaccinia virus expression system. The expressed protein bound T3 and DNA-cellulose with expected affinities, and the major binding site for the receptor protein was found to be located in the third intron of the RGH gene. This site displayed significantly higher affinity for the receptor protein than a previously described thyroid hormone response element (TRE) in the promoter of this gene, and also conferred stronger hormone responsiveness in vivo to a heterologous promoter. The data suggest that this novel TRE plays a major role in the regulation of rat growth hormone gene expression by thyroid hormones. Key words: c-erbA / growth hormone / thyroid hormone/ TRE/vaccinia

Introduction Thyroid hormone receptors (TR) with a nuclear localization have now conclusively been identified as members of a superfamily also encompassing the receptors for steroid hormones, vitamin D3, retinoic acid, and possibly other ligands (Sap et al., 1986; Weinberger et al., 1986; Forman et al., 1988; Izumo and Mahdavi, 1988; Evans, 1988; Munioz et al., in preparation). All members of this family contain a characteristic and conserved cysteine-rich domain demonstrated in several instances to be responsible for their DNA-binding capacity, and specificity for cis-acting hormone response elements. This domain is thought to fold into a structure similar to DNA-binding 'fingers' containing zinc atoms, as originally proposed for the eukaryotic transcription factor TFIIIA (Berg, 1986; Evans and Hollenberg, 1988; Freedman et al., 1988). A domain showing a lower degree of conservation between the members of this family, and responsible for binding of the respective ligands, is present towards the carboxy terminus of the receptor molecules, separated from the DNA-binding domain by a non-conserved stretch of amino acids that may act as a hinge between these two domains (for reviews, see Evans, 1988; Oxford University Press

Beato, 1989). The common structure of these receptors, and the successful construction of functional chimaeric receptors (Green and Chambon, 1987) suggest they all function similarly as shown in a detailed manner for the glucocorticoid and oestrogen receptors: i.e. sequence-specific binding to cis-acting regulatory sequences, leading to increased transcription of particular target genes through an as yet poorly understood mechanism. Binding of the hormone has been proposed to play a functional role at the levels of nuclear relocalization, dimerization, DNA-binding and transcriptional activation (Kumar and Chambon, 1988; Webster et al., 1988; Picard and Yamamoto, 1987). Little is known, in contrast to the situation for steroid hormones, about the putative thyroid hormone responsive elements (TREs) that mediate thyroid hormone regulation of gene expression. Difficulties encountered with receptor purification have hampered the analysis of physical interactions between the receptor and DNA. Direct target genes have been hard to identify since many of the effects of thyroid hormone are slow and indirect, and even fast responses have been shown to be blocked by inhibitors of protein synthesis (Oppenheimer and Samuels, 1983; Munioz et al., in preparation). Finally, multiple and often co-expressed forms of TR have been shown to exist, an observation of which the significance remains unclear (Sap et al., 1986; Weinberger et al., 1986; Thompson et al., 1987; Benbrook and Pfahl, 1987; Murray et al., 1988; Izumo and Mahdavi, 1988). Elucidation of the functional differences between these forms will require a detailed analysis of their individual DNA-binding specificities, and their abilities to activate transcription from different promoters. The rat growth hormone (RGH) gene serves as a primary model system for regulation of transcription by thyroid hormones, and primarily functional assays have so far led to the identification of an element in the promoter of this gene capable of directing T3 induction (Reed Larsen et al., 1986; Koenig et al., 1987; Flug et al., 1987; Glass et al., 1987). Although this response element has been the subject of extensive study, conflicting data remain as to its exact boundaries, and more importantly, as to whether it is sufficient to explain the regulation of RGH expression by thyroid hormone in vivo (for review, see Samuels et al., 1988). The TRE in the RGH promoter, or a palindromic derivative of it, has been shown to confer also regulation by retinoic acid and to be related to oestrogen-responsive elements (Glass et al., 1988; Umesuno et al., 1988). The identification and study of other TRE sequences might answer the question as to whether or not these are general phenomena. We have taken advantage of overexpressed chicken c-erbA/TR-ca protein to search systematically for the DNA sequence elements in the RGH gene that specifically bind the a-form of the thyroid hormone receptor, and subsequently asked whether the identified elements are able to function as TREs in vivo. The chicken a-receptor was

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expressed at high levels in eukaryotic cells using a vaccinia vector, allowing the preparation of crude nuclear extracts highly enriched in receptor protein that binds T3 as well as DNA. The approach solves the problems associated with receptor purification from tissues, such as limited availability and the associated ambiguity due to the co-occurrence of multiple receptor forms in one tissue or receptor preparation (Murray et al., 1988). A similar approach has recently led to the identification of a TRE in the LTR of Moloney murine leukemia virus (MoMuLV) (Sap et al., 1989). The data presented here show that the T3-receptor protein preferentially binds to a previously undetected site in the third

Fig. 1. Expression of the c-erbA protein in HeLa cells using a vaccinia virus vector. Left panel: Coomassie blue stained gel of nuclear extracts prepared from cells 24 h after infection. Lane a: uninfected cells; lane b: wt (control) vaccinia virus; lane c: c-erbA expressing virus. Right panel: immunoprecipitation from total [3 S]-methionine-labeled cells 24 h after infection. Lane a: marker proteins; lane b: wt virus infected cells, pre-immune serum; lane c: wt virus infected cells, anti-erbA antiserum; lane d: as lane c, 10-fold lower amount loaded; lane e: c-erbA recombinant virus infected cells, pre-immune serum; lane f: c-erbA recombinant virus infected cells, anti-erbA antiserum; lane g: as lane f, 10-fold lower amount loaded.

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Results High-level expression of the chicken c-erbA protein In all species examined (Sap et al., 1986; Thompson et al., 1987; Benbrook and Pfahl, 1987), the nucleotide sequence preceding the c-erbA/TR-a translation initiation codon strongly deviates from a consensus sequence for efflcient initiation of translation (Kozak, 1986). Furthermore, the RNA leader sequence in the c-erbA mRNA, containing multiple short open reading frames terminated by stop codons, also negatively interferes with translation. In order to remove these constraints on expression, we chose to modify appropriately the chicken c-erbA cDNA at this position, using site-directed mutagenesis to change the environment of the ATG initiator codon into a sequence more related to the Kozak consensus. In addition, a new restriction site was inserted upstream of the coding sequence so as to be able to remove all non-coding leader sequence from the cDNA, while leaving the coding sequence intact (see Materials and methods for details). The resulting modified cDNA (without the leader sequence) will be referred to as CEA-ll. The c-erbA coding sequence was subsequently introduced into a vaccinia virus vector, in order to obtain amounts of authentic thyroid hormone receptor protein sufficient for biochemical analysis. The resulting recombinant virus was able to direct the synthesis of high levels of c-erbA protein in HeLa cells, as illustrated in Figure 1. A Coomassie blue stained gel (left panel) of a high salt nuclear extract prepared from cells 24 h after infection shows the presence of one additional band of 46 kd (lane c) as compared to cells infected with wt virus (lane b), representing 1-2% of total protein in the nuclear extract. An abundant 46 kd protein could also be immunoprecipitated using anti-erbA antisera from cells infected with -

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Fig. 2. Characteristics of the vaccinia-expressed c-erbA protein. a: Scatchard analysis of T3 binding to nuclear extracts prepared from cells infected with the c-erbA expressing vector, as well as to control extracts from wt vaccinia virus infected cells. All points from the binding analysis to wt (control) extracts are superimposed at the origin of the graph. Binding was measured as described (Sap et al., 1986) except that 0.1 mg/ml BSA was included in the binding reactions (0.35 jig protein extract, equivalent to 0.5 x 104 cells in a total volume of 100 1u). b: DNA-cellulose binding of the T3 receptor expressed using the vaccinia vector, as monitored by the release of receptor-associated radiolabeled T3. After binding, the matrix was washed extensively with binding buffer and subsequently eluted stepwise with the same buffer containing increasing KCI concentrations. Curve I: DNA-cellulose; curve H: normnal cellulose; curve m: binding of an equivalent amount of labeled T3 to DNA-cellulose in the absence of added nuclear extract.

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the c-erbA expressing virus only (Figure 1, right panel). The total amount of c-erbA protein produced, judged from the Coomassie blue stained gels, amounts to - 1 Ag recombinant protein per 106 infected cells, a level within the range to be expected with this expression system. The c-erbA protein present in nuclear extracts from the vaccinia virus infected cells was subsequently tested for its

ability to bind T3, as well as DNA in a non-specific manner. As expected, Scatchard analysis of T3 binding to the nuclear extracts (Figure 2a) demonstrated high-affinity T3 binding activity in nuclear extracts from cells infected with the recombinant virus. No T3-binding activity was detectable in extracts from wt vaccinia virus infected cells under these conditions. The dissociation constant (Kv =

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Fig. 3. Co-immunoprecipitation of RGH gene fragments with the c-erbA protein. a: a plasmid containing a 5.8 kb genomic clone of the RGH gene (Barta et al., 1981) was restricted with Bgml, KpnI and XhoI, the fragments end-labelled with Klenow enzyme and incubated with wt (control) extract (lanes 1), or c-erbA containing extract (lanes 2) in the presence of different amounts of competitor DNA. Complexes were then precipitated with control (pre-immune; left panel) or anti-erbA (right panel) antibodies bound to Staph.A. Lane M: total input DNA for the assays; left panel: preimmune serum; right panel: anti-erbA antiserum (veA2, Goldberg et al., 1988). Cold competitor DNA used (f.c.): a, none, b, 100 ng/gl poly[d(IC)]; c, 20 ng/yl calf thymus DNA; d, 75 ng/ul calf thymus DNA. The location of the 630 bp (containing the c-erbA binding site described here) and 240 bp (promoter) fragments within the RGH gene is indicated in Figure 9a. b: co-immunoprecipitation assay as in a, but using pTZ19R (panel A, left), or equal amounts of the same plasmid containing the subcloned 630 bp BglJI-KpnI RGH fragment (pRGH630) (panel B, right), as cold competitor (instead of calf thymus DNA). Amounts of competitor used: lane 1, no competitor; 2, 50 ng; 3, 200 ng; 4, 400 ng; 5, 800 ng; 6, 1600 ng. Poly[d(I-C)] was present in all reactions at 0.1 mg/ml. Lane 0: total input DNA for the assays. Part c: localization of the c-erbA binding site within the 630 bp RGH fragment to a 200 bp XoII subfragment spanning the third intron of the gene. The 630 bp Bgll-KpnI fragment detected in previous assays was subcloned between the BamHI and KpnI sites in the polylinker of pTZ19R. The resulting plasmid, pRGH630, was restricted with XhoII, the fragments end-labeled using Klenow enzyme and subjected to co-immunoprecipitation analysis after binding to c-erbA containing nuclear extract. Increasing amounts of calf thymus DNA were used as unlabeled competitor (lanes a, none; lanes b, 10 ng/1l; lanes c, 20 ng/gl; lanes d, 40 ng/1l), and the bound complexes precipitated using pre-immune (lanes 1) or anti-erbA (lanes 2) serum. Poly[d(I-C)] was present in all reactions at 0.1 mg/ml. The 410 and 200 bp fragments are derived from the RGH gene, others are vector plasmid fragments.

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0.1 nM) agrees well with published data on the nuclear thyroid hormone receptor present in a wide variety of tissues (Oppenheimer and Samuels, 1983), and is slightly lower than that measured for the chicken c-erbA/TR-a protein produced by in vitro translation (Kd = 0.3 nM, Sap et al., 1986). From the Scatchard analysis, it can be calculated that 2 x 106 active T3 receptors are present per cell (assuming a 1:1 stoichiometry of binding of the hormone to the receptor), corresponding to a 100- to 1000-fold overexpression compared to the normal cellular T3 receptor levels (Oppenheimer, 1979). The specific activity of active receptor in the extracts is thus 30 fmol/4l or 43 fmol/,tg protein. Assuming (from the Coomassie blue stained gel) that the c-erbA protein represents 2% of the total protein in the extracts, it can be estimated that 10% of the total c-erbA protein in the extracts binds T3. Furthermore, DNA -cellulose chromatography shows that the c-erbA protein (as monitored by associated radiolabelled T3) was retained on DNA -cellulose but not on control cellulose, and could be eluted by salt concentrations between 0.1 and 0.25 M KCI (Figure 2b). -

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Detection of a c-erbA binding site in the third intron of the rat growth hormone gene The availability of nuclear extracts highly enriched in thyroid hormone receptor protein provided us with the opportunity to identify TREs on the basis of their expected interaction with the receptor. For all experiments described we used the crude nuclear extracts prepared from either c-erbAvaccinia infected cells or, as a control, similarly prepared extracts from cells infected in parallel with wt vaccinia virus. We directed our attention to the RGH gene since transcription of this gene is well documented to be directly controlled by thyroid hormones through a nuclear receptor. Furthermore, the existence of one or possibly several thyroid responsive elements in the promoter region of the RGH gene has been reported (see references above). In order to extend our analysis of sequence-specific binding by the c-erbA protein to the entire RGH gene, a plasmid clone representing the RGH gene was cleaved by a set of restriction enzymes, and the resulting mixture of fragments, after end-labelling

with Klenow enzyme, used for co-immunoprecipitation experiments. To this purpose, the mixture of RGH gene fragments was incubated with control (wt vaccinia infected cells) or c-erbA containing nuclear extracts in the presence of increasing amounts of unlabelled poly[d(I-C)] or calf thymus DNA as competitor, and subsequently subjected to immunoprecipitation using either anti-erbA or pre-immune serum. Figure 3a shows an analysis of the co-precipitating fragments under various conditions of stringency. It can be seen that, using anti-erbA antiserum, a 630 bp (Bglll-KpnI) fragment was preferentially precipitated from c-erbA containing extracts at the higher competitor DNA levels, suggesting a preferential binding of the c-erbA protein to sequences present in this fragment (Figure 3a right panel, lanes c-2 and d-2). This enrichment was not observed when wt extracts (lanes 1) or pre-immune serum (left panel) was used as a control. In these cases, larger fragments were preferentially precipitated, probably due to non-specific adsorption of the larger DNA fragments to the Staph. A beads used for the immune precipitation. The 630 bp fragment contains sequences internal to the gene, starting in the second intron, and ending in the fourth exon. Only 890

little enrichment could be observed of the 240 bp (Bglll-XhoI) fragment that contains 230 bp of sequence upstream of the cap site of the gene (a schematic representation of the RGH gene with restriction sites and fragments used is shown in Figure 9a). The specificity of this effect was confirmed by performing a similar co-immunoprecipitation experiment, but using as unlabelled competitor DNA either a vector plasmid (pTZ19R), or the same plasmid containing the subcloned 630 bp fragment that was preferentially precipitated (pRGH630). The plasmid containing the insert competed for binding to the c-erbA immune complex with an - 10-fold higher efficiency (Figure 3b). For proper interpretation of this difference, it has to be borne in mind that any putative binding site for the c-erbA protein present in the competitor plasmid would be expected to be vastly underrepresented with respect to non-specific low-affinity sites that make up the majority of the plasmid DNA. The localization of the expected c-erbA binding site within the 630 bp fragment was mapped further by performing co-immunoprecipitation analysis on the subcloned fragment. Under these conditions the preferential precipitation was more pronounced, and was limited to a 200 bp XhoII fragment that encompasses the third intron of the gene (Figures 3c and 9a). A DNase I footprinting experiment (Figure 4) confirmed the presence of a discrete binding site for the c-erbA protein within sequences contained in the fragment detected by preferential immunoprecipitation. Protection was observed on both strands over a length of -40 nucleotides in the presence of c-erbA containing extract, but not in the presence of a control extract. Analysis of the protected sequence reveals that it contains an imperfectly repeated sequence of 14 bp, the two repeats being separated by (and partially overlapping) a 15 bp near perfect palindrome (see Figures 7 and 9b). To study in detail the interaction between the nuclear protein in the extracts and the sequences in the region protected from DNase I digestion, methylation interference assays were done for both strands. Figure 4C shows that binding was prevented by methylation of the two Gs on the upper strand in a GGTCA motif (left panel, bold) and a G on the lower strand in the complementary sequence TGACC (right panel, bold). The GGTCA motif was found in the second repeat of the protected 40 nucleotide region.

Binding of the c-erbA protein to the third intron binding site is specific and occurs with higher affinity

than to a TRE in the promoter of the RGH gene In order to assess the specificity of receptor binding to the site in the third intron, complex formation between the c-erbA protein and the site in the third intron was monitored

by non-denaturing polyacrylamide gel electrophoresis (Figure 5). Using an RGH gene fragment containing the third intron binding site as a probe, several discrete retarded bands

could be observed in the presence of c-erbA containing extracts, whereas no retardation was seen with wt (control) extract under these conditions (Figure 5, lanes 1 -3). Titration with increasing amounts of c-erbA extract revealed that, at higher concentrations of added extract, all of the probe accumulated into the slowest migrating form, suggesting that complex formation may involve the binding of multiple c-erbA proteins to the fragment containing the

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recutting with Aval (in the insert, see Figure 9a)]. For footprinting. the resulting 350 bp fragments by gel electrophoresis. incubated or not with wt (control) or c-erbA containing nuclear extract, subjected to limited DNase I digestion and analysed on denaturing polyacrylamide gels. Methylation interference was examined by methylation of the labelled fragment followed by incubation with c-erbA extract, separation of free and complexed fragment by band shift analysis, and analysis of methylated guanosines on denaturing gels. Panel A: labeling using Klenow enzyme (upper strand). Lane M. Maxam-Gilbert G+A reaction; lane a. no extract added: lane 1. 10 jig wt (control) extract added; lane 2, 10 jig c-erbA extract. Panel B; labeling using T4 polynucleotide kinase (lower strand). Lane a, no extract; lane (1), 3 Mig wt (control) extract; lane (2), 3 Mig c-erbA extract; lane 1, 10 Mig wt (control) extract; lane 2, 10 Mig c-erbA extract. For clarity, the protected sequence shown represents the upper strand. Panel C; methylation interference assay on the upper and lower strands (left and right). Lanes t, no separation of bound and free fragment; lanes f, free fragment; lanes s, shifted fragment. kinase [after alkaline labeled

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Fig. 6. Gel retardation experiment comparing complex formation between c-erbA protein containing extracts and (panel A) a RGH promoter fragment (230 bp upstream of the cap site; Bgl-XoI), or (panel B) a fragment encompassing the preferential c-erbA binding site in the third intron described in the text (StuI-KpnI). Nuclear extracts from wt (lanes 2 and 4) or c-erbA recombinant (lanes 3 and 5) vaccinia virus-infected cells were incubated with the respective endlabelled fragments in the absence (lanes 2 and 3) or presence (lanes 4 and 5) of poly[d(I-C)] as non-specific competitor DNA (0.1 mg/ml f.c.). Complexes were then analysed by non-denaturing polyacrylamide gel electrophoresis (lanes 1: no extract added).

Fig. 5. Gel retardation assay showing competition of various unlabeled oligonucleotides for complex formation between c-erbA containing extracts and a labeled DNA fragment (StuI-KpnI) containing the c-erbA binding site in the third intron. A 100-fold molar excess of each oligonucleotide was used for the competition. See the text and Figure 7 for description of the oligonucleotides.

binding site (data not shown). Competition experiments (Figure 5; lanes 4-9) in which double-stranded synthetic oligonucleotides corresponding to the entire protected sequence (or parts of it) were used as competitors showed that the complex formation observed was specific, and not an artefact of non-specific DNA-binding affinity due to the high concentration of c-erbA protein in the reactions. Complex formation could be competed by an excess oligonucleotide of the complete DNase I protected sequence (C) and by an oligonucleotide corresponding to the second repeat only (A) but not by an oligonucleotide encompassing another fraction of the protected site, the central sequence with a palindromic character (P) (see also Figure 7 for a schematic representation of the oligonucleotides used). Moreover, an oligonucleotide (SAL) representing a thyroid responsive element in the LTR of MoMuLV (Sap et al., 1989) also efficiently competed, whereas one corresponding to the consensus for another DNA-binding protein, AP-1, did not. An oligonucleotide (PRO, partially homologous to the two repeats) corresponding to the RGH promoter TRE (Glass et al., 1987) competed, but inefficiently. In the co-immunoprecipitation experiments using the entire RGH gene, only weak retention of the 240 bp (BglH -XhoI) RGH promoter fragment containing the RGH promoter TRE (Glass et al., 1987) could be detected. Due to the nature of the assay used, the presence of a c-erbA binding site with higher affinity could be expected to mask binding to any lower affinity sites. We therefore also directly tested in a gel retardation assay the binding of the c-erbA protein to the promoter fragment, so as to assess more clearly the relative affinities of both sites for the receptor (Figure 6). Equal amounts of labeled fragments corresponding to sequences in the promoter of the RGH gene (Figure 6, panel 892

A), or containing the binding site in the third intron (Figure 6, panel B), were allowed to bind in parallel to c-erbA or control extracts. The promoter fragment showed three retarded bands with the control nuclear extract, and indeed an additional retarded band with the c-erbA protein. This complex migrates at a similar position to that observed with the intron fragment but is markedly less abundant, suggesting a lower affinity. Further dissection of the c-erbA binding site

The above competition results (Figure 5) already suggested that critical determinants for c-erbA binding must be localized in the second repeat (oligonucleotide A), and that the central palindrome (oligonucleotide P) plays a less crucial role. In agreement with this, a gel retardation experiment in which labeled oligonucleotides themselves were used as probes showed that sequences corresponding to the second repeat within the DNase I protected sequence (oligonucleotides B and T), but not the first (M), can form a complex with the receptor that is stable enough to be detected by gel retardation, whereas the central palindrome (P) does not (Figure 7). Furthermore, oligonucleotide A, although it can compete for complex formation, did not show binding under these conditions, indicating that sequences at the 5' of the repeat (possibly the AGGC motif present in both repeats) contribute to high affinity binding. Taken together, the results indicate that the major determinants for thyroid receptor binding are located in the second repeat, with no detectable role for the central palindrome, a result in agreement with the homology between the repeated sequences and the TRE in the promoter of the RGH gene (see Discussion and Figure 9b). Hormone responsiveness of the intron TR binding site Next, we assessed the ability of the intron binding site to function as a TRE in vivo, and its ability to mediate hormone induction as compared to the TRE in the RGH promoter under the same conditions. For this purpose, the C oligonucleotide as well as an oligonucleotide (PRO) corresponding to the previously described TRE in the promoter of the RGH gene (- 185 to - 157, Glass et al., 1987) were cloned in

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