Vol. August

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COMMUNICATIONS Pages

1167-1175

RAT LIVER c-erb A gl THYROID HORMONE RECEPTOR IS A CONSTITUTIVE ACTIVATOR IN YEAST (Saccharomyces cerevkiae): ESSENTIAL ROLE OF DOMAINS D,E AND F IN HORMONE-INDEPENDENT TRANSCRIPTION Hiroyuki Thyroid

Received

OHASHI,

Yong-Fan YANG, and Paul G. WALFISH’

Research Laboratory, Samuel Lunenfeid Research Institute of Mount Hospital, University of Toronto, 600 University Avenue, Toronto, Ontario, Canada M5G 1X5 June

28,

Sinai

1991

SUMMARY: To assess thyroid hormone receptor (TR)-mediated activation of transcription in yeast in the presence or absence of thyroid hormone (T3), we developed a co-expression system using a TR+l expression vector and a reporter plasmid containing a 16 base pair palindromic thyroid hormone response element (TRE) upstream from a proximal CYCl promoter that was fused to the 8-galactosidase lac Z gene of Escherichia colt’. Although TRgl functions as a repressor in most mammalian systems, using our system we observed a unique thyroid hormone-independent transcriptional response indicating that wild TR-~1 acted as a constitutive activator in yeast; the addition of 1pM T3 induced a moderate but significant (p < 0.01) 25-40% further increase in transcriptional activity. Using a series of rat TR-,.91 mutant constructs, we found that deletion of domain D and portions of E completely eliminated transcriptional activity, whereas truncations of domain F and E permitted a partial (20-40%) response compared to wild TR+l in the presence or absence of T3. These observations indicate that TR-~1 functions as an activator in yeast and that domains D,E and F play important interactive roles in its hormone-independent gene activation with the D domain likely being the most essential. Furthermore, our results suggest that the different transcriptional property of TR-~1 in yeast compared to mammalian cells i.e. activator vs repressor function, is likely determined by transcriptional factor differences which are dependent upon cellular origin. 0 1991Academic Press, Inc.

It has been established that the intracellular action of steroids and thyroid hormones are mediated by their ligand modulated nuclear hormone receptors (1). However, the precise nature of the interactions between thyroid hormone receptor (TR) and its response elements (TRE)

in the activation or repression of transcription

remains to be elucidated

(2,3). Several members of the steroid receptor superfamily e.g. vitamin D (4), glucocorticoid (5), estrogen (6), progesterone (7) have been expressed in yeast (Sacchromyces cerevisiae); the activation of these reporter genes occurs primarily in the presence of their ligands. Recently, our laboratory (8) has shown that rat liver TR-~1 can be expressed in yeast using a copper-responsive metallothionein promoter and ubiquitin-fusion protein technology (4,7). * To whom reprint requests should be addressed.

1167

0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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In the present report, we describe results on the activation of the rat TR-,91 gene in a yeast co-expression system in the presence or absence of thyroid hormone. Although in most mammalian cells TR acts as a repressor of transcription in the absence of hormone (912), we unexpectedly observed a unique hormone-independent

transcriptional

effect. To

further investigate this phenomenon, we used several truncated or deleted TR-~1 mutants to determine the role of domains D,E and F of the TR+l in regulating hormoneindependent

gene activation.

Our observations suggest that the function of TR+l

constitutive activator in our system was likely mediated by transcriptional

as a

factors specific

for yeast cells. MATERIALS

AND METHODS

Materials: Rat c-erb A ~1 cDNA in pTZ19R (13) vector was kindly provided from Dr. H.C. Towle (MN, USA). A polylinker plasmid Pmnhub-poly 5253, the ubiquitin expression plasmid YEp 46, a yeast reporter plasmid pC2, yeast Scerevisiae strain F762 (4) were kindly gift from Dr. T.R. Butt (Smith Kline & French Lab.,PA,USA). An antibody against C-terminal of human c-erb A protein (C-91) was kindly provided from Dr. S-Y. Cheng (NIH, Bethesda, Maryland, USA). Restriction enzyme, Nuclease BaZ 31, T4 DNA ligase and Pst I and Hind III linkers were purchased from Promega Biotec and Boehringer Manheim Biochemicals. [lsI]T3 (2200 Ci/mmol) was purchased from Du Pont-New England Nuclear. L-tryptophan, uracil, L-3,3’,5triiodothyronine (T3), L-thyroxine (T4), 3,3’,5triiodothyroacetic acid (TRIAC), L-3,3’,5’-triiodothyronine (rT3), glass beads (425600 microns) and o-nitrophenyl+D-galactopyranoside (ONPG) were purchased from Sigma Chemical Co. Wild-type TR$l Expression Vector and Preparation of Mutants: As shown in Fig 1 A, c-erb A gl cDNA was inserted just downstream of ubiquitin and CUP 1 promoter as described previously (8). To obtain truncated mutants, the Pst I site of rat thyroid hormone cDNA was cleaved and then digested with BaZ 31. After digestion blunted fragments were ligated with Pst I linker. Another mutant was derived by deletion of Nlte I fragment, followed by ligation with T4 DNA ligase. These truncated or deleted fragments were inserted into a YEp 46 expression plasmid as described previously (8). The prepared

A

Af I ! I

/Hind

III

6

Ncol I

& Schematic Outline of Expression Plasmids (A) Construct of Yeast Expression Vector, YEp-TRBl. The c-e& A gl cDNA was inserted

a ubiquitin expression plasmid YBp-46 which are responsive to copper sulfate. CUP1 is the yeast metallothionein promoter. ‘IRPl is the tryptophan selective marker. c-e& A gl is rat thyroid hormone receptor gl as described by Murray et al. (13). UB: ubiquitin. (B) Construct of Yeast Reporter Plasmid, YRpTRl. Two sequences of synthesized palindromic TRE were inserted into the upstream of proximal CYCl promoter. lac 2: structure gene of E. cofi with ,+galactosidase activity. URA3: selective marker of uracil. 1168

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truncated and deleted TR-~1 expression vector constructs were as follows: YEp-T424, a(Met425-Asp456); YEp-T414,~(Lys~l’-Asp~~); YEp-T324,~(Leu~~-Asp~~); YEp-TR-pD A(kdal -Gly380) and illustrated schematically in Fig 2A. Mutants were confirmed by DNA sequencing. These vectors were amplified in E.coli and purified before transformation to S. cerevisiae. Transformants were selected by tryptophan auxotrophy. Construction of Reporter Plasmid: Two copies of the palindromic TRE (5’TCAGGTCATGACCTGA-3’) as described by Glass et.af (15) was inserted into X/m 1 sites upstream from a proximal CYCl promoter of yeast reporter plasmid pC2 that was fused to p-galactosidase gene of E.coli. The resulting reporter plasmid containing palindromic TRE referred to as YRpTRl (Fig 1 B). Transformants were selected by uracil auxotrophy. Hormone Binding Studies: T3 binding studies were performed as described previously (8). In brief, yeast extracts were incubated at 4 C for 18 hours with [12’I]T3 (InM) in the absence or presence of a l,OOO-fold excess of unlabelled T3, followed by separation of free and bound [lz I]T3 using membrane filtration method (11). Non-specific binding was determined in the presence of a excess of unlabelled T3 and subtracted from total binding to calculate the specific [125I]T3 binding (16). The results were expressed as specific binding (metiSD, cpm/lOOpg protein). Transcription Assays: Yeast (S. cerevisiae) containing a receptor vector (TR-~1 or mutant) and the reporter plasmid YRpTRl was grown in minimal medium devoid of uracil and tryptophan. After enrichment of yeast culture medium to reach a cell density 0.5 at OD,,, T3 or one of several thyroid hormone analogs was added in the presence or absence of 1OOpM copper sulfate. After 4 hours of incubation, cells were harvested and washed with phosphate buffered saline (PBS). The pellet was suspended in Z buffer (0.1 M sodium phosphate buffer containing 0.01 M KCl, 1mM MgSO,, 0.05 M bmercaptoethanol) and lysed with glass beads (425-600 micron). After centrifugation (5000 rpm), the supernatant (1Opg of protein) was assayed for p-galactosidase activity as described by Miller (18) and the enzyme activity was expressed as meatiSD, Miller units per mg protein. Protein concentration was measured by Lowry’s method (19) using bovine serum albumin as a standard. Western blotting: Yeast extracts were resolved on 12% SDS-polyacrylamide gel. Subsequently, protein was transferred to nitrocellulose filters and affinity purified antibody against C-terminal of c-erb A protein (C-91) was used as described by Fukuda et al. (14) for performing Western blotting analyses. RESULTS

AND DISCUSSION

To assess the function of thyroid hormone receptors in S. cerevisiae, we performed studies involving co-expression of a TR-~1 expression vector and a reporter plasmid. In such previous studies using mammalian constitutive

cell systems, it was found that TR had functioned as a

repressor and T3 ligand binding relieved this repression (9-12). Unexpectedly

(see Table lA), we observed that YEp-TR@l

transcriptional

activity in the absence of added

T3 hormone was 15-fold higher (~~0.01)

than YEp-46 (control vector). Although

our

laboratory had previously shown that TR+l expression was augmented in yeast by 100pM copper sulfate (8), a slightly reduced (but not statistically significant) effect on Bgalactosidase transcriptional activity occurred in the presence of copper sulfate in this system (see Table 1A). Addition of 1pM T3 to wild-type TR+l 25-40% further increase in transcriptional concentration

induced a moderate but significant (p < 0.01) activity (see Table 1A and Fig 2B). At a final

of lpM, the relative potency of several thyroid hormone analogs was: TRIAC 1169

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Table IA Effects of T3 and Copper Sulfate on a Reporter GeneActivation by TR-~1 in Yeast

Vector

T3

cuso4

fl-Galactosidase Activity

(bW

(100rW

(Miller units/mg protein) 163+15

YEp-46 (control)

2,610&!60a

YEp-TRpl +

YEp-TRfil

3,340+260a*b

YEp-TR,H YEp-TRpl

+

+

2,510~400as

+

3,040f400~‘~

The T3 receptor ~1 expression vector YEp-TRpl or control vector YEp-46 was cotransfected with a reporter plasmid YRpTRl containing two copies of palindromic thyroid response element (TRE) upstream from a CYC 1 promoter. Transcriptional activities (agalactosidase activities) were obtained for 4 hours of culture in yeast. Statistical analysis was performed by paired Student’s t test and data were expressed mear&iD of 5 different experiments. a, ~~0.01, vs YEp-46 (control). b, ~~0.01 vs YEp-TR,gl, T3(-), C&O,(-). c, no significant, vs YEp-TRpl, T3(-), CuSO,(-). d, p< 0.01, vs YEp-TRpl, X3(-), CuSO,( +).

= T3 > T4 > rT3 (see Table 1B). These results are in accordance with the anticipated analog binding affinities to rat TR$l as previously reported (13). When other members of the steroid receptor superfamily have been studied in a yeast co-expression system, they were found to function primarily as constitutive repressors in the absence of hormone (4-7). The fact that TR-~1 functions as an activator in a yeast co-expression system, that is, with significant gene activation in the absence of ligand, is of obvious interest.

Our observation that TR-~1 is a constitutive

activator in yeast is in

agreement with a recent report by Privalsky et al. (12) who observed an identical phenomenon in yeast for not only human TR-81 but also the v-erb A oncogene. We have now extended the investigation

of the activator function of TR-~1 by assessing the possible

structural role of D,E and F domains and its mutants

(see Fig 2A)

in mediating

gene

activation in the presence or absence of thyroid hormone. Western blotting was performed using a C-terminal directed polyclonal antibody of c-et-bA (14) for measurement of wild and mutant TR+D expression. A single 52 Kd band was obtained for TR-~1, and 33Kd for TR,8D, in accordance with their predicted molecular weights (see Fig 3). The results of the above studies (meatiSD, Miller units/mg protein) are summarized in Fig 2B. Compared to the .YEp-46 control (163?15), the wild YEp-TRpl had marked (p