GAL4 activates gene expression in mammalian cells

Cell, Vol. 52, 161-167,

January

29, 1988, Copyright

GAL4 Activates

0 1988 by Cell Press

Gene Expression

Hit&hi Kakidani’ and Mark Ptashne Department of Biochemistry and Molecular Harvard University Cambridge, Massachusetts 02138

Biology

in Mammalian

Cells

does not. This activation was observed only with a promoter bearing a GAL4 binding site upstream of the transcriptional start site. We also describe a synergistic effect of the GR and GALd The accompanying paper by Webster et al. (1988) also shows that GAL4 activates transcription in mammalian cells.

Summary Results GAL4, a protein that activates transcription in yeast, is shown to activate the mouse mammary tumor virus promoter in mammalian cells. Activation depends upon a GAL4-binding sequence inserted upstream of the gene. Deletion mutants of GAL4 bearing one or both of the %ctivating regions” required for activation in yeast also activate transcription in mammalian cells. A derivative of GAL4 that blnds to DNA but cannot activate transcription in yeast also fails to activate transcription in mammalian cells. We also show that GAL4 and the glucocorticoid receptor activate the mouse mammary tumor virus promoter synergistically.

GAL4 is a sequence-specific DNA-binding protein that activates transcription in the yeast Saccharomyces cerevisiae. The proteinrecognizes four sites in the galactose upstream activating region UASo (Guarente et al., 1982; Yocum et al., 1984; West et al., 1984; Johnston and Davis, 1984; Bram and Kornberg, 1985; Giniger et al., 1985), and a near consensus of the naturally occurring sites, the “17-mer,” will also mediate GAL4’s stimulatory activity (Giniger et al., 1985). The part of GAL4 required for DNA binding is separable from the part(s) that, once tethered to DNA, activates transcription (Brent and Ptashne, 1985; Keegan et al., 1986): the fragment GAL4(1-147) which bears the first 147 amino acids of this 881 amino acid protein, binds to DNA but does not activate transcription, whereas eitherof two other segments (amino acids 148238, region I; and amino acids 768-881, region II) stimulates transcription when attached to a DNA-binding fragment (Ma and Ptashne, 1987a). The glucocorticoid receptor (GR), also a sequence-specific DNA-binding protein (Payvar et al., 1983; Scheidereit et al., 1983) activates transcription of mammalian genes (Karin et al., 1984; Renkawitz et al., 1984; Slater et al., 1985; Yamamoto, 1985; Miksicek et al., 1986; Jantzen et al., 1987). For example, in the presence of dexamethasone, GR binds to several glucocorticoid-responsive elements (GREs) upstream of the transcription start site in the mouse mammary tumor virus (MMTV) promoter and activates transcription (Scheidereit et al., 1983; Yamamoto, 1985; Chandler et al., 1983). In this paper we show that GAL4 and two deletion derivatives bearing one or both activating regions attached to GAL4(1-147) activate the MMTV promoter in mammalian cells, but GAL4(1-147) * Present address: Biotechnology ration, 2743-l Hayakawa, Ayase,

Research Laboratory, Kanagawa. Japan.

Tosoh Corpo-

Plan of the Experiment In a typical experiment we cotransfected mammalian cells with two plasmids: an ‘effector” plasmid that directs synthesis of GAL4 or one of its derivatives (see Figure lA), and a “reporter” plasmid that bears a UASo or a 1Fmer upstream of the start site of the MMTV promoter (see Figure 1B). The chloramphenicol acetyltransferase (CAT) gene, whose protein product is easily assayed (Gorman et al., 1982), was present just downstream of the transcription start site in each reporter plasmid. For one set of experiments the GREs were deleted from the MMTV promoter, and in another set these sites were present in addition to the GAL4 binding sites. For all transcription experiments we used the Chinese hamster ovary (CHO) cell line (Kaufman et al., 1985); these cells synthesize no GR, and so in experiments exploring the effects of this receptor we used a derivative of CHO stably transfected with a plasmid (Miesfeld et al., 1986) that directs constitutive synthesis of receptor. In yeast, transcriptional activation by GAL4 (although not DNA binding) depends on growth in galactose because galactose dissociates the inhibitor GALBO, which otherwise covers the activating surface(s) (Perlman and Hopper, 1979; Oshima, 1982; Johnston et al., 1987; Ma and Ptashne, 1987b); in experiments reported in this paper no GAL80 was present, and so galactose was not required for transcriptional activation by GAL4. Mammalian Gene Activation by GAL4 The experiments of Figures 2 and 3 show that an MMTV promoter in which the GREs indicated in Figure 1 were replaced by UASc, was activated by GAL4. Two deletion derivatives of GAL4 that activate transcription in yeast were also active; one (GAL4[1-147,788-8811) contains activating region II attached to the DNA-binding fragment, and the other (GAL4[1-238, 768-8811) contains both activating regions I and II attached to the DNA-binding fragment. Thus, cells transfected with a reporter plasmid encoding GAL4 or one of its active derivatives synthesized CAT at a level at least 20-fold above background (Figure 2, lanes 8, 10, and 11). In contrast, no detectable CAT activity was elicited by GAL4(1-147) (Figure 2, lane 9) nor by transfection with the reporter plasmid alone (lane 1). In the experiment shown in Figure 2, the GAL4 derivative bearing both activating regions directed more CAT activity than did the other two active GAL4 species (compare lanes 8, 10, and 11). In other experiments we usually detected no differences between the two GAL4 deletion derivatives.

A AdMLP pUC18

phAT2nvl4

1

I

fiEcoRI GAL4

pAG4

SV40 polyA DHFR

sequence

D --

I

-II

1-881 D

pAG147

1-147

pAG236

1-147,

768-881 D I= m--

pAG242

l-238,

B

768-88

1000

1

stu I

Sac I I

I

CAP GRE NFI 1

rrnnl

MMTV-LTR

pGMCS

bD

Pvu II: 1 b

I

I

CAT

TATA

pGMCSl17 pGMCS/UAS

I

pGMCSnGRE

pGMCSnGRE/UAS

UASG

N l&l

I

n nl

1. Structures

of the Effector

and Reporter

I 200 bp

.--& m

Figure

*

IlIlt1

Plasmids

(A) Effector plasmids. encoding GAL4 and various GAL4 derivatives. DNA fragments encoding GAL4 and its derivatives were inserted into the EcoRt site of a plasmid (pMT2AVA) that expresses the mouse dihydrofofate reductase (DHFR) gene driven by the adenovirus major late promoter (WMLP). The plasmid bears other components as shown, including the tripartite leader sequence from the AdMLP (dotted box) and the intron sequences from the immunoglobulin heavy chain gene (filled box). The stippled bars below denote the GAL4-derived coding sequences present on each plasmid. D refers to the DNA-binding region (residues 1-147); I and II are the two activation regions (residues 146-236 and 766-661). (6) Reporter plasmids. bearing the CAT gene fused to the MMTV promoter. The parentaf plasmid (pGMCS) beam the CAT gene fused downstream of the MMNLTR, which includes two GREs and a NFl binding site. Two additional GR binding sites lie between the Sac1 site and TATA, one of which overlaps the NFl site (Scheidereit et al., 1983); these Sites alone have barely detectabfe activity (Lee et af., W64; Yamamoto, personal communication). pGMCS/l7 was generated by inserting a single Wmer, carried on a 36 bp fragment, into the Sacl site 164 bp upstream of the transcriptional start site. pGMCWJAS was generated by inserting an intact UASo, carried on a 366 bp fragment, into the same site. pGMCSAGRE and pGMCSAGRE/UAS are derivatives of pGMCS and pGMCS/UAS, respectively, from which the GREs have been deleted.

each of which usually gave rise to slightly more activity than did the wild-type GAL4. In some experiments a small amount of CAT activity was stimulated by an effector plasmid encoding no GAL4 (e.g., Figure 2, lane 12); the RNA analysis presented below shows that unlike the GAL4stimulated activity, this activity did not arise from transcripts initiating near the MMTV transcriptional start site. The reporter plasmids lacking UASo produced only

barely detectable CAT activity in response to GAL4 or any of its derivatives (Figure 2, lanes 2 through 5). Figure 3 shows that GAL4 and its active derivatives elicited transcripts that initiated at the same position (Buetti and Diggelmann, 1993) as that stimulated by active GR in the wild-type MMTV promoter (lanes 98, and 9). In contrast, no transcripts initiating at the correct position in the MMTV promoter were elicited by effector plasmids en-

GAL4 163

Activates

Gene

Expression

in Mammalian

Cells

nGRE + UAS

nGRE ‘1

2

5

4

3

6’

‘7

8

9

IO

11

12'

2

I

9n Figure

2. Stimulation

of an MMTV

Promoter

Lacking

GREs

by GAL4

and Its Derivatives,

CHO cells were transiently transfected with a reporter plasmid (either pGMCSAGRE [lanes effector plasmid as indicated (see also Figure 1). CAT activities were assayed as described

nGRE I

‘1,234 1 I,: ; cy:

1

78 910-M II *

(’

by CAT Activity

I-S] or pGMCSAGRE/UAS in Experimental Procedures.

[lanes 7-121 and with an

coding only the DNA-binding portion of GAL4 or no GAL4 (Figure 3, lanes 7 and 10). Moreover, lanes 1 through 5 (Figure 3) show that reporter plasmids lacking UASo synthesized no detectable MMTV/CAT mRNAs, whether or not GAL4 or its derivatives were present. We have no direct demonstration that GAL4(1-147) which did not activate transcription in the experiments of Figures 2 and 3, was synthesized and bound to DNA. We regard this as a reasonable assumption, however, for the following reasons. We transfected our effector plasmids (which contain the SV40 ori region) into COS-1 cells (see Gluzman, 1981) and found that our GAL4 derivatives were visualizable by 35S labeling and immunoprecipitation. We detected GAL4(1-147) at a level about lo-fold higher than that of GAL4, and GAL4(1-147,768-881) and GAL4(1-238, 768-881) at an intermediate level (data not shown). Thus in this strain of animal cells, GAL4(1-147) is neither particularly underexpressed nor unstable. In yeast cells GAL4(1147) is stable and binds to the 1Fmer (Keegan et al., 1986).

nGRE+UAS

5-6

Measured

x.

GALA-GR Synergism For the experiment of Figure 4 we used a reporter plasmid bearing a 1Fmer inserted into the intact MMTV promoter (see Figure 1). Figure 4 shows, first, an unexpected result: insertion of the 1Fmer diminished the ability of the GREs to activate transcription in response to dexamethasone in the absence of GAL4 (compare lanes 1 and 7) a matter

Figure 3. RNAase Protection Mapping Stimulated by GAL4 and Its Derivatives

of MMTWAT

mRNA

in Cells

CHO cells were transfected with a reporter plasmid (see Figure 2) and an effector plasmid as indicated and, in addition, with a reference plasmid bearing the SW9 enhancer that synthesizes a-globin mRNA from

its own promoter. The upper bold arrow indicates the expected position of the 135 nucleotide MMTVlCAT transcript initiated from the correct site (see Figure l), protected by a radioactive probe after RNAase digestion (see Experimental Procedures). The lower bold arrow indicates the 95 nucleotide a-globin mRNA, measured to normalize the amount of RNA loaded on the gel. M refers to a labeled size marker, an Mspl digest of PBR3222.

Cell 164

GRE ‘1

2

3

4

5

6"7

GRE+17 mer 10 8 9

11

12'

DEX: I Figure 4. Stimulation

5a of an MMTV

Promoter

Bearing

CHOlGRl cells, which express rat GR constitutively, with an effector plasmid as indicated. In lanes marked cells was analyzed.

Both GREs and the 1Fmer

Sequence

were transfected with a reporter plasmid (pGMCS “+” cells were treated with 0.2 urn dexamethasone.

we return to in the Discussion. Second, in the absence of dexamethasone, GAL4 did not stimulate transcription from this 1Fmer (Figure 4, lane 11); in yeast the 1Fmer mediates GAL4-dependent transcriptional activation significantly less efficiently than does the intact UASo, and so this result is not particularly surprising. Third, however, the presence of both dexamethasone (and hence active GR) and GAL4 markedly stimulated transcription (Figure 4, lane 8). As a control, we found that addition of dexamethasone had no effect on the activity of GAL4 if the reporter plasmid bore a 17-mer but was deleted for the adjacent GREs (data not shown). Synergy between GAL4 and GR was also observed with a reporter plasmid bearing UASo plus GREs. Figure 5 shows, first, that similar to but more extreme than the case with the Vmer, insertion of UASo abolished the ability of the GREs to mediate stimulation by dexamethasone in the absence of GAL4 (lane 1). Second, GAL4 stimulated expression from this plasmid in the absence of dexamethasone (Figure 5, lane 5), a result similar to that seen in the experiment of Figure 2. Third, Figure 5 shows that the addition of dexamethasone to cells synthesizing GAL4 increased the CAT activity to a level about 4-fold higher than that seen with GAL4 alone (lane 2). Discussion Our experiments show that GAL4 and two deletion derivatives that activate gene expression in yeast activate gene expression in mammalian cells as well. This activation depends upon a GAL4 binding site inserted upstream of the transcriptional start site. As an additional control, a derivative of GAL4 that binds to DNA but cannot activate tran-

[lanes l-61 or pGMCW7 CAT activity in the extracts

[lanes 7-121) and of the transfected

scription in yeast also fails to activate transcription in mammalian cells. We estimate that GAL4, expressed transiently from the adenovirus major late promoter, stimulates a promoter bearing a UASe about as efficiently as the GR, expressed from the Rous sarcoma virus promoter and integrated into the CHO genome, stimulates the MMTV promoter bearing the wild-type GREs (data not shown). Other experiments indicated that a promoter bearing multiple GAL4 binding sites (17-mers) responds at least 10 times more efficiently to GAL4 (data not shown). Webster et al. (1988), in the accompanying paper, report that a single consensus 17 bp GAL4 binding site mediates GAL4 stimulation almost as efficiently as does UASo, whereas we find that UASo works much more efficiently than does a single 1%mer. This difference may be explained, at least in part, by the fact that Webster et al. (1988) use a consensus 17 bp element (Hollis and Ptashne, unpublished results) that differs at two positions in sequence from our 17-mer and that binds GAL4 more tightly. According to our current ideas, DNA-bound GAL4 stimulates transcription in yeast by contacting some other protein (Ptashne, 1999). It is possible that GAL4 interacts directly with RNA polymerase or with the TATAbinding protein (TFIID) (Sawadogo and Roeder, 1985; Reinberg et al., 1987); another possible target would be the NFl protein, which binds to a site centered at position -70 in the MMTV promoter (Nowock et al., 1985; Cordingley et al., 1987). Experiments in yeast show that activating regions of GAL4 (Ma and Ptashne, 1987a) as well as that of another activator, GCN4 (Hope and Struhl, 1988; Struhl, 1987) are stretches of acidic amino acids with no additional very striking similarity. Activating regions generated from random genomic DNA fragments are also negatively charged

7;5LL” Activates

Gene

Expression

in Mammalian

Cells

GRE+UAS 4

5

6’

(1988) have shown that, in yeast, two GAL4 binding sites stimulate transcription synergistically and that this synergism reflects cooperative binding of GAL4 molecules. Perhaps GR and GAL4 bind cooperatively; this cooperativity might reflect a direct interaction between these proteins or, more likely in our view, might be mediated by contact with a common, third element of the transcriptional machinery. Experimental

DEX:

I

‘+

J I 3 n

Figure UASo

5. Stimulation

of an MMTV

Promoter

Bearing

Both GREs

8 f n and

CHO/GRl cells were transfected with pGMCS/UAS and with an effector plasmid as indicated. Experimental details are as in Figure 4.

(Ma and Ptashne, 1987c), and mutational analysis of one activating region of GAL4 shows that the negative charge is important but is not the sole determinant of activity (Gill and Ptashne, 1987). Finally, Giniger and Ptashne (1987) constructed a gene encoding a 15 amino acid peptide designed so that it could form an amphipathic a-helix, one surface bearing negatively charged amino acids and another bearing hydrophobic amino acids; when attached to a DNA-binding domain this peptide activates transcription in yeast. It remains to be seen whether this novel activator will be effective in mammalian cells. We do not know why insertion of a GAL4 binding site in the MMTV promoter abolishes the ability of the latter to respond to GR in the presence of dexamethasone. In our construct using UA!&, the GREs shown in Figure 2 were moved about 360 bp upstream from their ordinary position, and this displacement per se might account for their decreased activity. In our construct using the lFmer, however, these GREs were moved upstream only about 30 bp, a distance that should have little effect on their activity (Majors and Varmus, 1983). In both of these experiments the GAL4 binding site was inserted within, and 2 bp from one end of, one of the GR binding sites (see Scheidereit et al., 1983), and this insertion might have damaged the response to GR. It is also possible that a protein present in CHO cells binds to the GAL4 regulatory sequences and blocks the effect of an upstream activator (Brent and Ptashne, 1984; Keegan et al., 1986). An inhibitory effect of one DNA element (virus-responsive element of the IFN-al promoter) on another (the SV40 enchanter) was recently reported by Kuhl et al. (1987). They observed, moreover, that the two elements work synergistically. We find a similar effect: GAL4 and dexamethasone (and hence active GR) together are considerably more active than the sum of the activities of either alone. Giniger and Ptashne

Procedures

Plasmid Constructions Effector Plasmids pMT2AVA, which is a VA-gene-lacking derivative of PMT2 (Bonthron et al., 1986) was provided by Ft. Kaufman. For the construction of pAG4, the 2.9 kb Hindlll fragment of pLKC15 (Ma and Ptashne, 1987a) was blunt-ended by Escherichia coli DNA polymerase Klenow fragment with deoxyribonucleotides and ligated to an EcoRl linker. Then it was inserted into pMT2AVA. pAG147 was made from pAG4 by replacing the Xhol-Mlul fragment in the coding sequence with the Xhol-Xbal fragment in pTGH21, which is a deletion derivative of pLPK76-7 (Keegan et al., 1986) and expresses GAL4(1-147) in E. coli (unpublished results). The Mlul and Xbal sites were filled in prior to ligation. pAG236 and pAG242 were constructed by replacing the Xbal-Mlul fragment of pAG4 with that of pMA236 and pMA242 (Ma and Ptashne, 1987a), respectively. Reporter Plasmids The parental plasmid pGMCS was provided by K. Yamamoto. For the construction of pGMCS117 and pGMCS/UAS, the synthetic nearconsensus 1Fmer and UASo of the GAL+GAL70 divergent promoter were separately inserted in the unique Sac1 site of pGMCS. The Sac1 site was blunt-ended by T4 DNA polymerase prior to ligation. The 17mer sequences along with the surrounding pUC18 polyiinker DNA were taken from pSV3 (Giniger and Ptashne, 1988) by Smal and Hincll digestion. UASo of the GALhGALlO promoter was taken out as a 365 bp Sau3Al-Ddel fragment as described by Guarante et al. (1982). This fragment was then attached to a Bglll linker, followed by digestion with Bglll and filling in with Klenow enzyme prior to ligation. pGMCSAGRE was made by deleting both the 540 bp Stul-Sac1 fragment and the 800 bp BamHl fragment; the latter contains a mammary tumor virus GRE downstream of the CAT gene (Miesfeld et al., 1986). pGMCSAGREI UAS was similarly constructed as described above. Reference Plasmid rrSVHNEa575, which bears the human a-globin promoter (up to POW tion -575) and the SV40 enhancer, was provided by F. Baas. DNA Transfectlon, Induction, and CAT Assay Cells were grown in a-MEM supplemented with 10% fetal calf serum (GIBCO) and 10 ug/ml each of adenosine, deoxyadenosine, and thymidine. For the transfection 5 pg each of reporter and effector plasmid was added to the serum-free medium in the presence of 250 pglml DEAE-dextran (Pharmacia) per culture dish (5 x lo5 cells). For analyzing RNA 0.5 pg of reference plasmid was also added to the medium. After 2 hr cells were treated with 10% dimethyl sulfoxide I” phosphate-buffered saline for 2 min followed by incubation in medium containing 0.1 mM chloroquine for an additional 3 hr. Cells were then rinsed, refed, and induced by0.2 PM dexamethasone where tndrcated After 60 to 62 hr cells were harvested, and half of the extract was subjected to the CAT assay according to the method of Gorman et al. (1982). Reactions were carried out at 37% for 2 hr with 0.1 WCi of [%]chloramphenicol (New England Nuclear). RNA Preparation and RNAase Plotectlon Mapping Total RNA was extracted 48 hr after transfection by a modificatton of the procedure of Chirgwin et al. (1979). Twenty micrograms of total RNA was hybridized with T7/Sp6 probes following the procedure of Zinn et al. (1983). Template DNA was prepared as follows. For the MMTWCAT probe, the Sacl-Pvull fragment of pGMCS (Figure 16) was cloned into the Sacl-Pvull sites of pSP73 (Promega-Biotec), The resulting plasmid, pSP73/Ml, was cleaved at the EcoRl site prior to in vitro transcription. For the human a-globin probe, a portion of the human genomic a-globin DNA sequence was inserted into SPG-PLl as described by

Cell 166

Charnay et al. (1984). The resulting plasmid, ized by Ncol prior to in vitro transcription.

pSP6Hua,

was linear-

Stable lhdectants Clone CHO/GFtl was established as follows: CHO-DUKX cells were transfected with 10 ug of pRSVGR (Mlfeld et al., 1986) and 1 pg of pSV2neo (Southern and Berg, 1982) by the calcium phosphate-mediated transfection procedure (Chen and Okayama. 1987). Transfectants were selected in the presence of 600 &ml 6418 (GIBCO). Of 13 clones isolated, 8 showed a functional GR phenotype as determined by dexamethasone-dependent CAT activity after transient transfection with pGMCS. CHO/GRl was one of these isolates. Acknowtedgments

November

2.5, 1987; revised

December

11, 1987.

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