MOLECULAR AND CELLULAR BIOLOGY, Dec, 1990, p. 6674-6682 0270-7306/90/126674-09$02.00/0 Copyright © 1990, American Society for Microbiology

Vol. 10, No. 12

Activation of Chromosomal Vitellogenin Genes in Xenopus Oocytes by Pure Estrogen Receptor and Independent Activation of Albumin Genes EDWARD A. McKENZIE, NIGEL A. CRIDLAND, AND JOHN KNOWLAND* Department of Biochemistry, South Parks Road, Oxford OX] 3QU, England Received 22 January 1990/Accepted 24 September 1990

We generated pure estrogen receptor protein in Xenopus oocytes by injecting them with estrogen receptor mRNA synthesized in vitro. A chromosomal vitellogenin gene, which normally responds to estrogen only in liver cells, is activated. Primer extension shows that initiation is accurate, and ribonuclease mapping shows that the first exon is correctly spliced out of the initial transcript. Long transcripts are produced, one being equal in length to poly(A)- vitellogenin mRNA. Immunochemical estimates of receptor levels in the oocyte nuclei suggest that pure receptor, acting alone, cannot activate oocyte vitellogenin genes unless unusually large amounts are present. However, when a receptor-free extract from liver cells is also injected, the amount of receptor required is reduced. Such an extract, but not pure receptor, can also activate albumin genes in oocytes.

Our understanding of how tissue-specific gene expression is achieved is still very limited. In the case of genes activated by steroid hormones, we have no satisfactory explanation of why different cells containing similar amounts of the same receptor respond in different ways. For example, adult Xenopus liver cells contain estrogen receptor and respond to estradiol by producing vitellogenin (for a review, see reference 29), but other cells which also contain receptor do not. Neither adult oviduct (22) nor even liver from premetamorphic tadpoles (11, 13) does so, although both contain receptor and indeed respond to estradiol in other ways (17-19). Hence, it is clear that a certain level of receptor is sufficient to allow activation of chromosomal vitellogenin genes in adult liver cells, but it is equally clear that the presence of a similar amount in other cell types is not sufficient, even if the other cell types are inherently estrogen responsive. These observations raise three specific questions about the activation of steroid-controlled genes which are also relevant to the general question of how transactivators contribute to tissue-specific gene expression. The questions are as follows. (i) Can a pure receptor, acting alone, ever activate its normal target in a foreign cell? (ii) If so, how much receptor is needed relative to the amount of receptor found in a cell in which the gene is normally active? (iii) Can other factors affect the extent of activation which the pure receptor can achieve? The answers to these questions are important because if a pure transactivator can activate its target in a foreign cell only if the transactivator is present in very large amounts, then it is not safe to conclude that normal activation depends solely on the presence of the transactivator. In the particular case of vitellogenin gene expression, we found previously that when extracts of liver nuclei enriched in receptor are injected into Xenopus oocytes, chromosomal vitellogenin genes are activated (14). Because oocytes do not divide, those results showed that it is possible to activate vitellogenin genes in nonhepatic cells without any major reorganization of chromatin that may accompany cell division. However, we were unable to test *

whether receptor alone can do so or estimate accurately the amount required. Here, we first generate pure receptor protein in oocytes by injecting receptor mRNA and show that the vitellogenin B2 gene can be activated. Next, we vary the input of receptor RNA and estimate the amount of pure receptor which, acting alone, causes the same activation that is found in liver cells. Then, by injecting receptor RNA followed by an extract of liver nuclei which has been treated to selectively inactivate receptor, we ask whether the amount of pure receptor needed for activation is affected when other factors are also added. Finally, we ask whether receptor-free extracts can activate a gene other than vitello-

genin. MATERIALS AND METHODS Receptor RNAs. Human estrogen receptor RNA was synthesized in vitro by using the plasmid HEO linearized at the BamHI site and T7 RNA polymerase (16). The template was then destroyed by adding 1 U of RQ1 DNase (Promega) per ,ug of DNA, incubating for 30 min at 37°C, and then repeating this digestion. Purified RNA (4 ,ug) was then capped by incubation for 90 min at 37°C in 100 RI of a solution containing 50 mM Tris hydrochloride (pH 7.9), 1.25 mM MgCl2, 6 mM KCl, 2.5 mM dithiothreitol, 0.5 mM GTP, 100 ,uM S-adenosyl methionine, 100 ,ug of bovine serum albumin per ml, 100 U of RNasin (Promega), and 3 U of guanylyl transferase (GIBCO-BRL). Capped RNA was purified by phenol extraction and ethanol precipitation. Antisense receptor RNA was synthesized by using the plasmid HEO linearized at the PvuI site and T3 RNA polymerase. The amounts and lengths of all the RNAs were measured by parallel transcriptions in the presence of known amounts of [ot-32P]CTP followed by Cerenkov counting and gel electro-

phoresis. Extracts of liver nuclei and photochemical inactivation of receptor. In vitro transcription extracts were prepared and UV irradiated in the presence or absence of 6-oxo-estradiol, as described previously (5). Injection and incubation of oocytes. Oocytes were injected in the cytoplasm by standard procedures (9) and incubated in either MBS (9) or JK1 (120 mM NaCl, 3 mM KCl, 2.4 mM

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NaHCO3, 3 mM MgCl2, 0.89 mM Ca(NO3)2, 1.1 mM CaCl2, 0.25 mM ZnCl2, 10 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid] [pH 7.4]). Salines for incubation also contained 3.5 nM estradiol, the concentration found in female liver (36). Each oocyte received 50 nl of RNA at 0.1 ,ug/,I unless otherwise stated. Viteliogenin probe. A BglII fragment of the vitellogenin B2 gene running from positions -42 to +220 (8, 30) was subcloned into the BamHI site of pSP64. The antisense plasmid was isolated and verified by DNA sequencing. This plasmid was linearized at the SmaI site of the polylinker and transcribed by using SP6 RNA polymerase to generate an antisense vitellogenin probe. A probe made in this way contains only 4 bases from the polylinker complementary to polylinker sequences from the receptor RNA, and direct tests showed that there is no hybridization between the probe and the receptor RNA. RNA blots, hybridization, RNase treatment, and washing. For Northern (RNA) blots, RNA was run on formaldehyde gels. For both dot-blots and Northern blots, filters were prehybridized overnight at 55°C in a solution containing 50 mM sodium phosphate (pH 6.5), Sx SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 1 mM disodium EDTA, 50% (vol/vol) formamide, Sx Denhardt solution, 200 p.g of herring sperm DNA per ml, and 0.1% sodium dodecyl sulfate (SDS); 5 ml per 100 cm2 of filter was used. RNA probes were then added to fresh mix and hybridized for 48 h. Filters were washed two times for 30 min (each) at room temperature in 2x SSC-0.1% SDS, once for 30 min at room temperature in 2 x SSC-1 jxg of RNase A per ml, two times for 30 min (each) at room temperature in 2x SSC-0.1% SDS, once for 30 min at 550C in 0.1x SSC-0. 1% SDS, and once for 30 min at 68°C in 0.1x SSC-0.1% SDS. The filters were then dried and autoradiographed at -70°C using preflashed film and intensifying screens. Ribonuclease mapping. RNA samples were dissolved in 30 ,ul of a solution containing 80% (vol/vol) formamide, 400 mM NaCl, 1 mM disodium EDTA, and 40 mM PIPES [piperazine-N,N'-bis(ethanesulfonic acid)] (pH 6.7). One microliter (-200,000 dpm) of uniformly labeled antisense vitellogenin probe was added, and the RNA was denatured at 85°C for 5 min. Following hybridization for 16 h at 400C, 300 RIu of a solution containing 300 mM NaCl, 5 mM disodium EDTA, 10 mM Tris hydrochloride (pH 7.5), 40 ,ug of RNase A per ml, and 2 ,ug of RNase T1 per ml was added. Digestion was for 1 h at 35°C. Tests showed that these conditions of hybridization and digestion were optimal for our purposes. Digestion was terminated by adding 20 ,ul of 10% SDS and 50 p,g of predigested proteinase K followed by incubation for 30 min at 37°C and phenol extraction. Albumin probe. The albumin plasmid pEMBL9-ALB, which contains the 2.6-kb EcoRI-BamHI fragment spanning the initiation site of the 68-kDa albumin gene (20), was used to generate a DNA probe by random-primed labeling using a Boehringer kit. The probe was denatured by boiling and hybridized to RNA dots as described above for RNA blots but at 42°C. Filters were washed as described above for RNA blots but without RNase. To remove the DNA probe, the filter was washed four times for 30 min (each) at 650C in 5 mM Tris hydrochloride (pH 8.0)-2 mM disodium EDTA0.1 x Denhardt solution and was then incubated two times for 30 min (each) at 370C in SP6 transcription buffer containing 5 U of RQ1 DNase per ml with 5 ml of the buffer per 100 cm2 of filter. The DNase treatment was essential to ensure complete removal of the albumin probe. Primer extension. A 31-nucleotide (nt) 5'-labeled synthetic

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primer, 5'-CCGCTAGACCGAGAAGCAGAGCAAGTAT GAT-3', which complements the end of the first exon of the vitellogenin B2 gene, was hybridized for 6 h to the RNA from 50 oocytes as described earlier (27), with a total volume of 100 ,ul. Following precipitation, the sample was dissolved in 55 ,ul of water and then adjusted to 100 ,ul containing 50 mM Tris hydrochloride (pH 8.2); 10 mM dithiothreitol; 6 mM MgCI2; 25 ,ug of dactinomycin per ml; 0.5 mM (each) dATP, dCTP, dGTP, dTTP; 10 U of avian myeloblastosis virus reverse transcriptase (Pharmacia); and 20 U of RNasin. The sample was then incubated at 42°C for 1 h. Tests using small amounts of RNA from female liver mixed with a large excess of oocyte RNA showed that the conditions of hybridization and extension described above were optimal for our purposes. Following phenol extraction and precipitation, the sample was redissolved in 100 ,ul of hybridization buffer (no additional primer), denatured, hybridized for 16 h, and again incubated with reverse transcriptase. After four rounds of hybridization and extension, the sample was extracted with phenol and precipitated with 10 ,ug of herring sperm DNA. The sample was then dissolved in 100 ,ul of 0.3 M KOH, incubated at 37°C for 16 h to hydrolyze RNA, adjusted to pH 5 by adding 77 p.1 of 1 M potassium acetate (pH 5.0), diluted twofold with water, precipitated with ethanol, redissolved, and precipitated again from ammonium acetate. The final pellet was run on a sequencing gel. A 1-h exposure was used to locate the unextended primer, which was then cut away to avoid obscuring the extended primer during a longer exposure. The hydrolysis step was included to destroy the RNA from the oocytes which would otherwise clog up the sequencing gel. Measurement of receptor levels in oocytes. Oocytes were homogenized in TBS (10 mM Tris hydrochloride, 150 mM NaCl [pH 8.0]) and centrifuged (10,000 x g, 30 s) to remove lipid and yolk. Using a dot-blot apparatus, the supernatant was applied to a Hybond-C membrane equilibrated in TBS, which was also used to rinse the wells after loading. Blocking of nonspecific sites by using TBS containing 0.2% Tween 20 for 3 h was followed by incubation overnight at 4°C with an anti-receptor rat monoclonal antibody (Abbott Laboratories) in TBST (TBS + 0.05% Tween 20). Following removal of excess antibody by three washes in TBST, the membrane was incubated overnight at 4°C with a second, 125I-labeled anti-rat-and-mouse polyclonal antibody in TBST. Excess antibody was removed as described above, and specifically bound 125I-labeled antibody was detected by autoradiography using preflashed films and intensifying screens. In preliminary experiments we varied the concentrations of both the first and the second antibodies to ensure that the measurements were quantitative. To follow the entry of receptor into the nucleus, oocytes were injected in the cytoplasm with 35S-labeled receptor made by in vitro translation (16) followed by precipitation with ammonium sulfate (36) and dialysis against transcription buffer (27). Receptor in the nuclear and cytoplasmic compartments (2) was extracted (25) and measured by scintillation counting. RESULTS Pure receptor can activate chromosomal vitellogenin genes in oocytes. The simplest way to generate pure receptor protein in an oocyte is to inject receptor RNA into the cytoplasm. Other workers have injected oocyte nuclei with DNA expression vectors encoding the human estrogen receptor protein (26), but we decided against this approach because such vectors may produce other proteins as well as

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FIG. 1. Activation of chromosomal vitellogenin genes in oocytes. Oocytes were injected with receptor RNA and either incubated in JK1 and sampled at 24-h intervals (row A, columns 1 through 6) or incubated in MBS and sampled at 48-h intervals (row B, columns 1, 3, and 5), starting at time zero in both cases. For each sample, 10 oocytes were extracted and RNA from S was applied to a Hybond-N filter. Row C, Signals obtained with 100 ng of total RNA from female liver (column 1), 100 ng of RNA from male liver (column 2), 5 uninjected oocytes (column 3), and oocytes injected with anti-sense receptor RNA, incubated in JK1, and sampled at 0, 48, and 96 h (columns 4, 5, and 6, respectively). Results with uncapped receptor RNA were also negative. The RNA probe covers positions -42 to +220 of the B2 gene but also contains short signals from the polylinker of the vector at either end. The signals detected were quantitated with a Quantimet image analyzer, and the results were plotted in arbitrary units. receptor. We synthesized estrogen receptor RNA in vitro by

using T7 RNA polymerase and the linearized plasmid HEO (16, 31) and added a cap. It is established that receptor RNA made in vitro translates into protein which can bind estradiol (16), and we confirmed this for our own work (data not shown). After injecting the RNA we assayed for expression of the vitellogenin B2 gene by using an RNA probe covering the initiation site of the gene. Vitellogenin transcripts were not detectable either in uninjected oocytes or immediately after injection, but they appeared 24 h after injection and accumulated steadily for 4 to 5 days (Fig. 1). In these experiments, we used two salines: MBS as traditionally used (9) and JK1, which is based on the ionic composition of Xenopus blood serum as measured by atomic absorption spectroscopy. We used the second saline mainly because the analyses revealed significant concentrations of zinc; in practice, however, oocytes behaved in much the same way in both. Uncapped RNA did not generate any vitellogenin transcripts, nor did antisense receptor RNA, which we injected as a control. These results show that the receptor RNA must be capped for functional receptor to be produced and that activation is not a nonspecific effect of injecting RNA. The results also confirm that the signals detected in the injected oocytes are due to transcription of the vitellogenin genes in the oocytes and not to hybridization of the probe to other RNA. We conclude that pure estrogen receptor protein activates the chromosomal vitellogenin genes in oocytes. Because it is already established that the receptor must be complexed with estradiol in order to bind to the estrogen response elements (15) and because oocytes contain enough estrogen to saturate the receptor (see Discussion), we assume that the activation is due to the estrogen-receptor complex.

Activation is specific to estrogen-responsive genes. It is essential to ask whether estrogen receptor generated in oocytes activates any genes which are not normally affected by estrogen. We therefore tested for activation of albumin genes, which, unlike vitellogenin genes, are transcribed in both male and female liver. In these experiments, it is important to consider carefully the effects of estrogen on the levels of both vitellogenin and albumin RNA, because it is widely believed that estrogen invariably stabilizes the former (1) and destabilizes the latter (12). However, we have recently shown that such stabilization of vitellogenin mRNA requires artificially high concentrations of estradiol and does not occur at the physiological concentrations which we use in all our experiments (21). The stability of albumin mRNA in relation to estrogen concentration has not been systematically investigated, but active destabilization of albumin mRNA has been demonstrated only at pharmacological concentrations of estradiol (12). Furthermore, it is firmly established that the transcription of albumin genes in liver is barely affected by estrogen (12), so that if destabilization of albumin mRNA does occur at normal estrogen concentrations, one would expect the steady-state levels of albumin RNA in male and female liver to be very different. However, this is not the case. Albumin transcripts are found at about the same level in both female and male liver (Fig. 2), in which the estradiol concentrations are, respectively, 3.5 and 0.18 nM (36). Thus, our findings show that normal concentrations of estradiol do not destabilize albumin mRNA. Albumin transcripts were not found in oocytes which had been injected with receptor RNA, even though vitellogenin transcripts were induced in the same oocytes (Fig. 2). In the liver samples, the albumin signals are much more intense than the vitellogenin signals, although the rates of albumin

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FIG. 2. Induction of vitellogenin transcripts in oocytes by receptor but not of albumin transcripts by receptor. Oocytes were injected with sense or antisense receptor RNA, and RNA from five oocytes was probed with a 2.6-kb DNA probe spanning the initiation site of the 68-kDa albumin gene. Row A, Albumin signals detected in female (column 1) and male (column 2) liver, uninjected oocytes (column 3), and oocytes injected with antisense RNA and incubated for 72 h (column 4). Row B, no albumin RNA was detected in oocytes injected with sense RNA and analyzed at time zero (column 1), 72 (column 2), and 120 h (column 3). Column 4 is empty. The albumin probe was then removed by a combination of washing and DNase treatment (see Materials and Methods), and the filter was reprobed with a vitellogenin probe made as indicated in the legend to Fig. 1. Row A', Vitellogenin RNA was found in the female liver (column 1) but not in the male liver (column 2), the uninjected oocytes (column 3), or the oocytes injected with antisense RNA (column 4). Row B', In the oocytes injected with sense RNA, vitellogenin RNA was not present at time zero (column 1) but was present at 72 (column 2) and 120 h (column 3).

and vitellogenin transcription are similar (12), because the albumin probe is 10 times longer than the vitellogenin probe. The high sensitivity of the albumin assay reinforces our conclusion that receptor does not activate albumin genes in oocytes. The results suggest that chromosomal genes activated by estrogen receptor in liver can also be activated in oocytes and that receptor does not affect genes which are not under estrogen control in liver. We have not so far tested whether vitellogenin genes are the only ones to be activated or whether other estrogen-regulated genes are also activated.

Viteliogenin transcription and processing in oocytes closely resembles transcription in liver cells. We compared the vitellogenin transcription induced in oocytes with that found in liver cells by using ribonuclease mapping. All samples contain a background of small fragments due to degradation of the probe (Fig. 3, lanes 3 and 4), but samples from oocytes injected with receptor RNA (Fig. 3, lanes 8 through 17) also contain protected fragments which are normally found only with female liver RNA (Fig. 3, lanes 2 and 19). One in particular (Fig. 3, arrow) is 53 nt long, increases in intensity with time (Fig. 3, compare lanes 8 through 10 and 12 through 17), and presumably represents the first exon of the gene (Fig. 1). We attribute the collection of fainter and shorter fragments at 40 to 50 nt to processing products. The oocyte signals are weaker than those in Fig. 1, because the dot-blot concentrates all the protected fragments into one spot whereas the mapping gel separates them. The presence of the 53-nt band and the fact that the overall spectrum of fragments does not change over time strongly suggest that vitellogenin transcription in oocytes initiates at the same chromosomal site that is used in liver cells and that the first exon is correctly spliced out of the initial transcript. In neither liver nor oocytes could we detect significant amounts of unspliced product, showing that the initial transcript is rapidly processed in both cases. To confirm that transcription initiates correctly and that

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the 53-nt fragment shown in Fig. 3 is due to the first exon, we used primer extension. Because we are looking for transcripts from a single-copy gene by using very few cells and against a huge background of RNA (4 ,ug per oocyte), we first optimized the protocol for our particular application (see Materials and Methods). In particular, we used four successive rounds of hybridization and extension to amplify the signals obtained. We found that the transcripts induced in oocytes do indeed initiate at the site which is used in liver cells (Fig. 4). The signals are, as expected, weak; but this result, especially when taken with those shown in Fig. 3, confirms that transcription of the B2 vitellogenin gene induced by pure receptor in oocytes initiates correctly. Liver samples contain the normal 6.3-kb vitellogenin signal due to poly(A)+ vitellogenin mRNA (Fig. 5). Oocytes injected with receptor mRNA produced vitellogenin signals at about 1.8, 4.9, and 5.4 kb. The longest of these, although produced in small amounts, is virtually identical in length to mature but poly(A)- vitellogenin mRNA, which is 5.6 kb long (7). Thus, it is clear that receptor-induced transcription of vitellogenin genes in oocytes initiates correctly and possible that subsequent events, which include accurate splicing of the first exon, result in the production of at least some full-length but poly(A)- vitellogenin mRNA. Pure receptor is needed in large amounts to activate vitellogenin genes in oocytes. In order to see how the amount of vitellogenin RNA produced is affected by the level of receptor, we injected serial dilutions of receptor RNA, assayed for vitellogenin RNA, and estimated the amounts of receptor by using a monoclonal anti-receptor antibody and a 125I-labeled second antibody. The amount of vitellogenin RNA which accumulates increases with the amount of receptor RNA injected, in both a 24- and a 72-h incubation (Fig. 6, rows A and B, respectively). With any given amount of receptor RNA the strength of the vitellogenin signal increases with time (Fig. 1 and 6, rows A and B). Like vitellogenin RNA, receptor becomes detectable when each oocyte receives 5 x 106 molecules or more of receptor RNA, but at higher inputs the amount produced is not greatly affected by the amount of RNA injected (Fig. 6, rows C and D). Nor does the amount of receptor produced from a given amount of RNA increase greatly with the time of incubation (Fig. 6, row E); a plausible explanation for these findings is that the receptor RNA is not very stable in oocytes, perhaps because it is poly(A)-. However, it seems that the receptor produced is fairly stable, and these results suggest that a pool of pure receptor in oocytes maintains the vitellogenin genes in an active state for a considerable time. To see how much of the receptor enters the nucleus and how quickly it does so, we injected oocytes with radioactive receptor made in vitro and separated the nucleus and cytoplasm at increasing times (2). In this way we can study the intracellular location of receptor both at early times, before any synthesized on injected RNA would be detectable immunochemically, and at later times. In liver cells, we find about half of the total receptor in the nucleus (35), and this is also true of oocytes, with the final distribution being reached at about 24 h (Fig. 6), the earliest time at which we have detected vitellogenin transcripts. Other workers have suggested that the proportion of total receptor in the liver nucleus is greater than 50% (10), but as we have previously pointed out (36), the binding data on which that suggestion was based do not give a linear Scatchard plot and so may not be reliable. However, the precise distribution of receptor does not affect our conclusion that if the concentration of receptor in the oocyte nucleus is high enough, the vitellogenin genes are activated

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FIG. 3. Ribonuclease mapping of vitellogenin transcripts induced in oocytes. Oocytes were injected with RNA, and batches of 10 were frozen at various times. The RNA from five oocytes was analyzed by ribonuclease mapping with a probe made as indicated in the legend to Fig. 1. Lane 1, Undigested probe; lanes 2 and 3, protected fragments found in female and male liver RNA (100 ng [each]). The strong band at 53 nt in lane 2 (arrow) represents the first exon of the vitellogenin B2 gene. Lane 4, Uninjected oocytes; lanes 5 through 7, oocytes injected with antisense RNA and analyzed at zero, 48, and 96 h, respectively; lanes 8 through 10, oocytes injected with sense RNA, incubated in MBS, and analyzed at 0, 48, and 96 h, respectively; lane 11, blank; lanes 12 through 17, oocytes injected with sense RNA, incubated in JK1, and analyzed at 0, 24, 48, 72, 96, and 120 h, respectively; lane 18, blank; lane 19, female RNA. The positions of DNA size markers are indicated on the right in nucleotides.

and that the products of transcription accumulate steadily. We estimate that for the oocyte vitellogenin B2 gene to be fully activated by pure human receptor, between 108 and 109 molecules have to enter the nucleus. By contrast, when vitellogenin genes are active in frog liver nuclei, only some 102 to 103 molecules of receptor are found in the nucleus

(36).

Activation of albumin genes in oocytes in the absence of receptor. We know that pure receptor cannot activate albumin genes in oocytes (Fig. 2). In order to see whether other factors can, we exploited our recent finding that receptor can be permanently inactivated by a simple photochemical procedure. When an extract of liver nuclei which accurately transcribes both vitellogenin and albumin genes in vitro is UV irradiated in the presence of 6-oxo-estradiol, receptor is selectively inactivated because of a reaction between excited 6-oxo-estradiol and the hormone-binding site. The extract

loses its ability to transcribe vitellogenin genes, but albumin transcription is unaffected (5). This is also true in vivo (Fig. 7). An extract which has been irradiated in the absence of 6-oxo-estradiol activates both albumin and vitellogenin genes in oocytes in a time-dependent manner (Fig. 7, rows A and A', columns 1 and 2). After enough irradiation in the presence of 6-oxo-estradiol to inactivate all the receptor, transcription of albumin genes continues but transcription of vitellogenin genes stops (Fig. 7, rows A and A', columns 3 and 4). When an extract is irradiated in the presence of 6-oxo-estradiol for increasing times so as to progressively inactivate the receptor, the number of albumin transcripts generated during a fixed incubation is unaffected, whereas the number of vitellogenin transcripts gradually falls to zero (Fig. 7, rows B and B'). Thus, for vitellogenin genes to be activated, there is an absolute requirement for receptor whether other liver factors are present or not, whereas

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FIG. 4. Mapping the initiation site of the B2 vitellogenin gene in and extended as described in Materials and Methods. Lane 1, RNA from female liver; lane 2, RNA from male liver; lane 3, RNA from 50 oocytes injected with receptor RNA and incubated for 4 days; lane 4, RNA from 50 uninjected oocytes; lane 5, DNA size markers. The arrow marks the position (53 nt) of the extended primer. oocytes. A synthetic primer was hybridized to RNA

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The number of albumin transcripts is clearly independent of the amount of receptor present (Fig. 7, row B), confirming that the expression of the albumin gene and the stability of its mRNA are not affected by receptor. Presence of other factors reduces the amount of pure receptor needed to activate oocyte viteliogenin genes. In order to see whether the presence of other liver factors affects the efficiency of the activation induced by receptor, we injected first receptor mRNA and then a photochemically-treated,

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Northern blot of vitellogenin transcripts generated in Oocytes were injected with receptor RNA and incubated for 72 h. RNA from 10 oocytes was run on an agarose gel, transferred to Hybond-N, and probed with the probe described in the legend to Fig. 1. Lanes 1 and 2, RNA from female and male liver, respectively; lane 3, RNA transcripts, with sizes indicated (kilobases), induced in oocytes. oocytes.

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receptor-free extract from liver nuclei into oocytes. Increasing the amount of receptor by injecting more mRNA increases the intensity of the vitellogenin signal (Fig. 6 and 7, row C, lanes 1 and 2). One would expect any effect of the extract, either positive or negative, to be more noticeable when the amount of receptor is insufficient to ensure maximal activation, and this is found in practice. Receptor-free extracts stimulate the activation induced by receptor (Fig. 7, row C), and when the intensities of the signals are compared by image analysis, we estimate that the receptor-free extract increases the overall expression of the vitellogenin genes by a factor of 5 when a smaller amount of receptor is present (Fig. 7, compare columns 1 and 3) and by a factor of only 2 when receptor is present at higher concentrations (Fig. 7, compare lanes 2 and 4). A full analysis of these effects requires a wider range of measurements, but our results so far suggest that factors in liver, other than receptor, contribute to the activation of chromosomal vitellogenin genes but are not able to induce transcription of those genes independently of receptor. Whether they are the same factors which contribute to albumin expression and exactly how they work remain unknown; but it is clear that whatever the factors do, their functions can be bypassed if a high enough level of receptor is present.

DISCUSSION The results reported here show that when pure human estrogen receptor protein is generated in Xenopus oocytes by translation of cloned receptor RNA, half of the receptor enters the nucleus and activates at least one vitellogenin gene. Initiation is accurate, and the early events of transcription faithfully mimic the process as it occurs in liver cells in that the first exon is correctly spliced out of the initial transcript. The products of transcription accumulate steadily for several days, and it is possible that full-length, poly(A)vitellogenin mRNA is produced. The presence of receptor is necessary to activate the chromosomal vitellogenin B2 gene, and at high concentrations receptor alone is sufficient to do so. Receptor-free extracts from liver cannot activate vitellogenin genes, but the presence of such extracts reduces the amount of receptor required to achieve a given degree of activation. Albumin genes, on the other hand, can be activated by receptor-free extracts of liver nuclei, and albumin gene activity is neither increased nor decreased by receptor. Our results contradict a recent report which states that chromosomal vitellogenin genes cannot be activated when cloned receptor is generated inside oocytes. Watson and Torres (32) injected a plasmid encoding the receptor we use here into the nuclei of oocytes. They detected newly synthesized receptor but found no vitellogenin protein. They concluded that although oocytes accumulate receptor in their nuclei, oocytes do not express endogenous vitellogenin genes. However, Watson and Torres did not report any direct measurements of receptor levels or any assays of vitellogenin RNA, and so the work which they describe cannot be compared with ours. It is not yet clear whether chromosomal vitellogenin genes in nonhepatic somatic cells can also be activated by pure receptor. Seiler-Tuyns et al. (24) transfected the Xenopus kidney cell line B3.2 with the human estrogen receptor plasmid HEO and, 18 to 24 h later, assayed for expression of endogenous A2 and Bi vitellogenin genes by primer extension using primers from the third exon of each gene. They did not detect any transcripts emanating from the endoge-

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0 40 20 HOURS AFTER INJECTION FIG. 6. Vitellogenin transcription in oocytes in relation to receptor in the nucleus. (A) Production of estrogen receptor protein and vitellogenin RNA. Rows A through D, Batches of 20 oocytes were injected with increasing concentrations of receptor RNA. Columns 1 through 6, Each oocyte received 5 x 104, 5 x 105, 5 x 106, 5 x 107, 5 x 108, or 5 x 109 molecules of RNA, respectively. RNA from 5 oocytes was probed as indicated in the legend to Fig. 1 after either 24 (row A) or 72 h (row B), and the receptor protein present in 10 oocytes was measured after 24 (row C) or 72 h (row D). Row E, Oocytes were injected with antisense receptor RNA and assayed for receptor after 24 (column 1) or 120 h (column 2) or with a fixed amount (109 molecules) of sense RNA and assayed for receptor at 24, 48, 72, and 120 h (columns 3 through 6, respectively). Row F, known amounts of receptor synthesized in vitro were applied as standards. Columns 1 through 6, 0.3 x

1o-3, 3 x 10-3, 0.03, 0.3, 3.0, and 30 fmol, respectively. By comparing the intensities of the spots we estimate the total amount of receptor present per oocyte to be approximately (in femtomoles per oocyte) as follows: row C, columns 3 through 6, 0.1, 0.8, 1, and 0.8, respectively; row D, columns 3 through 6, 0.5, 3.3, 3.3, and 3.3, respectively; row E, columns 3 through 6, 1, 3.3, 1, and 1.5, respectively. Columns 1 and 2 in rows C through E are essentially background. (B) Distribution of receptor between nucleus and cytoplasm in oocytes. Oocytes were injected with 35S-labeled receptor made in vitro, and the amount of receptor present in the nuclear and cytoplasmic compartments was measured by using 5 to 10 oocytes for each point. nous genes but did find accurate transcription of cotransfected vitellogenin plasmids, and concluded that receptor generated in the kidney cells does not seem to faithfully activate the chromosomal vitellogenin genes to any significant extent. However, as they point out, there are many possible explanations for these findings, including the simple one that receptor levels were too low, and they do not necessarily mean that nonhepatic somatic cells such as kidney cells are inherently incapable of responding to receptor by expressing chromosomal vitellogenin genes. This work extends our previous demonstration that extracts of liver nuclei enriched in estrogen receptor protein activate chromosomal vitellogenin genes when the extracts are injected into Xenopus oocytes (14). It appears from both our earlier and our present results that the vitellogenin gene activation which can be achieved in oocytes is similar to that found in liver cells. This emphasizes the quantitative efficiency of vitellogenin transcription in oocytes compared with that of current in vitro systems (3-5). For example, five oocytes contain 20 copies of the B2 gene (oocytes being tetraploid in DNA content) and can generate a detectable vitellogenin signal in 24 h whether activation is induced by pure receptor or by a complete in vitro transcription extract. To achieve this, each gene must be transcribed many times. Transcription in vitro using systems such as the one we previously described, in which 1 jig of DNA containing 2 x 1011 copies of a B2 sequence generates a similar signal in 3 h (5), appears to be much less efficient, although without knowing how many copies are transcribed, or how many times, it is not possible to assess the discrepancy precisely. However, despite the high yield of vitellogenin transcripts in oocytes, it does appear that when receptor operates by itself to trigger vitellogenin transcription, very large amounts are needed. The absolute amount needed in the oocyte nucleus

exceeds the amount needed in a liver nucleus by a factor of about 106, which represents, in concentration terms, a factor of about 103. It seems unlikely that the reason for this difference is simply that the receptor we have used is very inefficient at activating chromosomal vitellogenin genes. HEO is closely homologous to Xenopus receptor (33), binds to estrogen response elements, and activates vitellogenin promoters in plasmids both in vivo and in vitro. The mutation at position 400 (Gly->Val [28]) known to be present in HEO is not relevant here for three reasons. First, although the mutation reduces the stability of the unoccupied receptor at 25°C, the mutation does not reduce the stability of the occupied receptor. Second, in the experiments described here, the receptor is synthesized inside mature oocytes, in which the concentration of estradiol (30 x 10-9 to 70 x 10-9 M [6]) is more than enough to ensure that even the elevated level of receptor generated here is permanently occupied and therefore functional. Third, the mutation does not affect the DNA-binding and transcription activation properties of the estradiol-receptor complex. As vitellogenin transcription in vivo normally occurs only when the estrogen response unit in the vitellogenin gene is occupied by receptor, the requirement for a high level of pure receptor to achieve activation implies that receptor cannot interact easily with its target in oocytes. This in turn suggests that the estrogen response unit (ERU) is much less accessible in oocytes than in liver cells, consistent with the view that the accessibility of cis-acting elements to transacting factors plays an important part in gene expression. Our results also suggest that the amount of receptor needed for the ERU to be occupied is reduced when other liver factors, which cannot by themselves activate vitellogenin genes, are present. They may mean that the other factors exert their effects by increasing the effective concentration

ACTIVATION OF OOCYTE VITELLOGENIN AND ALBUMIN GENES

VOL. 10, 1990

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FIG. 7. Activation of albumin and vitellogenin genes in oocytes by extracts of Xenopus liver which either do or do not contain estrogen receptor. In vitro transcription extracts from female liver nuclei were UV irradiated either with no additions or in the presence of 6-oxo-estradiol to inactivate receptor to the extent required and were then injected into oocytes. Batches of 10 oocytes were extracted, and the RNA from 5 was analyzed. Samples were probed first for albumin and then for vitellogenin RNA as described in the legend to Fig. 2. Rows A through B', Inactivation of receptor eliminates transcription of vitellogenin genes but not of albumin genes. Row A, Albumin probe. An extract was irradiated with no additions, and oocytes were incubated for 24 (column 1) or 72 h (column 2), or the same extract was irradiated for 110 min in the presence of 43 nM 6-oxo-estradiol to completely inactivate receptor. Row A', The samples in row A were reprobed with a vitellogenin probe; row B, albumin probe. Oocytes were analyzed 72 h after being injected with extract which had been irradiated in the presence of 43 nM 6-oxo-estradiol for 0, 11, 27, and 110 min to inactivate 0 (column 1), 30 (column 2), 70 (column 3), and 100%S (column 4), respectively, of the receptor; row B', the samples in row B were reprobed with a vitellogenin probe; row C, factors other than receptor increase the extent of vitellogenin transcription induced by receptor. Columns 1 and 2, Oocytes were injected with either 5 x 106 (column 1) or 5 x 108 (column 2) molecules of receptor RNA; columns 3 and 4, oocytes were first injected with 5 x 106 (column 3) or 5 x 108 (column 4) molecules of receptor RNA and, 24 h later, with extract which had been irradiated to inactivate 100% of the receptor. All samples were probed for vitellogenin after a total incubation of 72 h.

of the ERU, and a possible explanation for our findings is that the other factors modify the organization of chromatin so as to increase the accessibility of the ERU to receptor. Others, too, have suggested that the organization of chromatin plays an important part in the accessibility of vitellogenin genes to transactivators such as receptor (3). A systematic examination of the initiation of vitellogenin transcription in vitro shows that, at least in liver extracts, estradiol, its receptor, and the ERU are all required for accurate initiation (3-5). Hence, liver extracts give a qualitatively accurate picture of vitellogenin transcription in liver cells, although the quantitative efficiency of such transcription systems is unknown. However, extracts of B3.2 kidney cells also initiate vitellogenin transcription accurately in vitro in the absence of added estradiol or receptor whether the template contains an ERU or not (3), although intact B3.2 cells do not contain receptor and do not express their own vitellogenin genes (24). To account for these observations, Corthdsy et al. (3) suggest that vitellogenin genes in the chromatin of intact kidney cells are not available for transcription at all but that the same genes in liver chromatin are and that vitellogenin gene activity in liver depends on a

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balance of both positive and negative trans-acting factors. Our work, which suggests that liver contains at least one factor which increases the accessibility of the ERU in the intact chromatin of a nonvitellogenic cell to receptor, is compatible with those suggestions. However, our work also indicates that receptor may be able to interact with and activate even very inaccessible genes if receptor concentration is high enough. A general implication of our work is that quantitative considerations, as well as qualitative ones, must be taken into account when investigating the basis of tissue-specific gene expression. The simple finding that a particular gene, normally active in one type of cell, can be activated in a foreign cell by a single transactivator cannot be taken as evidence that normal activation is achieved by a single factor operating independently of other control systems if the amount required in the foreign cell greatly exceeds the amount needed in the normal cell. In this context it would be interesting to know whether unusually high levels of MyoD can activate muscle-specific genes in cells which do not respond to normal levels (23), and also how the minimum level of MyoD needed to activate muscle-specific genes in cells which do respond to MyoD (34) compares with the amount present in a normal muscle cell. ACKNOWLEDGMENTS We thank P. Chambon for the receptor plasmid, G. U. Ryffel for the albumin plasmid, W. Wahli for vitellogenin plasmids, R. B. Sim for help with immunoassays, S. Bradbury for image analysis, J. Kench for atomic absorption spectroscopy, C. V. E. Wright and A. M. Thorburn for helpful comments on the manuscript, and G. Sullivan for technical assistance. E.A.M. and N.A.C. thank the Medical Research Council for postgraduate studentships. This work was supported by the Cancer Research Campaign and by the Research and Equipment Committee of the General Board of the Faculties, Oxford University. LITERATURE CITED 1. Brock, M. L., and D. J. Shapiro. 1983. Estrogen stabilizes vitellogenin mRNA against cytoplasmic degradation. Cell 34: 207-214. 2. Contreras, R., D. Gheysen, J. Knowland, A. van de Voorde, and W. Fiers. 1982. Evidence for the direct involvement of DNA replication origin in synthesis of late SV40 RNA. Nature (London) 300:500-506. 3. Corthesy, B., J.-R. Cardinaux, F.-X. Claret, and W. Wahli. 1989. A nuclear factor I-like activity and a liver-specific repressor govern estrogen-regulated in vitro transcription from the Xenopus laevis vitellogenin Bi promoter. Mol. Cell. Biol. 9:5548-5562. 4. Corthesy, B., R. Hipskind, I. Theulaz, and W. Wahli. 1988. Estrogen-dependent in vitro transcription from the vitellogenin promoter in liver nuclear extracts. Science 239:1137-1139. 5. Cridland, N. A., C. V. E. Wright, E. A. McKenzie, and J. Knowland. 1990. Selective photochemical treatment of oestrogen receptor in a Xenopus liver extract destroys hormone binding and transcriptional activation but not DNA binding. EMBO J. 9:1859-1866. 6. Fortune, J. E. 1983. Steroid production by Xenopus ovarian follicles at different developmental stages. Dev. Biol. 99:502509. 7. Gerber-Huber, S., D. Nardelli, J.-A. Haefliger, D. N. Cooper, F. Givel, J.-E. Germond, J. Engel, N. M. Green, and W. Wahli. 1987. Precursor-product relationship between vitellogenin and the yolk proteins as derived from the complete sequence of a Xenopus vitellogenin gene. Nucleic Acids Res. 15:4737-4760. 8. Germond, J.-E., P. Walker, B. ten Heggeler, M. Brown-Luedi, E. de Bony, and W. Wahli. 1984. Evolution of vitellogenin genes: comparative analysis of the nucleotide sequences down-

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24. Seiler-Tuyns, A., A.-M. Merillat, S. Nardelli-Haeffiger, and W. Wahli. 1988. The human estrogen receptor can regulate exogenous but not endogenous vitellogenin gene promoters in a Xenopus cell line. Nucleic Acids Res. 16:8291-9305. 25. Smith, R. C., and J. Knowland. 1984. Protein synthesis in dorsal and ventral regions of Xenopus laevis in relation to dorsal and ventral differentiation. Dev. Biol. 103:355-368. 26. Theulaz, I., R. Hipskind, B. ten Heggeler-Bordier, S. Green, V. Kumar, P. Chambon, and W. Wahli. 1988. Expression of human estrogen receptor mutants in Xenopus oocytes: correlation between transcriptional activity and ability to form proteinDNA complexes. EMBO J. 7:1653-1660. 27. Thorburn, A., and J. Knowland. 1988. A novel nuclear transcription system which responds correctly to cloned estrogen receptor. Nucleic Acids Res. 16:10469-10476. 28. Tora, L., A. Mullick. D. Metzger, M. Ponglikitmongkol, I. Park, and P. Chambon. 1989. The cloned human oestrogen receptor contains a mutation which alters its hormone binding properties. EMBO J. 8:1981-1986. 29. Wahli, W. 1988. Evolution and expression of vitellogenin genes. Trends Genet. 4:227-232. 30. Walker, P., J.-E. Germond, M. Brown-Luedi, F. Givel, and W. Wahli. 1984. Sequence homologies in the region preceding the transcription initiation site of the liver estrogen-responsive vitellogenin and apo-VLDLII genes. Nucleic Acids Res. 12: 8611-8626. 31. Walter, P., S. Green, G. L. Greene, A. Krust, J. M. Bornert, J. M. Jeltsch, A. Staub, E. Jensen, G. Scrace, M. Waterfield, and P. Chambon. 1985. Cloning of the human estrogen receptor cDNA. Proc. Natl. Acad. Sci. USA 82:7889-7893. 32. Watson, C. S., and T. Torres. 1990. Expression and translocation of cloned human estrogen receptor in the Xenopus oocyte does not induce expression of the endogenous oocyte vitellogenin genes. Mol. Endocrinol. 4:565-572. 33. Weiler, I. J., D. Lew, and D. J. Shapiro. 1987. The Xenopus laevis estrogen receptor: sequence homology with human and avian receptors and identification of multiple estrogen receptor messenger ribonucleic acids. Mol. Endocrinol. 1:355-362. 34. Weintraub, H., S. J. Tapscott, R. L. Davis, M. J. Thayer, M. A. Adams, A. B. Lassar, and A. D. Miller. 1989. Activation of muscle-specific genes in pigment, nerve, fat, liver and fibroblast cell lines by forced expression of MyoD. Proc. Natl. Acad. Sci. USA 86:5434-5438. 35. Westley, B. R., and J. Knowland. 1978. An estrogen receptor from Xenopus liver possibly connected with vitellogenin synthesis. Cell 15:367-374. 36. Wright, C. V. E., S. C. Wright, and J. Knowland. 1983. Partial purification of estradiol receptor from Xenopus liver and levels of receptor in relation to estradiol concentration. EMBO J. 2:973-977.