Hormonal Regulation of TSEI-Repressed Genes:Evidence

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MOLECULAR AND CELLULAR BIOLOGY, JUlY 1989, p. 2837-2846 Vol. 9, No. 7 0270-7306/89/072837-10$02.00/0 Copyright C) 1989, American Society for Microbiology Hormonal Regulation of TSEI-Repressed Genes: Evidence for Multiple Genetic Controls in Extinction MATHEW J. THAYER AND R. E. K. FOURNIER* Department of Molecul(ar Medicine, Fred Hiutchinson Cancer Research Center, 1124 Columbia Street, Seattle, Washington 98104 Received 9 January 1989/Accepted 26 March 1989

Somatic cell hybrids formed by fusing hepatoma cells with fibroblasts generally fail to express liver functions, a phenomenon termed extinction. Previous studies demonstrated that extinction of the genes encoding tyrosine aminotransferase, phosphoenolpyruvate carboxykinase, and argininosuccinate synthetase is mediated by a specific genetic locus (TSEI) that maps to mouse chromosome 11 and human chromosome 17. In this report, we show that full repression of these genes requires a genetic factor in addition to TSE1. This conclusion is based on the observation that residual gene activity was apparent in monochromosomal hybrids retaining human TSEI but not in complex hybrids retaining many fibroblast chromosomes. Furthermore, TSE1- repressed genes were hormone inducible, whereas fully extinguished genes were not. Analysis of hybrid segregants indicated that genetic loci required for the complete repression phenotype were distinct from TSE1.

Tissue-specific gene expression in mammalian cells is ing that single fibroblast chromosome are extinguished for primarily regulated at the level of transcription (8). A par- both serum albumin and alcohol dehydrogenase gene activ- ticular gene may account for a large fraction of total tran- ity (A. C. Chin and R. E. K. Fournier, submitted for publi- scription in one cell type yet be virtually silent in other cell cation). This locus (TSE2) is apparently identical to one lineages. Furthermore, expression of many tissue-specific previously assigned to an L-cell marker chromosome termed genes is controlled by humoral agents, and the hormonal M1 (24). responses of a given gene may differ in different tissues. Deletion hybrids retaining fragments of human chromo- Although the regulation of tissue-specific and inducible gene some 17 have been used to show that the TAT, PEPCK, and expression clearly involves interactions between trans- AS extinction phenotypes behave as a single trait (15). That acting factors and specific nucleotide sequences in target is, TSEI maps to a specific site on 17q distal to D17S4, and genes, the developmental events that establish these differ- this region seems to affect expression of all three genes in a entiated phenotypes have yet to be defined. coordinate manner. This is interesting because TAT, Genetic tests can be used to identify trans-acting factors PEPCK, and AS share other forms of gene control. Devel- involved in the regulation of tissue-specific gene activity. opmentally, all three genes are activated in the liver within a Davidson et al. (7) first observed that somatic hybrids few hours of birth (27, 28, 33), and this process requires the formed by fusing different cell types fail to express tissue- function of a mouse chromosome 7-linked locus designated specific products, a phenomenon they termed extinction. csdr-l (16, 31). Furthermore, humoral agents modulate This phenomenon proved to be both general, occurring in expression of these genes in similar ways: both glucocorti- virtually all intertypic hybrid crosses, and bidirectional, coids and cyclic AMP (cAMP) induce PEPCK (29), TAT affecting the differentiated products of both parental cells (30), and AS (22) expression in liver. Other studies suggest (32, 37, 40). Subsequently, Weiss and co-workers discovered that cAMP stimulates the initial synthesis of PEPCK shortly that hybrid segregants that had lost parental chromosomes after birth (28), and both glucocorticoids and cAMP have could reexpress previously extinguished functions (39, 40). been implicated in the postnatal induction of TAT (11, 12, These observations suggested that intertypic hybrids could 33). provide a system with the potential to define genetic factors In this report, we show that basal expression of the TAT, that affect tissue-specific gene activity in trans. With the PEPCK, and AS genes was reduced but not abolished in the development of methods for constructing karyotypically presence of fibroblast TSEJ. Furthermore, these TSEI- simple hybrids with predetermined genotypes (10, 17), that repressed genes were partially responsive to hormonal potential has been largely realized. agents that normally induce their transcription. In marked Recent studies have identified two distinct genetic loci that contrast, neither residual activity nor hormone inducibilty are involved in extinction of liver gene activity in hepatoma could be demonstrated in genotypically complete hepatoma hybrid cells (13, 15, 23, 24). The first such locus, tissue- x fibroblast hybrids. These data indicate that multiple con- specific extinguisher 1 (TSEI), is a discrete genetic entity trols are involved in extinction and that complete transcrip- that resides on mouse chromosome 11 and human chromo- tional repression of the TAT, PEPCK, and AS genes re- some 17. TSEI extinguishes tyrosine aminotransferase quires a genetic factor(s) in addition to TSEI. (TAT) (13), phosphoenolpyruvate carboxykinase (PEPCK) (15), and argininosuccinate synthetase (AS) (this report) MATERIALS AND METHODS expression in trans, but it has no effect on expression of most other liver A Cell lines and culture conditions. FAO-i is an HPRT-, genes. second extinguisher locus has been ouabain-resistant derivative of the highly differentiated rat mapped to mouse chromosome 1; rat hepatoma cells retain- hepatoma line H4IIEC3 (25); its properties have been reported (13). The microcell hybrid clone FH(17)I was con- * Corresponding author. structed by transferring human chromosome 17 from diploid 2837 2838 THAYER AND FOURNIER MOL. CELL. BIOL. fibroblasts into TK- FTO-2B rat hepatoma recipients (14) SSC-0.1% SDS for 15 min at room temperature, and in two and selecting the TK+ phenotype in hypoxanthine-amino changes of 0.lx SSC-0.1% SDS at 65°C for 30 min each. pterin-thymidine. FHB(17)1 was a backselectant population Autoradiography was for 1 to 5 days, using Kodak XAR or generated from FH(17)1 by selection in medium containing XRP film (Eastman Kodak Co.) and a single intensifying 30 ,ug of bromodeoxyuridine per ml; these cells no longer screen. The blots were stripped for reuse by boiling for 2 min retain human chromosome 17. The FF-series whole-cell in double-distilled water. hybrids were prepared by fusing FAO-1 rat hepatoma cells Southern blot analyses. High-molecular-weight cellular with diploid fibroblasts from C57BL/6J mouse embryos. The DNA was extracted from each cell line as described previ- properties of these clones have been reported (13). ously (18). DNA (5 ,ug) was digested to completion with All cells were cultured in a 1:1 mixture of Ham F12- EcoRI (New England Bio-Labs, Inc.) and run through 0.7% Dulbecco modified Eagle medium supplemented with 10% agarose gels in 0.04 M Tris acetate-2 mM EDTA. The DNA fetal bovine serum (GIBCO Laboratories). FH(17)1 was was transferred to Zetabind or Magna Nylon 66 (Fisher maintained in medium containing hypoxanthine-aminopter- Scientific Co.) by standard techniques (4, 36). Prehybridiza- in-thymidine. Antibiotics were not used, and the cells were tion and hybridization conditions were as described above free of mycoplasma, as judged by staining with the fluoro- for RNA blot analyses. chrome Hoechst 33258 (2). TAT immunofluorescence. Cells were seeded onto 12- Plasmids. Plasmids were labeled to specific activities of 0.5 mm-diameter glass cover slips and cultured in complete x 109 to 1.0 x 109 cpm/,ug by random priming. The rat medium in the presence or absence of 10-6 M dexametha- PEPCK cDNA clone pPCK10 was provided by R. Hanson sone, 5 x 10-4 M dBtcAMP-1 mM theophylline, or both for (42). The rat TAT cDNA clone pcTAT-3 was obtained from 24 h. The cells were then fixed and stained essentially as G. Schutz (34). The human cx-tubulin cDNA pKa-1 was described by Mevel-Ninio and Weiss (21), as follows. The provided by N. Cowan (5). The rat argininosuccinate syn- cover slips were rinsed with phosphate-buffered saline and thetase cDNA clone pASR was from D. Mathieu-Mahul (19). fixed in 3% formaldehyde for 1 min. The formaldehyde RNA dot blots. (i) Time course analyses. Monolayers of solution was diluted 1:1 with methanol and aspirated, and cells in late exponential growth were exposed to 10-6 M pure methanol was added. After 20 min at 4°C, the cover dexamethasone or 5 x 10-' M dibutyryl cAMP (dBtcAMP) slips were rinsed in phosphate-buffered saline and incubated plus 1 mM theophylline for 2, 4, 8, 16, 24, or 36 h. with rabbit anti-rat TAT antiserum for 30 min at 37°C in a Cytoplasmic RNAs (3 ,ug) extracted from the cells were humidified chamber. After two rinses with phosphate- applied in duplicate to Zetabind membranes and hybridized buffered saline, bound immunoglobulin was stained by incu- to cDNA probes as described previously (3). After autora- bation with fluorescein-conjugated secondary antibody diography, each dot was cut out, and radioactivity was (sheep anti-rabbit immunoglobulin G; Organon Teknika). quantitated by liquid scintillation counting. Each time point After three rinses in phosphate-buffered saline, the cover represents the amount of specific hybridization normalized slips were mounted with buffered glycerol and examined to the a-tubulin control. under phase-contrast and epifluoresence illumination, using (ii) Dose-response curves. Monolayers of cells in late ex- a Zeiss Axiophot photomicroscope. X ponential growth were exposed to 5 x 10-6 , 10-5, 5 x 10-4, or 5 x 10-3 M dBtcAMP for 4 h, and cytoplasmic RNA RESULTS was extracted and analyzed by dot blotting as described above. Each point was normalized to the value for the Properties of the cell lines. FH(17)1 is a hepatoma microcell x-tubulin control, and values are expressed relative to max- hybrid that selectively retains human chromosome 17 de- imal induction in each cell line. rived from diploid fibroblasts. Its isolation and properties (iii) Cycloheximide experiments. Monolayers of cells in late were similar to those of the HF(17)-series clones described exponential growth were exposed to 5 x 10-4 M dBtcAMP previously (13). This particular hybrid was used as a proto- plus 1 mM theophylline for 0, 2, or 4 h in the presence or type in the studies reported here because the population had absence of 10 F.M cycloheximide. This concentration of a stable karyotype and contained few segregant cells. cycloheximide inhibited protein synthesis by >90% at both 2 FH(17)1 cultures at early passage were challenged with and 4 h of incubation, as judged by incorporation of medium containing bromodeoxyuridine, and TK- clones [3H]leucine into trichloroacetic acid-precipitable material. that had segregated human chromosome 17 arose at a Cytoplasmic RNA was extracted and analyzed by the dot frequency of 11%. Approximately 100 such clones were blot procedure described above. Each measurement repre- pooled to form a mass population of backselected cells sents hybridization corrected for the cx-tubulin control. designated FHB(17)1. The presence or absence of human Northern (RNA) blot analyses. Cytoplasmic RNA (5 jig) chromosome 17 in FH(17)1 and FHB(17)1, respectively, was was extracted from each cell line (9) and size fractionated on confirmed by cytogenetic analysis and by Southern marker 1.2% agarose-formaldehyde gels. The RNAs were trans- analysis, using cloned probes from four loci on human ferred to Zetabind nylon membranes (AMF Cuno) by capil- chromosome 17: MYHI, D17SJ, D17S4, and ERBAI (data lary transfer, baked for 2 to 4 h at 80°C, and UV cross-linked not shown). FH(17)1 populations retained a single copy of an by standard techniques (4). The blots were prehybridized for apparently intact human chromosome 17 in >90% of the 5 min to several hours in hybridization buffer (50% formam- cells; FHB(17)1 cells had segregated that single human ide, 1% bovine serum albumin (fraction V), 1 mM EDTA, 0.5 chromosome. M sodium phosphate [pH 7.2], 7% sodium dodecyl sulfate The FF-series clones FF3-3 and FF5-1 are intertypic [SDS]). Hybridizations were for 17 to 24 h at 42°C in fresh hybrids formed by fusing highly differentiated rat hepatoma hybridization buffer containing 1 x 108 to 8 x 108 cpm of cells (FAO-1) with diploid mouse embryo fibroblasts; these randomly primed [32P]-labeled cDNA probe (specific activ- hybrids have been characterized extensively (3, 13). Cyto- ity, 0.5 x 109 to 1.0 x 109 cpm/,ug). The filters were washed genetic analysis has shown that these hybrids are karyotyp- in 2x SSC (SSC is 0.15 M NaCl plus 0.015 M sodium ically complete: they retain virtually all parental chro- citrate)-0.1% SDS for 15 min at room temperature, in 0.lx mosomes. Liver-specific gene activity is extinguished in VOL. 9, 1989 HORMONAL EFFECTS AND EXTINCTION 2839

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410wo 1.25 _ _O 4 0.62 _ _~_ :_ 0.31 - _ _ _~ 0.16 0.08 0.04' 0..0212__L .- , FIG. 1. Quantitation of basal PEPCK, TAT. and AS mRNA expression in a TSE1+ microcell hybrid and its backselectant. Serial twofold dilutions of cytoplasmic RNA from FH(17)1 and FHB(17)1 were applied to nylon membranes and hybridized with labeled cDNA probes from the PEPCK, TAT, AS, and oA-tubulin (TU) genes. these hybrids, as judged by expression of 20 different liver that segregants alone could not account for the residual gene mRNAs. Finally, TAT and serum albumin mRNA levels in activity in FH(17)1, as segregants would contribute equally these clones are repressed 500- to 1,000-fold relative to to the accumulation of all three mRNAs. Furthermore, the levels in parental hepatoma cells. -100-fold reduction of PEPCK mRNA expression suggested Basal expression of PEPCK, TAT, and AS mRNAs in the that fewer than 1% of cells in this population were segre- presence of fibroblast TSEI. As indicated above, extinction in gants, a value in agreement with the frequency of TK- cells whole-cell hybrids is a quantitatively large effect, with determined by selective challenge with bromodeoxyuridine. tissue-specific mRNA levels being reduced 500- to 1,000- Thus, residual TAT, AS, and possibly PEPCK gene expres- fold. In contrast, extinguished mRNAs are generally re- sion was detected in the presence of fibroblast TSEJ. We pressed only 10- to 20-fold in microcell hybrid clones. These next assayed whether gene activity could be induced by the differences are largely due to the fact that microcell hybrids positive factors that control expression of these genes in contain many more segregant cells than are generally present liver. in whole-cell hybrid populations. These segregants have lost Hormonal induction of TSEJ-repressed genes. The effects the donor chromosome under selection, are reexpressing of TSEI on TAT, PEPCK, and AS mRNA accumulation are previously extinguished functions, and are dying in the largely mediated at the level of gene transcription, as judged selective medium. The segregants typically constitute 5 to by nuclear run off assays (unpublished data). Furthermore, 15% of cells in microcell hybrid populations at steady state, both glucocorticoids and cyclic nucleotides induce expres- and this is sufficient to account for the residual levels of gene sion of these genes by increasing the rate of transcription activity detected in these clones (13). The presence of initiation (29, 30). Therefore, we determined whether either segregants in most microcell hybrids has precluded an accu- of these positive regulators of PEPCK and TAT gene tran- rate assessment of the true magnitude of TAT, PEPCK, and scription could induce gene activity in the presence of AS gene repression by fibroblast TSEI. fibroblast TSEJ. To circumvent this problem, we quantitated basal expres- The kinetics of PEPCK, TAT, and AS mRNA accumula- sion of TAT, PEPCK, and AS mRNAs in a microcell hybrid tion in cells induced with dexamethasone or cAMP were that was largely free of reexpressing segregant cells. Serial determined by an RNA dot blot assay. Cytoplasmic RNA twofold dilutions of FH(17)1 and FHB(17)1 cytoplasmic was extracted from the primary clone [FH(17)1] and its RNAs were applied to nylon membranes, and the filters backselectant [FHB(17)1] after various intervals in the pres- hybridized with labeled PEPCK, TAT, AS, and cx-tubulin ence of either dexamethasone or dBtcAMP plus theophyl- cDNA probes (Fig. 1). Specific hybridization of the PEPCK, line. The RNA (3 ,ug) was applied to nylon membranes and TAT, and AS probes to FHB(17)1 RNA was detected over a hybridized with radioactively labeled cDNA probes. Dupli- wide concentration range. In contrast, hybridization of these cate dots from each time point were quantitated by liquid probes with FH(17)1 RNA was detected only at significantly scintillation counting. Each value represents the amount of higher RNA concentrations. Hybridization of the a-tubulin specific hybridization to the TAT, PEPCK, or AS cDNA probe was similar at equivalent concentrations of FH(17)1 probe normalized for hybridization to the ot-tubulin control, and FHB(17)1 RNA. We estimate that PEPCK mRNA levels whose expression remained constant throughout the time in FH(17)1 were decreased 64- to 128-fold relative to those of course of the experiment in both cell lines. its FHB(17)1 backselectant, whereas TAT and AS mRNA Results of the dexamethasone inductions are shown in levels were reduced 8- to 16-fold. Thus, it appeared that Fig. 2. In the absence of TSEJ [i.e., in FHB(17)1], induction TSEJ had a greater effect on PEPCK mRNA expression than of all three mRNAs was detected within 2 to 4 h of hormone on accumulation of TAT or AS mRNA. This result indicated addition, and steady-state levels increased 10- to 20-fold 2840 THAYER AND FOURNIER MOL. CELL. BIOL.

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1z _"W w _m ___ ) 10 20 30 40 Llk TIME (hours) FIG. 2. Dexamethasone induction of PEPCK, TAT, and AS mRNAs in a TSE1+ microcell hybrid (L) and its backselectant (A). U Cells were incubated in medium containing 10-6 M dexamethasone U for various intervals, and RNA was prepared and analyzed as I described in Materials and Methods. RNA concentrations were normalized to a-tubulin mRNA levels, which remained constant throughout the experiment. mA within 24 h. This induction phenotype is typical of rat hepatoma lines of the H4IIEC3 family. However, a different FIG. 3. cAMP induction of and AS mRNAs in a response was observed in hepatoma cells retaining fibroblast PEPCK, TAT, TSE1+ microcell hybrid (U) and its backselectant (A). Cells were TSEJ. Whereas both TAT and AS mRNA levels increased in incubated in medium containing 5 x 10-4 M dBtcAMP-1 mM FH(17)1 cells treated with dexamethasone, induced levels theophylline for various intervals and analyzed as described in were only 15 to 30% those of the FHB(17)1 backselectant; Materials and Methods. RNA concentrations were normalized to that is, dexamethasone-induced gene activity was decreased o-tubulin mRNA levels. Insets show the early kinetics of induction in cells containing TSEJ. Induction of PEPCK mRNA under in the TSE1+ microcell hybrid [FH(17)1] after incubation in medium these conditions was barely detected. From these results, it with dBtcAMP plus theophylline for 0, 0.5, 1.0. 1.5, 2, 3, 4, 5, 6, 8, appeared that TAT and AS expression was partially dexa- and 10 h. methasone inducible in the presence of fibroblast TSEJ, whereas PEPCK gene activity was refractory to glucocorti- dBtcAMP plus theophylline (Fig. 3). Although basal levels of coid induction under these conditions. Furthermore, the PEPCK mRNA differed by about 100-fold in FH(17)1 versus magnitude of the TAT and AS responses in FH(17)1 was FHB(17)1, cAMP treatment abolished this difference: within such that these responses could not have been due solely to 2 h of cAMP addition, PEPCK mRNA levels were compa- segregant cells in the population. These induction pheno- rable in the two cell lines. Similar results were obtained for types were very different from those of karyotypically com- the TAT and AS genes; the differences in basal expression in plete whole-cell hybrids, which were refractory to glucocor- the primary hybrid and its backselectant ('10-fold) were ticoid induction (13; see below). eliminated by incubating the cells in the presence of cyclic Induction of these genes by cAMP was even more dra- nucleotide. Thus, cAMP induced expression of the PEPCK, matic. A rapid increase in steady-state levels of all three TAT, and AS genes to the same high levels in the presence mRNAs occurred in both the primary hybrid and its back- or absence of fibroblast TSE1. selectant when cells were incubated in medium containing dBtcAMP dose response. To determine whether the ability VOL. 9, 1989 HORMONAL EFFECTS AND EXTINCTION 2841

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-6 -5 -4 -3 -6 -5 -4 -3 -6 -5 -4 -3 log [cAMP] (M) FIG. 4. cAMP dose response in a TSE1+ microcell hybrid (-) and its backselectant (A). Cells were incubated in medium containing the indicated concentrations of dBtcAMP for 4 h, and RNA was prepared and analyzed as described in Materials and Methods. RNA levels are expressed relative to maximum induction of PEPCK (A), TAT (B), and AS (C) achieved in each cell line. of the cells to respond to suboptimal concentrations of tration of cycloheximide inhibited protein synthesis by cAMP was somehow altered by the presence of fibroblast >90% at both times, as judged by incorporation of TSEJ (resulting in lower basal expression), dose-response [3H]leucine into acid-precipitable material. PEPCK and TAT experiments were performed. The FH(17)1 primary hybrid mRNAs were induced in both the primary hybrid and its and its FHB(17)1 backselectant were incubated in the pres- backselectant after 2 and 4 h in medium containing dBt- ence of various concentrations of dBtcAMP for 4 h, and cAMP, and induction was not affected by cycloheximide cytoplasmic RNA was extracted. Levels of PEPCK, TAT, (Fig. 5). AS mRNA was induced 3- and 10-fold in FH(17)1 and AS mRNAs were quantitated as described above. In- cells after 2 and 4 h in medium containing dBtcAMP, ductions of all three genes were virtually identical in the two whereas little induction in the backselectant was detected at cell lines at dBtcAMP concentrations between 0.05 and 5 these early times. Again, the presence of cycloheximide in mM (Fig. 4). We conclude that the ability of the cells to the medium had no effect on the induction of AS mRNA. respond to cAMP was not altered by fibroblast TSEI. Thus, protein synthesis was not required for induction of To determine whether changes in endogenous cAMP these genes in either the presence or the absence of fibro- levels might be responsible for the decrease in basal PEPCK, blast TSEJ. Therefore, transcriptional activation of extin- TAT, and AS mRNA expression in the presence of fibroblast guished genes by dBtcAMP must be a primary response TSEJ, cAMP concentrations within the cells were measured. mediated through preexisting factors. Whole-cell extracts from induced (0.5 mM dBtcAMP, 4 h) Induction of TAT protein in cells containing fibroblast and uninduced cells were prepared, and the cAMP concen- TSE1. The results summarized above indicate that TAT trations in the extracts were determined by radioimmunoas- expression was reduced -10-fold in the presence of fibro- say (RIANEN; Dupont, NEN Research Products). This blast TSEJ but that gene activity could be induced with assay detected both cAMP and dBtcAMP with equal effi- either glucocorticoids or cAMP. The magnitude of these ciency. The concentration of cAMP in uninduced FH(17)1 responses was such that it seemed likely that most cells of cells was 17.2 pM/mg of cell protein, and this increased to the hybrid population were responding to the inducer. How- 121 pM/mg of protein after incubation of the cells in medium ever, it remained possible that a subpopulation of highly containing 0.5 mM dBtcAMP for 4 h. Corresponding values inducible cells accounted for the inducible phenotype. To for uninduced FHB(17)1 cells were 16.9 and 188 pM/mg of distinguish between these possibilities, we determined the protein. These results indicated that intracellular cAMP fraction of cells expressing TAT under inducing conditions concentrations were not affected by the presence of fibro- by immunofluorescence. blast TSEJ; the two cell lines contained similar cAMP FH(17)1 and its backselectant were plated onto cover slips concentrations both before and after induction. and incubated with dexamethasone, dBtcAMP, or both for Induction of extinguished genes does not require protein 24 h. The cells were fixed and stained, using a TAT-specific synthesis. Glucocorticoid induction of most genes is a pri- primary antibody and a fluorescein-labeled secondary anti- mary response; i.e., protein synthesis is not required (41). body. No specific labeling of either the primary clone or its Similarly, cyclic nucleotide induction of PEPCK mRNA backselectant was detected with uninduced cells. Thus, TAT synthesis does not require protein synthesis (29). To deter- specific activities of <40 mU/mg of protein could not be mine whether active versus TSEI-repressed genes could be visualized under these conditions. In contrast, TAT-specific discriminated on the basis of protein synthesis requirements immunofluorescence was readily detected after induction for induction, cycloheximide experiments were performed. with dexamethasone or dBtcAMP. In cAMP-induced cells, Cells were induced with dBtcAMP in the presence or the intensities of TAT immunofluorescence were similar in absence of 10 ,uM cycloheximide for 2 or 4 h. This concen- the primary hybrid and its backselectant, and >90% of the 2842 THAYER AND FOURNIER MOL. CELL. BIOL.

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cells in each population were clearly expressing TAT (Fig. for 8 h, and RNA was extracted and analyzed as described in 6). In dexamethasone-treated cultures, TAT-specific immu- Materials and Methods. FAO-1 parental hepatoma cells nofluorescence was evident in >80% of the cells of either expressed PEPCK, TAT, and AS mRNAs, and expression FH(17)1 or FHB(17)1 populations. In this case, however, the was inducible by both glucocorticoids and cAMP (Fig. 7). In intensity of staining was clearly less in the primary hybrid contrast, PEPCK and TAT mRNAs were not detectable in than in the backselectant. The primary hybrid did contain a mouse embryo fibroblasts under any conditions. AS mRNA small fraction of cells (=1%) with backselectant levels of was expressed at low levels in MEF cells (<5% hepatoma fluorescence; we presume that these cells were segregants. levels), but this expression was not inducible by hormones. In the presence of both inducers, >90% of cells in each FF5-1 cultures at early passage and all FF3-3 populations population were highly fluorescent. As TAT was clearly assayed displayed similar phenotypes: PEPCK and TAT induced in most FH(17)1 cells by either dexamethasone or mRNA expression was extinguished, and neither dexam- cAMP, induction of gene activity was necessarily occurring ethasone nor cAMP induced detectable accumulation of in cells that retained fibroblast TSEJ. either mRNA. Extinction of basal and inducible PEPCK and Extinction of inducible and basal expression in whole-cell TAT mRNA expression was also observed in four other hybrids. Previous studies indicated that both basal and karyotypically complete hybrid clones (data not shown). dexamethasone-inducible TAT expression were extin- These results are in accord with previously published obser- guished in karyotypically complete hepatoma x fibroblast vations (13). Thus, whole-cell hybrids and microcell hybrids hybrids (13). Extinction in this context corresponded to a have demonstrably different extinction phenotypes. >500-fold reduction in steady-state levels of TAT mRNA The extinction phenotype of FF5-1 changed between early (3). This behavior contrasts with that of microcell hybrids, as and late passage (Fig. 7). While neither basal nor inducible described above. To more fully document these apparent PEPCK or TAT expression was detected at passage 5, differences, we compared the glucocorticoid and cyclic continued cultivation resulted in the generation of a hybrid nucleotide induction phenotypes of microcell versus whole- population that accumulated PEPCK and TAT mRNAs cell hybrids. when treated with cAMP. Significantly, these populations FF5-1 and FF3-3 are intertypic hepatoma x fibroblast did not induce either mRNA in response to dexamethasone. hybrids with essentially complete karyotypes; each contains Thus, the extinction phenotype of late-passage FF5-1 popu- approximately 50 chromosomes derived from its rat hepa- lations, which had likely segregated several fibroblast chro- toma parent plus 20 to 30 mouse fibroblast chromosomes. mosomes, resembled that of TSEI-containing microcell hy- Detailed karyotyping has shown that these hybrids retain brids. each of the 20 different mouse chromosomes in >90% of the Unlike PEPCK or TAT mRNAs, AS transcripts were cells (13). Furthermore, mouse chromosome 11 was present expressed in both parental cell types, but expression was at in 23 of 24 FF5-1 metaphases and 35 of 36 FF3-3 metaphases least 20- to 50-fold greater in hepatoma cells than fibroblasts. examined. Most of the cells in each hybrid population (23 of Karyotypically complete hybrids expressed fibroblast levels 23 and 30 of 35 metaphases for FF5-1 and FF3-3, respec- of AS mRNA, and expression was inducible by cAMP but tively) retained only a single copy of that particular mouse not by dexamethasone. Late-passage FF5-1 populations chromosome. partially reexpressed the dexamethasone-inducible pheno- Expression of PEPCK, TAT, and AS mRNAs in these type. hybrids under various inducing conditions was assayed by Finally, TAT immunofluorescence assays were performed RNA blot hybridization. The cells were incubated in serum- to determine the fraction of cells in whole-cell hybrid popu- free medium containing dexamethasone, dBtcAMP, or both lations that expressed a TAT-inducible phenotype. In FF3-3 VOL. 9, 1989 HORMONAL EFFECTS AND EXTINCTION 2843

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FIG. 6. TAT immunofluorescence of microcell and whole-cell hybrids. Cells were incubated in medium containing dexamethasone plus dBtcAMP for 24 h, fixed, and stained as described in Materials and Methods. Shown are phase-contrast (A, C, and E) and epifluorescence (B, D, and F) images of a TSEIJ microcell hybrid [FH(17)1; A and B], a karyotypically complete hybrid (FF3-312; C and D), and a hybrid segregant (FF5-113; E and F). The latter contains both inducible and noninducible cells. 2844 THAYER AND FOURNIER MOL. CELL. BIOL.

\1f:lH 1v:i-:i,) .) I [ 1.- l I DISCUSSION 1)(' (l IX l) (' 1I) (PII) I D ( V I) S DC IC(' 11 Tissue-specific gene activity is generally suppressed in a intertypic hybrids formed by fusing different cell types of equal ploidy (reviewed in reference 6). The extinction phenotype displayed by such hybrids is remarkable in that most, if not all, tissue-specific transcription is suppressed (3). However, extinction is reversible, and the tissue-specific 'pl genes of one parent tend to be reexpressed as chromosomes of the other are lost (39, 40). These phenomena have formed a basis for attempts to identify genetic factors that regulate tissue-specific gene expression in trans. TSEJ was the first trans-acting locus of this type to be b defined (13). Although clearly involved in PEPCK, TAT, and AS extinction in hepatoma microcell hybrids, TSEJ seems to require the activity of other genetic factors for complete repression of these genes. This conclusion is supported by two main observations. First, the magnitude of PEPCK, TAT, and AS mRNA repression was less in microcell hybrids (10- to 100-fold) than in whole-cell hybrids (>500- to 1,000-fold), and segregant cells alone could not account for the residual expression in microcell hybrid clones. Second, the PEPCK and TAT genes could be induced by dexametha- c sone and dBtcAMP in TSEJ-containing microcell hybrids but not in karyotypically complete whole-cell hybrids. Fur- thermore, whole-cell hybrids acquired an extinction phenotype similar to that of microcell hybrids as fibroblast chromosomes were lost. Several observations suggest that conversion of whole-cell hybrids from the noninducible to the inducible phenotype involved the segregation of genetic loci distinct from TSEJ. First, most cells in fully extinguished hybrid populations retained only a single copy of mouse chromosome 11, as did d microcell hybrids and hybrid segregants. Thus, TSEJ gene dosage cannot account for the different extinction phenotypes of these clones. Second, hybrid segregants displayed an induction phenotype that was more similar to that of microcell hybrids (TSE1+) than parental hepatoma cells (TSEl-). For example, cAMP and dexamethasone induced similar levels of PEPCK mRNA in hepatoma cells, but only cAMP induced significant PEPCK mRNA accumulation in microcell hybrids. Hybrid segregants (e.g., FF5-1, passage 13) displayed the latter phenotype. Similarly, dexametha- FIG. 7. Expression of PEPCK (a), TAT (b), AS (c), and a- sone induced more AS mRNA than did cAMP in hepatoma tubulin (d) mRNAs in hepatoma x fibroblast hybrids. Northern cells, but the reverse was true in microcell hybrids and blots of RNAs from uninduced cells (lanes B) and cells treated with hybrid segregants. Third, although the induction phenotype dexamethasone (lanes D), cAMP (lanes C), or both (lanes DC) for 8 of hybrid FF5-1 changed between passages 5 and 13, basal h were prepared and analyzed as described in Materials and Meth- expression of AS mRNA was constant (and low) during this ods. interval. This finding suggests that TSEJ was not being segregated from the cells. Finally, TAT immunofluorescence data clearly demonstrated that whole-cell hybrids contained both cAMP-inducible as well as noninducible cells; TSEI- populations induced with cAMP and dexamethasone, only 1 containing microcell hybrids contained only cAMP-inducible to 2% of cells exhibited TAT-specific immunofluorescence cells. (Fig. 6C and D). In contrast, late-passage (p13) FF5-1 Although cAMP reversed TSEI-mediated extinction in populations contained 20 to 30% TAT' cells after induction microcell hybrids, it is unlikely that TSEJ functions through (Fig. 6E and F). Thus, whole-cell hybrids consisted of the cyclic nucleotide induction pathway. Results presented subpopulations of inducible as well as noninducible cells. here show that neither intracellular cAMP levels nor cAMP- This was in direct contrast with the behavior of microcell inducible gene transcription was affected by fibroblast TSEJ. hybrids, which consisted predominantly of TSE1- Furthermore, TSEI affected both basal and glucocorticoid- extinguished yet cAMP-inducible cells (Fig. 6A and B). inducible TAT expression, and neither of these activities is Since TAT was expressed in >90% of FH(17)1 cells under cyclic nucleotide dependent (1). Finally, cAMP-responsive similar inducing conditions, karyotypically complete hybrids elements lie within 120 base pairs of the PEPCK cap site (26, must retain a genetic factor(s) in addition to TSEI that 35), whereas sequences required for TSE1 regulation map results in a more complete extinction phenotype. further upstream (unpublished observations). Thus, TSEI VOL. 9, 1989 HORMONAL EFFECTS AND EXTINCTION 2845 and cAMP affect PEPCK, TAT, and AS gene transcription Cell. Res. 104:255-262. in mechanistically distinct ways. 3. Chin, A. C., and R. E. K. Fournier. 1987. A genetic analysis of The extinction phenotype of TSEJ-containing microcell extinction: trans regulation of 16 liver-specific genes in hepatoma-fibroblast hybrid cells. Proc. Natl. Acad. Sci. USA 84: hybrids is interesting. The PEPCK, TAT, and AS genes of 1614-1618. these cells are in a functional state that is different from that 4. Church, G. M., and W. Gilbert. 1984. Genomic sequencing. of either parent. These loci are neither silent, as they are in Proc. Natl. Acad. Sci. USA 81:1991-1995. fibroblasts, nor fully active, as in hepatic cells. However, 5. Cowan, N. J., P. R. Dodner, E. U. Fuchs, and D. W. Cleveland. expression of all three genes can be restored to hepatoma 1983. Expression of human alpha-tubulin genes: interspecies levels in response to a strong transcriptional inducer such as conservation of 3' untranslated regions. Mol. Cell. Biol. 3: cAMP. This suggests that TSEJ-repressed genes are compe- 1738-1745. tent for transcription but that a factor(s) required for full 6. Davidson, R. L. 1974. Gene expression in somatic cell hybrids. basal activity is not functioning in the presence of fibroblast Annu. Rev. Genet. 8:195-218. 7. Davidson, R. L., B. Ephrussi, and K. Yamamoto. 1966. Regula- TSEI. According to this model, the TSEI-sensitive factor(s) tion of pigment synthesis in mammalian cells as studied by would no longer be limiting for transcription under inducing somatic hybridization. Proc. Natl. Acad. Sci. USA 56:1437- conditions. 1440. Although TSEJ is likely to alter the array of transcription 8. Derman, E., K. Krauter, L. Walling, C. Weinberger, M. Ray, factors that interact with the PEPCK, TAT, and AS gene and J. E. Darnell. 1981. Transcriptional control in the produc- promoters in hybrid cells, no evidence for a direct interac- tion of liver-specific mRNAs. Cell 23:731-739. tion between the TSEJ product(s) and these genes has been 9. Favaloro, J., R. Freisman, and R. Kamen. 1980. Transcription obtained. Furthermore, it is not clear whether TSEI func- maps of polyoma virus-specific RNA: analysis by two-dimen- tions by generating a negative factor (necessarily leaky) that sional nuclease S1 gel mapping. Methods Enzymol. 65:718- 749. represses transcription or by neutralizing the expression or 10. Fournier, R. E. K., and J. A. Frelinger. 1982. Construction of function (or both) of a positive factor required for gene microcell hybrid clones containing specific mouse chromo- activity. In this regard, it is interesting that growth hormone somes: application to autosomes 8 and 17. Mol. Cell. Biol. extinction seems to operate via repression of the gene 2:539-552. encoding GHF-1, a primary activator of growth hormone 11. Ghisalberti, A. V., J. G. Steele, M. H. Cake, M. C. McGrath, gene transcription (20). Whether this is the sole mechanism and I. T. Oliver. 1980. Role of adrenaline and cyclic AMP in operating in growth hormone extinction has yet to be deter- appearance of tyrosine aminotransferase in perinatal rat liver. mined (38). In any case, these results suggest that a primary Biochem. J. 190:685-690. function of extinguisher loci may be to regulate expression of 12. Holt, P. G., and I. T. Oliver. 1969. Studies on the mechanism of genes encoding tissue-specific transcription factors. Further induction of tyrosine aminotransferase in neonatal rat liver. the Biochemistry 8:1429-1437. insight into this mechanism of gene control will require 13. Killary, A. M., and R. E. K. Fournier. 1984. A genetic analysis isolation of individual TSE loci and their products. of extinction: trans-dominant loci regulate expression of liver- Finally, the results presented here demonstrate that ex- specific traits in hepatoma hybrid cells. Cell 38:523-534. tinction is a polygenic phenomenon. Not only are different 14. Killary, A. M., T. G. Lugo, and R. E. K. Fournier. 1984. tissue-specific genes controlled by distinct extinguisher loci Isolation of thymidine kinase deficient rat hepatoma cells by (13, 24), but repression may require the action of more than selection with bromodeoxyuridine, Hoechst 33258, and visible one trans-acting locus. We do not yet know whether the light. Biochem. Genet. 22:201-213. two-layered extinction phenotype displayed by the PEPCK, 15. Lem, J., A. C. Chin, M. J. Thayer, R. J. Leach, and R. E. K. TAT, and AS genes will prove general, nor is the genetic Fournier. 1988. Coordinate regulation of two genes encoding hepatoma micro- gluconeogenic enzymes by the trans-dominant locus Tse-J. basis of full repression apparent. Indeed, Proc. Natl. Acad. Sci. USA 85:7302-7306. cell hybrids retaining most chromosomes of the murine 16. Loose, D. S., P. A. Shaw, K. S. Krauter, C. Robinson, S. complement have been screened for PEPCK, TAT, and AS England, R. W. Hanson, and S. Gluecksohn-Waelsch. 1986. extinction phenotypes, and only chromosome 11 (carrying Trcans regulation of phosphoenolpyruvate carboxykinase (GTP) TSEJ) has been implicated. This may indicate that full gene, identified by deletions in chromosome 7 of the mouse. repression can only be achieved in the presence of TSE1 or Proc. Natl. Acad. Sci. USA 83:5184-5188. that the second-level phenotype itself is polygenic. Genetic 17. Lugo, T. G., B. Handelin, A. M. Killary, D. E. Housman, and resolution of these questions will require the analysis of cell R. E. K. Fournier. 1987. Isolation of microcell hybrid clones populations with carefully constructed hybrid genotypes. containing retroviral vector insertions into specific human chromosomes. Mol. Cell. Biol. 7:2814-2820. 18. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular ACKNOWLEDGMENTS cloning: a laboratory manual. Cold Spring Harbor Laboratory, We thank our colleagues for many helpful discussions, especially Cold Spring Harbor, N.Y. Hal Weintraub and Jon Cooper for their comments on the manu- 19. Mathieu-Mahul, D., D. Q. Xu, S. Saule, J. R. Lidereau, F. script. We are also grateful to N. Cowan, R. Hanson, D. Mathieu- Galibert, R. Berger, M. Mauchauffe, and C. J. Larsen. 1985. An Mahul, and G. Schutz for providing reagents. 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