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Hormonal Regulation of TSEI-Repressed Genes:Evidence

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Hormonal Regulation of TSEI-Repressed Genes:Evidence 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 re- ported (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.
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