the of chemistry vol. 264, no. issue of …the journal of biological chemistry 0 1989 by the...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc,

Vol. 264, No. 26, Issue of September 15, pp. 15634-15641.1989 Printed in U. S.A.

Loss of Transcriptional Repression of Three Sterol-regulated Genes in Mutant Hamster Cells*

(Received for publication, April 27, 1989)

James E. Metherall$, Joseph L. Goldstein, Kenneth L. Luskeys, and Michael S. Brown From the Departments of Molecular Genetics and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235

Two genes that encode enzymes in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and HMG-CoA synthase, and the gene encoding the low density lipoprotein (LDL) receptor are repressed when sterols accumulate in animal cells. Their 5”flanking regions contain a common element, designated sterol regulatory element-1 (SRE- 1). In the HMG-CoA synthase and LDL receptor promoters, the SRE-1 enhances transcription in the absence of sterols and is inactivated in the presence of sterols. In the HMG-CoA reductase promoter, the region containing the SRE- 1 represses transcription when sterols are present. In the current studies, we show that the SRE-1 retains enhancer function but loses sterol sensitivity in mutant Chinese hamster ovary cells that are resistant to the repressor, 25- hydroxycholesterol. In the absence of sterols, the mutant cells produced high levels of all three sterol-regulated mRNAs, and there was no repression by 25- hydroxycholesterol. When transfected with plasmids containing each of the regulated promoters fused to a bacterial reporter gene, the mutant cells showed high levels of transcription in the absence of sterols and no significant repression by sterols. When the SRE-1 in the LDL receptor and HMG-CoA synthase promoters was mutated prior to transfection into the mutant cells, transcription was markedly reduced. Thus, the 25- hydroxycholesterol-resistant cells retain a protein that enhances transcription by binding to the SRE-1 in the absence of sterols, but they have lost the function of a protein that abolishes this enhancement in the presence of sterols. Mutation of a 30-base pair segment of the HMG-CoA reductase promoter that contains the SRE- 1 did not reduce transcription in the mutant cells, indicating that this promoter is driven by elements other than the SRE-1. Nevertheless, this promoter failed to be repressed by sterols in the mutant cells. These data suggest that a common factor mediates the effects of sterols on the SRE-1 in all three promoters and that this factor has been functionally lost in the 25-hydroxycholesterol-resistant cells.

The buildup of sterols within animal cells leads to a simul- taneous reduction in the input of cholesterol from external

* This work was supported in part by Research Grant HL20948 from the National Institutes of Health and a grant from the Perot Family Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of Postdoctoral Fellowship CA 08398 from the Nat.iona1 Institutes of Health.

Established Investigator of the American Heart Association.

and internal sources. Uptake of cholesterol is reduced through a decline in the number of cell surface receptors for low density lipoprotein (LDL).’ Synthesis of cholesterol is reduced through a decline in the activities of several enzymes, most notably 3-hydroxy-3-methylglutaryl-coenzyme A (HMG- CoA) synthase and HMG-CoA reductase. Conversely, when the demand for sterols increases, there is a coordinate increase in the number of LDL receptors and in the enzymes of cholesterol biosynthesis (1).

Much, although not all, of this sterol-mediated regulation is achieved at the level of gene transcription. The 5”flanking region of each of the three sterol-regulated genes contains at least one copy of a sequence resembling CACCCCAC (or its inverse complement), which has been designated sterol regulatory element-1 (SRE-1) (2-6). The promoters from all three genes have been placed upstream of the coding region of a reporter gene (bacterial chloramphenicol acetyltransferase or CAT), and the chimeric plasmids have been introduced into Chinese hamster ovary (CHO) cells by transfection. All three promoters produce relatively large amounts of mRNA when the cells are grown in the absence of sterols. The addition of an oxygenated sterol such as 25-hydroxycholesterol, or a mixture of 25-hydroxycholesterol plus cholesterol, represses this transcription. When the sequence corresponding to the SRE-1 is altered by in uitro mutagenesis prior to transfection, sterol-mediated repression is markedly reduced (2-6).

In all three promoters, the SRE-1 is located adjacent to sites that bind positively acting transcription factors. These factors have been identified as Spl in the case of the LDL receptor (3,4) and nuclear factor-1 in the case of HMG-CoA reductase (5 , 7, 8). The positive factor or factors that activate HMG-CoA synthase transcription have not been identified. Although mutation of SRE-1 leads to a loss of sterol-dependent regulation of all three genes, the absolute levels of transcription differ. Mutation of the SRE-1 in the LDL receptor (3) and HMG-CoA synthase (6) promoters prevents the increase in transcription which normally occurs in the absence of sterols and thus leads to a constitutively low level of transcription. In these promoters, the SRE-1 acts as a conditional positive element, enhancing transcription in the absence of sterols. The result with the HMG-CoA reductase promoter is just the opposite: mutations of the SRE-1 region lead to a constitutively high level of transcription which is not repressed by sterols ( 5 ) . Thus, in this promoter, the SRE-

The abbreviations used are: LDL, low density lipoprotein; LDLR, LDL receptor; CAT, chloramphenicol acetyltransferase; CHO, Chinese hamster ovary; HMG CoA, 3-hydroxy-3-methylglutarylcoen- zyme A; HSV, herpes simplex virus; SRE-1, designation for sterol regulatory element-1, a DNA sequence that mediates sterol repression of certain genes; SRD-1 and SRD-2, designation for two different mutant cell lines that are resistant to 25-hydroxycholestero1; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

15634

Transcriptional Regulation in Sterol-resistant Cells 15635

1 may act as a conditional negative element, repressing transcription in the presence of sterols.

Little is known about the putative proteins that mediate the SRE-1-dependent enhancement and repression of transcription. To identify these proteins genetically, we have begun to study mutant cells that have lost the ability to repress transcription in the presence of sterols. Several lines of 25-hydroxycholesterol-resistant cells have been isolated by others (9-12), and several complementation groups have been defined (9, 12). These cells were selected by growth in the absence of lipoproteins under conditions in which the cells are totally dependent on cholesterol biosynthesis. Under these conditions, 25-hydrox~ycholesterol is lethal because it blocks cholesterol synthesis, leading to cholesterol depletion and cell death. Mutagenized cells that no longer repress cholesterol synthesis will grow in the presence of 25-hydroxycholesteroL In general, the mutant cells show a coordinate resistance to suppression of HMG-CoA synthase and HMG-CoA reductase activity when incubated with either 25-hydroxycholesterol or plasma LDL (11).

Chang and Chang (113) devised a selection for reversion to 25-hydroxycholesterol sensitivity. They showed that the polyene antibiotic amphotericin will kill cells that synthesize cholesterol. Killing is based on the ability of the antibiotic to bind cholesterol in the outer membrane and to render the cells permeable. Cells that suppress cholesterol synthesis in the presence of 25-hyd~:oxycholesterol are resistant to amphotericin killing. By this method, Chang and Chang (13) selected revertants of 25-hydro:uycholesterol-resistant cells which had regained sensitivity to 25-hydroxycholesterol. These cells simultaneously regained their sensitivity to sterol-mediated repression of HMG-CGA synthase and HMG-CoA reductase, suggesting the operation of a common mechanism. Sinensky et al. (14) showed that 25-hydroxycholesterol-resistant cells fail to diminish the incorporation of [35S]methionine into immunoprecipitable HMG-CoA reductase in the presence of 25-hydroxycholesterol. I t seems likely that the 25-hydroxycholesterol-resistant cells fail to repress sterol-regulated mRNAs when they are incubated with 25-hydroxycholesterol, but this phenomenon has not yet been demonstrated by direct mRNA measurements.

In the current studies, we describe the isolation and char- acterization of two 25-hydroxycholesterol-resistant cell lines. We show that these cells have lost the ability to repress the mRNA for the LDL receptor, HMG-CoA reductase, and HMG-GOA synthase. Moreover, these cells no longer repress transcription of transfected chimeric plasmids containing the promoters of any of the three genes, demonstrating that their defect is at the transcriptional level. We further show that in the mutant cells, the sterol regulatory element loses sensitivity to sterols but retains its ability to enhance transcription of the LDL receptor and HMG-CoA synthase genes.

EXPERIMENTAL PROCEDURES

Materials-We obtained amphotericin and nitrosourea from Sigma, 25-hydroxycholesterol from Steraloids, Inc., and cholesterol from Alltech Associates, Inc. Mevalonolactone was purchased from Fluka Chemical Co. and converted to the sodium salt as described (15). Human and newborn calf lipoprotein-deficient serum (d < 1.215 g/ml) and human LDL (ct 1.019-1.063 g/ml) were prepared as described previously (16). LDL was radiolabeled with 1251 as described (16). Compactin was kindly provided by Akira Endo (Tokyo Noko University, Tokyo, Japan). Plasmids (pRSV CAT, pSV3 Neo, pTK CAT, and various chimeric promoter-CAT constructs) and other materials were obtained from sources described previously (2-6).

Cell Growth-All cells were grown in monolayer a t 37 "C in an atmosphere of 5% CO,. CH:O-7 cells were derived from CHO-Kl cells by adaptation to continuous growth in lipoprotein-deficient serum

and were maintained in medium A (a 1:l mixture of Ham's F-12 medium and Dulbecco's modified Eagle's minimum essential medium containing 100 units/ml penicillin, 100 wg/ml streptomycin, 2 mM glutamine, 10 mM HEPES, and 10% (v/v) newborn calf lipoprotein- deficient serum). The sterol-resistant cell lines were maintained in medium A containing 1 pg/ml 25-hydroxycholesterol.

Mutagenesis and Isolation of 25-Hydroxycholestero1-resistant Cells-Two 25-hydroxycholesterol-resistant cell lines, SRD-1 and SRD-2, were isolated from pools of CHO-7 cells that had been mutagenized with nitrosoethylurea (SRD-1 cells) or y-irradiation (SRD-2 cells). To isolate SRD-1 cells, five sets of CHO-7 cells were plated on day 0 a t 3 X IO5 cells/75 cm2 flask in medium A. On day 1, the medium was replaced with fresh medium containing 0.4 mg/ml nitrosoethylurea. After 24 h, the cells were washed three times with phosphate-buffered saline and refed with medium A. The mutagenized cells were grown for 7 days to permit the expression of altered phenotypes. On day 8 after mutagenesis, pools of cells were plated into 6-10 dishes a t 3 X lo5 cells/lOO-mm dish in medium A. On day 9, the cells were refed with medium A supplemented with 0.3 pg/ml 25-hydroxycholesterol. On day 13-20, depending on cell growth, the concentration of 25-hydroxycholesterol was increased to 0.5 pg/ml. Approximately 1 week later, surviving colonies were isolated with cloning cylinders and transferred to 24-well Linbro plates in 1 ml/ well medium A containing 0.5 pg/ml25-hydroxycholestero1. The cells were allowed to proliferate for 7-10 days with refeeding every 2-3 days. Once healthy cultures were established, the concentration of 25-hydroxycholesterol was increased to 1 wg/ml.

T o obtain SRD-2 cells, CHO-7 cells were harvested from stock flasks by trypsinization, centrifuged, washed, and resuspended in phosphate-buffered saline. The cell suspension (5 X lo6 cells in 0.5 ml) was exposed to 600 rads of y-irradiation derived from a '37Cs source using a Mark 1 irradiator (J. L. Shepherd Co.). The cells were immediately divided into eight pools of 5 x IO5 cells and plated into 100-mm dishes in medium A. Sterol-resistant cell lines were selected essentially as described above for SRD-1 cells.

Amphotericin Killing Assay-On day 0, cells were plated a t 2-5 X lo5 cells/lOO-mm dish in medium A. On day 2, the cells were fed medium A containing various additions as indicated. On day 3, 10- 14 h following the additions, cells were refed with a 1:l mixture of Ham's F-12 medium and Dulbecco's modified Eagle's medium containing 100 units/ml penicillin, 100 pg/ml streptomycin, 1% calf lipoprotein-deficient serum with or without 50 pg/ml amphotericin in dimethyl sulfoxide (0.5% (v/v), final concentration). The cells were incubated 3-5 h, washed with phosphate-buffered saline, and treated for 20-30 min with a hypotonic solution (Dulbecco's modified Eagle's medium/H20, 1:4, v/v) to facilitate killing. Twenty to 30 min later the cells were rinsed with phosphate-buffered saline and refed with medium A. On day 4, the cells were washed, fixed, and stained with 0.1% (v/v) crystal violet.

Assays-The activity of HMG-CoA reductase was measured in detergent-solubilized cell extracts as described (16). Reductase activity is expressed as pmol of ["CIHMG-CoA converted to ['4C]mevalonate/min/mg of detergent-solubilized protein. LDL receptor activity was assayed as the receptor-dependent degradation of '251-LDL by cell monolayers a t 37°C as described previously (16). The incorporation of ["Cloleate into cholesteryl [14C]oleate by cell monolayers was measured as described previously (16).

DNA Transfection and (3418 Selection-Cells were seeded a t 5 X lo5 cells/lOO-mm dish. On the following day, they were transfected with DNA that had been precipitated with calcium phosphate (17). The cells were incubated with the DNA for 12-16 h and then refed with medium A. Two days later, the cells were fed with medium A supplemented with 700 pg/ml G418, and selection was maintained for 12-14 days.

For studies of promoter constructs, cells were transfected with coprecipitates of the indicated test plasmid (9.5 p g ) , pSV3 Neo (0.5 pg), and pRSV CAT (5 wg) except for transfections in Fig. 5, which did not receive pRSV CAT. G418-resistant colonies (200-1000 indi- vidual clones) were pooled and expanded in mass culture in the presence of G418 and used for experiments. Pooled cell lines were seeded (2 X IO5 CHO-7 cells or 5 X lo5 SRD-1 and SRD-2 cells/100- mm dish) on day 0 in medium A. On day 2, the cells were refed with medium A containing either no additions or a mixture of 25-hydroxy cholesterol (0.6 pglrnl) plus cholesterol (10 pg/ml). On day 3, the cells from 10 dishes were harvested in 4 M guanidinium thiocyanate containing 6.25 g/liter lauroylsarcosine, 36 mM sodium citrate, and 100 mM 2-mercaptoethanol. Total RNA was purified by centrifuga- tion through CsCl (18). The RNA pellet was dissolved in water and

15636 Transcriptional Regulation in Sterol-resistant Cells quantified by ahsorhance a t 260 nm.

RNA Analyses-To measure endogenous hamster HMG-CoA reductase mRNA and HMG-CoA reductase promoter-CAT chimeric mRNA, total cellular RNA was subjected to quantitative S1 nuclease analysis as described previously (19, 20). Endogenous hamster LDL receptor mRNA was assayed by S1 nuclease analysis using a probe derived from a genomic clone containing exon 2 (kindly provided by Richard Bishop and David W. Russell, University of Texas South- western Medical Center, Dallas, TX). Primer extension assays for the detection of pSV3 Neo mRNA were performed with a 40-nucleotide synthetic DNA primer complementary to nucleotides +261 to +300 of the mRNA (21). Primer extension analysis of pRSV CAT, pSyn CAT and pLDLR CAT mRNA was performed with a 40- nucleotide synthetic DNA primer that hybridizes to nucleotides +400 to +439 of the CAT mRNA (22). S1 nuclease analysis of pTK CAT mRNA was performed using an end-labeled EcoRI fragment that extends from nucleotide position -60 within the thymidine kinase promoter to position +312 within the CAT gene (2). Endogenous hamster HMG-CoA synthase mRNA was measured by extension with a 60-nucleotide synthetic DNA primer corresponding to nucleotides +41 to +I00 of the cDNA sequence (23). Endogenous ribosomal protein S17 mRNA was measured by extension with a synthetic DNA primer that hybridizes to nucleotides +94 to +125 of the published sequence (24). The authenticity of the major product of the S17 mRNA extension (142 base pairs) was verified by extensions using a synthetic oligonucleotide that hybridizes to nucleotides +21 to +47 of the published sequence. The primer extension assays were performed as described by Sudhof et al. (2, 3). S1 nuclease-resistant or primer-extended products were subjected to electrophoresis on de- naturing polyacrylamide gels that were dried and exposed to x-ray film at -20 “C with intensifying screens. Densitometry was performed with a Hoefer scanning densitometer (model GS 300).

RESULTS

For the current experiments, two lines of mutant 25-hydroxycholesterol-resistant cells were isolated independently as described under “Experimental Procedures.” One line, designated SRD-1, was mutagenized with nitrosoethylurea, which produces a preponderance of point mutations (25). The other cell line, SRD-2, was mutagenized with y-irradiation, which favors deletions (26). Following mutagenesis, cells were isolated based upon their ability to survive growth in the absence of LDL and in the presence of 1 pg/ml 25-hydroxycholesterol, a concentration that kills 100% of the wild-type CHO-7 cells.

Fig. 1, column 2, demonstrates that the SRD-1 and SRD-2

No

-MeV. 1 +MeV. I -MeV. 1 +MeV. Additions

Compactin 25-Hydroxycholesterol

CHO-7

SRD-1

SRD-2

1 2 3 4 5 FIG. 1. Resistance of SRD-1 and SRD-2 cells to 25-hydrox-

ycholesterol. On day 0 of cell growth, cells were seeded at 2 X 10” (CHO-T and SRD-1) or 4 X lo” (SRD-2) cells/100-mm dish in 10 ml of‘ medium A. On day 1, the medium was changed to medium A containing no additions, 1 pg/ml 25-hydroxycholesterol, or 100 PM rompartin in the absence (-) or presence (+) of 50 mM sodium mevalonate ( M a c ) as indicated. The cells were refed every second day. On day 7, the cells were washed, fixed, and stained with crystal violet.

cells grew in the presence of 1 pg/ml 25-hydroxycholesterol, whereas the wild-type CHO-7 cells were killed. Growth of the wild-type cells was restored by mevalonate, indicating that 25-hydroxycholestero1 was acting through inhibition of mevalonate synthesis (column 3) . All three cell lines were killed by compactin, a competitive inhibitor of HMG-CoA reductase (column 4), and growth of all three lines was restored with mevalonate (column 5) . These results suggest that the resistance of the SRD-1 and SRD-2 cells is attributable to a failure of 25-hydroxycholesterol to inhibit mevalonate synthesis.

Amphotericin, a polyene antibiotic, kills cells by binding to cholesterol in the plasma membrane (13). Fig. 2, column 2, shows that the SRD-1 and SRD-2 cells as well as the parental CHO-7 cells were sensitive to the killing effect of amphotericin when grown in lipoprotein-deficient serum. 25-Hydroxy- cholesterol protected the CHO-7 cells against killing by amphotericin, apparently by reducing cholesterol synthesis and thereby depleting cholesterol from the cell membrane (column 3) . In contrast, 25-hydroxycholesterol did not protect the SRD-1 and SRD-2 cells against killing by amphotericin (column 3) , a finding that is consistent with an inability to repress cholesterol synthesis in these cells. The further addition of compactin blocked cholesterol synthesis and protected the SRD-1 and SRD-2 cells from amphotericin killing (column 4) . When grown in the presence of 25-hydroxycholesterol and compactin, the CHO-7 cells remained resistant to amphotericin even when LDL was added (column 5 ) . We attribute this finding to the ability of 25-hydroxycholesterol to suppress LDL receptors in these wild-type cells (see below). The SRD- 1 and SRD-2 cells were rendered sensitive to amphotericin when incubated with LDL, suggesting that 25-hydroxycholesterol did not suppress LDL receptors in these mutant cells (column 5) .

Fig. 3 shows that the HMG-CoA reductase activity of SRD- 1 and SRD-2 cells was resistant to suppression by 25-hydroxycholesterol (panels A and D). Enzyme activity was also not repressed to a normal extent by LDL-derived cholesterol (panel B and E). High levels of mevalonate repressed HMG- CoA reductase activity in the wild-type CHO cells but not in the mutant cells (panels C and F ) . Each of the three agents studied in Fig. 3 is known to suppress HMG-CoA reductase activity by multiple mechanisms, including repression of gene transcription and enhanced degradation of the enzyme (1, 19). The partial decrease in HMG-CoA reductase activity in the SRD-1 and SRD-2 cells is consistent with the possibility

- + ” + + Amphotericin - - + + + 25-Hydroxychol. - - - + + Compactin - - - - + LDL J

1 2 3 4 5

FIG. 2. Failure of 25-hydroxycholesterol to protect SRD-1 and SRD-2 cells from killing by amphotericin. Cells were set up for experiments on day 0 as described in the legend to Fig. 1 and refed on day 2 with medium containing various combinations of 25- hydroxycholesterol(1 pg/ml), sodium mevalonate (0.1 mM), and LDL (40 pg of protein/ml). On day 3, the cells were subjected to amphotericin killing as described under “Experimental Procedures.” On day 4, cells were stained with crystal violet.


LDL Mevalonate

IS I C

Z S - H y d r ~ x y ~ h o l e ~ i e r ~ i (pg!ml) LDL (pg prolemlml) Mevalonate ImM)

FIG. 3. Regulation of HMG-CoA reductase act ivi ty in CHO- 7, SRD-1, and SRD-!2 cells by 25-hydroxycholestero1, LDL, and mevalonate. On day 0, cells were plated at 3 X lo4 (CHO-7) or 7 X lo4 (SRD-1 and SRD-2) cells/60-mm dish in the absence (CHO cells) or presence of 1 pg./ml25-hydroxycholestero1 (SRD-1 and SRD- 2 cells). On day 1, the cells were refed with medium of identical composition. On day 2, all cells received 2 ml of medium A with no added 25-hydroxycholes~erol. On day 3, cells received 2 ml of medium A and the indicated concentrations of 25-hydroxycholesterol, LDL, or sodium mevalonate. After incubation for 6 h a t 37 "C, the cells were harvested for measurement of HMG-CoA reductase activity. The 100% of control values were: panels A and B, 1648 pmol min" mg of protein" for CHO-7 cells and 1437 for SRD-1 cells; panels D and E , 1728 for CHO-7 cells and 882 for SRD-2 cells; panels C and F, 1192 for CHO-7 cells, 2427 for SRD-1 cells, and 1260 for SRD-2 cells. Each value is the average of duplicate or triplicate incubations.

TABLE I Sterol-mediated suppression of LDL receptor activity in CHO,

SRD-1, and SRD-2 cells Cell monolayers were :jet up on day 0 in 60-mm dishes as described

in the legend to Fig. 3. On day 1, replicate dishes received 2 ml of medium A in the absence (-sterols) or presence (+sterols) of 0.6 pg/ ml 25-hydroxycholesterol plus 10 pg/ml cholesterol as indicated. On day 2, the cells were refed with medium of identical composition. On day 3, each monolayer received 2 ml of Dulbecco's modified Eagle's medium (without glutamine) containing 2 mg/ml bovine serum albumin, 10 pg protein/ml '251-LDL (259 cpm/ngprotein in Experiment A; 263 cpm/ng protein in Experiment B) in the absence or presence of 500 pg of protein/ml of unlabeled LDL. After incubation for 5 h a t 37 "C, high affinity degradation of lZ5I-LDL was determined by sub- tracting the values for nonspecific degradation (+unlabeled LDL) from the values for total degradation (no unlabeled LDL). Each value is the average of triplicate incubations.

L

Y - L D L degraded % s~~~~~~~~~~ --Sterols +Sterols 100 X (Q - b)/ Cell line

( 4 ( b ) ng 5 h" mgprotein"

a

Experiment A CHO 1002 67 93 SRD-1 3925 3217 18

Experiment B CHO 1010 46 95 SRD-2 1643 1358 17

that these cells are completely unresponsive to one mechanism such as repressio'n of gene transcription, whereas they remain sensitive to oth.er mechanisms such as enhanced degradation of the enzyme.

Addition of 25-hydroxycholestero1 plus cholesterol to CHO- 7 cells depressed LDL receptor activity by 93-95%, as measured by the rate of receptor-dependent degradation of Iz5I-

LDL (Table I). In the SRD-1 and SRD-2 cells, the sterols reduced LDL receptor activity by only 18%, indicating a

marked resistance of the mutant cells to sterol-dependent repression of LDL receptors.

One action of 25-hydroxycholesterol was preserved in the SRD-1 and SRD-2 cells, namely the stimulation of cholesteryl ester synthesis (Table 11). This action does not require gene activation or new protein synthesis (27,28). When the mutant cells were grown in the absence of lipoprotein, the rate of incorporation of [14C]oleate into cholesterol ['*C]oleate was markedly elevated. We attribute this finding to an elevated rate of cholesterol synthesis which results from overexpres- sion of HMG-CoA synthase and reductase in the mutant cells (see below). Under these conditions, the addition of 25-hydroxycholesterol increased the rate of cholesteryl ester synthesis in the CHO-7 cells but not in the SRD-1 or SRD-2 cells. To abolish the cholesterol overproduction, the mutant cells were treated with compactin, which dramatically reduced the rate of cholesterol ester synthesis (Table 11). The subse- quent addition of 25-hydroxycholesterol to the compactin- treated cells now stimulated cholesteryl ester synthesis to a similar degree in all three cell lines, confirming that 25- hydroxycholesterol can be taken up into the mutant cells.

To test directly for repression of mRNA levels, we incubated the wild-type and mutant CHO cells with a mixture of 25- hydroxycholesterol plus cholesterol and then assayed the amounts of the three sterol-regulated mRNAs by S1 nuclease protection or by primer extension (Fig. 4). 25-Hydroxycholes- terol suppressed the HMG-CoA reductase mRNA in CHO-7 cells by 63% (panel A ) , but there was no detectable suppression in SRD-1 or SRD-2 cells. The LDL receptor mRNA in CHO-7 cells was suppressed by 83% in the presence of sterols (panel B ) , and again there was no effect on the LDL receptor mRNA in the two mutant cell lines. The mRNA for HMG- CoA synthase (panel C) gives two major bands in this primer extension assay because of alternative splicing of an exon in the 5"untranslated region (29). Sterols repressed both forms of HMG-CoA synthase mRNA by more than 90% in the wild- type CHO cells. The sterols caused no detectable repression of the synthase mRNA in the SRD-1 or SRD-2 cells (panel C). As a control for the specificity of these repressions, we measured the amount of mRNA for a ribosomal protein, S17. Sterols caused no consistent change in this mRNA (panel D).

In addition to their resistance to repression by exogenous

TABLE I1 Sterol-stimulated incorporation of ["Cloleate into cholesteryl esters

in CHO, SRD-1, and SRD-2 cells Cells were set up on day 0 in 60-mm dishes as described in the

legend to Fig. 3 except that none of the cells received sterols. On day 2, all cells received 2 ml of medium A containing 0.1 mM sodium mevalonate in the absence or presence of 10 p~ compactin as indicated. On day 3, the cells were refed with medium of identical composition. On day 4, the cells received the same medium as on day 3 in the absence (-1 or presence (+) of a mixture of 2 pg/ml 25- hydroxycholesterol plus 10 pg/ml cholesterol as indicated. After incubation for 5 h a t 37 "C, each monolayer was pulse labeled for 2 h with 0.2 mM [14C]oleate/albumin (6482 dpm/nmol) after which the cells were harvested for measurement of their cholesteryl ['4C]oleate content. Each value is the mean of triplicate incubations.

Cholesteryl ['4C]oleate formed

Cell line -Compactin +Cornpactin

-Sterols +Sterols "Sterols +Sterols

CHO-7 nmol h" mg protein"

1.0 4.1 0.26 1.2

SRD-1 38.3 26.6 0.85 4.3

SRD-2 10.6 9.5 0.61 1.5

15638

c. /gm

Transcriptional Regulation in Sterol-resistant Cells

147nt -

FIG. 4. Regulation of endogenous mRNA for the LDL receptor, HMG-CoA reductase, and HMG-CoA synthase in SRD-1 and SRD-2 cells. Cells were set up for experiments on day 0 as described in the legend to Fig. 3 and refed on day 2 either in the absence (-) or presence (+) of a mixture of 0.6 pg/ml25-hydroxycholesterol plus 10 pg/ml cholesterol. After incubation for 24 h, total RNA was isolated, and aliquots (20-40 pg) were subjected to either quantitative S1 nuclease analysis for HMG-CoA reductase mRNA ( A ) and LDI, receptor mRNA ( B ) or primer extension analysis for HMGCoA synthase mRNA (C) and ribosomal protein S17 mRNA ( I ) ) as described under "Experimental Procedures." The gels were exposed to x-ray film for 12-48 h at -70 "C. For quantification, the relative amounts of mRNA products were determined by densitometry and normalized in reference to the signal produced by the S17 mRNA. % Suppression by sterols was calculated from the normalized data. Virtually identical results were obtained in eight separate experiments. nt , nucleotides; nd, none detected.

sterols, the mutant cells have elevated levels of mRNA for HMG-CoA reductase, LDL receptor, and HMG-CoA synthase when grown in lipoprotein-deficient serum lacking exogenous sterols (Fig. 4). The increase was most pronounced for HMG- CoA synthase mRNA, which was present a t greater than 30- fold higher levels in the SRD-1 and SRD-2 cells as compared with the CHO-7 cells (panel C). Similar results were consistently observed in eight experiments.

I t is likely that the lack of sterol-mediated reduction of mRNA levels in the SRD-1 and SRD-2 cells is attributable to a failure of the SRE-1 elements in these genes to repress transcription. To test this hypothesis, we prepared recombi- nant plasmids containing fragments of each regulated promoter attached to the coding sequence for a reporter mRNA encoding bacterial CAT. The only component of these plasmids which is derived from the sterol-regulated genes is the promoter, and therefore any effect of sterols on mRNA levels must he caused by transcriptional changes. Pools of perma- nently transfected cells were incubated in the absence and presence of sterols, after which the amount of CAT mRNA was measured. Fig. 5 shows an experiment with a plasmid containing a 508-base pair fragment of the HMG-CoA reductase promoter, which contains a single copy of SRE-1. When this plasmid was expressed in CHO-7 cells, there was a 63% suppression of CAT mRNA in the presence of sterols (Fig. 5, lanes I and 2). A much smaller degree of suppression was seen in the SRD-1 cells (4%) and SRD-2 cells (20%). We then studied another plasmid that was identical except that a 30- base pair sequence that included the SRE-1 had been scram- bled by mutagenesis (plasmid pD). The CAT mRNA produced by this plasmid was not suppressed by 25-hydroxycholesterol in the CHO-7 cells (lanes 7 and 8) or in the SRD-1 or SRD-2

-300 -250 -200 -150 -400 -50 +1 5' ' I I I I I ' 3'

pRed CATV 1 -- C O ' H.1

SRE! 1 ii " 1

404nt- ""0"""- " "__ I - .. 8 a s = 7 a a = f ? RedCAT I - """" 1 mRNA

447nt - Relolmue Expresston ~l.O~.37~2.4~23~1.5~1.2~1.4)l.8~1.4~i.i ]2.212.0 % S Y P P T ~ L ~ O [ 63% I 4 % I 20%1 0% I 2 1 % 9%

300nt - - - - - - - - - _ _ - - c pSV3 Nea m R N A

FIG. 5. Sterol-mediated suppression of transfected HMG- CoA reductase promoter-CAT chimeric constructs. A schematic diagram of the hamster HMG-CoA reductase promoter region (5) is shown at the top of the figure. Bores denote regions of the promoter which affect transcription; the position of SRE-1 is indicated. CHO- 7, SRD-1, and SRD-2 cells were transfected with pSV3 Neo, pRSV CAT, and the indicated HMG-CoA reductase promoter-CAT chimeric plasmid and selected for resistance to G418. A mixture of 200-1000 stably transfected colonies was propagated in mass culture. Total RNA was prepared from cells grown in the absence (-) or presence (+) of 0.6 pg/ml hydroxycholesterol plus 10 pg/ml cholesterol and subjected either to quantitative S1 nuclease analysis using a probe specific for HMG-CoA reductase mRNA or to primer extension analysis using an oligonucleotide specific for pSV3 Neo mRNA. The gels were exposed to x-ray film for 12-48 h at -70 "C. For quantification, the relative amounts of reductase-CAT mRNA produced from the bands denoted by the asterisks (*) were determined by densitometry, and the data were normalized to the signal produced by the SV3 Neo mRNA. % Suppression by sterols was calculated from the normalized data. The slight stimulation by sterols in lunes 7 and 8 was not consistently observed. The mean values for % Suppression in three experiments for pRed CAT-1 were 73, 14, and 7% in CHO-7, SRD-1, and SRD-2 cells, respectively. For pD, the corresponding mean values were 0, 7, and 9%. nt , nucleotides.

cells (lunes 9-12) (see legend to Fig. 5 for mean values for multiple experiments).

Fig. 6 shows a similar set of experiments performed with a plasmid containing a 144-base pair segment of the LDL receptor promoter, which also contains a single copy of the SRE-1. Sterols reduced the amount of mRNA transcribed from this plasmid by 68% in CHO-7 cells (lunes I and 2), but produced no significant suppression in the SRD-1 or SRD-2 cells (lunes 3-6). Mutation of the SRE-1 abolished suppression by sterols in the CHO-7 cells (lunes 7 and 8). The SRD- 1 cells transcribed this mutant plasmid at a low rate, and there was no significant repression by 25-hydroxycholesterol (lunes 9 and 10). In the SRD-2 cells, the amount of CAT mRNA produced from this plasmid was not sufficient to be measured (lunes I I and 12).

In previous studies, we have shown that a 42-base pair sequence from the LDL receptor containing the SRE-1 con- fers sensitivity to sterol-mediated repression when inserted into the promoter for herpes simplex virus (HSV) thymidine kinase (Refs. 2-4). This fragment contains two imperfect repeats of 16-base pairs, designated repeats 2 and 3. Repeat 2 contains the SRE-1. Repeat 3 is the site for the binding of a positive transcription factor, Spl. Fig. 7 shows that this chimeric promoter drove transcription in CHO-7 cells and was repressed by 64% in the presence of sterols (lunes 1 and 2). A similar degree of expression was observed in the SRD-1 and SRD-2 cells, but there was virtually no suppression by


5' L I I i 3' -450 -100 -50 +1

1

-144 -161/-152

TATA 4 1 3 .'

Plosmtd pLDLR CAT-I -l64/-152 Cell Lrne CHO-7 SRD-I SRD-2 CHO-7 SRO-I SRD-2

+ - + - + - + - + - + I 2 3 4 5 6 7 8 9 1 0 4 1 1 2

l47nt-

400 n l - e o n a - -. L RSV CATmRNA - LDLR CAT mRN0

FIG. 6. Sterol-mediated suppression of transfected LDL receptor (LDLR) promoter-CAT chimeric constructs. A schematic diagram of the human LDL receptor promoter region (2-4) is shown at the top of the figure. Boxes denote regions of the promoter which affect transcription; the position of SRE-1 is indicated. CHO- 7, SRD-I, and SRD-2 cells were transfected with pSV3 Neo, pRSV CAT, and the indicated LDL receptor promoter-CAT chimeric plasmid and selected for resistance to G418. Total RNA was prepared from pools of stably transfected cells grown in the absence (-) or presence (+) of sterols as described in the legend to Fig. 5. Total RNA was subjected to primer extension analysis using oligonucleo- tides specific for either pSV3 Neo mRNA or CAT mRNA from the LDL receptor and RSV promoter constructs. Autoradiography and quantitation were performed as described in Fig. 5. The significance 0 1 the slight stimulation by sterols in lanes 7 and 8 and in lanes 9 and 1 0 is quedonahle owing to the low levels of expression. The mean values for % Suppression in three experiments for pLDLR CAT-1 were 64, 2, and 4% in CHO-7, SRD-1, and SRD-2 cells, respectively. For plasmid -161/-152, the mean values were 0% for CHO-7 and 1% for SRD-1 cells. nt, nucleotides; nd, none detected.

FIG. 7. Sterol-mediated suppression of transfected HSV thymidine kinase (TIC)-CAT plasmids containing repeats 2 and 3 from the human LDL receptor promoter. A schematic diagram of the LDL receptor promoter-HSV thymidine kinase-CAT chimeric constructs (2, 4) is shown at the top of the figure. CHO-7, SRD-I, and SRD-2 cells were transfected with pSV3 Neo, pRSV CAT, and the HSV thymidine kinase-CAT plasmid containing the indicated insertion from the LDL receptor promoter. Total RNA was prepared from pools of stably transfected cells grown in the absence (-1 or presence (+) of sterols as described in the legend to Fig. 5. Total RNA was subjected either to quantitative S1 nuclease analysis using a probe specific for HSV thymidine kinase-CAT mRNA or to primer extension analysis using an oligonucleotide specific for pSV3 Neo mRNA. Autoradiography and quantitation were performed as described in the legend to Fig. 5. The average values for % Suppression in two experiments for pA were 69,5, and 6% in CHO-7, SRD-1, and SRD-2 cells, respectively. For PC, the corresponding mean values were 9,8, and 2%. For pG, mRNA levels were too low to be accurately quantified. For pK, t.he corresponding mean values were 66, 4, and 6%. nl, nucleotides; nd, none detected.

sterols (lanes 3-6). Plasmid PC contains a mutated copy of repeat 2 and an intact copy of repeat 3. This plasmid was expressed at a relatively low level in all three cell lines, and there was no major repression by sterols (lanes 7-12). When repeat 3 was mutated (plasmid PC), expression was markedly reduced or not detectable in the absence of sterols (lanes 13- 18). When the plasmid contained only repeat 2, it was expressed, and a 69% repression was achieved in the wild-type cells (lanes 19 and 20). Again, the two mutant cell lines failed to show significant repression (lanes 21-24). Previous studies by McKnight (30) have shown that sequences of greater than 32 base pairs inserted at position -60 in the HSV thymidine kinase promoter reduce transcription unless they contain a positive element. This blocking effect presumably explains the low level of transcription of the pG plasmid, in which the positive element, repeat 3, is mutated. Even though it lacks repeat 3, the pK plasmid was expressed because the construct is short enough so that it does not interfere with the positive elements already present in the HSV thymidine kinase promoter (2).

Fig. 8 shows an experiment performed with cells transfected with a plasmid containing a 366-base pair fragment from the promoter for hamster HMG-CoA synthase. This promoter contains three elements that participate in sterol-dependent regulatory events (6). Two of these elements show a strong resemblance to the SRE-1 sequences identified in the HMG- CoA reductase and LDL receptor promoters. These are designated SRE-1 in Fig. 8. The other sequence, designated SRE- 2, does not show a convincing sequence resemblance to the SRE-1. When a promoter containing all three of these sequences was introduced into CHO-7 cells, transcription was observed in the absence of sterols, and this was repressed by 64% when sterols were added (Fig. 8, lanes 1 and 2). In the SRD-1 and SRD-2 cells, this construct gave relatively high levels of expression in the absence of sterols, and no significant repression when sterols were added (lanes 3-6).

5, -350 -300 -250 -200 -450 -400 -50 , '1 3'

SREP SREl SRE4 lCGTCCCq Icbcccrgcl -3

TATA 1 pSynCAT-1 ' ' - ,

- - - - - - - - - - 'pSV3NeornRNe

FIG. 8. Sterol-mediated suppression of transfected HMG- CoA synthase promoter-CAT chimeric constructs. A schematic diagram of the hamster HMG-CoA synthase promoter region (6) is shown at the top of the figure. Boxes denote regions of the promoter which affect transcription; the positions of SRE-1 are indicated. CHO-7, SRD-1, and SRD-2 cells were transfected with pSV3 Neo, pRSV CAT, and the indicated HMG-CoA synthase promoter-CAT chimeric plasmid and selected for resistance to G418. Total RNA was prepared from pools of stably transfected cells grown in the absence (-1 or presence (+) of sterols as described in the legend to Fig. 5. Total RNA was subjected to primer extension analysis using oligo- nucleotides specific for either pSV3 Neo mRNA or CAT mRNAs from the HMG-CoA synthase and RSV promoter constructs. Auto- radiography and quantitation were performed as described in Fig. 5. The mean values of % Suppression in three experiments for pSyn CAT-1 were 69, 0, and 0% in CHO-7, SRD-1, and SRD-2 cells, respectively. For pW, the corresponding mean values were 7, 0, and 3%. nt. nucleotides.

15640 Transcriptional Regulation in Sterol-resistant Cells

TABLE I11 Contribution of SRE-1 to expression of promoter-CAT constructs

i n transfected hamster cells in the absence of sterols Each value represents the average of three experiments. The transfected promoter-CAT constructs were as

follows: hamster HMG-CoA reductase, pRed CAT-1 (normal SRE-1) and pD (mutated SRE-1); hamster HMG- CoA synthase, pSyn CAT-1 (normal SRE-1) and pW (mutated SRE-1); and human LDL receptor, pLDLR CAT- 1 (normal SRE-1) and -161/-152 (mutated SRE-1). Conditions for transfection and cell growth in the absence of sterols were as described in the legends to Figs. 5, 6, and 8. Quantification of relative mRNA concentrations was performed as described in the legend to Fig. 5.

Transfected promoter- CAT construct

Recipient cell line

Relative mRNA concentration

SRE-1 (a) Normal

SRE-1 (b) Mutated

Enhancement of transcription by

SRE-1 (a /b)

HMG-CoA reductase CHO-7 SRD-1 SRD-2

HMG-CoA synthase CHO-7 SRD-1 SRD-2

LDL receptor CHO-7 SRD-1 SRD-2

We have shown previously that the ability of the HMG- CoA synthase promoter to undergo enhanced expression in the absence of sterols is reduced when 4 conserved adenines in the two copies of SRE-1 are changed to cytosines (plasmid pW in Fig. 8) (Ref. 6). When this plasmid was transfected into CHO-7 cells, relatively low transcription was observed in the absence of sterols, and there was no significant further reduction when sterols were added (lanes 7 and 8). In the SRD-1 and SRD-2 cells, transcription of this plasmid was also reduced in the absence of sterols, and again there was no significant repression by sterols (lanes 9-12).

DISCUSSION

The current experiments demonstrate that two lines of 25- hydroxycholesterol-resistant CHO cells have lost the ability to repress transcription of the LDL receptor, HMG-CoA synthase, and HMG-CoA reductase-three genes that are controlled by a sterol regulatory element. The 25-hydroxycholesterol-resistant cells, SRD-1 and SRD-2, were isolated with a technique that has been used by others to select similar cells that fail to repress cholesterol synthesis (9-11). In studies of such cells, Chang and Chang (13) showed that sterol resistance to suppression of HMG-CoA synthase and HMG-CoA reductase, as measured by enzymatic activity, could be cor- rected simultaneously by a single reversion event, implying a defect in a common factor that controls both of these genes. The current data extend these findings by showing that the gene for the LDL receptor is controlled by a similar factor. This factor is likely to be a protein that participates in the chain of events by which 25-hydroxycholestero1 causes SRE- 1-dependent repression of transcription. The defect might reside in a protein that binds 25-hydroxycholesterol, in a protein that binds to the SRE-1, or in another protein that couples these two events.

The current results in the sterol-resistant cells are in agree- ment with previous experiments in wild-type CHO cells which suggest that the SRE-1 in the HMG-CoA reductase promoter behaves differently than it does in the other two promoters (2-6). In the HMG-CoA synthase and LDL receptor promoters, the SRE-1 enhances transcription in the absence of sterols and loses this function in the presence of sterols (2-4, 6). In the HMG-CoA reductase promoter, the region of DNA

-fold increase 1.0 1.5 0.7 2.9 1.4 2.1 1.4 1.9 0.7

1.0 0.23 4.3 2.8 0.80 3.5 1.8 0.23 7.8

1.0 0.20 5.0 4.4 0.53 8.3 1.6 c0.2 3 . 0

which contains the SRE-1 is not required for high level transcription in the absence of sterols. Thus, mutation of a 30-base pair sequence containing the SRE-1 does not lower transcription appreciably (Fig. 5). However, this mutation does prevent sterol-mediated repression of transcription, suggesting that the SRE-1 may exert a sterol-dependent negative effect on transcription in the HMG-CoA reductase promoter.

To illustrate the differences among the three promoters, in Table I11 we have compared the transcription rates of the chimeric promoter plasmids containing an intact or mutated SRE-1. All of the measurements in Table I11 were made in the absence of sterols. In the HMG-CoA reductase promoter, mutation of the region containing SRE-1 had little or no effect in the wild-type and mutant cells, confirming that in the absence of sterols the SRE-1 does not have a required positive role in this promoter. A different result was seen with the other two promoters. When the SRE-1 in the HMG-CoA synthase and LDL receptor promoters was mutated, transcription fell by more than &fold in the wild-type CHO cells. A dramatic fall in transcription was also observed in the SRD- 1 and SRD-2 cells, indicating that the SRE-1 element played a major positive role in transcription in these cells in the absence of sterols. Thus, these mutant cells are likely to retain a protein that binds to the SRE-1 in the LDL receptor and HMG-CoA synthase promoters and enhances transcription, but the action of this protein is no longer blocked by sterols.

Even though the mechanism for sterol-mediated regulation of HMG-CoA reductase transcription appears to differ from the mechanism for HMG-CoA synthase and the LDL receptor, sterol-dependent effects on all three promoters were lost in the SRD-1 and the SRD-2 cells. It seems likely that each of these mutant cells has lost the function of a single protein that is required for all sterol-dependent effects on transcription. This putative protein might be one that binds to the SRE-I, or it might be a protein that modulates the activity of other proteins that bind to the SRE-1.

Several lines of evidence indicate that the defect in the SRD-1 or SRD-2 cells does not involve defective cellular uptake of 25-hydroxycholesterol. First, although the cells lost the ability to repress transcription, they retained the ability to respond to 25-hydroxycholesterol with a stimulation of cholesterol esterification (Table 11), an event that does not


require transcriptional regulation (27,28). Second, the mutant cells have high levels of mRNA for HMG-CoA reductase, HMG-CoA synthase, and the LDL receptor when grown in lipoprotein-deficient serum. Under these conditions, the rate of cholesteryl ester synthesis is extremely high in the mutant cells, implying cholesterol overproduction (Table 11). In normal cells, such cholesterol overproduction does not occur because as sterols accumulate within the cell, they depress the cholesterol biosynthetic enzymes (1, 27). The fact that the three mRNAs were high in the mutant cells implies that these cells have lost the ability to respond to their own endogenously synthesized sterol. Consistent with this finding was the failure of exogenous mevalonate to suppress HMG- CoA reductase activity normally in these cells (Fig. 3).

An unexpected finding in the current studies was the observation that the mutant SRD-1 and SRD-2 cells contained >30-fold more mRNA for HMG-CoA synthase than the wild- type CHO cells when grown in the absence of sterols (Fig. 4), an observation that was reproduced on multiple occasions. When the mutant cells were transfected with chimeric plasmids containing the known elements of the HMG-CoA synthase promoter, the overproduction of mRNA was only 1.8- 2.8-fold (see Table 111). This might indicate that the HMG- CoA synthase gene co'ntains another enhancer element that is not included in the 366-base pair promoter fragment that was present in the chimeric plasmid. Alternatively, it is pos- sible that the mRNA for HMG-CoA synthase became more stable in the mutant ccells, and this stabilization was not seen with the CAT mRNA produced by the chimeric plasmid. Such an observation would suggest a previously unsuspected role for sterols in regulating the turnover of HMG-CoA synthase mRNA. Studies of the synthesis and degradation of the HMG- CoA synthase mRNA in the mutant cells will be required to resolve this issue.

Acknowledgments-W'e thank Edith Womack and Lavon Sanders for excellent help with the tissue culture work. Deborah Noble and Gloria Brunschede provided excellent technical assistance.

REFERENCES 1.

2.

3.

Goldstein, J. L., and Brown, M. S. (1984) J. Lipid Res. 25, 1450-

Siidhof, T. C., Russell, D. W., Brown, M. S., and Goldstein, J. L.

Siidhof, T. C., Van Der Westhuyzen, D. R., Goldstein, J. L.,

1461

(1987) Cell 48 , 1061-1069

Brown, M. S., and Russell, D. W. (1987) J. Biol. Chem. 2 6 2 ,

4. Dawson, P. A., Hofmann, S. L., van der Westhuyzen, D. R., Siidhof, T. C., Brown, M. S., and Goldstein, J. L. (1988) J. Biol. Chem. 263,3372-3379

5. Osborne, T. F., Gil, G., Goldstein, J. L., and Brown, M. S. (1988) J. Biol. Chem. 263, 3380-3387

6. Smith, J. R., Osborne, T. F., Brown, M. S., Goldstein, J. L., and Gil, G. (1988) J. Biol. Chem. 263,18480-18487

7. Gil, G., Osborne, T. F., Goldstein, J. L., and Brown, M. S. (1988) J. Biol. Chem. 263,19009-19019

8. Gil, G., Smith, J. R., Goldstein, J. L., Slaughter, C. A., Orth, K., Brown, M. S., and Osborne, T. F. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,8963-8967

9. Sinensky, M., Logel, J., and Torget, R. (1982) J. Cell Physiol. 113,314-319

10. Chen, H. W., Cavenee, W. K., and Kandutsch, A. A. (1979) J. Biol. Chem. 2 5 4 , 715-720

11. Chang, T-Y., and Limanek, J. S. (1980) J. Biol. Chem. 2 5 5 ,

12. Leonard, S., and Sinensky, M. (1988) Biochim. Biophys. Acta

13. Chang, T-Y., and Chang, C. C. Y. (1982) Biochemistry 21,5316-

14. Sinensky, M., Torget, R., and Edwards, P. A. (1981) J. Biol.

15. Brown, M. S., Faust, J. R., Goldstein, J. L., Kaneko, I., and Endo,

16. Goldstein. J. L.. Basu. S. K.. and Brown, M. S. (1983) Methods

10773-10779

7787-7795

947,101-112

5323

Chem. 256,11774-11779

A. (1978) J. Biol. Chem. 2 5 3 , 1121-1128

Enzymol. 98,'241-260 '

17. van der Eb. A.. and Graham. F. (1980) Methods Enzymol. 65, , . 826-839

18. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter,

19. Gil. G.. Faust. J. R.. Chin. D. J., Goldstein. J. L., and Brown, M. W. J. (1979) Biochemistry 18 , 5294-5299

S . (1985) Cell 41,' 249-258 '

20. Osborne, T. F., Goldstein, J. L., and Brown, M. S . (1985) Cell

21. Beck. E.. Ludwig. G., Auerswald. E. A., Reiss, B., and Schaller, 42,203-212

H. (1982) Gene'(A&.) 19,327-336 '

. .

22. Alton, N. K., and Vapnek, D. (1979) Nature 282,864-869 23. Gil, G., Goldstein, J. L., Slaughter, C. A., and Brown, M. S. (1986)

24. Chen, I-T., Dixit, A., Rhodes, D. D., and Roufa, D. J. (1986) Proc.

25. Thacker, J. (1986) Mutat. Res. 160 , 267-275 26. Drinkwater, N. R., and Klinedinst, D. K. (1986) Proc. Natl. Acad.

27. Goldstein, J. L., and Brown, M. S. (1976) Curr. Topics Cell Regul.

28. Chang, T-Y., and Doolittle, G. M. (1983) Enzymes 16,523-539 29. Gil, G., Smith, J. R., Goldstein, J. L., and Brown, M. S. (1987)

30. McKnight, S. L. (1982) Cell 31,355-365

J . Biol. Chem. 261,3710-3716

Natl. Acad. Sci. U. S. A. 8 3 , 6907-6911

Sci. U. S. A . 83,3402-3406

11,147-181

Proc. Natl. Acad. Sci. U. S. A. 8 4 , 1863-1866

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