5′ end of hmg CoA reductase gene contains sequences responsible for cholesterol-mediated inhibition of transcription

Cell, Vol. 42, 203-212, August 1985, Copyright © 1985 by MIT

0092-8674/851080203-10

$02.00,'0

5' End of HMG CoA Reductase Gene Contains Sequences Responsible for Cholesterol-Mediated Inhibition of Transcription Timothy F. Osborne, Joseph L. Goldstein, and Michael S. Brown Department of Molecular Genetics University of Texas Health Science Center at Dallas 5323 Harry Hines Boulevard Dallas, Texas 75235

Summary Cholesterol homeostasis is maintained by feedback inhibition of transcription of the gene encoding HMG CoA reductase. To study this mechanism, we joined the 5' end of the hamster reductase gene to the coding region for chloramphenicol acetyltransferase (CAT). The chimeric gene produced high levels of CAT activity in mouse L cells; sterols suppressed expression by 70% to 90%. Sequences responsible for both promotion and inhibition of transcription were distributed over 500 bp extending 300 bp upstream of the reductase transcription initiation sites. Any sizable deletion within this region decreased CAT expression in vivo and CAT mRNA transcription in vitro. This region contains five hexanucleotide repeats (CCGCCC or GGGCGG) that occur in promoters of viral and cellular housekeeping genes. Every reductase-CAT plasmid that showed transcriptional activity also showed inhibition by sterols, indicating that the sites for promotion and inhibition of transcription are closely associated. Introduction The cholesterol synthetic pathway is regulated by feedback suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase), an enzyme that is an integral protein of the endoplasmic reticulum membrane (Brown and Goldstein, 1980). Suppression is mediated in part by inhibition of transcription of the reductase gene (Luskey et al., 1983). Suppression occurs when cells develop elevated levels of cholesterol and other isoprenoid products derived from mevalonate, the product of the enzyme. This regulatory system is an example of the general phenomenon by which end products of biosynthetic pathways inhibit transcription of genes for ratecontrolling enzymes. The mechanism for such negative regulation in eukaryotic cells is not known. As a first step in exploring the molecular basis for the regulation of HMG CoA reductase, we have isolated a 4.8 kilobase (kb) cDNA for the hamster enzyme (Chin et al., 1984) and the corresponding genomic sequences, which consist of 20 exons spread over 25 kb (Reynolds et al., 1984). With these probes, we showed that lipoproteinderived cholesterol and related sterols such as 25-hydroxycholesterol inhibit the synthesis of reductase mRNA, as indicated by a decrease in the rate of incorporation of

[3H]uridine into total hybridizable reductase RNA (Luskey et al., 1983). The 5'flanking and untranslated regions of the hamster reductase gene exhibit several unusual features, some of which may be related to the mechanism of negative feedback regulation. For example, the 5' flanking region does not contain a classic TATA or CCAAT box, but it does contain a region of 265 bp that includes five copies of the hexanucleotide sequence CCGCCC or its inverse complement GGGCGG (Reynolds et al., 1984). This hexanucleotide sequence is known to be important for transcription of the herpes simplex thymidine kinase gene (McKnight and Kingsbury, 1982) and the early region genes of the SV40 virus (Fromm and Berg, 1982). Transcription of reductase mRNA is initiated at multiple sites that range over 98 bp, a finding that probably reflects the lack of a functional TATA box. The 5' untranslated region of the reductase gene contains a large intron ("~3.5 kb) that has multiple 5' splice sites, any one of which can be spliced to a common 3' acceptor site. Depending on the location of the 5' donor splice site, the mRNA transcripts contain a 5' untranslated leader sequence that ranges from 68 to 670 nucleotides and contains up to eight AUG codons upstream of the AUG that initiates translation of the reductase protein (Reynolds et al., 1984; 1985). All of the above observations have been made with cDNA and genomic clones isolated from UT-1 cells, a strain of Chinese hamster ovary cells that were selected for growth in the presence of compactin, a competitive inhibitor of the reductase (Chin et al., 1982). UT-1 cells have a 300-fold elevated rate of reductase transcription and a 15-fold increase in the number of copies of the structural gene (Luskey et al., 1983). A similar multiplicity of transcription initiation sites and 5' splice sites has been observed in reductase mRNA from normal hamster liver through the use of $1 nuclease mapping and primer extension analyses (Reynolds et al., 1984). We have sought to determine whether the 5' flanking region of the reductase gene contains the promoter sequences responsible for transcription and whether these flanking sequences also mediate the repression of transcription by cholesterol. For this purpose, we have constructed chimeric genes that contain various parts of the 5' flanking region of the reductase gene placed immediately upstream of the coding sequence of the bacterial gene encoding chloramphenicol acetyltransferase (CAT) (Gorman et al., 1982b). These recombinant plasmids have been introduced into mouse L cells by calcium phosphatemediated transfection with pSV3-Neo, a plasmid that confers resistance to the antibiotic G418 (Southern and Berg, 1982). Permanently transfected G418-resistant cells were tested for the expression of CAT enzymatic activity and reductase promoter-CAT chimeric mRNA after growth in the absence or presence of sterols that are known to repress transcription of HMG CoA reductase. The results indicate that the 5' flanking region of the reductase gene contains a 500 bp sequence that serves as an efficient

Cell 204

-758 to -7q5

"670 tO -640

5.5 kb

Ecl

5' - 4420

5' -'102d -t001

-881

-862 -812

-513

-756

-25

Plasmid -'1420

Rel oti ve Promoter Activity

Percent Suppression by Sterols

pRed CAT-3

100

75 %

pRed CAT-2

t7

81%

pRed CAT -1

28

61%

-23

-1420

-515

l

I

-t02t

-515

I

I l

-9~i9

I

-759

-515

I, -515

I -I021

I .... -875

pRed CAT-9

5.0

42%

pRed CAT-7

5.4

28 %

pRed CAT-6

1.5

pRed CAT- 4

0.6

pRedCAT-5

0.7

pRed CAT - 8

0.07

-513

deletion . . . . I -699

I

-875

t

,;+

V -25

-515

I

-699

I

I

-669

-515

Figure1. Structu~ and Activity of ReductasePromote~CAT Chimeric Genes The DNA fragment from the 5' end of the hamster reductaee gene used to construct pRedCAT-1 to -9 is represented at the top by the solid bar and bounded by the indicated restriction endonuclease sites. Nucleotide positions -1420 to - 2 3 of the reductase promoter region are numbered in relation to the A of the methionine initiation codon (Reynolds et al., 1984). The exact end point of the Eco RI site at -1420 is estimated from partial sequencing and detailed restriction mapping. The "-,3.5 kb intron is omitted from the numbering scheme. The hexanucleotide repeat sequences homologous to the SV40 promoter are represented by open (CCGCCC) or dotted (GGGCGG) boxes. Arrows denote the positions of the transcription initiation sites for HMG CoA reductase that were characterized previously (Reynolds et al., 1984; 1985). The CAT coding sequence is denoted by the cross-hatched bar, and the direction of CAT transcription is indicated by the arrow. The structure of each pRedCAT construct is shown. The columns on the right give the average CAT enzymatic activity obtained in several experiments with each plasmid (see Table 1). The data are expressed as a percentage of the mean activity obtained with pRedCAT-3, the plasmid with the largest amount of reductase sequence+ The mean percent suppression by 25-hydroxycholesterol plus cholesterol for each plasmid is also shown.

transcriptional promoter as well as a site for cholesterolrnediated inhibition of transcription.

Results Localization of Regulatory Regions for Reductase Transcription Figure 1 shows the general features of the 5' end of the HMG CoA reductase gene and the structures of the various reductase-CAT chimeric plasmids that were used in this study. In describing this region, we use a numbering scheme in which the A of the AUG initiator codon of the reductase protein is designated as position +1 (Reynolds et al., 1984). The sequence upstream of the initiator codon is numbered according to the sequence of pRed-227, the reductase cDNA that was described earlier (Chin et al., 1984; Reynolds et al., 1984). Thus, the numbering scheme omits the sequence of the 3.5 kb intron that is spliced out of the mRNA. The arrows in Figure 1 denote the multiple transcription initiation sites, which occur in two clusters: one cluster at nucleotides -738 to -713, designated l a - d (70% of total initiation sites); and another cluster at nucleotides -670 to -640, designated 2-5 (30% of total; Reynolds et al., 1984; 1985). The five repetitions of the hexanucleotide sequence CCGCCC or its inverse complement GGGCGG are indicated by the open and dotted boxes, respectively.

The indicated segments of the reductase gene were placed in front of the coding region of the CAT gene in pSV0-CAT, a plasmid vector that contains the CAT structural gene and is devoid of any defined eukaryotic promoter or enhancer elements (Gorman et al., 1982b). These recombinant plasmids were transfected into mouse L cells together with the pSV3-Neo plasmid, which confers resistance to the antibiotic G418. Pools of G418-resistant colonies (200-500 colonies) were propagated in mass culture. To initiate each experiment, aliquots of the mass cultures were seeded into dishes and incubated in the absence or presence of a mixture of sterols (25-hydroxycholesterol plus cholesterol), which are known to inhibit transcription of the reductase gene (Luskey et al., 1983). After a predetermined interval, the cells were harvested and CAT activity was measured. The results of individual experiments with each reductase-CAT chimeric plasmid are shown in Table 1. The mean results of several experiments with each plasmid are summarized on the right side of Figure 1, where the mean level of CAT enzymatic activity is expressed as a percentage of the mean result obtained with pRedCAT-3. Plasmid pRedCAT-3, which has the largest amount of reductase sequence, including the 3.5 kb intron, consistently produced the highest level of CAT activity (Table 1 and Figure 1). CAT activity was reduced by an average of 75% when the pRedCAT-3 transfected cells were in-

Cholesterol Regulatory Region of HMG CoA Reductase Gene 2O5

Table 1. Comparison of CAT Activity in Mouse L Cells Transfected with Various Reductase Promoter-CAT Chimeric Genes CAT Activity, nmole per rain per mg protein Plasmid Transfected into Cells

- Sterols

+ Sterols

% Suppression by Sterols

Experiment 1 pRedCAT-1 pRedCAT-2 pRedCAT-3 pRedCAT-6

29 9.7 58 0.68

6,9 1.5 10 0.57

76% 85o 83% -

Experiment 2 pRedCAT-1 pRedCAT-2 pRedCAT-3 pRedCAT-6

22 10 56 0.86

8.8 2.3 19 0.70

60% 77% 66% --

Experiment 3 pRedCAT-1 pRedCAT-5 pRedCAT-7 pRedCAT-9

11 0.43 3.4 1.4

3.7 0.25 2.6 0.9

66% 24% 36%

Experiment 4 pRedCAT-1 pRedCAT-4 pRedCAT-5 pRedCAT-7 pRedCAT-9

14 0.26 0.33 2.7 2.6

5.5 0.19 0.22 2.2 1.6

61% -19% 38%

Experiment 5 pRedCAT-1 pRedCAT-2 pRedCAT-7 pRedCAT-9

13 9.5 3.2 4.2

5.9 1.7 1.9 2.1

55% 82% 40o 50%

Experiment 6 pRedCAT-1 pRedCAT-4 pRedCAT-5 pRedCAT-8

12 0.44 0.44 0.04

6.6 0.24 0.45 0.09

45% ----

Mouse L cells were transfected with pSV3-Neo and the indicated plasmid, followed by selection for resistance to G418. A mixture of 200-500 transfected colonies was propagated as a mass culture. For each experiment, six 60 mm dishes were seeded on day 0 at 3.5 x 104 cells per dish in 3 ml of medium A with 10o FCS. On day 2, the dishes were washed with phosphate-buffered saline, and the medium was changed as follows. Each dish received medium A with 10% lipoprotein-deficient serum in the absence ( - sterols) or presence of a mixture of cholesterol and 25-hydroxycholesterol at a final concentration of 12 and 0.2/~g/ml, respectively (+ sterols). The dishes were re-fed on day 4 with the same medium and harvested by scraping on day 5. The cells from three identical dishes were pooled and processed for determination of CAT activity. Each value is the average of duplicate determinations.

cubated with sterols, indicating that positive and negative regulatory regions were both present in this plasmid. Elimination of the 3.5 kb intron, as in pRedCAT-2, reduced expression to about 17% of the level seen with pRedCAT3, but suppression with sterols was still obtained. Although the intron a p p e a r e d to increase CAT expression when it was present together with upstream sequences, the intron itself did not contain promoter activity, as indicated by the low level of expression obtained with pRedCAT-6 (Table 1 and Figure 1). Plasmid pRedCAT-1, which contains only the s e q u e n c e from - 5 1 3 to -1021, was at least as active as pRedCAT-2 and still showed suppression by sterols. Thus, the important reductase promoter elements and the sequences responsible for regulation by sterols both reside in the region between positions - 5 1 3 and -1021. This region contains the multiple transcription initiation sites and the multiple copies of the C C G C C C hexanucleotide and its complement.

When we attempted to localize the promoter activity further by deleting sequences in the - 5 1 3 and -1021 region, nearly all promoter activity was lost. For example, deletion of the 102 base pairs between positions - 9 1 9 and -1021, as in pRedCAT-9, severely reduced the expression of CAT (Table I and Figure 1). The small a m o u n t of activity that was expressed a p p e a r e d to remain sensitive to suppression by sterols. Similar results were obtained with pRedCAT-7, which was deleted up to position -759. A further deletion to position - 6 6 9 (pRedCAT-8) essentially eliminated all promoter activity. The value of CAT activity for pRedCAT-8 represents the background level in the assay. These findings suggest that an important element of the reductase promoter resides in the region between - 9 1 9 and -1021, which includes the most upstream copy of the G G G C G G hexanucleotide. However, this region alone is not sufficient for promoter activity because pRedCAT-4, which retains the - 9 1 9 to -1021 sequence but lacks the - 6 9 9 to - 8 7 5 sequence, showed little ex-

Cell 206

Table 2. SteroI-Mediated Suppression of CAT Activity in Mouse L Cells Transfected with pRedCAT-2, but Not pRSV-CAT CAT Activity, nmole per min per mg protein Plasmid Transfected into Cells

- Sterols

% Suppression by Sterols

+ Sterols

Experiment 1 pRedCAT-2 pRSV-CAT

7.8 7.5

1.9 7.6

76% 0%

Experiment 2 pRedCAT-2 pRSV-CAT

9.7 5.4

2.0 4.0

79% 26%

Experiment 3 pRedCAT-2 pRSV-CAT

5.7 11

1.1 18

81% 0%

Experiment 4 pRedCAT-2 pRSV-CAT

8.6 5.4

2.5 8.6

71% 0%

Mouse L cells were transfected with pSV3-Neo and the indicated plasmid. Mass populations of G418-resistant cells (200-500 individual colonies) were ncubated in the absence or presence of sterols, and CAT activity was measured as described in the legend to Table 1.

presslon. In addition, the isolated - 8 7 5 to - 6 9 9 seq u e n c e (pRedCAT-5) has little i n d e p e n d e n t promoter activity, but its deletion severely reduced CAT expression (pRedCAT-4). Considered together, the findings of Table 1 and Figure 1 suggest that the promoter for FIMG CoA reductase is distributed rather diffusely in the 500 bp region between positions - 5 1 3 and -1021, and that m a n y regions within this s e q u e n c e function in concert to promote efficient transcription of the reductase mRNA. The s e q u e n c e s necessary for sterol-mediated inhibition of transcription are also contained within this region and may overlap with the elements of the basal promoter.

Characterization of SteroI-Mediated Suppression of pRedCAT-2 Expression We next performed a series of detailed studies with cells transfected with pRedCAT-2. This plasmid lacks the 3.5 kb intron, but it still shows relatively high levels of CAT expression and suppression by sterols. To ensure that suppression of CAT activity in the pRedCAT-2 transfected cells was not an artifact of nonspecific suppression of all transfected genes, we transfected cells with pRSV-CAT, which contains the CAT g e n e under the control of the promoter from the long terminal repeat of Rous sarcoma virus (Gorman et al., 1982a). When cells were grown in the absence of sterols, the pRedCAT-2 and pRSV-CAT constructs produced c o m p a r a p l e levels of CAT activity. However, when sterols were a d d e d to the medium, the expression of pRedCAT-2 was suppressed, whereas the pRSV-CAT was not affected (Table 2). To isolate individual clones of pRedCAT-2 transfected cells, we seeded an average of 0.5 cells in each well of 96well microtiter dishes. Seventeen out of 23 G418-resistant clones synthesized detectable levels of CAT activity. There was a variation of 140-fold in the absolute level of expression (range, 0.1 to 14 n m o l e per min per mg protein). However, all clones showed suppression of CAT

,of '°

4

W,,h St ro,s

o I 0

24

48

7'2

Time After Removal Of Lipoproteins (hours) Figure 2. Induction of CAT Activity in Cloned Mouse L Cells Transfected with pRedCAT-2 and Incubated in the Absence of Sterols On day 0, replicate 60 mm dishes of a cloned isolate of mouse L cells permanentlytransfectedwith pRedCAT-2 were seeded at 5 x 104cells per dish in medium A containing 10% fetal calf serum. On day 2, each washed monolayer received medium A containing 10% lipoproteindeficient serum in the absence (no sterols) or presence of a mixture of cholesterol and 25-hydroxycholesterol at a final concentration of 12 ~g/ml and 0.2 ~g/ml, respectively (with sterols). Triplicate dishes of cells were harvested at the indicated time and pooled for determination of CAT activity as described in Table 1. when sterols were included in the culture m e d i u m (data not shown). One representative clone was grown in bulk and used for studies of the correlation of reductase and CAT activities. Figure 2 shows the'time course of induction of CAT activity in the cloned pRedCAT-2 transfected cells that were initially grown with lipoproteins and then incubated in the absence or presence of sterols. When the cells were grown in the presence of fetal calf serum, which contains low density lipoprotein-cholesterol, CAT activity was relatively low (zero time in Figure 2). When the lipoproteins were removed, CAT activity rose 5-fold at 24 hr and remained high over the following 48 hr. In contrast, when sterols were a d d e d to the m e d i u m at zero time, CAT activ-

Cholesterol Regulatory Region of HMG CoA Reductase Gene 207

|0

Table 3. LDL-mediated Suppression of CAT Activity in Mouse L Cells Transfected with pRedCAT-2 Addition to Medium

CAT Activity, nmole per rain per mg protein 6.4 1.4 1.5

78°/o

Experiment B None Sterols LDL

9.7 1.1 3.1

-89% 68%

770/0

Experiments were performed as described in the legend to Table 1 except that a cloned isolate of mouse L cells permanently transfected with pRedCAT-2 was used. On day 2, each monolayer received medium A containing 10% lipoprotein-deficient serum with one of the following additions: none; a mixture of cholesterol and 25-hydroxycholesterol at a final concentration of 12 and 0.2/~g/ml, respectively (sterols); or 100/~g protein/ml of human LDL. The dishes were re-fed on day 4 with the same medium and harvested by scraping on day 5. The cells from three identical dishes were pooled and processed for CAT activity, Each value is the average of duplicate determinations.

ity rose only slightly (Figure 2). A similar suppression of CAT activity was achieved when the pRedCAT-2 transfected cells were incubate~d with plasma low density lipoprotein, which delivers cholesterol by receptor-mediated endocytosis (Table 3). Figure 3A compares the suppression of CAT activity and HMG CoA reductase activity in the cloned pRedCAT-2 transfected cells as a function of the concentration of cholesterol and 25-hydroxycholesterol in the culture medium. Cells growing in the presence of fetal calf serum were switched to m e d i u m containing lipoprotein-deficient serum and varying amounts of a mixture of cholesterol and 25-hydroxycholesterol at a ratio of 20:1. After a 48 hr incubation, the cells were harvested for determination of CAT and reductase activities. The sterol mixture suppressed CAT activity and reductase activity at similar concentrations, although the extent of suppression was greater for reductase than for CAT (see Discussion). Most of the suppression occurred at a cholesterol concentration below 0.5 Hg/ml and a 25-hydroxycholesterol concentration b e l o w 0.025/~g/ml. Similar results were obtained with a mass population of pRedCAT-3 transfected cells (Figure 3B).

SteroI-Mediated Suppression of pRedCAT-2 mRNA We used the $1 nuclease technique to measure the a m o u n t of the reductase-CAT chimeric m R N A in ceils that had been transfected with pRedCAT-2 and grown in the absence and presence of sterols (Figure 4). As a probe, we used a 528 bp double-stranded restriction fragment of g e n o m i c DNA that extended upstream of the known transcription initiation sites. The probe was 5'-end-labeled only on the strand c o m p l e m e n t a r y to the mRNA. When this probe was hybridized with RNA from pRedCAT-2 transfected cells grown in the absence of sterols, multiple $1 nuclease-protected fragments were seen, suggesting

--

60

<

4O

~ IM

2O

B. pRedCAT-3

\

% Suppression

Experiment A None Sterols LDL

~

A. pRedCAT-2

•

/CAT ~

0

0.50 I. 0

I

I 0.025

/CAT

t.0 I

I // I 0.05 0

0.50 I

I 0.025

t . 0 Cholesterol I

I 0.05

I 25-Hydroxycholesterol

Sferol (,u.g/ml )

Figure 3. SteroI-MediatedSuppression of HMGCoA ReductaseActivity and CAT Activity in Mouse L Cells Transfected with pRedCAT-2 or pRedCAT-3 On day 0, replicate 60 mm dishes of a cloned isolate of mouse L cells permanently transfected with pRedCAT-2 (A) or a mixed culture of mouse L cells that had been transfected with pRedCAT-3 (B) were seeded at 5 x 104 ceils per dish in medium A containing 10% FCS. On day 2, the dishes were washed and re-fed medium A containing 10% lipoprotein-deficient serum and varying amounts of a mixture of cholesterol and 25-hydroxycholesterol at a ratio of 20:1. The final concentration of each sterol is shown on the abscissa. The cells were refed on day 3 and harvested on day 5. Cell extracts were processed for HMG CoA reductase activity and CAT activity as described in the legend to Table 1. Extracts processed for HMG CoA reductase activity were preincubated with "activation buffer" prior to assay. The 100% of control values for CAT were 14 nmole per min per mg protein and 26 nmole per min per mg protein for (A) and (8), respectively. The 100% of control values for reductase were 479 and 894 pmole per min per mg protein, respectively.

that the probe was hybridizing to mRNAs that initiate at multiple sites (Figure 4, lane C). When the cells were grown in the presence of sterois (Figure 4, lane D), all of the protected fragments were reduced by more than 75% (as measured by densitometric scanning of the gel), indicating that transcription from all of these initiation sites was suppressed by sterols. To determine whether the initiation sites used in the pRedCAT-2 transfected cells corresponded to initiation sites used for production of reductase m R N A in hamster cells, we performed a similar $1 nuclease analysis using RNA isolated from UT-1 cells (Figure 4, lanes A and B). Determination of transcription initiation sites for reductase m R N A in UT-1 cells is complicated by the presence of multiple initiation sites and multiple 5' splice donor sites for intron 1, s o m e of which are close to the initiation sites. No single end-labeled probe can detect all transcription initiation sites in UT-1 cells (Reynolds et al., 1985). The Barn HI site that is labeled for the $1 nuclease analysis in Figure 4 is spliced out of m a n y mRNAs that initiate at sites 1a-d, which are the major initiation sites in UT-1 cells (Reynolds et al., 1985). Thus, this probe quantitatively underestimates the mRNAs that initiate at these upstream sites, but it does permit accurate quantitation of mRNAs that initiate at sites 2-5. In mRNAs produced from pRedCAT-2, no splicing occurs in this region as determined with $1 nuclease analysis using uniformly labeled single-stranded probes (data not shown). Thus, the Bam

Cell 208

L Cells Transfected With UT-1

Cells

p.g RNA

10

1

A

B

Red CAT-2 None

10 10

10

C

E

Sterols

5 2 8 nt - -

-,,,

230 n t - - ~

=

D

-- probe

-~

1 2 7 nt --

la -d

.e0uc,os. Promo,or

2:345

BamHl

tilt

CAT

3'

,¢ I / / / / / / H / / / / / / / / / / /

i

- 1021

-513

x- protected 1 2 7 - 2 3 0 nt

Figure 4. $1 Nuclease Mapping of Transcription Initiation Sites of the Reductase Promoter-CAT Chimeric Gene in Cloned Mouse L Cells Transfected with pRedCAT-2 and Incubated with or without Sterols UT-1 cells (lanes A and B) were grown in the presence of 40 /~M compactin and 10% lipoprotein-deficient serum. A cloned isolate of mouse L cells permanently transfected with pRedCAT-2 (lanes C and D) and a mass population of nontransfected mouse L cells (lane E) were grown in the absence or presence of sterols as described in Table 1 except that cells were plated on day 0 at 1 x 105 cells per 100 mm dish. Poly(A)÷ (lanes A and B) or total cytoplasmic RNA (lanes C, D, and E) were prepared, and the indicated amount of RNA was annealed to a 5'-end-labeled 32p-DNA reductase probe from genomic subclone B1 and processed for $1 nuclease analysis. The position of the 32P-end-labeledprobe with respect to the reductase-CAT fusion gene and a diagram of the observed $1 resistant fragments is shown at the bottom. An autoradiogram of a 5% polyacrylamide/8 M urea gel displaying the Sl-resistant fragments is pictured at the top. The five predominant reductase initiation sites previously observed in UT-1 cells (Reynolds et al., 1984; 1985) are indicated by arrows at the bottom of the figure; nt, nucleotide.

~- probe 5 2 8 nt

HI site is retained in all mRNAs that are produced in the pRedCAT-2 transfected cells. With these quantitative considerations in mind, a comparison of Lanes A and C in Figure 4 indicates that the spectrum of initiation sites in the pRedCAT-2 transfected cells generally resembles the spectrum of sites observed in UT-1 cells. To ensure that the suppression of transcription was specific to the reductase-CAT chimeric gene, we analyzed RNA transcribed from the co-integrated neo gene, which encodes the structural gene for aminoglycoside 3'-phosphotransferase. For this purpose, we performed $1 nuclease mapping using a DNA probe specific to the coding region of the neo gene. When RNA was isolated from cells grown in the absence and presence of sterols, there was no difference in the amount of neo-specific RNA (data not shown). In V i t r o A s s a y s of R e d u c t a s e

Promoter

Three pRedCAT plasmids were incubated with a HeLa cell extract prepared as described by Manley et al. (1980) and the 5' ends of the synthesized mRNAs were mapped with the $1 nuclease technique (Figure 5). Plasmids pRedCAT-2 and pRedCAT-1 (lanes C and D) both directed the in vitro production of mRNAs whose 5' ends were essentially identical to the 5' ends of the mRNAs produced in vivo from pRedCAT-2 in intact mouse L cells (lanes A and B). On the other hand, pRedCAT-7, which had reduced promoter activity in intact mouse L cells (Table 1 and Figure 1), had virtually no promoter activity when assayed in vitro (Figure 5, lane E). The finding of similar patterns of heterogeneous initiation sites used both in vivo and in

vitro suggests that it may be possible to study the sterolmediated inhibition of reductase transcription in cell-free extracts. Discussion

These studies reveal that DNA sequences responsible for promotion and inhibition of HMG CoA reductase transcription reside within a 500 bp segment that extends 300 bp upstream of the multiple transcription initiation sites, i.e., from position - 5 1 3 to -1021 (Figure 1). When DNA fragments containing this sequence were placed in front of the CAT gene, CAT protein was produced, as in plasmids pRedCAT-1, -2, and -3. CAT activity was reduced by 70% to 90% when the transfected cells were incubated in the presence of sterols or LDL. Measurements of mRNA by the $1 nuclease technique confirmed that the sterolmediated suppression was caused by a reduction in the amount of reductase-CAT mRNA, presumably owing to a decrease in the rate of transcription. The positive promoter function within the -513 to -1021 region was quite strong. Thus, in the absence of sterols, the amount of CAT activity generated from the pRedCAT plasmids was similar to that generated when transcription of the CAT gene was driven by the RSV long terminal repeat promoter (Table 2). The latter is felt to be an especially strong promoter in mammalian cells (German et al., 1982a). Deletion experiments (Table I and Figure 1) suggested that both the positive promoter and the sterol-regulated negative elements of the reductase gene are distributed over the 500 bp region between -1021 and

Cholesterol Regulatory Region of HMG CoA Reductase Gene 209

Figure 5. $1 Nuclease Analysis of RNA Transcribed In Vivo and In Vitro from pRedCAT R e c o m b i n a n t Plasmids

I i, vivo I in

vitro

IABCDE 528 n t - ~

alp ~

~

~

-Probe

230 nt -

127 nt la-d

2 545 5I

51

I

-1420

I

- 402"1

I

-759

I

pRedCAT-2

I

pRedCAT- 1

A clonedisolateof mouse L cells permanently transfected with pRedCAT-2was grown in the absence (lane A) or presence (lane B) of sterols, and cytoplasmic RNA (50 ~g) from each culturewas analyzedby the 5' nuclease $1 methodas describedin Figure4. 2.5/~gof pRedCAT-2(laneC), pRedCAT-1(laneD), and pRedCAT-7 (lane E) were transcribed in a whole cell extract from HeLa cells, and onefourth of the RNA productwas subjectedto a 5' nuclease$1 analysisas above.A schematic representationof the reductasepromoter region is shown at the bottom. The thin black lines representthe sequenceof the promoter region retained in each of the plasmids. All other symbols and notations correspond to those in Figure4. The gel was exposedto x-ray film for 16 hr with aid of an intensifyingscreen; nt, nucleotide.

pRedCAT-7 I -5"13 x protected 127- 230nt x probe 528 nt

-513. Reductase transcription seemed to require the whole segment; deletion of any part of the region severely reduced the expression of pRedCAT mRNA and CAT activity. It is likely that the activity of the reductase promoter and its susceptibility to inhibition are affected by the sites at which the chimeric plasmid integrates into the genome. Thus, when individual clones of cells transfected with pRedCAT-2 were isolated, the level of CAT expression varied over 140-fold. However, in each case in which expression was detected, suppression by sterols was also observed, suggesting that the promoter and sterol-regulatory functions are closely coupled. So far, we have not observed a reductase promoter-CAT construct that produced CAT activity without susceptibility to sterolmediated inhibition. The close association between the promoter and sterol-regulatory elements is also suggested by the deletion experiments (see below). The mechanism for sterol-mediated inhibition of the pRedCAT constructs appeared to be similar to that for inhibition of the endogenous reductase gene in mouse L cells. Thus, CAT activity and endogenous reductase activity were reduced in parallel when increasing concentrations of sterols were used (Figure 3). In general, the extent of suppression of reductase activity was greater than the extent of suppression of CAT activity (Figure 3). In part, this relates to the ability of sterols to enhance the rate of degradation of HMG CoA reductase protein, an effect that potentiates the fall in reductase activity but not CAT activity (Faust et al., 1982; Tanaka et al., 1983; Chin et al., 1985). However, it is possible that the endogenous reductase promoter may also show a greater degree of suppression than that seen with the pRedCAT plasmids. We do not yet know precisely the extent of reduction in endoge-

nous reductase mRNA levels when sterols are added to mouse L cells. The -513 to -1021 region of the reductase promoter contains multiple copies of the hexanucleotide repeat CCGCCC or its complement GGGCGG, which are also present in the SV40 promoter and in the thymidine kinase gene of herpes simplex virus. Removal of these repeats from the SV40 promoter (Fromm and Berg, 1982) and from the thymidine kinase promoter (McKnight and Kingsbury, 1982) reduces the efficiency of transcription in vivo. Dynan and Tjian (1983) have identified a transcription factor, SP1, that binds to the hexanucleotide repeats of the SV40 promoter region and is required for initiation of SV40 RNA synthesis in vitro. SP1 has also been shown to bind specifically to the same hexanucleotide repeats in a presumptive promoter in monkey DNA (Saffer and Singer, 1984; Gidoni et al., 1984). It seems likely therefore that SP1 might bind to the reductase promoter and facilitate transcription. On the other hand, it is unlikely that inhibition of reductase transcription would be mediated by an influence of sterols on the level of SP1 protein in the cell. This conclusion follows from the observation that transcription of the n e o gene driven by the SV40 early promoter was not inhibited by sterols (data not shown). The promoter regions of a number of cellular housekeeping genes, in addition to HMG CoA reductase, have recently been shown to contain multiple copies of the GC hexanucleotide box. These genes include human adenosine deaminase (Valerio et al., 1985), mouse and human dihydrofolate reductase (McGrogan et al., 1985; Masters and Attardi, 1985), human superoxide dismutase (Levanon et al., 1985), chicken U1 RNA (Roebuck and Stumph, 1985), and mouse HPRT (Melton et al., 1984). Like the HMG CoA reductase gene, the multiple GC boxes in these

Cell 210

genes are spread over several hundred nucleotides in the 5' flanking region, raising the possibility that the promoter activity for these genes will also be distributed over this region. The e n d o g e n o u s HMG CoA reductase gene and the pRedCAT-2 plasmid both use multiple initiation sites for m R N A production, a finding that has a parallel in transcription from the SV40 late promoter (Buchman et al., 1981). The initiation sites used in the reductase-CAT constructs in vivo and in vitro were generally similar to those used in UT-1 cells, the cell line from which the reductase gene was isolated (Figures 4 and 5). The role, if any, of the 3.5 kb intron in the 5' untranslated region in regulation of reductase expression remains to be elucidated. The construct that contained this intron, pRedCAT-3, showed a higher level of expression in the induced state than did the constructs that lacked the intron. The intron itself did not seem to have promoter function because constructs that contain this intron but lacked the - 5 1 3 to -1021 sequence (i.e., pRedCAT-6 in Table 1 and Figure 1) did not produce CAT activity. It is possible that this intron contains a cis-acting transcriptional enhancer that augments expression of the - 5 1 3 to -1021 promoter region. Alternatively, the increased expression of the intron-containing construct may relate to more efficient splicing. The 3.5 kb intron has variable 5' splice donor sites, one of which is located at position - 6 6 9 , which is close to the downstream transcription ~initiation sites (Reynolds et al., 1985). All of the 5' splice sites are joined to a common 3' acceptor site at position - 2 3 . Plasmids pRedCAT-1 and pRedCAT-2 contain the 5' donor site at - 6 6 9 , but lack the acceptor site at - 2 3 . In studies of cytoplasmic m R N A produced from pRedCAT-2, we did not find evidence that the splice donor site at - 6 6 9 was used, as indicated by $1 nuclease protection using uniformly labeled single-stranded DNA probes that covered this region. The inability to use this 5' splice site to produce cytoplasmic mRNA may account for the smaller amount of CAT activity expressed by pRedCAT-1 and -2 as compared with pRedCAT-3. The extent to which the current findings on HMG CoA reductase can be generalized to other eukaryotic genes that undergo negative feedback regulation by metabolic end products remains to be determined. It se~ms reasonable to suspect that the 5' flanking region may be involved in negative regulation in other systems in addition to the reductase. This region has already been implicated in positive control of gene expression by such agents as steroid hormones, heavy metals, and regulators of the genes for interferons and heat-shock proteins (reviewed in Kessel and Khoury, 1983). Moreover, it is well established that the inhibition of early SV40 transcription is mediated by the binding of SV40 large tumor antigen to DNA in the 5' flanking region of the early SV40 transcription unit (Myers et al., 1981; Hansen et al., 1981). In the HMG CoA reductase system, the next step will be to determine the precise sequences in the 5' region that mediate feedback inhibition and whether trans-acting factors, such as sterol-binding repressor proteins, are involved.

Experimental Procedures Materials [14C]Chloramphenicol(40-60 mCi/mmole) and DL-3-hydroxy-3-methyl[3-14C]glutaryl coenzyme A (55 mCi/mmole) were purchased from New England Nuclear. [7-32P]ATP (>5000 Ci/mmole) was obtained from ICN. Enzymes used in plasmid constructions were obtained from New England Biolabs and Boehringer Mannheim Biochemicals. G418 sulfate (Geneticin) was purchased from GIBCO Laboratories. Plasmid pSV3-Neo, which contains a bacterial gene that confers resistance to G418 (Southern and Berg, 1982), was obtained from Bethesda Research Laboratories. Plasmid pUC-13 and acetyl CoA (lithium salt) were obtained from Pharmacia PL Bioohemicals. Cholesterol and 25hydroxycholesterol were purchased from AIItech Associates and Steraloids, Inc., respectively. E1 and B1, genomic Eco RI and Barn HI restriction fragments of hamster HMG CoA reductase, were described previously (Reynolds et al., 1984). Plasmids pRSV-CATand pSV0-CAT (Gorman et al., 1982a, 1982b) were kindly provided by Bruce Howard. Newborn calf lipoprotein-deficient serum (d > 1.215 g/ml) and human I_DL (d 1.019-1.063 g/ml) were prepared by ultracentrifugation (Goldstein et al., 1983). Plasmid Constructions Standard protocols were used for all recombinant DNA technology (Maniatis et al., 1982). Plasmids pRedCAT-1 and pRedCAT-2(Figure 1) were constructed by insertion of hamster genomic reductase sequences into pSV0-CAT,a recombinant plasmid that contains the betalactamase gene (ampR),the origin of replication from pBR322, and the coding sequence for chloramphenicol acetyltransferase (Gorman et al., 1982b). Plasmid pSV0-CATcontains a single Hind III site 5' to the CAT structural gene; it has no defined eukaryotic promoter or enhancer sequences. Plasmid pRedCAT-1 was prepared from genomic subclone B1, which spans nucleotide position -1021 to -513 of the hamster reductase gene (Reynolds et al., 1984). Reductase sequences are numbered in a negative fashion with regard to the A nucleotide of the AUG initiator codon at position +1 (Reynolds et al., 1984). The sequence of the 3.5 kb intron in the 5' untranslated region is omitted from the numbering scheme. The staggered ends of the B1 subclone were filled with deoxyfibonucleoside triphosphates as catalyzed by the Klenow fragment of DNA polymerase I. After attachment of a Hind III linker octanucleotide (New England Biolabs), the DNA fragment was inserted into the Hind III site of pSV0-CATin the orientation shown in Figure 1. Plasmid pRedCAT-2was derived from pRedCAT-1 by tacking onto pRedCAT-1 approximately 400 bp of additional 5' flanking DNA contained in genomic subclone El. Therefore, pRedCAT-2contains the sequence spanning nucleotide positions -1420 to -513 of the hamster reductase gene fused to CAT. Plasmid pRedCAT-3 was constructed by isolating the CAT gene from pSV0-CATas a Hind Ill-Barn HI fragment and filling in the ends as described above. After attachment of a Sal I linker octanucleotide (New England Biolabs), the DNA fragment was inserted into the unique Sal I site of pRed-3. Plasmid pRed-3 was constructed from pBR322 and contains 5' flanking DNA from the hamster reductase gene (nucleotide position -1420 to -23). The Barn HI site at -23 of pRed-3 was filled in and a Sal I linker was added, thus leaving the 3' junction of the 3.5 kb intron 1 intact. Plasmid pRedCAT-4 was made by deletion of the unique 178 bp Sma I-Nae I fragment (nucleotide position -875 to -699) from pRedCAT-1. Plasmid pRedCAT-5was constructed by inserting the 178 bp Sma I-Nae I fragment into the Hind III site of pSV0-CATafter attachment of Hind III linker octanucleotides to the blunt-ended fragment. Plasmid pRedCAT-6was made by deletion of the Bam HI fragment (nucleotide position -1021 to -513) from pRedCAT-3. Plasmids pRedCAT-7,pRedCAT-8,and pRedCAT-9are deletion mutants of pRedCAT-I. They were constructed by Ba131 nuclease resection (Osborne et al., 1981)after digesting pRedCAT-1with Nde I, which cuts once in the vector sequence 5' to the reductase promoter insert. A Sal I linker octanucleotide was inserted to mark the 5' end point of the deletion, and the deleted reductase promoter-CAT sequence was excised from the pSV0-CAT vector after Pst I cleavage and inserted

Cholesterol Regulatory Region of HMG CoA Reductase Gene 211

into the Sal I-Pst I sites of pUC-13. The 5' endpoints of the deletions were estimated by restriction endonuclease cleavage analysis, and the exact endpoints were positioned by DNA sequencing (Sanger et al., 1977) after cloning into M13mp18 (Messing, 1983).

Cell Growth, BNA Transfection, and G418 Selection All cells were grown in monolayer culture at 37°C in an atmosphere of 5%-7% CO2. Mouse L cells (t/c) were obtained from Richard Axel and maintained in medium A (Dulbecco's modified Eagle medium containing 100 U/ml penicillin and 100 #g/ml streptomycin) supplemented with 10% (v/v) fetal calf serum. Cells were seeded at 5 x 10s per 100 mm dish and transfected on the following day with one of the pRedCAT plasmids (4.5 ~g) together with pSV3-Neo (0.5 ,~g) by the calcium phosphate coprecipitation technique (van der Eb and Graham, 1980). The cells were incubated with the DNA for 12-16 hr and then switched to medium A. 48 hr later the cells were fed medium A supplemented with 10% FCS and 700 ~g/ml G418. Selection with G418 was maintained for 12-14 days. Resistant colonies were pooled (200-500 individual clones), expanded in mass cultures in the presence of G418 (350 #g/ml), and cloned by limiting dilution of the mass culture. The nonselectable CAT marker was expressed in 74% to 80% of the G418resistant clones. Cells were plated for individual experiments as described in the figure legends. UT-1 cells, a line of compactin-resistant Chinese hamster ovary cells, were grown in Ham's F-12 medium supplemented with 40 ~M compactin and 10% lipoprotein-deficient serum (Chin et al., 1982).

Clarice Grimes, Claudia Stewart, and Debra Noble-Schlesinger provided excellent technical assistance. This research was supported by a research grant from the National Institutes of Health (HL 20948). T. E O. is the recipient of a postdoctoral fellowship from the National Institutes of Health (HL 06867). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received December 19, 1984; revised April 22, 1985

References Aviv, H., and Leder, P. (1972). Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Prec. Natl. Acad. Sci. USA 69, 1408-1412. Berk, A J., and Sharp, I? A. (1977). Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of $1 endenucleasedigested hybrids. Cell 12, 721-732. Brown, M. S., and Goidstein, J. L. (1980). Multivalent feedback regulation of HMG CoA reductase, a control mechanism coordinating isoprenoid synthesis and cell growth. J. Lipid Res. 21, 505-517. Buchman, A., Burnett, L., and Berg, IR (1981). In DNA Tumor Viruses, Part II, Revised, J. Tooze, ed. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory), pp. 799-841.

Enzymatic Assays The activity of HMG CoA reductase was measured in detergentsolubilized cell extracts as described (Goldstein et al., 1983). In some experiments, each extract was incubated prior to assay for 20 rain at 3-/°C in "activation buffer" in order to allow complete dephosphorylation of reductase (Gil et al., 1985). Reductase activity is expressed as the pmole of [14C]HMG CoA converted to [14C]mevalonate per rain per mg of detergent-solubilized protein. CAT activity was measured in freeze-thawed cell extracts as described by Gorman et al. (1982b). CAT activity is expressed as the nmole of [14C]chloramphenicol converted to acetylated forms of [14C]chloramphenicol per rain per mg of cell protein. Each assay tube (0.15 ml) contained 2 to 10/~g protein and 3 to 4.3 nmole [14C]chloramphenicol ('~ 1.3 x 105 cpm/nmole) in a buffer containing 0.47 M Tris-chlodde (pH 7.8) and 0.53 mM acetyl CoA. The tubes were incubated for 10 to 60 rain at 37°C. All assays were linear with respect to time of incubation and concentration of extract protein. The acetylated forms of [~4C]chloramphenicol were separated from unreacted [14C]chloramphenicol by thin layer chromatography, detected by autoradiography, and quantified by liquid scintillation spectrometry (Gorman et al., 1982b). Protein was measured by a modification of the method of Lowry et al. (1951).

Chin, D. J., Gil, G., Faust, J. R., Goldstein, J. L., Brown, M. S., and Luskey, K. L. (1985). Sterols accelerate degradation of HMG CoA reductase encoded by a constitutively expressed cDNA. Mol. Cell. Biol. 5, 634-641. Chin, D. J., Gii, G., Russell, D. W., Liscum, L., Luskey, K. L., Basu, S. K., Okayama, H., Berg, P., Goldstein, J. L., and Brown, M. S. (1984). Nucleotide sequence of HMG CoA reductase, a glycoprotein of the endoplasmic reticulum. Nature 308, 613-617.

In Vitro Transcription Assays of in vitro transcription were performed with the indicated plasmid (2.5/~g of the covalently closed circular form) in a whole cell extract from HeLa cells (Manley et al., 1980). The Hela extract (obtained from Bethesda Research Laboratories) was used as described by the supplier. The DNA template was destroyed after the transcription reaction by the addition of 10 ~g of DNAse (Worthington Biochemicals, grade DPFF) and 10 U of RNasin (Promega Biotec), followed by incubation at 37°C for 20 rain. The RNA product was extracted and analyzed by $1 nuclease mapping as described below.

Fromm, M , and Berg, R (1982). Deletion mapping of DNA regions required for SV40 early region promoter function in vivo. J. Mol. Appl. Genetics 1, 457-481.

Sl Nuclease Mapping of mRNA Cytoplasmic RNA (Reynolds et al., 1984) and poly(A) + RNA (Aviv and Leder, 1972) were prepared as described in the indicated reference. Nuclease $1 mapping of the 5' termini of reductase mRNA was performed by the method of Berk and Sharp (1977) with modifications as described by Reynolds et al. (1984). All gels shown in the figures were exposed to x-ray film for 12-24 hr; the gel in Figure 5 was exposed to film with the aid of an intensifying screen. Acknowledgments We thank Gary Reynolds and Kenneth Luskey for helpful discussions.

Chin, D. J., Luskey, K. L., Anderson, R. G. W., Faust, J. R., Goldstein, J. L., and Brown, M S. (1982). Appearance of crystaltoid endoplasmic reticulum in compactin-resistant Chinese hamster cells with a 500-fold elevation in 3-hydroxy-3-methylglutaryl CoA reductase. Prec. Natl. Acad. Sci. USA 79, 1185-1189. Dynan, W. S., and Tjian, R. (1983). The promoter-specific transcription factor Spl binds to upstream sequences in the SV40 early promoter. Cell 35, 79-87. Faust, J. R, Luskey, K. L, Chin, D. J., Gotdstein, J. L., and Brown, M. S. (1982). Regulation of synthesis and degradation of 3-hydroxy-3methylglutaryl coenzyme A reductase by low density lipoproteins and 25-hydroxycholesterol in UT-1 cells. Prec. Natl. Acad. Sci. USA 79, 5205-5209.

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