Characterization of the phosphoenolpyruvate carboxykinase (GTP

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc

Vol. 261, No. 21, Issue of July 25, pp. 9714-9720, 1986 Printed in U.S.A.

Characterization of the Phosphoenolpyruvate Carboxykinase (GTP) Promoter-regulatory Region 1. MULTIPLE HORMONE REGULATORY ELEMENTS AND THE EFFECTS OF ENHANCERS*

(Received for publication, December 24, 1985)

Anthony Wynshaw-Boris$, Jay M. Shorts, David S. LooseSU, and Richard W. HansonII From the Department of Biochemistry, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106

Transcription of the gene for cytosolic Phospho- enolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) from rat liver is increased by cAMP and glucocorticoids and decreased by insulin. A PEPCK- thymidine kinase (TK) chimeric gene was transfected into FTO-2B rat hepatoma cells, which were TK-deficient. Previous studies showed that a cAMP regulatory element is located at the 5' end of the PEPCK gene. In this report, we demonstrate that the 5' end of the gene also contains a glucocorticoid regulatory element, but not one for insulin. Regions of the PEPCK gene that contain these regulatory elements were attached to the Herpes simplex virus TK structural gene containing its own promoter. The hormone regulatory elements within the 5' flanking region of the PEPCK gene conferred cAMP and glucocorticoid responsiveness on the TK gene after transfection into FTO-PB cells. Like viral enhancer elements, these regulatory elements functioned properly when placed in either orientation at various positions 5' or 3' to TK. The presence of the SV40 enhancer element upstream from the PEPCK- TK gene had little effect on the basal level of expression or hormonal regulation of the chimeric gene.

Hormonal regulation of gene expression is a primary mechanism by which eukaryotic organisms integrate cellular re- sponses to environmental and metabolic conditions. Many of the hormonally regulated genes that have been studied to date are controlled by alterations in the level of transcription. The mechanism of this regulation is best understood for genes controlled by steroid hormones, where a hormone-receptor complex binds to defined regions of these genes (1, 2). How- ever, little is known of the mechanism(s) by which non-steroid hormones regulate gene transcription, or how multiple hormones regulate the transcription of a single gene.

The gene for cytosolic phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK') from the rat is a good model

* This work was supported in part by Grants AM-21859 and AM 24451 from the National Institutes of Health. The costs of publication

This article must therefore be hereby marked "aduertisement" in of this article were defrayed in part by the payment of page charges.

accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 4 Trainees on the Metabolism Training Program AM 07319 from

the National Institutes of Health. (j Present address: Vector Cloning Systems, 3770 Tansy St., San

"

Diego, CA 92121. 7 Present address: Department of Pharmacolom. University of

Texas Health Science Center, Houston, TX 77225." 11 To whom correspondence should be addressed. The abbreviations used are: PEPCK, phosphoenolpyruvate car-

boxykinase; TK, thymidine kinase; BtZcAMP, dibutyryl cyclic AMP; HAT, 10" M hypoxanthine, 4 X lo-' M aminopterin, 1.6 X M thymidine; PIPES, 1,4-piperazinediethanesulfonic acid; kb, kilobase pair; bp, base pair.

for studies of the regulation of gene transcription by a variety of hormones. The transcription of the gene for PEPCK is stimulated by CAMP, glucocorticoids, and thyroid hormones and depressed by insulin in the liver (3-5, for review, see Ref. 6). The PEPCK gene is contained within a 7.0-kb BamHI restriction fragment (7), and the start site of transcription has been localized (7, 8). A chimeric gene was constructed which contained the PEPCK transcriptional start site and 548 bp of 5' flanking sequence fused to the structural gene of Herpes simplex virus thymidine kinase (TK). After transfection into FTO-2B rat hepatoma cells (8, 9), the transcription of the PEPCK-TK chimeric gene was regulated by Bt2cAMP (S), indicating that the cAMP regulatory element for the PEPCK gene is contained within a 621-bp fragment at the 5' end of the gene.

In this report, we demonstrate the presence of cAMP and glucocorticoid regulatory elements in the immediate 5' flanking region of the PEPCK gene. However, the same 621-bp segment of DNA does not appear to contain an insulin regulatory element. The orientation dependence and distance dependence of the hormone regulatory elements of the PEPCK gene on the TK promoter, as well as the effect of these elements when placed 3' to the TK gene, were determined. We also examined the effect of the SV40 enhancer element on the expression and regulation of the PEPCK-TK chimeric gene.

EXPERIMENTAL PROCEDURES

Materials-Bt2cAMP, restriction enzymes, DNA ligase, DNA polymerase, calf intestinal alkaline phosphatase, polynucleotide kinase, and S1 nuclease were purchased from Boehringer Mannheim. The- ophylline and bovine insulin were purchased from Calbiochem-Behr- ing. Dexamethasone was from Sigma and was dissolved and stored in 100% ethanol at -20 'C. [T-~'P]ATP (7000 Ci/mmol) was purchased from ICN or Amersham Corp. Formamide (MCB Biochemical) was deionized before use with Bio-Rad Ag-50-XA8 resin. All buffers and salts were purchased from Sigma or Fisher and were of the highest purity available. Cell culture medium and sera were from Flow Lab- oratories or GIBCO. pOPF (10) was a gift of Dr. Richard Flavell (Biogen Research Corp) and pTK-109 (11) was a gift from Dr. Steven McKnight (University of Washington). FTO-2B rat hepatoma cells were previously described (8, 9) and were provided by Dr. Keith Fournier (University of Southern California). These cells are TK- deficient; after transfection' with TK-containing plasmids, cells ex- pressing TK can be selected in HAT medium (12).

Plasmid Construction-Digestion with restriction enzymes, trans- formation of Escherichia coli, and purification of plasmid DNA were carried out as described (8, 13). DNA fragments were isolated by electroelution from agarose gels (13). Ligations were carried out in 20-pl reaction mixtures consisting of 20 mM Tris-C1 (pH 7.5), 2 mM MgC12, 0.5 mM ATP, 2 mM dithiothreitol, and 1 unit of T4 DNA ligase. Vector DNA (200 ng) was incubated with a 5-20-fold molar excess of insert DNA at room temperature for at least 6 h.

The first series of plasmid vectors contained the 621-bp BanHI- BglII fragment of the PEPCK gene (Fig. lA). This fragment contains

9714

Multiple Hormone Regulatory Elements of the PEPCK Gene 9715

"*',"E p0PF-BBRl now fragment of DNA polymerase I by standard procedures (13). The O l l D C B " " " ' p0FFF-BBI-J PuuII-PuuII fragment of the PEPCK 5' flanking sequence (-416 to

-61 of the BamHI-BglII fragment in Fig. 1A) was inserted at the BamHI site of pTK-109 in the same orientation (pTK-109-PP(+)) or in the opposite orientation (pTK-lOg-PP(-)) relative to the TK transcriptional unit a t this site (Fig. ID). The identity of pTK-109- PP(+) and pTK-lOg-PP(-) was confirmed by sequencing these plasmids (data not shown). Similarly, the PuuII-PuuII fragment of the PEPCK 5' flanking sequence was inserted at the HindIII site of pTK- 109 in the same orientation (pTK-109 3'-PP(+)) or in the opposite

PEPCK sequences 3' to the gene (Fig. 1D).

EcoRI

Pw Ne0 P w pTK-109-PPItI orientation IpTK-109 3'-PP(-)) relative to TK. This places the

The SV40 sequences, including enhancer elements, were removed

religated to form pPCTK-GA(-SV40) (Fig. 1E) and pOPF(-SV40) (Fig. IF).

Cell Transfection and Hormonal Treatment-The growth and transfection of FTO-2B cells have been described in detail previously (8). HAT-resistant colonies were pooled and grown in sufficient

day prior to hormonal treatment. Cells were changed to medium without fetal calf serum and incubated for 4 h in the presence or absence of hormones. To measure the effects of CAMP or glucocorticoids on these cells, 0.5 mM Bt,cAMP plus 1 mM theophylline or 1 WM dexamethasone, respectively, were added. To measure the effect of insulin, transfected cells were simultaneously treated with 0.5 mM BhcAMP, 1 mM theophylline, and 50 nM insulin, since insulin at these concentrations blocks the increase in transcription of the PEPCK gene caused by BkcAMP (4).

A. 5 M guanidinium isothiocyanate (15) and centrifuged through a cush- RNA Isolation and Analysis-RNA from cells was extracted with

shown. It contains the PEPCK transcriptional start site (direction of m~~~ was analyzed by s1 nuclease mapping (17) using 5 , 32p end-

chimeric gene containing the BamHI-BglII fragment of the PEPCK of 50 mM ~ ~ i ~ - ~ 1 ( P ~ 8), 0.1 mM EDTA ( P ~ 8), and 200 units of calf

TK gene. It contains the SV40 Hind111 fragment from 5107 to 982, at 37 "C, then at 52 "C, an additional 2 0 ~ units of phosphatase were including the SV40 enhancer element. This plasmid was constructed added, and the incubations repeated. phosphatase was inactivated at from POPF as described (8). C, POPF was linearized at the unique 65 ac for 20 min, extracted, and precipitated. The phosphate-free

inserted in both orientations relative to the intact TK gene to give twice for 30 min each at 37 "C in 50 mM Tr is -H~l

Hind111 fragment with enhancer element. 0, PTK-109 was digested and at least a 5-fold molar of [y-32p]ATp. Kinase was inacti- either at the BamHI site of the HindIII site. The PuuII-PuuII frag- vated at 65 oc for 15 min, and the DNA was cleaved with a restriction merit Of the 5' flanking sequence (-416 to -61) was inserted enzyme such that the labeled fragment corresponding to the tran- in either orientation at the BamHI site to give pTK-lOg-PP(+) and scriptional site ofpEpCK, PEPCK-TK, or TK would be labeled pTK-109-PP(-), Or in either orientation at the site to give at only one end. The correct end-labeled fragment was isolated by pTK-109 3'-PP(+) and pTK-109 3'-PP(-). These plasmids contain electroelution from agarose gels (8), no SV40 sequences. E, P P C T K - ~ A ( - S V ~ ~ ) was m d e from PPCTK- tal RNA (50 was precipitated with 0.5-1 x 105 cpm of end- 6A by complete HindIII digestion, which removes sv40 sequences- labeled probe (25-50 ng). ~ ~ ~ l ~ i ~ acids were resuspended in 10 of tion, removing all SV40 sequences. mM EDTA, pH 6.5), denatured at 72 "C for 10-15 min, and hybridized

for 16 h at 52 "C. Samples were precipitated and separated by elec- the PEPCK mRNA start site, 73 bp of the 5' untranslated region, trophoresis on 8% denaturing acrylamide gels (19). The gels were and 547 bp of 5' flanking sequence (8). pPCTK-6A (Fig. 1B) includes fixed, dried, and exposed to XAR film (Kodak). Autoradiographs were a PEPCK-TK chimeric gene consisting of the BamHI-BglII pro- scanned densitometrically to quantitate mRNA levels for PEPCK, moter-regulatory fragment of the PEPCK gene fused to the BgZII site TK, and PEPCK-TK. in the 5' untranslated region of the TK gene (+50). The mRNA Droduced from this transcriptional unit codes for TK, but its produc- RESULTS

D from pPCTK-6A and pOPF by HindIII digestion and the vector

mPClK-61

Ero R I m

"v pTI-109

'"&h. pT*-109 3"PP(+I P W l quantities to isolate RNA. Cells were changed twice weekly, and 1

E

1. vectors used to transfect FTo-2B A 621-b~ BQmHI-WI fragment a t the 5' end Of the gene ion of 5.7 M cesium chloride (16). PEPCK, TK, or PEPCK-TK

transcription is shown by the armw), 73 bp of5' untranslated region, labeled probes (18). The 5' phosphates were removed from the ends and 548 bP of 5' flanking sequence. B, pPCTK-6A is a PEPCK-TK of the DNA by incubation of 20 Wg of DNA in 50-~1 reaction mixtures

gene fused to the 5' untranslated region Of the Herpes simp1ex V'ruS intestinal alkaline phosphatase. Samples were incubated for 15 min

BamHI site, and the BamH1-Bg'l fragment Of the gene was ends were labeled with [y-32P]ATP with polynucleotide kinase (4-10

pOPF-BB(+) and poPF-BB(-)' POPF contains the sv40 mM MgC12, 5 mM dithiothreitol, 0.1 mM spermidine, 0.1 mM EDTA, 7.6),

F, POPF("SV40) was made from POPF by diges- 80% formami&, 1 x PIPES buffer (0.5 M NaC1, 10 m~ PIPES, 1


PEPCK

- “-373

73“ - -.r

Barn HI CAP BgllI - Barn HI CAP BqlP AvaI - 548 73 540 73 300

PEPCK mRNA Y A A A PEPCK-TKmRNA “ A A A PEPCK-TK mRNA Y A A A

FIG. 2. S1 nuclease analysis of mRNA isolated from cell line, PTK-1, which was transfected with pPCTK-6A. RNA was isolated from this cell line after a 4-h treatment with serum-free medium (control), BWAMP (0.5 mM) and theophylline (1 mM) (CAMP), dexamethasone (1 p ~ ) or BWAMP, theophylline and insulin (50 nM) (CAMP + insulin). The mRNA start site for PEPCK (left panel) or PEPCK-TK (r%ht panel) was determined by SI nuclease mapping with 5‘ 32P end-labeled probes, as shown in the inset a t the bottom. This fragment also maps the start site of transcription of PEPCK-TK mRNA. However, the level of this mRNA is only 5-10% that of PEPCK mRNA (data not shown) and does not significantly alter the amount of mRNA detected with PEPCK probes. For mapping the start site of PEPCK mRNA, the PEPCK gene was labeled at the BgDI site in the 5’ untranslated region of the PEPCK gene. PEPCK mRNA protected a 73-nucleotide fragment. For mapping the start site of the PEPCK-TK gene, the chimeric gene was labeled a t an AvaI site 300 bp 3’ to the BgAI site where the chimeric gene was made. PEPCK-TK protected a 373-nucleotide fragment, comprised of 300 nucleotides which were homologous to the TK gene and 73 nucleotides which were homologous to the PEPCK gene.

(Table I). This agrees with previous work using whole animals (20) or cells in culture (21,22). The levels of TK mRNA were insensitive to hormonal treatment in cells transfected with the intact TK gene in pOPF (Table I). In cells transfected with pPCTK-6A, the chimeric PEPCK-TK gene was regulated by BbcAMP and dexamethasone, but not by insulin. Glucocorticoids caused a 4-5-fold increase in PEPCK-TK mRNA in uncloned cells, whereas Bt,cAMP, caused a 5-6- fold induction in specific mRNA (Table I), in agreement with previous studies. In clonal cell lines, the PEPCK-TK gene was also sensitive to cAMP and glucocorticoids, whether the clone contained two copies of the gene as in PTK-A7A or greater than 30 copies of the gene as in PTK-A6B (Table I). However, insulin added together with Bt2cAMP did not block the increase in PEPCK-TK mRNA normally mediated by the cyclic nucleotide (Table I). Pretreatment of these cells with insulin for 4 h had no effect on the Bt2cAMP stimulation of

PEPCK-TK mRNA levels (data not shown). The simplest explanation of these data is that the 621-bp promoter-regulatory region at the 5’ end of the PEPCK gene contains both cAMP and glucocorticoid regulatory elements, but not an insulin regulatory element.

Effects of Enhancer Elements on TK and PEPCK-TK-In order to determine if the SV40 enhancer elements altered the expression and regulation of either the TK or PEPCK-TK genes, we measured the levels of TK or PEPCK-TK mRNA in pooled cultures of cells transfected with pOPF, pOPF(-SV40), pPCTK-6A, or pPCTK-GA(-SV40) (Fig. 1). In the absence of hormonal stimulation the amount of PEPCK-TK mRNA was similar in cells transfected with either plasmid (Fig. 3). The largest difference in basal levels of expression was a 50% increase when the SV40 enhancer was linked to the PEPCK-TK gene. In this cell line, there were a significant number of HAT-resistant colonies which were TK-positive revertants of FTO-2B cells that could ac- count for the slight difference in expression. In all other cell lines transfected with either plasmid the levels of PEPCK- TK mRNA were nearly identical. Also, the amount of the TK mRNA was comparable in cells transfected with either pOPF or pOPF(-SV40) (Fig. 3). The PEPCK-TK gene was induced by 5-6-fold by BbcAMP and about 4-fold by glucocorticoids in cells transfected with pPCTK-GA(-SV40) (Fig. 3), which is virtually identical to the level of induction of the chimeric gene in cells transfected with pPCTK-6A (see Table I). These results demonstrate that the SV40 enhancer element has little effect on the expression and regulation of linked PEPCK-TK genes.

The Effect of Position and Orientation of the Hormone Regulatory Elements of PEPCK on the TK Gene Containing Its Own Promoter-Fragments of the PEPCK promoter-regulatory region which contain sequences responsive to cAMP and dexamethasone were placed a t -650 or -109 from the 5’ end, or approximately +MOO, near the 3‘ end of the TK gene (Fig. 1, C and D). These fragments of the PEPCK gene were most effective at conferring hormonal responsiveness to the TK gene (23). RNA was isolated from hormonally treated, HAT-resistant colonies after transfection with these plasmids and quantitated by S1 nuclease mapping. In all plasmids tested, TK mRNA initiated at the proper start site of transcription, 50 bp 5’ to the BgZII site within the TK gene (data not shown). To minimize the variation in expression level and hormonal inducibility due to the site of integration of transfected DNA, we pooled 100-200 HAT-resistant colonies as mass cultures for RNA isolation, each of which contained multiple copies of the transfected genes. All of these cells were responsive to Bt2cAMP and dexamethasone, based on changes in the level of PEPCK mRNA transcribed from the endogenous gene (data not shown).

The Effect of Orientation at -650 of TK-The vectors pOPF-BB(+) and pOPF-BB(-) contain the BamHI-BgZII fragment in the same and opposite orientation relative to TK transcription, respectively, placed approximately 650 bp 5’ to the TK gene (Fig. 1C). BbcAMP increased the level of TK mRNA 4-5-fold in cells transfected with either pOPF-BB(+) or pOPF-BB(-) (Fig. 4). Treatment of these cells with dexamethasone caused about a 4-fold increase in TK mRNA, regardless of the orientation of the BamHI-BgZII fragment. TK-specific mRNA from cells transfected with pOPF was not altered by hormonal treatment.

The Effect of Orientation at -109 of TK-The hormonal regulation of the TK gene in pOPF-BB(+) and pOPF-BB(-) suggested that the hormonal regulatory elements in the PEPCK gene acted transcriptionally, since no PEPCK se-

Multiple Hormone Regulatory Elements of the PEPCK Gene TABLE I

Analysis of hormone regulatory elements present at the 5‘ end of the PEPCK gene: qwntitation of PEPCK, TK, and PEPCK-TK mRNA

FTO-2B cells were treated for 4 h with 0.5 mM Bt2cAMP plus theophylline (1 mM) (Bt,cAMP), Bt,cAMP plus theophylline and 50 nM insulin (Bt,cAMP + insulin), or with 1 p M dexamethasone (Dex). The levels of specific mRNA were determined by densitometrically scanning autoradiographs of S1 nuclease mapped RNA using procedures outlined under “Experimental Procedures.” Fold induction was calculated by dividing the level of specific mRNA in hormonally treated cells by the level of mRNA in control cells. The numbers presented are the averages of at least two separate S1 nuclease mapping experiments. TK-3B and TK-4A are clonal cell lines derived from cells transfected with pOPF. PTK-A7A and PTK-A6B are clonal cell lines derived from cells transfected

9717

with pPCTK-6A (see ref. 8 for details). PEPCK mRNA

Cell line

TK-1 TK-3B TK-4A PTK-1 PTK-2 PTK-3 PTK-A7A PTK-A6B

Transfected Plasmid

-

pOPF pOPF pOPF pPCTK-6A pPCTK-6A pPCTK-6A pPCTK-6A pPCTK-6A

Bt2cAMP :%::,’ -fold induction

7.6 1.0 7.9 1.9 6.0 1.0 5.0 1.1 6.2 0.7 9.9 1.6 5.2 1.5 6.5 1.0

Dex __

4.0 3.5 2.9 3.2 2.9 3.0 2.7 3.7

pOPF pOPF pPCTK-6A pPCTK-6A (- S V 4 0 1 (-SV40)

FIG. 3. The effect of the SV40 enhancer sequences on the expression of TK or PEPCK-TK genes. Cells were transfected with pOPF, pOPF(-SV40), pPCTK-6A, or pPCTK-GA(-SV40). HAT-resistant cells were pooled for each plasmid. RNA was isolated from cells after a 4-h treatment with serum-free medium (filled bars), Bt2cAMP (0.5 mM) and theophylline (1 mM) (open bars), or dexamethasone (1 PM) (hatched bars). S1 nuclease mapping was used to determine the levels of TK mRNA (in cells transfected with pOPF or pOPF(-SV40)) or PEPCK-TK mRNA (in cells transfected with pPCTK-6A or pPCTK-GA(-SV40)). Autoradiographs were scanned densitometrically, and for each treatment the mean & S.E. was determined.

quences were included in the TK mRNA. However, the BarnHI-BglII fragment of the PEPCK gene does contain the PEPCK promoter and start site of transcription, in addition to the hormonal regulatory elements. Therefore, we tested the hormonal responsiveness of a smaller fragment of the PEPCK promoter-regulatory region, from -416 to -61, which did not contain the PEPCK transcriptional start site or TATA box.

TK mRNA PEPCK-TK mRNA

-fold induction -fold induction 1.0 1.0 1.0 0.8 0.8 0.8 1.0 1.0 1.1

6.4 5.6 4.7 5.2 5.7 4.3 5.3 6.5 4.5 4.2 4.4 2.7 5.8 6.2 5.0

This fragment of DNA was ligated to the TK gene at -109, in either orientation, to give pTK-109-PP(+) and pTK-109- PP(-) (Fig. 10).

The amount of TK mRNA was increased by treatment with both Bt2cAMP or dexamethasone in cells transfected with either pTK-109-PP(+) or pTK-109-PP(-) (Fig. 4). Bt2cAMP caused an approximate 4-fold increase in TK mRNA in cells transfected with pTK-lOg-PP(+), and a 3.6-fold increase in cells transfected with pTK-109-PP(-). Glucocorticoid treatment induced TK-specific mRNA 3-fold in cells transfected with either plasmid. TK-specific mRNA was unaltered by hormonal treatment in cells transfected with pTK-109.

The Effect of Orientation a t the 3’ End of TK-The PuuII- PuuII fragment of the PEPCK gene (-416 to -61) was inserted in both orientations relative to transcription, into a unique Hind111 site located 3’ to the TK gene in pTK-109 to give pTK-109 3’-PP(+) and pTK-109 3’-PP(-) (Fig. 1D). This places the PuuII-PuuII fragment of the PEPCK gene approximately 1.8 kb 3’ to the TK promoter or 4.8 kb 5’ to TK.

The levels of TK mRNA were increased approximately 2- fold by either Bt2cAMP or dexamethasone treatment in cells transfected with pTK-109 3’-PP(+) or pTK-109 3’-PP(-) (Fig. 4). Although the extent of induction of TK mRNA by these hormones was less than noted for cells transfected with constructs containing the PEPCK hormone response elements 5’ to the TK gene, the TK genes in pTK-109 3’-PP(+) and pTK-109 3’-PP(-) were hormonally responsive. Since the TK gene in cells transfected with pTK-109 was insensitive to hormonal stimulation, placing the PuuII-PuuII fragment of PEPCK promoter-regulatory region 3’ to the TK gene conferred hormonal responsiveness to the TK gene. Thus, the hormone regulatory elements at the 5’ end of the PEPCK gene are active from a variety of locations relative to a heterologous promoter.

DISCUSSION

The Hormonal Regulation of the PEPCK Gene-Both Bt2cAMP and glucocorticoid regulatory elements are located in the 5’ flanking region of the gene for PEPCK, within a 350-bp fragment. These elements retained their activity when linked to the TK gene containing its own promoter. The effects of the hormone regulatory elements on T K were

9718 Multiple Hormone Regulatory Elements of the PEPCK Gene

Relotive Position in TK Gene Relotive Densitometric Units

900 600 300 +I 300 I800 +2400 1 2 3 4 5

pOPF

I

-650 + 50 pOPFBB(+)

pOPFBB(-)

pTK-109

pTK-109-PP(+)

+-0 w

-109 -50

- 4 1 6 ! + 5 0

-61%+50 pTK-I09-PP(-)

"661 - pTK-109 3'PP(+)

pTK-109 3'PP(-l +50 +l8&%?416

FIG. 4. Effect of the PEPCK promoter-regulatory region on hormonal control of the intact TK gene. FTO-2B cells were transfected with the plasmids shown in the left-hand side of the figure. HAT-resistant cells were pooled for each plasmid. RNA was isolated from cells after a 4-h treatment with serum-free medium (filled bars), or medium containing Bt,cAMP (0.5 mM) and theophylline (1 mM) (open bars), or dexamethasone (1 PM) (hatched bars). S1 nuclease mapping was used to determine the levels of mRNA initiating from the TK start site. Autoradiographs were scanned densitometrically, and the values were expressed as densitometric units for a specific hormonal treatment divided by the densitometric units for RNA from control cells. A representative experiment is shown. It was repeated at least twice for the RNA samples shown, and confirmed in at least two separate mass cultures of transfected cells.

independent of orientation and distance when placed 5' to the gene and were also active 3' t o TK in either orientation. In the accompanying paper (23), the sequences in the PEPCK gene required for the regulation of the PEPCK gene by cAMP and glucocorticoids were further defined.

The effects of cAMP and glucocorticoids on the transcription rate of the gene for PEPCK have been demonstrated using isolated hepatic nuclei from intact animals (3) and from cells in culture (4). However, cAMP or glucocorticoids may also alter PEPCK gene expression at a post-transcriptional step, as previously suggested (24, 25). The responsiveness of the chimeric plasmid pTK-109-PP(+) and pTK-109-PP(-) to both Bt2cAMP and glucocorticoids demonstrates that these hormones act transcriptionally in these plasmids, since no PEPCK sequences are represented in T K mRNA. This is an important consideration, since the mouse methallothionein I gene is regulated post-transcriptionally by glucocorticoids when transfected into HeLa and L cells (26).

Since insulin treatment blocks the CAMP- or glucocorticoid-mediated increase in transcription of PEPCK (4), it is interesting that sequences responsive to insulin do not appear to be located within 2.1 kb of 5' flanking sequence of the PEPCK gene: while the cAMP and glucocorticoid regulatory elements are contained within 400 bp of the start site of transcription. The insulin regulatory element could be either further than 2.1 kb 5' to the gene or within the gene and further than 73 bp 3' to the start site of transcription. It is unlikely that the insulin regulatory element is further upstream since there is a transcriptionally active region at this part of the genome with an unknown function, which is not regulated by in~u l in .~ The PEPCK gene may contain intra- genic sequences involved in the regulation of transcription by insulin, as was shown in the genes for immunoglobulins (27- 30), chicken T K (31), and &globin (32, 33). Alternatively, we may not have found the optional conditions for insulin regu-

E. A. Park, A. Wynshaw-Boris, and R. W. Hanson, unpublished observation.

D. S. Loose, H. Meisner, J. Cook, and R. W. Hanson, unpublished data.

lation of the transfected chimeric gene. The cAMP and glucocorticoid regulatory elements from the

PEPCK gene can also regulate a heterologous promoter. There are 109 bp of 5' flanking sequences in pTK-109, in- -cluding all of the elements required for maximal T K mRNA production in frog oocytes (11). pOPF contains about 650 bp of 5' flanking sequences including sequences upstream of -109 which may be part of the TK promoter. The CAMP and glucocorticoid regulatory elements of PEPCK were effective when linked to either of these intact promoters, suggesting that the hormone regulatory elements were independent of T K promoter functions in these vectors. In support of this, the addition of PEPCK sequences to pOPF or pTK-109 in the plasmids described in this paper had no effect on transfection efficiency and the presence of PEPCK sequences had little effect on basal T K mRNA levels in transfected cells (data not shown). It is possible that these hormone regulatory elements could stimulate T K gene expression more effectively if they were inserted in place of the proper promoter elements in the TK gene. This positional enhancement of inducible response elements was recently demonstrated with the inter- feron genes (34).

Hormonal Regulatory Elements and Enhancer Elements- Like the PEPCK gene, other inducible genes have been shown to contain regulatory elements which could be placed in a number of locations relative to a gene and retain their activity. The glucocorticoid regulatory elements in the long terminal repeat of mouse mammary tumor virus were the first regulatory elements shown to be active on a heterologous gene from a variety of positions (1,35). More recently, similar properties were demonstrated for the inducible element in the fi-inter- feron gene (34), the regulatory elements in the human methallothionein IIA gene (36) and mouse methallothionein I gene (37, 38). Positional flexibility is also a property of viral enhancer elements which can markedly affect the expression of linked genes from a variety of locations (39, 40; for a review, see Ref. 41). This common property of positional flexibility has been used to imply a similar mechanism of action between regulatory elements of inducible genes and enhancer elements (1 ,34,35) .


Hormone regulatory elements and viral enhancer elements have other properties in common. The activity of these elements depends upon the binding of trans-acting factors to DNA sequences. Factors which bind enhancer elements have been identified by competition experiments (42). They are constitutively active and do not appear to depend upon specific cellular treatments. Hormonal regulation of gene expression also involves the binding of specific factors to hormonal regulatory elements in target genes (1, e), but these factors are active only in the presence of hormones. This model is based upon the regulation of gene expression by steroid hormones, and may also extend to peptide hormones. Recently, Yamamoto and co-workers (43) found that the Moloney murine leukemia virus iong terminal repeat contains both enhancer elements and glucocorticoid regulatory elements, which have additive effects on linked chloramphenicol ace- tyltransferase genes after transient expression. It is possible, then, that these elements share a common mechanism of action to modulate gene transcription.

Evidence presented in this paper suggests, however, that hormone regulatory elements and viral enhancers may modulate gene transcription by different mechanisms. The SV40 enhancer element had no effect on the expression or regulation of linked PEPCK-TK genes, which contains hormone response elements. If both elements acted via the same mechanism, the basal level of PEPCK-TK gene expression should be higher in cells transfected with genes linked to the enhancer, since it is constitutively active. The factors which interact with the SV40 enhancer are present in FTO-2B cells, since the transfection efficiency of PEPCK-TK or TK genes linked to the SV40 enhancer is 10-fold greater than the transfection efficiency of unlinked genes in these Sim- ilar results were found with T K genes (44) or Harvey sarcoma virus ras genes (45) when stably transfected into cells. These experiments seem contradictory to those using the Moloney murine leukemia virus long terminal repeat (43). It is possible that differences were observed because the PEPCK, TK, or ras genes were stably transfected into cells, while the long terminal repeat activities were assayed after transient expression. Enhancer elements frequently increase gene expression transiently (39, 40, 46-48). Conversely, since the Moloney murine leukemia virus long terminal repeat is a complete unit, the enhancer element and hormone response element could be in the proper position necessary for interaction, while the artificially constructed linkage of PEPCK sequences to SV40 sequences may not be optimal for interaction.

Positional flexibility is a property which is common to a variety of DNA sequence elements which affect a diverse range of genetic events, in species from bacteria to humans. In bacteria, for instance, the inversion of DNA sequences by site-specific recombination is stimulated by elements which act in an orientation- and distance-independent manner (49, 50). Silencer sequences in yeast decrease the expression of linked genes from a variety of locations (51). Enhancer elements have also been identified in specific cellular genes which appear to play some role in tissue-specific gene expression (27-30). Regulatory elements may simply bind trans- acting factors bi-directionally and from a variety of locations relative to the processes which they affect, but the result of that binding is different for different element-factor combi- nations. We are presently examining the relationship between hormone-response elements and viral enhancers so that we may better understand their mechanisms of action.

A. Wynshaw-Boris, D. S. Loose, and R. W. Hanson, unpublished data.

Acknowledgments-We wish to thank Drs. Yaacov Hod, Alan Goodridge, David Samols, and John Nilson for their useful comments on the manuscript. We also acknowledge the excellent technical assistance of Heidi Short.

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