BIOCHEMICAL

Vol. 175, No. 3, 1991

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 866-871

March 29, 1991

TWO &-ACTING

REGULATORY SEQUENCES IN THE PEROXISOME PROLIFERATOR-

RESPONSIVE ENHANCER REGION OF RAT ACYL-CoA Takashi Department

Osumil,

Jin-Kun

Wenz,

Shinshu

of Biochemistry,

Received

February

and Takashi

University

Nagano

390,

OXIDASE

GENE

Hashimoto

School

of Medicine,

Matsumoto,

Japan

7, 1991

To clarify the mechanism of transcriptional induction of the rat liver acylCoA oxidase gene by hypolipidemic agents, we searched for &s-acting regulatory sequences in the 5’ upstream region of the gene by transfection studies. We found that the sequence between -639 and -472 acts as a peroxisome proliferator-responsive, tissue-specific enhancer. Footprint analysis revealed two protein binding sites in this region. One of these sequences exhibited a positive, whereas the other a negative, regulatory activity in transcription assays. 0 1991 Academic Press, Inc.

Rat

liver

peroxisomes

peroxisomal

matrix

administration highly

organized

oxidation

enzymes

of gene

transcription

the

in

the

present

acyl-CoA

oxidase

transfection

induced

event. The (10).

the

of

the

initial

enzyme

1 To whom all

correspondence

Copyright AU rights

of

the p-

at the

characteristics

the

in this

peroxisomal

to occur

P-oxidation

level

were genes of the

B-oxidation

also

(9).

In

gene

of

system,

by

H4IIEC3, found to should

AND METHODS was be

used for susceptible

address: Institute of Hebei, People’s Republic AOX, acyl-CoA (EC 2.3.1.28);

transfection studies, to the induction by a

Faculty of Science, Hyogo 671-22, Japan.

Basic Medicine, of China 050017. oxidase (EC 1.3.3.6); kb, kilobase(

$1.50

0 1991 by Academic Press. Inc. of reproduction in any form reserved.

the

be addressed.

address: Department of Life Science, 2167 Shosha, Himeji, of Technology,

Abbreviations: acetyltransferase 0006-291X/91

intimately

mechanism

of the

DNA sequences of

by the

involved

molecular

the

analysis.

Reuber rat hepatoma, because this strain was

z Present zhuang,

inducible

a-regulatory

MATERIALS

Present Institute

the

common sequence

for

An

no doubt

was demonstrated

Several regions

is

in

fashion

(l-3).

cDNAs and genes

induction

we searched (AOX),

genes

located

parallel

agents

To elucidate the

enzymes

in a closely

corresponding

and sequenced

5’ upstream

fl -oxidation

hypolipidemic

of the

(4-9).

study,

three

markedly

cellular

we cloned

the

various

regulation

induction,

noted

are of

coordinated

and

866

Hebei

Province,

Hiaeji Shi

CAT, chloramphenicoi

jia

Vol.

175,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

peroxisome proliferator, ciprofibrate, in cultures (11). Throughout the present study, we used the bacterial chloramphenicol acetyltransferase (CAT) A promoterless starting plasmid, pTZSVOcat, and SV40 gene as a reporter. enhancer/promoter-containing plasmid, pTZSV2cat, were constructed by inserting the CAT structural gene and the transcriptional regulatory regions of pSVOcat and pSV2cat (12), respectively, in the polylinker region of pTZ18U. An AOX gene upstream sequence from positions -4.3 kb to 34, the latter being located between the major cap site and the initiation codon (6), was inserted in front of the CAT structural gene of pTZSVOcat, yielding the “-4.3k” construct. Other plasmids containing various regions of the AOX gene upstream sequence restriction enzymes and/or sequential were generated by digestion with A neomycin resistance-conferring plasmid, deletion with exonuclease III. pSTneoB, was provided by Y. Kitagawa, with the permission of H. Kondo. A lawas supplied by G. MacGregor. galactosidase expression vector, pCMVp (13), To improve the efficiency of gene transfection to H4IIEC3 cells, we employed a modified calcium phosphate method, as follows. The cells were recovered from confluent cultures by trypsinization, and suspended in Dulbecco’s Modified Eagle’s Medium containing 10% fetal bovine serum at a density of ca. 4 x 106 cells/ml. The cell suspension (1.0 ml) was mixed with 1.0 ml of suspension of DNA/calcium phosphate precipitates containing 20-24 fig of closed circular plasmid DNA. The cell/DNA mixture was divided into two equal parts, and each part was added to 2 ml of the serum-containing medium in a 6-cm tissue culture dish. The cells were incubated for 6 hr under 5% COz at 37°C in the presence of 80 yM chloroquine. After removal of the precipitates and washing twice with 3 ml each of the serum-free medium, the cells were cultured for three days with or without 0.5 mM ciprofibrate, as described In most cases, pCMVfl was cotransfected as an internal standard for the (11). efficiency of transfection. we used a mixture of 10 u g of the For stable transfection experiments, After plasmid to be tested and 2 fi g of pSTneoB for each 6-cm dish. selection was performed for 12 days in the transfection as described above, Transformants obtained in each dish (usually 50presence of 800 ,ugg/ml G418. 200 colonies) were used for the induction studies (ll), without cloning. Gorman (12). The react ion CAT assay was performed as described by conditions were adjusted so that the chloramphenicol acetylation did not exceed 50%, to assure linearity of the reaction. The results were normalized when pCMVP was used as an on the basis of the amount of protein, and, internal standard, further normalization was performed on the basis of /? galactosidase specific activities. Averages of relative CAT activities obtained in at least two independent series of experiment are shown. RESULTS AND DISCUSSION We first of

sequences

site

tried

(Fig.

(-4.3k), kb and

the

1A).

to

AOX gene

within

The

construct

containing

kb all

decrease

in

the

exhibited

2-

to 3-fold

resulting

in

A-12sl-20

had

the

expression

an

no activity sequence

at an uninduced

the 5’ most

levels

whole

as the in

irrespective state,

-472

whereas 867

sequence

sequence

the

induction

between

upstream

and -129 that

region, moderate

progressed.

Remarkably, presence of the

cap

up to -2.5

though

deletion

the

this

was deleted

ratio. of

of

of

of CAT expression,

only high

regulatory

region

by ciprofibrate.

activity

the

transcriptional

4.3-kb

observed

induction

unexpectedly

almost Thus,

were

the

the

the

high

activities

a significant

inducer.

in which

supported

exhibited

the

locate

rat

and two mutants -1.3

roughly

On is

between

A-412/-129 the inducer,

the

presence

other or

essential -128

They

hand,

absence only

and -20

of for

seems

Vol.

BIOCHEMICAL

175, No. 3, 1991

A

Cm

-4.3k

AND BIOPHYSICAL 6

RelAct

AcCm

RESEARCH COMMUNICATIONS

Ret Act. 1.0

2.6

-111111

8.1

7.6 1.7

-2.5k

1.5

-623

4.6

12.1

1.0 -1.3k

-2.5kA4n,-,2g

-

-2.5kA-,26,-20

.

-

1.2

-596

3.3

7.1

0.3

2.0

-576

3.5 0.1

’

11.4 1.0

-530

0.3

1.9

C

Rel.Act.

-1.3kA-an/-m

C I

-

A-516/-129,

[

2

A-sw-129,

[

i”

4-666/-129,

[

:

A-n6/-129,

[

:

Fig. 1. Transfection studies with the constructs containing various AOX gene upstream regions linked to the CAT gene. Thick bar indicates the AOX upstream region contained in each construct. The 5’ end of the region contained is shown with negative number on the left, together with the region deleted (represented by “A m/n”, where q and n mean the start and end points of the Figures are not to scale. Results of CAT assays are shown on the deletion). The uninduced (upper lane) and induced (lower lane) activities are right. given for each construct. Cm, chloramphenicol; AcCm, acetylated chloramphenicol. CAT activities are given as average relative values, taking as 1.0 the uninduced activities of -1.3k (in _A), -639A-47z/-129 (in B), and -1.3kA-47z/-lzs (in C).

to

have

a function

copies

of

of putative

binding

Essentially except

that

the (an

shown).

We verified, the

effect

results

from

We attempted deletion

transient

experiments,

up to -640, conditions inducibility deletion These

results

correct

down

the

stable

dropped from

-473

activation reproduced

faithfully the

contains

factor,

SPl

stable for

(data

using

sequence

not

could

efficient

A

CAT

(data

the

not

transformants, of CAT

amounts

the

shown).

required the

(9).

transformants)

reflected

region

several

transformants,

essential

mapping

employing

upstream

without significant (Fig. 1B). When

were

the

positions

narrow

region

with

was

in

CAT assays

the

severely

transcriptional

the

experiments

proceeded

obtained -1.3k

by RNase protection

to

sequential

of

observed

latter

transcription

were

upstream

only

of

mRNA initiated

of the

results

sequence

The

promoter.

site

similar

expression that

basal

for

induction

the

-4i’z./-129

COnStrUCtS.

be deleted

from

by In

the

5’ side

decrease in the CAT activity under induced was further dissected, the the 5’ sequence In contrast, when the between -578 and -530. to

was in

the

5’

observed the

direction, between

experiments

marked -516

and

using

stable

decrease -559

(Fig.

in

the 1C).

transformants

Vol.

175,

No.

BIOCHEMICAL

3, 1991

I

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

D C D $nll$ Q FBBFO

-

I

a al 5

A I TCAGGACAATGAACCCTTTCccG**cGTGACCTTTGTCCT

-597

I

B -534

I

II

~*TCAAc*GTTcc*GTAAATTCCCCT*TT*GccTcTTc*cTccGcccG*

Fig. 2. DNase I footprint assay of the putative enhancer region. The -SW/-472 probe was prepared by 3’-end labeling of -647/-472 (u3AI/&I) fragment with Klenow fragment and [a -32P]dCTP, followed by digestion with &-&I at -597. The assay was carried out using liver extract prepared as described (14) from control and di(2-ethylhexyl)phthalate-induced (15) (lanes “D”) rats. After DNase I digestion, the samples were subjected to polyacrylamide gel electrophoresis to separate the protein-bound (lanes “B”) and protein-free (lanes “F”) probes, which were then analyzed by denaturing polyacrylamide gel electrophoresis. All other experimental conditions were as described (16). TOP right, footprint pattern of the whole region. Left, magnification of the upper part of the footprint obtained in two different experiments. In lane marked 2X, twice the amount of sample as in 1X was analyzed. GtA, MaxamGilbert sequencing ladder detecting guanine and adenine of the probe fragment. Bottom, nucleotide sequence of the fragment. Two protected sequences (sites A and B) are shown with square brackets. Underlined sequences marked I and II are closely similar to C3P element (17) and a half of the binding sequence for APF/HNFl/LF-Bl (18), respectively, both of which have been suggested to be involved in liver-specific gene expression. Double underlined sequence is a motif commonly found in the three inducible P-oxidation genes (9).

(data

not

seemed

to

shown). be

Possible sequence AOX

basal

orientation.

Thus,

located enhancer

(data

not

sequences

between

-578

function

of

shown).

promoter Furthermore,

when

This placed it

essential and the

region

sequence on

for

the

inducible

expression

-516. was conferred

either

similarly

5’ acted

869

examined the or

on

with

3’ SV40

the

-639/-472

inducibility side,

basal

and promoter.

on in

the

either It

was

Vol.

175, No. 3, 1991

BIOCHEMICAL

AND BJOPHYSICAL RESEARCH COMMUNICATIONS

C,,,

-636 -c

-473-126

&cm

IWAct.

1

6 ....ii.: ..

’

c

---I[

Fig. 3. Transcriptional regulatory activities of sites A and B sequences. Double-strand synthetic oligonucleotides corresponding to these sites (-578/ -553 and -543/-520, respectively) were linked to the basal AOX promoter. Experiments were carried out with stable transformants using plasmids containing the far upstream sequence (-2.5k/-1.3k). Transient experiments gave similar results. Data are given as in Fig. 1, taking the uninduced activity of -639A-a7z/lzg as 1.0.

also

active

inducible the

in

certain

expression

region

has

responsive

was only

examining

searched

the

employing

rat

One

A)

of

with

non-hepatic

H4IIEC3.

origins,

These

liver-specific,

but

results

peroxisome

(site

d-regulatory

of

nuclear

(Fig.

liver was

Indistinguishable

the

suggest

that

proliferator-

protein

2).

nuclear

elements factors

When

the

extract,

rather

weak,

results

were

in

with

first

sequence,

one

were

(site

extracts

from

by

by DNase was

regions

other the

region,

fragment

protected

the

obtained

the

-597/-472

two

whereas

to

this

I

analyzed detected.

B) was

strong.

control

and the

rats.

To

examine

the

functions

A (-578/-553),

promoter induction expression lowered

the

only

region

essential flanking

The

resulting by the

It

elements

the

CAT expression.

B has a negative

induction.

in

3).

is

in

site

noteworthy

that

the

the

minimal

induction

sequences

might

which

or both,

presence

or

absence

of

higher

when

repressing Thus, and both

these

sites,

was more

have

for

some effects. 870

severe

induction necessary

implying inducible

by

span that

on the

ratio for

when combined, the

the

basal

A, significantly

A has a positive,

are

above),

but of

B sequence

effect site

role,

site

to site

higher

basal

activated

level

The

sequence

inducer,

linked

an apparently

(see

the

the

of the

dramatically

construct).

but

in

the

upstream

to

the

sites

contained

just

sequence

A

due

requirement also

binding

plasmids

A sequence.

regulatory

for

satisfy

protein

The site

on the activity,

expression,

exhibited

test

at most about 2-fold, the result with the -639

had no effect

basal

these

B (-543/-520),

(Fig.

either

was (cf.

three

site

of the AOX gene

transcription

itself

of

we constructed

transcription, of site

the

of

for

binding

analysis

site

seen

a function

footprint

the

cells

enhancer.

We next

induced

cultured

than whereas

significant most of the

these

sequence

expression,

though

Vol.

175,

No.

The

sequences

nuclear been

3, 1991

factors shown

region

Fig.

respond motif

we were

AND

sites 2,

to

peroxisomal

peroxisome

shared

hormone

receptor

regulation

above

are

None of

by the

5’

these

a paper

that

transcription superfamily

(19).

precise

known

factors,

however, of

the

preparation

the

cloning

factor Possible

functions

contained

regions

During reported

COMMUNICATIONS

certain

Also

flanking

genes.

and the

to

proliferators.

B-oxidation of

RESEARCH

similarity

bottom).

peroxisome

aware

BIOPHYSICAL

have

proliferator-responsive

the AOX gene described

(see

to

manuscript, steroid

of these

a sequence

inducible

BIOCHEMICAL

which role

of the

of

hepatic have in

the

this

two

other

of

this

of a putative belongs this

protein-binding

to

the

protein

in sites

to be investigated.

ACKNOWLEDGMENTS This work was supported in part by Grants-in-Aid for Scientific the Ministry of Education, Science, and Culture of Japan, grants Medical Research Foundation, the Fugaku Trust for Medicinal Yamanouchi Foundation for Research on Metabolic Disorders.

Research from from the ON0 Research and

REFERENCES 1. Reddy, J.K., Warren, J.R., Reddy, M.K., and Lalwani, N.D. (1982) Ann. N. L. Acad. Sci. 386, 81-110 2. Hashimoto, T. (1982) Ann. N. Y. Acad. Sci. 386, 5-12 3. Osumi, T., and Hashimoto, T. (1984) Trends Biochem. sci. 9, 317-319 4. Osumi, T., Ishii, N., Hijikata, M., Kamijo, K., Ozasa, H., Furuta, S., Miyazawa, S., Kondo, K., Inoue, K., Kagamiyama, H., and Hashimoto, T. (1985) J- -Biol. Chem. 260, 8905-8910 5. Miyazawa, S., Hayashi, H., Hijikata, M., Ishii, N., Furuta, S., Kagamiyama, H., Osumi, T., and Hashimoto, T. (1987) J. Biol. Chem 262, d 8131-8137 6. Osumi, T., Ishii, N., Miyazawa, S., and Hashimoto, T. (1987) J. Biol. Chem. 262, 8138-8143 7. Ishii, N., Hijikata, M., Osumi, T., and Hashimoto, T. (1987) J- Biol. Chem 262, 8144-8150 8. Hijikata, M., Ishii, N., Kagamiyama, H., Osumi, T., and Hashimoto, T. (1987) J- Biol Chem 262, 8151-8158 9. Hijikata, tie=-K., Osumi, T., and Hashimoto, T. (1990) J. Biol. Chem. 265, 4600-4606 10. Reddy, J.K., Gael, S.K., Nemali, M.R., Carrino, J.J., Laffler, T.G., Reddy, M.K., Sperbeck, S.J., Osumi, T., Hashimoto, T., Lalwani, N.D., and Rao, M.S. (1986) Proc Nat1 Acad Sci . U . S . A . 83, 1747-1751 11. Osumi, T., Yokotax anda=to, T. (1990) J. Biochem. 108, 614-621 12. Gorman, C.M., Moffat, L.F., and Howard, B.H. (1982) Mol. ------c Cell Biol. 2, 1044-1051 13. Foecking, M.K., and Hofstetter, H. (1986) Gene 45, 101-105 14. Gorski, M., Carneiro, M., and Schibler, Il. (1986) m 47, 767-776 15. Osumi, T., and Hashimoto, T. (1978) J- Biochem. 83, 1361-1365 16. Brenowitz, M., Senear, D.F., and Kingston, R.E. (1989) In Current Protocols b Molecular Biology (Ausubel, F.M., Brent, R., Kingston, R., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K., Eds.), Vol. II, 12.4.1-12.4.16, John Wiley b Sons, New York 17. Leff, T., Reue, K., Melian, A., Culver, H., and Breslow, J.L. (1989) JBiol. Chem. 264, 16132-16137 18. Herbomel, P., and Yaniv, M. (1989) In Tissue Specific Gene Expression (Renkawitz, Ed.), pp. 165-184, VCH Publishers, Weinheim, Germany 19. Issemann, I., and Green, S. (1990) Nature 347, 645-650 871