Vol.

187,

No.

September

2, 1992

BIOCHEMICAL

BIOPHYSICAL

AND

RESEARCH

COMMUNICATIONS Pages

16, 1992

778-782

INSULIN-RESPONSIVE TYROSINE AMINOTRANSFERASE TRANSCRIPTION REQUIRES MULTIPLE PROMOTER REGIONS Douglas ‘Program

Carmichael’

and John

in Cell, Molecular

‘Department

Received

July

SUMMARY: regions

This

which

encoding

study

mediate

parts

of two

transient

basal

and insulin-sensitive

regions:

requires

expression.

Thus,

components

required

suggest

the insulin

transcription

Tennessee

used

of Tennessee,

37996

transfection

aminotransferase

sensitivity

that

University

Biology,

1992

tyrosine

at least Insulin

27,

and Developmental

of Biochemistry, Knoxville,

Koontz’*’

a region

cannot

for basal

at -3600

Basal

and a region

from

the gene requires

-208

to +62.

not required

results

for basal

of any of the from

this

and glucocorticoid-induced

of the proximal

the DNA

expression

modification

Previous

on basal regions

from

by direct

transcription.

different

transcription

of the promoter

act solely

effects

to determine

EC 2.6.1.5).

at least one region

insulin

require

(TAT;

analysis

laboratory

TAT Q 1992 Academic

promoter.

Press, Inc.

Cis-acting including

insulin-responsive

regions

phosphoenolpyruvate

phosphate crystallin

dehydrogenase (4). amylase

concensus Insulin hepatocytes

inhibits

carboxykinase (GAPDH)

(2). human

(51, haptoglobin

insulin-responsive

have

sequence

transcription

(8) and in a hepatoma

been

found

in a variety

(I),

glyceraldehyde-3-

(PEPCK) growth

hormone

(6) and c-fos

(7).

has yet been

found

of the TAT

gene

cell line (9.10).

(3), 61-

However,

in primary

of genes,

no

in those

regions

cultures

In this study,

of fetal

we

The abbreviations used are: TAT, tyrosine aminotransferase; PEPCK, phosphoenolpyruvate carboxykinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CAT, choramphenicol acetyl transferase; HSV TK, Herpes simplex virus thymidine kinase; DME, Dulbecco’s Modified Eagle’s medium; DEAE, diethyl aminoethyl; bp, base pairs; BE, basal element; GRE, glucocorticoid responsive element. 0006-291X/9? Copyright All rights

$4.00 0 1992 by Academic Press. of reproduction in any form

Inc. reserved.

778

Vol.

187,

transiently in control

No.

2, 1992

BIOCHEMICAL

transfected

that

cell line with

of the chloramphenicol

insulin-responsive

CAT

AND

BIOPHYSICAL

plasmids

bearing

acetyltransferase

expression

requires

RESEARCH

mutant

(CAT)

multiple

gene.

COMMUNICATIONS

TAT

promoters

We report

cis-acting

promoter

that

regions.

METHODS PLASMIDS: (See fig.1). pBLCAT2, pTC3.92, pTC3.92A and pBETK were generously provided by G. Schutz; these latter three are identical to pTATCAT-3922/ + 62, pTATCAT-3922A-3660/-3543 and TTCI 24 as described in (1 I). pBLCAT2 contains the Herpes simplex virus thymidine kinase (HSV TK) promoter region from -105 to + 52 in control of the CAT gene. pBE.208 and pTC3.67A were constructed by standard cloning methods (12) using available restriction sites. CELL CULTURE AND TRANSIENT TRANSFECTIONS: KRC-7 rat hepatoma cells (9.10) were grown in Dulbecco’s Modified Eagle’s medium (DME) + 10% iron-supplemented bovine calf serum at 37°C and 5% CO,. Transfections were performed using a modified diethyl aminoethyl-dextran (DEAE-dextran) technique (13). Briefly, rapidly growing cells were removed from dishes with trypsin and transfected for 25 minutes at room temperature in suspension with 0.5 mg/ml DEAE-dextran at a total DNA concentration of 25 ug/ml. After transfection, cells were aliquoted into IOOmm dishes with DME + 5% serum, allowed to attach for 6 hours, then shocked with 20% dimethyl sulfoxide in DME at room temperature for 5 minutes. Cells were washed and fed with DME + 10% serum for 24 hours and withdrawn from serum for 24 hours before 12 hour treatments with 10 nM insulin. Cells were harvested at the beginning and end of the treatment period by scraping into Tris-buffered saline (25 mM Tris-HCI pH 7.4, 137 mM NaCI, 5 mM KCI, 0.7mM CaCI,, 0.5 mM MgCI,, and 0.6 mM Na,HP04.7H20). CAT ASSAYS: Cell extracts were prepared in 250 mM Tris-HCI, 5 mM EDTA and 0.5% Triton X-100, then heated to 60°C for 5 minutes to inactivate endogenous CAT inhibitors (1). CAT assays were performed as described in (14) and were normalized to protein as determined by Bradford assays (15). BE

TAT

p

CAT ----w--

N3l

1 I

I



p

I

I

CAT m-------m--m



Promoter the

start

pTC3.67A

I

g+ _......_. ____. . . . . ..... . . . . . . ... . ......... ..... ...TKK&2q Fiaure 1. represents

pTC3.92

I

I

reaions site

of the plasmids of transcription.

779

used

in this

pBETK study.

The

arrow

Vol.

187,

No.

BIOCHEMICAL

2, 1992

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Mean basal CAT activities for pTC3.92 and pBETK at the end of the treatment period were 300 pM/mg/min. and 2700 pM/mg/min., respectively. Percent insulin inhibition for each plasmid was calculated by comparing the change in CAT activity seen in insulin-treated cells during the treatment period with the change seen in untreated cells. RESULTS

AND

Basal -3543,

DISCUSSION

activity

relative

significant pBETK promoter, fig.3).

activity

fig.2).

basal Point

levels

to the start

basal basal,

equal

element

in this from

Together, is sufficient

these

when

Insulin

the 12 hour

data

to that

off assays

(9,101.

show

upstream basal

that

decrease

period

reported

in the TAT

TAT

basal

in

expression

this

117bp

promoter

to basal

exhibits

(See fig.3).

expression,

and

TAT

promoter.

CAT

from

pTC3.92

The

TAT

for basal

of the proximal expression magnitude

gene

of this

by Northern

is retained

effect

is

and nuclear

in pTC3.67A.

which

rh

160.

loo-

Y

60.

5

60. 40.

I’

4 0

T

/

,



I

/

I

I

nRFTY

nRI CAT9 0

I

Basal

0

IOnM

insulin

Figure 2. The HSV TK oromoter does not substitute for the TAT Dromoter regions required for insulin’s effect. The fold change in CAT expression from pBETK produced by insulin is identical to the insulin effect seen on pBLCAT2. Basal means were determined from each replicate’s value relative to pBLCAT2’s basal mean in that experiment. Insulin-treated means and standard deviations were calculated from each replicate’s value relative to that plasmid’s basal mean in that experiment. Shown are the results from 3 representative experiments, n = 9. 780

run-

has a

-

160.

2*

(See

in fig.3).

200

t

cells

a 3.92kbp

to reduce

of -208

in basal

Insulin-responsiveness

from

in which

to

confers in KRC-7

A plasmid

208bp

seen on the endogenous

-3660

and pTC3.92A

the BE is required

than

region

is deleted

pTC3.92

been

(I 1).

to no more

a > 50%

treatment

equivalent

have

pTC3.92n

This

spanning

HSV TK promoter

(Compare

region

region

(1 I).

this region

(See pBE.208

linked

causes

when

immediately

activity

a 117bp

site of transcription

is abolished

mutations to those

requires

to the heterologous

activity

basal

gene

In addition,

(BE) is placed

significant

over

of the TAT

Vol. 187, No. 2, 1992

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2.5,

pTC3.92 0

pTC3.67A basal

I.206

0

pTC3.92A

1 OnM insulin

Fiaure 3. Insulin inhibition of basal TAT transcrintion reouires more than 208bn of the oroximal oromoter. in addition to the basal element. Basal and insulin-treated values are significantly different by the Student’s T test at the 0.05 alpha level of confidence for both pTC3.92 and pTC3.67A. Basal means were determined from each replicate’s value relative to pTC3.92’s basal mean in that experiment. Insulintreated means and standard deviations were calculated from each replicate’s value

relative to that plasmid’s basal mean in that experiment. from 3 representative experiments, n = 9.

large

internal

inhibit

CAT

deletion expression data

These

sufficient

from from

show

to mediate

separate

regions

-2950

that

to -800

pTC3.92A

the basal

element

effect.

At least

insulin’s

to -208

of the critical

(See fig.3).

pBE.208,

Those

is also required.

and -800

function

-2950

(Compare part(s)

are the results

However,

or pBETK

insulin

(See

figs.

may be required, a part

regions

-3670

regions

of three

in figs.1

is not replaced

2).

but is not

to -3660,

and pBE.208

fails to 3 and

of one or more

span

pTC3.67A

of those

Shown

-3543

to

and 3).

The

by the HSV TK

promoter. These distinct,

findings

are similar

widely-separated

to those

regions

were

for PEPCK found

(1) and GAPDH

to be required

(2), in which

for the insulin

response. These

data

modification would

Previous

during

induction

-52 and -111 element

glucocorticoid-induced

from

of TAT

TAT

Thus,

transcription

transcription

effects require

solely

transcription

suggest requires

promoter

781

acts

expression (16)

insulin’s

insulin

basal

basal

this laboratory

of the proximal

(GRE).

that

of the minimal

the significant

findings

of glucocorticoid

responsive

the possibility

of any component

be present

between

also negate

in addition on both regions

by direct apparatus,

from

pBE.208.

that

insulin’s

a cis-acting

which

inhibition region

to the glucocorticoid basal

and

of the proximal

promoter

Vol.

187,

No.

in addition required

BIOCHEMICAL

2, 1992

AND

to the BE and GRE, respectively. apparently

do not overlap

RESEARCH COMMUNICATIONS

BIOPHYSICAL

However,

for the two

the proximal

different

insulin

ACKNOWLEDGMENTS This work was supported institutional support grant BRSG RR-07088. Schutz for plasmids used in this study.

regions

effects.

by NIH 30512 and We wish to thank

an G.

REFERENCES 1.

Forest, (1990)

C.D., O’Brien, R.M., Lucas, MO/. Endo. 4:1302-1310.

P.C.,

2.

Alexander-Bridges, Cell. Biochem.

3.

Prager, 1685.

4.

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5.

Keller, Genes

6.

Campos,

7.

Stumpo, D., Stewart, 263:1611-1614.

8.

Cake, Bioch.

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Moore,

P.S. and Koontz,

J.W.

(1989)

Arch.

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Moore,

P.S. and Koontz,

J.W.

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Boshart, M., Weih, 61:905-916.

12.

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Banerji,

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Seed,

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Bradford,

M. (1976)

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Cheatham, Press.

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D., Gebremedhin,

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S., Rosenberg, and Development

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T., Howard,

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T., Gilman,

M.H., Ho, K.K., 182:429-435.

Shelly,

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J., Borras,

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Meisler,

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E. and Yeoh,

J. (1988) Anal.

Carmichael,

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Biochem. D.,

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M. (1990)

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Eur. J.

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3:1724-1732.

R.E.K.

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12:1789-7797.

and Schutz,

J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Manua/, Cold Spring Harbor Laboratory Press. L. and Schaffner,

D.K.

87:3353-3357.

G. and

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L., Milward,

and Granner,

G. (1990)

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67:271-277. 72:248-254.

Peterson,

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Pan,

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In

Insulin-responsive tyrosine aminotransferase transcription requires multiple promoter regions.

This study used transient transfection analysis to determine the DNA regions which mediate basal and insulin-sensitive transcription from the gene enc...
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