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.
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