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Figure 3 Effects of various kinase inhibitors on the expression of c-Myc induced by thapsigargin Wild-type MEFs were pretreated
with kinase inhibitors for 30 min and then incubated with 1 μM of Tg for 1 hr. (A) is a representative of RT-PCR analysis. (B),
Quantitative data showing the average fold change at mRNA levels after Tg treatment obtained from three independent experiments.
PD98059, ERK kinase inhibitor; U73122, phospholipase C inhibitor; KT5720, protein kinase A inhibitor; LY294002,
phosphatidylinositol 3-kinase inhibitor; SB203580, p38 MAPK inhibitor.
KO
WT
KO
KO
WT
KO
KO
WT
KO
WT
WT
KO
WT
KO
Fed
Fasted
Tubulin
?
LAP
Alb-Cre mouse liver 3mo old
Ave
WT
KO
Fasted
Fed
0.242092
0.2384
0.304234 0.133067
Lip/Lap in Alb-Cre live r 3m o old m ice
0.35
0.3
lip-lap/Tubulin
Lip/Lap/Tubulin
WT Fast
0.237405
KO Fast
0.449906
WT Fast
0.213841
KO Fast
0.166527
WT Fast
0.27503
KO Fast
0.590061
KO Fast
0.010443
WT Fed
0.267081
KO Fed
0.172589
WT Fed
0.336391
KO Fed
0.06439
WT Fed
0.111728
KO Fed
0.188072
KO Fed
0.107217
0.25
0.2
WT
0.15
KO
0.1
0.05
0
Fasted
Fed
Nutritional s tatus
Amino Acid Deprivation Induces the Transcription Rate of the
Human Asparagine Synthetase Gene through a Timed Program of
Expression and Promoter Binding of Nutrient-responsive Basic
Region/Leucine Zipper Transcription Factors as Well as Localized
Histone Acetylation*
Hong Chen‡, Yuan-Xiang Pan‡, Elizabeth E. Dudenhausen, and Michael S. Kilberg§
From the Department of Biochemistry and Molecular Biology, Genetics Institute, and Shands Cancer Center,
University of Florida College of Medicine, Gainesville, Florida 32610
Expression of human asparagine synthetase (ASNS),
which catalyzes asparagine and glutamate biosynthesis,
is transcriptionally induced following amino acid deprivation.
Previous overexpression and electrophoresis
mobility shift analysis showed the involvement of the
transcription factors ATF4, C/EBP , and ATF3-FL
through the nutrient-sensing response element-1
(NSRE-1) within the ASNS promoter. Amino acid deprivation
caused an elevated mRNA level for ATF4,
C/EBP , and ATF3-FL, and the present study established
that the nuclear protein content for ATF4 and
ATF3-FL were increased during amino acid limitation,
whereas C/EBP -LIP declined slightly. The total
amount of C/EBP -LAP protein was unchanged, but
changes in the distribution among multiple C/EBP LAP forms were observed. Overexpression studies established
that ATF4, ATF3-FL, and C/EBP -LAP could
coordinately modulate the transcription from the human
ASNS promoter. Chromatin immunoprecipitation
demonstrated that amino acid deprivation increased
ATF3-FL, ATF4, and C/EBP binding to the ASNS promoter
and enhanced promoter association of RNA polymerase
II, TATA-binding protein, and TFIIB of the
general transcription machinery. A time course revealed
a markedly different temporal order of interaction
between these transcription factors and the ASNS
promoter. During the initial 2 h, there was a 20-fold
increase in ATF4 binding and a rapid increase in histone
H3 and H4 acetylation, which closely paralleled the increased
transcription rate of the ASNS gene, whereas
the increase in ATF3-FL and C/EBP binding was considerably
slower and more closely correlated with the
decline in transcription rate between 2 and 6 h. The data
suggest that ATF3-FL and C/EBP act as transcriptional
suppressors for the ASNS gene to counterbalance the
transcription rate
A). Overexpression of a cDNA encoding C/EBP -LAP
and -LIP was used to identify the migration of these two isoforms (not shown). C/EBP -LAP
migrated as three primary bands, as described under
“Results,” so each band was quantified separately. A and C shows representative
autoradiograms, whereas the graphs in B and D are the summary
of three independent experiments, from which the data are depicted as the averages S.D.
UT2
1.20580783
1
CPA1
1.22264028
CPA2
1.48452357
22 alone 1
1.75321144
22 alone 2
1.41421356
43 alone 1
1.3707828
43 alone 2
1.68179283
CPA +22 1
1.74110113
CPA +22 2
1.36131412
CPA +43 1
1.24401165
CPA +43 2
1.59107297
UT1
1.28788163
UT2
C-myc
1.43395525
CPA2
5.65685425
22 alone 1
1.89211529
22 alone 2
1.8403753
43 alone 1
3.01049349
43 alone 2
3.16016525
CPA +22 1
2.44528056
CPA +22 2
3.01049349
CPA +43 1
6.08391501
CPA +43 2
5.54043787
cmcy
egr1
1.10290391
1.14394081
1.35358192
3.54540475
1.5837125
1.8662453
1.52628782
3.08532937
1.55120762
2.72788703
1.41754231
2.72788703
5.65685425
WT MEFs treated with CPA and PLC
inhibitors
1
CPA1
Ave
UT
CPA
22 alone
43 alone
CPA +22
CPA +43
Egr-1
CPA normalized to tubuliin
(Power)
UT1
4
3.5
3
2.5
c-myc
egr-1
2
1.5
1
0.5
0
UT
CPA
22
alone
43
alone
Treatment
CPA
+22
CPA
+43