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.

WT

KO

WT

WT

WT

WT

WT

KO

KO

KO

KO

KO

KO

KO

LAP

? Tubulin

Ave Fasted FedWT 0.242092 0.2384KO 0.304234 0.133067

Lip/Lap/TubulinWT Fast 0.237405KO Fast 0.449906WT Fast 0.213841KO Fast 0.166527WT Fast 0.27503KO Fast 0.590061KO Fast 0.010443WT Fed 0.267081KO Fed 0.172589WT Fed 0.336391KO Fed 0.06439WT Fed 0.111728KO Fed 0.188072KO Fed 0.107217

Lip/Lap in Alb-Cre liver 3mo old mice

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Fasted Fed

Nutritional status

lip-lap/Tubulin

WTKO

Fasted Fed

Alb-Cre mouse liver 3mo old

Amino Acid Deprivation Induces the Transcription Rate of theHuman Asparagine Synthetase Gene through a Timed Program ofExpression and Promoter Binding of Nutrient-responsive BasicRegion/Leucine Zipper Transcription Factors as Well as LocalizedHistone 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 electrophoresismobility shift analysis showed the involvement of thetranscription factors ATF4, C/EBP , and ATF3-FLthrough the nutrient-sensing response element-1(NSRE-1) within the ASNS promoter. Amino acid deprivationcaused an elevated mRNA level for ATF4,C/EBP , and ATF3-FL, and the present study establishedthat the nuclear protein content for ATF4 andATF3-FL were increased during amino acid limitation,whereas C/EBP -LIP declined slightly. The totalamount of C/EBP -LAP protein was unchanged, butchanges in the distribution among multiple C/EBP -LAP forms were observed. Overexpression studies establishedthat ATF4, ATF3-FL, and C/EBP -LAP couldcoordinately modulate the transcription from the humanASNS promoter. Chromatin immunoprecipitationdemonstrated that amino acid deprivation increasedATF3-FL, ATF4, and C/EBP binding to the ASNS promoterand enhanced promoter association of RNA polymeraseII, TATA-binding protein, and TFIIB of thegeneral transcription machinery. A time course revealeda markedly different temporal order of interactionbetween these transcription factors and the ASNSpromoter. During the initial 2 h, there was a 20-foldincrease in ATF4 binding and a rapid increase in histoneH3 and H4 acetylation, which closely paralleled the increasedtranscription rate of the ASNS gene, whereasthe increase in ATF3-FL and C/EBP binding was considerablyslower and more closely correlated with thedecline in transcription rate between 2 and 6 h. The datasuggest that ATF3-FL and C/EBP act as transcriptionalsuppressors for the ASNS gene to counterbalance thetranscription rate

A). Overexpression of a cDNA encoding C/EBP -LAPand -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 summaryof three independent experiments, from which the data are depicted as the averages S.D.

Ave cmcy egr1UT 1.10290391 1.14394081CPA 1.35358192 3.54540475 5.6568542522 alone 1.5837125 1.866245343 alone 1.52628782 3.08532937CPA +22 1.55120762 2.72788703CPA +43 1.41754231 2.72788703

1.20580783

1

1.22264028

1.48452357

1.75321144

1.41421356

1.3707828

1.68179283

1.74110113

1.36131412

1.24401165

1.59107297

1.28788163

1

1.43395525

5.65685425

1.89211529

1.8403753

3.01049349

3.16016525

2.44528056

3.01049349

6.08391501

5.54043787

UT1

UT2

CPA1

CPA2

22 alone 1

22 alone 2

43 alone 1

43 alone 2

CPA +22 1

CPA +22 2

CPA +43 1

CPA +43 2

UT1

UT2

CPA1

CPA2

22 alone 1

22 alone 2

43 alone 1

43 alone 2

CPA +22 1

CPA +22 2

CPA +43 1

CPA +43 2

C-myc

Egr-1

WT MEFs treated with CPA and PLC inhibitors

00.5

11.5

22.5

33.5

4

UT CPA 22alone

43alone

CPA+22

CPA+43

Treatment

CPA normalized to tubuliin

(Power)

c-mycegr-1