An insulin induced DNA binding protein for the human growth hormone gene

An insulin-induced DNA-binding protein for the

human growth hormone gene.

D Prager, … , S Gebremedhin, S Melmed

J Clin Invest.

1990;

85(5)

:1680-1685.

https://doi.org/10.1172/JCI114620

.

The control of gene transcription is usually mediated by transacting transcriptional factors

that bind to upstream regulatory elements. As insulin regulates transcription of the growth

hormone (GH) gene, we tested nuclear extracts from unstimulated and insulin-stimulated

Chinese hamster ovarian (CHO) cells for binding to four human GH (hGH) gene promoter

oligonucleotide fragments identified as target-binding sequences by DNAse I footprinting.

Using a mobility shift assay, an insulin-induced DNA-binding protein was identified. This

protein binds to two upstream overlapping oligonucleotide sequences. Binding activity is

present at low levels in unstimulated CHO cells and is stimulated by insulin treatment with a

time course suggesting that protein synthesis is required. Incubation of the cells with

cycloheximide and puromycin confirmed that de novo protein synthesis is necessary for the

increased binding activity. Competition with excess unlabeled specific competitor

oligonucleotides prevented binding, while unrelated similar-sized oligonucleotides failed to

compete for binding, indicating that the observed DNA-protein complex formation is specific.

A protein of approximately 70-80 kD was detected by gradient gel electrophoresis. In

conclusion, insulin-mediated DNA-protein binding has been identified on the upstream hGH

promoter, suggesting a trans-active role for insulin in mediating polypeptide hormone gene

expression.

Research Article

Rapid Publication

An

Insulin-induced DNA-binding Protein for

the Human

Growth Hormone Gene

Diane Prager,SabaGebremedhin, andShlomo Melmed

DivisionofEndocrinology & Metabolism, Cedars-Sinai Medical Center-UCLA School ofMedicine, Los Angeles,California90048

Abstract

The controlof genetranscriptionis usually mediated by trans-actingtranscriptionalfactors that bindtoupstreamregulatory elements.As insulinregulatestranscriptionof thegrowth hor-mone(GH) gene, we tested nuclear extracts from unstimulated and insulin-stimulated Chinesehamsterovarian (CHO) cells

forbindingtofour human GH(hGH)gene promoter oligonu-cleotide fragments identified as target-binding sequences by DNAse Ifootprinting.Using a mobilityshiftassay, an insulin-induced DNA-binding protein was identified. This protein bindstotwoupstreamoverlappingoligonucleotidesequences.

Binding activity ispresent at lowlevels inunstimulated CHO

cells andisstimulatedby insulintreatmentwithatimecourse

suggestingthatprotein synthesis is required. Incubationof the cells with cycloheximide and puromycin confirmed that de novoprotein synthesis is necessary fortheincreased binding activity.Competition withexcessunlabeledspecific competitor oligonucleotides prevented binding, while unrelated similar-sizedoligonucleotides failedtocompetefor binding, indicating

that the observed DNA-protein complexformationisspecific.

Aproteinof 70-80kD was detected by gradient gel electro-phoresis. In conclusion, insulin-mediated DNA-protein binding has beenidentifiedonthe upstreamhGHpromoter,suggesting

a trans-active role forinsulin in mediating polypeptide hor-monegeneexpression. (J. Clin.Invest. 1990.85:1680-1685.) transcription factor-mobilityshift assay-polypeptidegene

Introduction

Multiple,

sequence-specific DNA-protein

interactionsoccur in

distinct regulatory regions ofgenes, the resultsof which deter-minethedegreeof transcriptional activation (1). Purification ofthese trans-acting factorshasallowed biochemical charac-terizationaswellaspromoter-selective transcription (2-4).

In-sulin influences the transcriptional rate ofseveral cellular genes(5-10). Although insulin-responsive, consensus DNA sequence(s)havenotyet beenidentified,anuclearproteinthat

bindstotheinsulin-stimulated c-fosgene has been described

(11). Deletional analysis has been used to localize minimal

Addresscorrespondence to Dr. Shlomo Melmed, Division of Endocri-nology & Metabolism, Room B131, BeckerBuilding, Cedars-Sinai

Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048. Receivedforpublication 20 April 1989 and in revised form 30 January 1990.

sequences required for inducible regulation. Insulin-respon-sive elements have been described for the rat phosphoenolpy-ruvatecarboxykinasegene(PEPCK)1 (12), human glyceralde-hyde-3-phosphate dehydrogenase gene(13), andmouse amy-lase genes(6).Werecently reportedthat the 5'flanking region of the humangrowth hormone (hGH) gene containsan

insu-lin-responsive element (14), in addition to previously

docu-mented tissue-specific sequences (15). Wepostulated thatan

IREwouldinteract with ubiquitous trans-active nuclear pro-teins. The hGH gene therefore serves as a useful model to

analyze insulin-mediated transcriptionalcontrol mechanisms.

The ovarycontainsabundant insulin receptors(16),and insu-lin has beenshowntoregulatetheexpressionof several genes in both normal ovarian tissue (17) and in Chinese hamster ovary(CHO) cell lines (18). AsCHO cellsare ahomogenous

cell line containing large numbers of insulin receptors (19),

theywereusedas arelativelyabundantsourceof nuclear

pro-teinfortheseexperiments. These CHO nuclearextracts were

tested forinsulin-responsive proteins bindingtothehGHgene

5'flanking region.The results show that this cell type contains nuclearbinding activitydetectablebymobility shift assay that

binds in a

sequence-specific

manner to the hGH promoter.

Thistrans-actingprotein is responsivetoinsulintreatment.

Methods

Protein extracts. CHO cells were grown to confluence on 225-cm2

plates in Ham's F12 medium in the presence of 10% (vol/vol) fetal calf serum(GibcoLaboratories, Grand Island, NY),penicillin (100 U/ml), andstreptomycin(100

,ug/ml).

Forinsulin treatment, the mediumwas replaced with Ham's F12 containing 1%BSA. Insulin (Humulin R) wasobtainedfrom Eli Lily and Co. (Indianapolis, IN). 14 nM insulin was added to theincubations for 2-16 h. Nuclear extracts were pre-pared byamodification of the method of Dignametal.(20).Briefly,

cellswerewashed with cold PBS and scraped into 5 ml PBS. After

centrifugation(500 g for 5 min) cellswereresuspended in hypotonic buffer (10 mM Tris[pH7.9], 1.5 mMMgCl2, 10mMKCI, 0.5 mM DTT) and keptonicefor 10 min. Cells were then homogenized with a glass douncer (type B) and the nuclei sedimented bycentrifugation

(1,000 g for5min). Nucleiwereresuspendedin20 mMTris (pH 7.9), 20% glycerol, 1.5 mMMgCI2,0.5 mM DTT, and4 MKCIto afinal concentration of 0.3 M KCI. After mixing for 30 min at4VCthe

suspensionwascentrifuged(13,000g for15min) anddialyzed against

20 mM Tris (pH7.9), 20% glycerol,0.1 MKCI,0.2mMEDTA,and 0.5 mM DTT. Thedialysatewasthencentrifuged (13,000 gfor 15

min), aliquotted, and storedat-70'C. Effective cell lysiswas con-firmed bymicroscopy.Allextractswereprepared in the presence of 10

mM sodium molybdate. Cycloheximide and puromycin were

pur-1. Abbreviations usedin thispaper:CHO, Chinese hamster ovary; hGH, human growth hormone; PEPCK, phosphoenolpyruvate car-boxykinase.

J. Clin. Invest.

©TheAmerican Society for Clinical Investigation, Inc.

0021-9738/90/05/1680/06 $2.00

chased fromSigma Chemical Co. (St. Louis, MO). Protein concentra-tionwasdetermined bythemethodof Bradford (21).

DNAfragments andoligonucleotides. The sequences usedin the bindingassays aredepicted in Fig. 1.Complementary oligonucleotide sequencessynthesizedatthe UCLAMolecularBiology Institute (core facility)wereannealed. Thesequencescontained5'protrudingends to facilitate labeling with klenow polymerase anddeoxyribonucleoside triphosphates. Labeled double-strandedoligonucleotideswerepurified bypolyacrylamide gel electrophoresis and elution of DNA fragments, followed by ethanolprecipitation.

Gel shiftassays. Binding reactions wereperformed with 3-5 ng DNA, 1-4 jg poly (dI-dC), and the indicated amounts of protein extractinabuffercontaining10 mMTris-HCI (pH 7.5), 50mMNaCi, 5mMMgCl2,1 mMEDTA, 50mMKCI,I mMDTT, and5%glycerol (22).Reaction mixwasincubatedat roomtemperaturefor15minthen loadedonto 4-6%polyacrylamide gelsandelectrophoresedat10V/cm for 90minin 0.5XTBE(IXTBE=50mMTris, 50 mM boric acid, and1 mMEDTA)at4VC. The gelsweredriedandautoradiographed. CompetitorDNA(100-fold molarexcess)wasadded 10 min before labeledDNAand theincubation continued forafurther15min before gelloading.For someexperiments 3-25%polyacrylamide gradientgels were prepared(23). Samples fromatypicalbinding reactionwerethen electrophoresedat 10 V/cmat4°Ctogether withprestained high

mo-lecularweight markers(Bethesda Research Laboratories, Gaithers-burg, MD) until nofurthermigrationwasobserved. 1X TBEbuffer wasused.Gelswerethen processedasdescribed.

Results

An insulin-inducible DNA-binding protein binds to an up-stream sequence. The mechanism of insulin effects on the

hGH promoter was examined using the synthesized

oligonu-cleotide fragments depicted in Fig. 1. Footprinting experi-ments on the 5' hGH have indicated that sequences -290/-264,

-273/-252,

-140/-100,

and

-93/-66

are pro-tected (15) and correspond to oligonucleotides A,

B,

and D

depicted in Fig. 1. Nuclear extracts from unstimulated and

insulin-stimulated CHO cells were assayed for binding to the

four oligonucleotide fragments. Equal amounts of both

un-stimulatedandstimulatedextracts were incubated with each

radiolabeled oligonucleotide probe and tested for binding

ac-tivity in mobilityshift assays. Specific binding was only dem-onstratedusingtwooftheoligonucleotide fragments, A and B

(Fig. 2). Bindingactivity ofnuclear extracts derived from con-trolCHO cellswas barely detectable. In contrast, CHO nuclear extractsderived fromcellstreated with 14 nM insulin demon-strated an enhanced DNA-binding activity as evidenced by a retarded DNA-protein complex, B3, depicted in Fig. 2. The

largercomplexes were not appreciably altered by insulin

treat-EcoRI FnuDII

- 4

-497

-3"

A

ment. Boilingthe extractsbefore their inclusion in the assay

abolished DNA-protein complexformation. The results indi-catethe presence of an insulin-inducible DNA-binding protein that binds to two upstream overlapping oligonucleotide

se-quences, -290/-264 and -273/-252. Incubation of CHO nuclear extract with oligonucleotides C and D failed to show specific DNA-protein binding (data notshown).

To demonstrate the specificity of the observed nuclear protein binding to these DNA regions, competition reactions wereperformed after a 10-min preincubation with excess un-labeled competitor oligonucleotide fragments (Fig. 2, -290/264+A,B and -273/252+A,B). Both unlabeled

oligo-nucleotidesAand B prevented theformationof the indicated retarded complex when incubated with nuclear extract and

radiolabeled oligonucleotide fragmentsAand B.This respec-tive cross-competition of unlabeled oligonucleotide A for binding to oligonucleotide B and vice versa implies that the protein recognizes the same sequence. Unlabeled, unrelated oligonucleotides did not prevent binding (data not shown),

indicatingthat the binding is specific for oligonucleotides A and B.

Kineticsofinsulin-induced protein bindinginCHOextract.

CHO cells were incubated in serum-free medium with or without insulin (14 nM) for the indicated time periods (Fig. 3). The arrow indicates the enhanced binding of the insulin-in-duced protein to the -290/-264 oligonucleotide after 8 h treatment, with no further increase after 16 h. In the absence of

insulin treatment, negligible binding was observed. The ob-served kinetics of the insulin-induced binding activity may

reflectnewproteinsynthesis.To test forthisrequirement,cells wereincubated withprotein synthesisinhibitors, either cyclo-heximide (10

,uM)

orpuromycin (100 mM), for 16 h before harvest. Cycloheximide treatment diminished the observed DNAbinding inboth control andinsulin-treatedgroups. Cells werethenincubated with 100 mMpuromycin for 16h.

Puro-mycintreatment also blocked DNA-protein complex

forma-tion, confirming that the appearance of thisbinding activity requires protein synthesis (Fig. 4). Neither puromycin nor

cy-cloheximide appeared toalter the nonspecific larger

protein-DNAcomplexes,

indicating

aselective effect of theseagentson

theinsulin-regulated

protein-DNA

complex.

The insulin-induced

DNA-binding

protein.Tocharacterize

therelativesize of the insulin-induced

DNA-binding

protein, UV-protein cross-linkingstudieswereperformedinvitro. Ex-tracts incubated with

oligonucleotide

probes A and B were

electrophoresed in lowmeltingagarosegelsand irradiatedat

305nMfor30 minat4°C.After

autoradiography

thespecific

B11

+2

-2W

B

-273 -252

C

-132

D

*~ -W

Figure1.hGHpromoter

region with

oligonucleo-tides(A-D)shown.

8a

. + + ++

v cm In cmea

cmu 4

cmu

D ND N o _{P C} en

ao

raw_cm o

Ns N N

1 2 3 4 5 6

78891

10o1

.+t

9

F_-Figure2.InsulintreatmentofCHO cells inducesanuclearprotein-DNA

complex:

localization ofbindingsites between -290 and -252. Lane 1, -290/-264probealone;lanes 2 and4,-290/-264 probe,5 tg CHO extract, 4 ggpoly (dI-dC);lanes 3 and 5, -290/-264 probe, 5 ,gg insu-lin-treated (14 nM) CHO extract,4ggpoly(dI-dC);lane6, -273/-252 probe alone;lanes 7 and 9, -273/-252 probe, 5,ggCHO extract, 4 ,g poly(dI-dC);lanes8and10,-273/-252probe,5 ug insulin-treated CHOextract(14 nM),4Mg poly (dI-dC).The-290/-264 and -273/-252 probeswereincubated with5Mgof CHO nuclearextractthathadbeenboiledat950C for 5 min, followedby5 minonice. 32P-labeled -290/-264 and-273/-252 (I ng),5 ug insulin-treated CHOnuclearextract, and 4

Mg poly

(dI-dC)wereincubated with100-foldexcess indi-catedcompetitoroligonucleotides (AandB,seeFig. 1).ArrowsandB3 indicate boundcomplex.F, free DNA. B3 indicates insulin-induced boundcomplex.

DNA-protein complexes of interestwereexcisedand electro-phoresed on a 12% SDS-polyacrylamide gel. As two bands of 50 and 70 kD wereidentified inthe gelsof the insulin-treated extracts, the possibility ofan adjacent protein being

cross-linked could not be excluded. Therefore, to further confirm the size of the DNA-binding protein,themobilityshift assay was performed using a 3-25% gradient gel (23) with protein markers(Fig.5). The gradient allows DNA-protein complexes tomigrateattheirtruemolecularweights,which can be com-paredwiththeprestainedmarkers. Theinsulin-inducedDNA

binding proteinappeared tomigratewith adifferentmolecular

weight depending on which oligonucleotide probe was used.

Mobility shiftassays allow resolution of complexes formed by

different proteins recognizinganidenticalsequence as long as the proteins differ significantly in their molecular mass and

charges, thus allowing altered electrophoresis. Alternatively,

two _{different proteins recognizing} twodifferent sequences within the same probe may generate an identical complex.

Competition studies indicatethat theprotein(s)recognize the

sameDNA sequence, and the presenceoftwoproteins cannot beexcluded. Also, the relative contributionof oligonucleotide size(21 vs. 26 bp)tothe complexformation needs to be

con-sideredininterpretingtherelativeprotein size.

Discussion

Although

insulin

has been shown to directly regulate the

ex-pression ofseveral genes at the transcriptional level, no single

insulin-responsive DNA sequencehas yet been identified.

Transfection studiesusinganhGHpromoter

chloramphenicol

acetyl

transferase reporterconstructwereusedto localize

in-sulin-responsive element(s)

within 500

bp

ofthe GH promoter

(14).

In

addition,

DNAse Ifootprinting of the hGH promoter revealed threemajor protected regions (15).Theproximaltwo

protectedsites bind thetissue-specifictrans-actingfactor, Pit I

(GHF-I) (3).

The

third,

distal site isprotected

by

ubiquitous

factors

thought

tobe involved in cAMP andproteinkinase C

signal

transduction

(2).

Usingamobilityshiftassaywe identi-fiedaninsulin-induced DNA-protein complex bindingtothe

-290/-252

region

ofthe GHpromoter,whichcorrespondsto

thedistal

_footprinted

siteonthe hGH promoter. Incubation of the

protein

extract with excess cold specific

oligonucleotide

sequencesabolished binding,whereas similar-sized unrelated

sequencesdid notcompete for

binding, confirming

the

speci-ficity of the observedcomplex formation. Boilingtheextract

before its inclusion in the reactions prevented

DNA-protein

complex formation, suggestingthat these nuclear factorsare

proteins. Novel DNA-protein interactions were detected

_by

electrophoresisin thepresenceofhighionicstrengthbuffer.

The observed insulin-inducedbinding activitycould repre-senta nonbindingor lowaffinityform of the factor that un-dergoes insulin-mediated modification togenerate a high

C

2h

4h

6h

8h

mn

rm

rm

rm

-273/-252

₁₂

₁

1

2

3 4

...

4."

-N-

_W

-290/-264

5

6

7

8

F

Figure3.Kinetic analysis of insulin-induced DNA-protein complex formation. CHO cellsweregrown toconfluenceinHam'sF12+10% (vol/vol)fetalcalfserum and thendeinduced inHam'sF12 and 1% BSA. 14 nMinsulinwasadded for the indicated timeperiods, and eachtime pointhad amatchinguntreatedcontrol(left). 32P-labeled

-290/-264 oligonucleotidewasincubated with5 ,uginsulin-treated CHO nuclearextract(timecourseasindicated), and4,g poly

(dl-dC).Arrow,Insulin-induced boundcomplex; F, freeDNA. LaneC, 32P-labeled -290/-264 probealone.

This relatively long lagperiod suggests that denovo protein

synthesis is required. To confirm this requirement for new

proteinsynthesis, cells were incubated with cycloheximide and

puromycin,twodifferent protein synthesis inhibitors,for 16 h

before harvest. Both treatments,respectively, diminished the

binding ofthe complex in control and insulin-treated cells,

suggesting

the presenceofalabileprotein.Asmany transcrip-tion factors are phosphoproteins (24), this insulin-mediated DNA-proteincomplex may be regulated by aposttranslational phosphorylation modification.

The ratPEPCKgene,murine c-fosgene, andmurine amy-lase-2.2 genecontaininsulin-responsivepromoters (6, 11, 12).

Insulin dependence of an amylase gene in diabetic transgenic

micewasanalyzed,andinsulin-responsive sequences were

re-portedin the 5'region of thisgene(6).Twoinsulin-sensitive DNA-binding proteins have been identified on the human

glyceraldehyde-3-phosphate dehydrogenase gene. The up-stream binding site interacts with aninsulin-sensitive

DNA-binding protein in 3T3 adipocytes. This sequence is

suppos-edly present in a number of insulin-sensitive genes. The downstreamelementinteracts withatrans-acting factorthatis

inducedfourfold by insulin in 3T3 adipocytes(13).Alignment

ofthe promoterregionsof thesethree genes and the hGH gene reveals homologous regions as indicated in Table I. Nuclear

proteinsextracted from a varietyofcelltypes contained

bind-ing factors detectablebymobility shiftassay that bound in a

sequence-specific

manner to thehGHpromoter.Althoughthe

DNA-binding protein is not tissue specific, its quantitative regulationbyinsulinappears to be cell specific.

Figure4.Effect of puromycinoninsulin-inducednuclear protein-DNAcomplexbindingto the -290/-264 and -273/-252 se-quences.CHO cellswereincubated with 100 mMpuromycin for16 hbefore harvest. All lanes contain5 ugofnuclear extract and 4 ig

poly(dI-dC).Lanes1-4 wereincubated with the -273/-252 probe. Lanes5-8wereincubated withthe -290/-264 probe. Lanes I and 5,probe, poly(dI-dC), CHOextract; lanes2and 6, probe, poly (dl-dC), insulin(14nM)-treated nuclearextract;lanes 3 and7,probe, poly(dI-dC), puromycin-treatedextract; lanes4 and 8,probe,poly

(dI-dC),puromycin, and insulin-treatedextract. Arrows, Insulin-in-ducedDNA-protein complex.

DNA-protein complex migrationisdeterminedby pH,salt

concentration, complex conformation, and the molecular

massoftheproteins. Thedifferences incomplexsizeobserved

with the gradientgel and oligonucleotides Aand B maybe

indicative oftwo different proteins recognizing an identical

sequence. However, the DNA-binding competition experi-mentssuggest that theprotein isrecognizingthe identical

se-quence.

The hGH gene wasreportedtocontainsequences

interact-ing with two DNA-binding factors (-308/-235) mediating

TableLSequenceHomologyinthePromoterRegionofFour

Insulin-responsiveGenes

Gene 5' Sequence 3' Match Strand

GH -287 ATGGCCTGCGG -277 Coding

c-fos -296 ATGTCCTAATA -306 6/11 Noncoding

PEPCK -202 GAGGCCTCAGG -88 7/11 Noncoding

PEPCK -78 GAGGCCTCAGG -202 7/11 Noncoding

Amy-2.2 -223 ATGGCCTCAGA -223 8/11 Coding Amy-2.2 -185 ATGGCCTCAGA -175 8/11 Coding

Depictedsequences(14, 11, 12, 6)werealigned withthe 11 base

pairsof the hGHpromoterregion (-287/-277).

I

.0 a

J.--s-- ...

-273/-252

-290/-264

II

1

2

3

4

5

6

7

8

Work inthe authors'laboratory was supported in part by grants from the National Institutes of Health(DK-34824) and by a grantfrom theJuvenile DiabetesFoundation. D. Prager is a recipient of an Ameri-canDiabetesAssociationCalifornia Research Fellowship.

97.4

68

Figure5.Nuclearprotein sizing bygradientgelelectrophoresis.After incubation withtheindicatedoligonucleotideprobes,DNA-protein

complexeswereseparatedon3-25%gradient polyacrylamide gels

to-gether withprestainedhighmolecularweight proteinstandards. Lanes 1-4 wereincubatedwiththe-273/-252probe.Lanes5-8 wereincubated withthe-290/-264 probe.Alllanes contain5,gg of

CHOextractand 4Mgpoly(dI-dC).Lanes1, 2, 5,and7,probe, poly

(dI-dC),andCHOnuclearextract. Lanes3, 4,6,and8, probe,poly

(dI-dC), insulin (14nM)-treatednuclearextract.Arrows,

Insulin-reg-ulatedprotein,molecularmass70-80kD.Protein sizes(kD)are in-dicatedontheleft.

positiveandnegative transcriptionalcontrol(25).One of these factorsbindingbetween

-275/-257

wasreportedtobe related

to major late transcription factor, the upstream stimulatory

factor that activates adenovirus major late promoter

tran-scription. The other factor detected with the-308/-235

frag-mentacted as a dominant repressor of gene expression.

Al-thoughtherelationshipbetween these factors and the

insulin-induced DNA-binding protein is presently unclear, insulin does suppress the GH promoter even though it induces this

DNA-bindingfactor. Therefore,thistrans-actingfactor could beactingas arepressor oftranscription.

DNA-binding transcription factors have distinct binding

and transcriptional activation domains (26). Itshould there-fore bepossibletotestthefunctional

significance

ofthis

insu-lin-mediated DNA-binding activity to these elements of the hGH gene promoter.

Acknowledaments

The authors are grateful to Grace G. Labrado for excellent typing assistance.

References

1.Maniatis, T., S. Goodbourn, andJ. A.Fischer.1987. Regulation

of inducible andtissue-specificgeneexpression.Science(Wash.DC).

236:1237-1244.

2. Imagawa, M., R. Chiu, and M. Karin. 1987. Transcription factor AP-2 mediates induction by twodifferent signal-transduction

path-ways:protein kinase C and cAMP. Cell. 51:251-260.

3. Ingraham, H.A.,R. Chen, H. J.Mangalam, H. P. Elshotz, S.E.

Flynn, C. R. Lin, D.M.Simmone, L. Swanson, and M. G. Rosenfeld. 1988. A tissue-specifictranscription factor containingahomeodomain species pituitary phenotype. Cell. 55:519-529.

4.Courey, A. J., and R. Tjian. 1988. Analysis of Spl in vivo reveals multiple transcriptional domainsincludinganovelglutamine-rich

ac-tivation motif.Cell. 55:881-898.

5.Sasaki, K., and D. K. Granner. 1988. Regulation of phospho-enolpyruvate carboxykinasegenetranscription by insulin and cAMP: reciprocal actionsoninitiation andelongation. Proc.Natl.Acad. Sci. USA. 85:2954-2958.

6.Osborn, L., M. P.Rosenberg, S. A. Keller, C. N. Ting, and M. H. Meisler. 1988. Insulin response ofahybrid amylase/CAT genein transgenic mice. Biol. Chem.263:16519-16522.

7. Strauss, D. S., and C. D. Takemoto. 1987. Insulin negatively regulates albumin mRNA at thetranscriptional and post-transcrip-tional level inrathepatoma cells. J. Biol. Chem. 262:1955-1960.

8.Yamashita, S., and S. Melmed. 1986. Insulin regulation of growthhormonegenetranscription.J.Clin.Invest. 78:1008-1014.

9. Podlecki, D. A., R. M.Smith, M. Kao, P. Tsai, T. Huecksteadt, D. Brandenburg, R. S. Lasher, L. Jarett, and J. M. Olefsky. 1987. Nuclear translocation of the insulin receptor: apossible mediator of

insulin's long-term effects. J. Biol. Chem. 262:3362-3368.

10.Kahn, C. R. 1985. The molecular mechanism of insulin action. Annu. Rev.Med. 36:429-451.

11.Stumpo, D. J., T. N.Stewart, M. Z. Gilman, and P. J. Black-shear. 1988. Identification of c-fossequencesinvolvedin inductionby insulin andphorbolesters.J. Biol. Chem. 263:1611-1614.

12.Magnuson, M., P. Quinn, and D. Granner. 1987. Multihor-monalregulation ofphosphoenolpyruvate carboxykinase chloram-phenicol acetyltransferase fusiongenes. J. Biol. Chem.

262:14917-14920.

13. Alexander, M. C., M.Lomanto, N. Nasrin, and C. Ramaika. 1988. Insulin stimulatesglyceraldehyde-3-phosphate dehydrogenase

geneexpression throughcis-actingDNAsequences.Proc.Natl.Acad. Sci. USA. 85:5092-5096.

14. Prager, D., andS. Melmed. 1988. Insulinregulates expression of the human genein transfected cells. J. Biol. Chem.

203:16580-16585.

15.Lefevre, C., M. Imagawa, S. Dan, J. Grindlay,M.Bodner,and

M. Karin. 1987.Tissue-specific expression of thehumangrowth

hor-monegeneisconferredinpartby bindingofaspecific trans-acting factor. EMBO(Eur. Mol.Biol.Organ.)J. 6:971-981.

16.Poretsky, L., andM. F.Kalin. 1987. The gonadotropicfunction

of insulin. Endocr. Rev. 8:132-140.

17. Caro, J. F., and R. L. Rosenfield. 1988. Insulin-like growth factorIand Insulinpotentiateluteinizing hormone-induced androgen synthesis by rat ovarian thecal interstitial cells. Endocrinology.

123:733-739.

18.Bhandari, B., R.H.Wilson, and R.E.Miller. 1987.Insulin and

dexamethasone stimulatetranscriptionofanamplified glutamine

syn-thetasegeneinchinese hamsterovarycells.Mol. Endocrinol. 1:403-407.

1684 D.Prager,S.Gebremedhin,andS.Melmed

l

I

19. Podskalny, J., A.McElduff, and P.Gordon. 1984. Insulin re-ceptors onChinese hamster ovary (CHO) cells: altered insulin binding toglycosylationmutants.Biochem. Biophys.Res. Commun. 125:70-75.

20.Dignam, J. D., R. M. Lebovitz, and R. G.Roeder. 1983. Accu-ratetranscriptioninitiation byRNApolymeraseIIinasolubleextract from isolated mammalian nuclei. NucleicAcids Res. 11:1475-1489.

21. Bradford,M. M. 1976. Arapidandsensitive methodfor the quantification of microgram quantitiesofproteinutilizing the princi-ple of protein dyebinding. Anal.Biochem. 72:248-254.

22. Distel, R. J., H.S.Ho, B.S.Rosen, D. L. Groves,and B. M. Spiegelman. 1987. Nucleoprotein complexes that regulate gene ex-pression in adipocyte differentiation: directparticipation of c-os. Cell. 49:835-844.

23.Rodriguez, R., and W. T. Schrader. 1989. A reversible native gel electrophoresis method for studying the composition of steroid receptor-DNA complexes. The Endocrine Society, 71st Annual Meet-ing, Seattle, WA. A568.

24. Gonzalez, G. A., and M. R. Montminy, 1989. Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 13. Cell. 59:675-680.

25.Peritz, L. N., J. B. Fodore, D. W. Silversides, P. A. Cattini, J. D. Baxter, and N. L. Eberhardt. 1987. The human growth hormone gene contains both positive andnegativecontrol elements. J. Biol. Chem. 263:5005-5007.

26.Mitchell,P. J., andR.Tjian. 1989.Transcriptional regulation in mammalian cells bysequence-specific DNA binding proteins. Science(Wash. DC). 245:371-378.