CCAAT/enhancer-binding protein mRNA is translated into multiple proteins with different transcription activation potentials

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CCAAT/enhancer-binding protein mRNA is translated into multiple proteins with different transcription activation potentials

OSSIPOW, Vincent, DESCOMBES, Patrick, SCHIBLER, Ulrich


The CCAAT/enhancer-binding protein (C/EBP) alpha is a leucine zipper protein that is preferentially expressed in certain cell types, such as adipocytes and hepatocytes. Here we show that C/EBP alpha mRNA is translated into two major proteins, C/EBP-42 and C/EBP-30, that differ in their content of N-terminal amino acid sequences. These results are best explained by a ribosome-scanning mechanism in which a fraction of ribosomes ignore the first two AUGs and initiate translation at an AUG located 351 nt downstream of the first one.

Because C/EBP-30, the translation product initiated at the third AUG, is devoid of the potent transcription-activation domain contained in C/EBP-42, the former protein stimulates transcription from the mouse albumin promoter much less efficiently than the latter. The gene encoding the liver-enriched transcriptional-activator protein LAP (C/EBP-beta) has also been shown to issue two proteins, LAP and the liver-enriched transcriptional-inhibitory protein LIP, with different transcription-activation potentials. The production of multiple proteins from a single mRNA is not only shared between [...]

OSSIPOW, Vincent, DESCOMBES, Patrick, SCHIBLER, Ulrich. CCAAT/enhancer-binding

protein mRNA is translated into multiple proteins with different transcription activation potentials.

Proceedings of the National Academy of Sciences, 1993, vol. 90, no. 17, p. 8219-8223

DOI : 10.1073/pnas.90.17.8219

Available at:

Disclaimer: layout of this document may differ from the published version.


Vol.90, pp. 8219-8223, September 1993 Biochemistry

CCAAT/enhancer-binding protein mRNA is translated into multiple proteins with different transcription activation potentials

(ribosomescanning/transcription activation/livergeneexpression)






Departmentof MolecularBiology,UniversityofGeneva,30QuaiErnestAnsermet, CH-1211Geneva4, Switzerland CommunicatedbySteven L.McKnight,May17,1993



protein (C/EBP) ais a leucine zipper protein that is preferentiafly expressed incertain celltypes, such as adipocytesandhepa- tocytes.Hereweshow thatC/EBPamRNAistranslatedinto twomajor proteins, C/EBP-42andC/EBP-30,thatdiffer in their contentofN-terminalaminoacid sequences. Theseresults are best explained byaribosome-sanningmechanisminwhich afraction of ribosomesignorethefirsttwoAUGs and initiate translation at an AUG located 351ntdownstreamof the first one. Because C/EBP-30, the translationproduct initiatedat the thirdAUG, is devoid of the potenttranscription-activation domain contained inC/EBP-42,theformerproteinstimulates transcription from the mouse albumin promoter much less efficiently than the latter. The geneencodingtheliver-enriched transcriptional-activator protein LAP


has also beenshownto issuetwoproteins,LAPandtheliver-enriched transcriptional-inhibitory protein LIP, with different tran- scription-activation potentials. The production of multiple proteins from a single mRNA is not only shared between different C/EBP family members but also appears to be conserved invertebrate evolution.



protein(C/EBP) leucine zip- perproteinfamily comprisesanumber ofindividualmembers of whichtwo,


and theliver-enrichedtranscription- al-activator proteinLAP


arehighly expressed in liver (1-4). During postnatal liver differentiation, both C/EBPand LAPreachhighestlevels interminally differen- tiated hepatocytes (ref. 5, this paper). The genes encoding these twoproteins donot have acomplexstructure in that they aredevoid ofintrons (2, 4).Despite this simple struc- ture, protein expression has proven more complex than anticipated. Recently, a single LAP mRNA species was found to be translated into two proteins, LAP and the liver-enriched transcriptional-inhibitory protein LIP. The lattercontains the DNA-binding and dimerization domains but is devoid ofthe transcription-activation domain. Thus LAPand LIP exhibitantagonistic activities (5).

Several authors have also observed two protein species with different molecular masses (42 kDa and 30 kDa) in immunoblot experiments by usingC/EBP antibodies (2, 6).

Here we show that C/EBP-30 is a bona fide translation product of C/EBP mRNA. This protein lacks the potent N-terminaltranscription-activationdomain ofC/EBP-42 (7) and therefore cannot stimulate transcription efficiently.


ConstructionofC/EBPExpression Vectors. The recombi- nantDNAtechniques describedbySambrooketal.(8)were used.CMV-C/EBPFL(for full length) vector was obtained

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by inserting aXba I-EcoRIC/EBPgenomic fragment into pSCTGAL X556(9) openedwithBamHI and EcoRI. CMV- C/EBP-42vectorwasobtainedby insertingaBamHI-Hind3 fragment from pMSV-C/EBP (3) into pSCT GAL X556 openedwith BamHI and Pvu II. CMV-C/EBP-30 vectorwas obtainedby inserting a Taq I-Hind3 C/EBP genomicfrag- ment intopSCT GAL X556 opened withBamHI and Pvu II.

CMV-C/EBP-20 was obtained by inserting a Not I-Hind3 C/EBP genomic fragment into pSCT GAL X556 opened with BamHI and Pvu II.

Transfection ofCOSCells. One day beforetransfection,106 cells wereplated onto a10-cm dish. Cells were rinsed twice with TBS(25 mM Tris, pH 7.4/137 mM NaCl/5 mMKCI/0.7 mMCaCl2/0.5mMMgCl2/0.6mMNa2HPO4) and incubated with 10 ,ug of plasmid DNA in 2 ml of TBS containing DEAE-dextran at 555 ,g/ml (500 kDa, Sigma) for 1 hrat room temperature. The DNA solution was then aspirated, and cells were incubated in TBS/25% (vol/vol) dimethyl sulfoxide for2 min. After thedimethyl sulfoxide shock,cells wererinsed three times with TBS and grownfor2 days in DMEM/10%fetal calf serum.

SynthesisofAzido-dUTP-SubstitutedProbe.Thefollowing twooligonucleotides wereused:


(ii) 3'


For synthesis of a double-stranded, radiolabeled, azido- dUTP-substituted probeencompassingthealbuminpromoter element D(10), 100ngeachof the 10-merand 26-mer were hybridized and incubated in a mixture (20-,l final volume) containing0.1 mM azido-dUTP, 0.25 mMdGTP/dCTP, 13 nM[32P]dATP(3000Ci/mM;1Ci=37GBq),10 mMTris(pH 7.4),10mMMgCl2,and 5 unitsofKlenow enzyme. After 20 minat room temperature 0.25 mMof unlabeled dATPand dTTP were added, and theincubation wascontinuedfor 5 min.Unincorporateddeoxynucleotideswereseparated from the DNAprobeby gel filtration.

UVPhotocrosslinkingandImmunoprecipitations. For pho- tocrosslinkingratliver nuclearproteinswereincubatedonice in a 15 ul ofbindingreactioncontaining 25 mM Hepes (pH 7.6),5mMMgCl2,34 mMKCI, 50 ngofpoly(dIdC),and 2.3 ngofazido-dUTP-substitutedD-site probe. After 30 min the reactionwasirradiated for1minat 254 nm at adistance of5 cm with a Waldmann UV handlamp. For SDS/PAGE the UV-irradiatedsamplewasmixedwith an equal volumeof2x loading buffer, products were resolved on an SDS/10%

polyacrylamide protein gel (11), and radiolabeled species werevisualized by autoradiography. Forimmunoprecipita- tions 4-fold larger binding reactions were assembled. The Abbreviations: C/EBP, CCAAT/enhancer-binding protein; LAP, liver-enrichedtranscriptional-activator protein;LIP, liver-enriched transcriptional-inhibitory protein; CAT, chloramphenicol acetyl- transferase.


8220 Biochemistry: Ossipow et al.

UV-crosslinked reactionwasbroughtup to a


vol with abuffercontaining 1% sodium deoxycholate, 10 mM Tris (pH 8.0), 150mMNaCl, 1mMEDTA, and 2


of C/EBPorLAP antiserum. After 1 hrat room temperature 125 ,1 ofa10%

suspension of protein A-Sepharose beads (Sigma, P 3391) in the same buffer wasadded, andincubation was continued for 75min. The beadswerepelleted, washed three times with the samebuffer, and resolved on SDS/10% polyacrylamide gelas above.

Two-Dimensional Electromobility-Shift Assay SDS/PAGE Mapping.Azido-dUTP-substituted D-site radiolabeled probe (6 ng)wasincubated on icein abinding reaction (see above) with 5 x 105 nuclei. After 45 min the nuclei were pelleted, supernatant was transferred to a new tube, and 1 ,ug of sonicated salmon sperm DNA was added. After another 45 min on ice, thereaction was loaded on a 6% nondenaturating polyacrylamide gel and run for 6 hr at 120 V at 4°C. The separated complexes were UV-crosslinked in the gel for 2 min at 254 nm at a distance of 5 cm. Thepolyacrylamidestrip containing the crosslinked material was placed horizontally on anSDS/10%polyacrylamide gel. After migration, radio- labeled species were visualized by autoradiography.

OtherTechniques. Theprocedures used for the preparation of nuclei from rat liver or tissue-culturecells, immunoblot- ting, transfections of hepatoma cells, chloramphenicol ace- tyltransferase (CAT) assays, and electromobility-shift assay were described (5). Allcellswere grown inDMEM/10%fetal calfserum.


Accumulation ofC/EBPDuring PostnatalLiver Differen- tiation. To examine the accumulation of C/EBP during hepatocyte differentiation, liver nuclei wereprepared from rat embryos 1 day before birth or rats were sacrificed at different times after birth and examinedbyimmunoblotting.

Two protein species, C/EBP-42 and C/EBP-30, were ob- served in all nuclearlysates,and the relativeconcentrationof both proteins increase with the age of the animals. The maximal levelsof bothproteinsareonly reached in adultrats (3 mo)(Fig. 1Right), suggesting thatC/EBPmay havearole in the establishment and/or maintenance ofterminal hepa- tocytedifferentiation. As suggested by UV-crosslinkingex- periments (see below) and immunoblot experiments with antisera against the C-terminal 140 amino acids ofC/EBP (P.D., unpublished result), liver nuclear extracts contain

2 3 4 5 6 2 3 4 5 6

4Pqi 4...,,-.C/EBP42

*W 4-C/EBP30

FIG. 1. ExpressionofC/EBP-42andC/EBP-30inratliverduring terminal differentiation.Liver nuclearlysateswerepreparedfromrat fetuses 1 daybefore birth(lane 1), newborn rats (lane 2), orrats sacrificed 2days (lane 3), 10days (lane 4), 25days (lane 5),and 90 days (lane 6) after birth. One-halfOD260units of eachlysate was fractionatedon anSDS/12% polyacrylamide gelandanalyzedwith

anti-C/EBP serum by immunoblotting. (Left) Coomassie blue- stained gel. (Right)Autoradiogram.

similaramountsof C/EBP-42 andC/EBP-30.Moreover,the ratio of thesetwoproteins doesnot appear tobesubject to developmental regulation, as has been reported for the C/EBP relatives LAP and LIP (5).

C/EBP-30 Is a Bona Fide Translation Product ofC/EBP mRNA. Thec/ebp gene encodes a single, unspliced, 2.7-kb mRNA (2, 6). It is, therefore, probable that C/EBP-30 is either a proteolytic fragment of C/EBP-42 or a translation product of C/EBP mRNA. These possibilities were ad- dressedinatransfection experiment with COS cells, which donotaccumulate detectableamountsofendogenous C/EBP (Fig. 2, lane 1). SeveralC/EBP expression vectors(Fig. 2) were constructed in which the transcription of C/EBP mRNAs is driven by the cytomegalovirus


promoter. Two days after transfection, the cells were har- vested and analyzed by immunoblotting for the presenceof nuclear C/EBP proteins. Transfection of CMV-C/EBP FL vector, which includes 5' nontranslated sequences encoded by the c/ebp gene, results in the production of both C/EBP-42 and C/EBP-30 at a ratio very similar to that seen inliver nuclei (Fig. 2, lanes 2 and 6). In contrast, CMV-C/

EBP-42 issues mainly C/EBP-42 (Fig. 2, lane 3). In this expression vector, the sequences surrounding the first in- frame ATG have been converted to CCACCATGG, which perfectly matches the Kozak consensus sequence CCRC- CATGG (for review, see ref. 12). As anticipated, this se- quence appears to be more efficiently recognized by ribo- somes thanthe nonoptimal Kozak sequence CCCCCATGG,

1 2 3 4 5 6

C/EBP42- am _



CMVf |N|o


1 351

.2 639 708 1059












FIG. 2. C/EBP-30 is atranslation product ofC/EBP mRNA.

One-fourth OD26o unit of nuclear lysates from COS cells were transfected withconstructs(shownbeneathlanes)at10 ,ug perdish;

numbers are innucleotides with 1 as the firstcoding nucleotide.

Constructswereanalyzed by immunoblottingwithanti-C/EBP se- rum.Lanes: 1,control(KS+) plasmid;2,CMV-C/EBPFL vector;

3,CMV-C/EBP-42vector;4, CMV-C/EBP-30vector;5, CMV-C/

EBP-20 vector; 6, 1 OD260 of adult rat liver nuclear lysate was includedas acontrol.Filmwasexposed13timeslongerfor lane 6 thanfor lanes 1-5.

Proc. Natl. Acad. Sci. USA 90



presentwithin the native


gene.Becausethe accumulationofC/EBP-30 dependsonthe sequencecontext of the first AUG, proteolytic degradation is unlikely to accountforitsgeneration.Morelikely,this truncatedprotein is initiated at a downstream AUG by ribosomes that have ignored thefirst AUG in thescanningprocess.Todetermine which downstream AUG serves asthe initiation codon for C/EBP30, the proteins encodedbytwo additional expres- sion vectors,


andCMV-C/EBP-20 (see Fig.

2) have been examined. Fig. 2, lanes 4 and 5, shows that transfectionofthese vectorsresults in the accumulationof truncatedC/EBP proteinswith apparent molecularmassesof 30kDa and 20kDa, respectively. We thus conclude that the AUGlocated 351ntdownstream of the first AUG is used for the initiation of C/EBP-30. A 20-kDa protein species, de- tected as aminor speciesinliver nuclei with the antiserum against C-terminalC/EBP sequences (datanot shown), ac- cumulates innuclei fromCOScells transfected with CMV- C/EBP 20 (Fig. 2, lane 5). However, this protein is not efficiently produced in COS cells transfected with the ex- pression vectors CMV-C/EBP FL, CMV-C/EBP-42, and


Therefore,theAUG located639ntdown- streamofthefirstAUG isapparently onlywell-recognized by ribosomes when the upstream sequences harboringalterna- tive AUGcodons aredeleted.

These results strongly support atranslational mechanism for the creation of C/EBP-30 (and the minor C/EBP-20 species). Were the production oftwo C/EBP proteins bio- logically significant, this mechanism would have been ex- pected to be conserved during vertebrate evolution. We, therefore, compared liver nuclear extracts fromchicken, rat, mouse, and human(hepatomacellnuclei)inanimmunoblot experiment. As shown in Fig. 3 Left, all nuclear lysates contain




atroughly similar ratios.

As expected, only C/EBP-42 is revealed by an antiserum raised against a peptide present within the N-terminal C/EBP-42 sequences (Fig. 3, Right). Chicken C/EBP-42 does not reactwith thisantibodybecause theepitopeis not conserved between mammalian species and chicken (see protein sequence in ref. 13). Twodifferently sized C/EBP proteins havealso been observed in in vitro translations of Xenopus C/EBPmRNA (14). The apparentconservation of two(or more)C/EBP proteinsduring vertebrate evolution is consistent with theirphysiological significance.

1 2 3 4 1 2 3 4

C/EBP-42andC/EBP-30HaveDifferentTrans-Activation Potentials.Thetrans-activation domainsofC/EBPhave been mapped in detail (7). Aparticularlystrong,perhapsa-helical activationdomainhasbeenuncovered within the sequences lacking in C/EBP-30. We thus anticipated that C/EBP-30 would be a less potentactivator oftranscription thanC/EBP- 42.Cotransfection experimentswithanalbumin-CAT target gene and variousCMV-C/EBP expressionvectorsin human HepG2 hepatoma cellsconfirmed this notion. Fig. 4 shows thatC/EBP-42(Fig. 4, lanes 1 and 2)activatestranscription ofaCAT-reportergene carrying the mouse serumalbumin promotermuch moreefficientlythanC/EBP30(Fig.4, lanes 5 and 6). Transfection ofCMV-C/EBP FL vector, which produces both C/EBP-42 and C/EBP-30, also results in stimulated target geneexpression,althoughto alesserextent thantransfection of


42vector(Fig. 4,lanes 3 and4). However,assuggestedbytheexperiments ofFig. 4, a more dramatic overexpression of C/EBP-30 attenuates transcription activation by C/EBP-42. Cotransfection ofa constantamountofCMV-C/EBP-42withincreasedamounts ofCMV-C/EBP-30(lanes8-11)results in the lossoftarget geneactivation at the twohigherCMV-C/EBP-30 expression vector concentrations. As expected, little CAT activity is seenin thecotransfectionswithincreasedamountsof CMV- C/EBP-30 alone(lanes 12-14).

C/EBP-42, CEBP-30, LAP, andLIPArethe


Site- D-Binding Liver Nuclear Proteins. The C/EBP family com- prises members with similar DNA sequencespecificity. We, therefore, wished to determine the relative contribution of thefour already identifiedmembers,C/EBP-42,C/EBP-30, LAP, and LIP, to the albumin site-D-binding activities in liver nuclei. To this end we used a UV-photocrosslinking strategy to identify individual site-D-binding proteins. As shown in Fig. 5, lane 1, SDS/gel electrophoresis ofphoto- crosslinked protein-DNAcomplexes revealsfour major spe- ciesinaliver nuclearproteinextract.Immunoprecipitation of thecomplexesidentifiedthemajor site-D-bindingproteins as

C/EBP-42, C/EBP-30,

LAP, and LIP(Fig.5, lanes 2 and 3).

Owingtothehighsequenceconservation within the dimer- ization regions of the differentC/EBPfamily members, the fourmajor proteinsencodedby thec/ebpand lap genes could form as many as 10 homo- and heterodimers. Unequivocal demonstration of dimeric proteins in vivo is a difficult en- deavorbecausecelllysis and extraction of nuclear proteins may result in artificial rearrangement of in vivo-assembled

2 3 4 5 6 7 8 9 10 11 12 13 14

_ _ :~~~~~~~


C/EBP42-+ ' -




FIG. 3. Accumulation ofC/EBP-42 and C/EBP-30 invarious vertebrate species. Human HepG2 cell nuclear lysate (0.5 OD260 unit,lane1)and liver nuclearlysatesfromrat(0.16OD260unit, lane 2),mouse(0.16OD260 unit,lane 3),and chicken (1OD260 unit, lane 4)wereseparatedon anSDS/12% polyacrylamide gel and analyzed by immunoblottingwithanti-C/EBPserumraised against recombi- nantC/EBP-42 protein (Left)orwithanantiserum raisedagainsta

peptidepresentwithin theN-terminalregionofC/EBP-42 (Right).

FIG. 4. Trans-activation potentials ofC/EBP-42andC/EBP-30.

CAT assays withHepG2cellscotransfected with 6,gof a reporter plasmid containing themouse albumin promoter region fused to a CAT gene(alb-CAT)andC/EBP expressionvectors.Lanes: 1and 2, 1 Mg and 4 ,g,respectively, ofCMV-C/EBP-42vector;3and 4, 1 tgand 4 ug,respectively, ofCMV-C/EBPFL vector; 5and 6, 1 ,ug and 4 pg,respectively, ofCMV-C/EBP-30 vector; 7, 4Mg of control plasmid; 8-10, results obtained in cotransfections with CMV-C/EBP-42vector(0.11ug perdish)andCMV-C/EBP-30vector with 7 Agof alb-CAT. Thefollowinglanes indicate the quantities of CMV-C/EBP-30 expressionvectorsused. Lanes: 8, 0.1 ,ug; 9, 1 ug;

10,10Mg;11-13,results withC/EBP-30expression vector only with alb-CATat0.1Mug(lane11)or at1Mg(lane12)or at10MAg(lane 13);

14, 10 ugof KS+ controlplasmid.

Biochemistry: Ossipow



8222 Biochemistry: Ossipow etal.

2 3 1 2 3 4 5 6 7 8

(LAP)2 ).

LAP.LIP - (LIP)2 ).

C/E ...:...


EBP42 . e L.AP-*....*...

C/EBP30- ' I.

LIPP * unbound DNA > _.

FIG. 5. Identification of the majoralbumin siteD-binding pro- teins. A liver nuclear extract (4.5 Mg or 18 Mg ofproteins for immunoprecipitations) was incubated with a radiolabeled, azido- dUTP-substituted albumin site-Dprobe (2.3ngor9.3ngfor immu- noprecipitations). After UVirradiation, crosslinked protein-DNA complexeswereseparatedon anSDS/12% polyacrylamide gel(lane 1). Toidentifythe D-site-binding proteins, photo-crosslinked pro- tein-DNA complexes were immunoprecipitated with either anti- LAP serum(lane 2)oranti-C/EBPserum(lane 3)andseparatedas described above.

dimers. Toassessthisrisk,weperformedapilot experiment inwhich twopurified recombinantproteins, LAPand LIP, weremixedfromconcentrated stocksolutions,inwhichmost of theseproteinsexist inhomodimeric forms(F. Fleuryand U.S., unpublishedobservation). Afterincubation for 10 min atdifferent temperatures with orwithout DNA,the dimers were analyzed by electrophoretic mobility-shift assay. At 37°C,heterodimerizationoccurs


whetherornot an excessof


site isincludedinthereaction(Fig.

6 Upper, lanes 5 and 8). At 20°C heterodimerization


occursin theabsence ofDNA(Fig.6Upper,lanes 4 and7).

No heterodimerization was seen at 0°C, irrespective of whether DNA was included during the incubation (Fig. 6 Upper,lanes 3 and6).Duringisolation of nuclei in low-salt buffers at 0°C, one would therefore not anticipate major homo- and heterodimer rearrangements. Furthermore, the enormous (=600-fold) excess of genomic




proteinswithin thenucleus (E. Falvey, L.Marcacci,andU.S., unpublishedobservation)islikelyto stabilizeinvivo-assembled homo- andheterodimericC/EBP proteins.Tokeepthe saltconcentrationlow,C/EBP-related proteins were extracted from nuclei by incubation with a double-strandedoligonucleotidespanning the albumin site D.

This results in the direct transfer ofDNA-binding proteins from nuclei onto the added binding site (15). Theprotein- DNA complexes thus obtained were analyzed by a two- dimensional gelelectrophoresis system. In thefirst dimen- sion,thecomplexeswereresolvedaccordingtoconventional electromobility-shiftassaytechniquesonanativepolyacryl- amidegel.Thepolyacrylamide stripcontainingtheseparated protein-DNA complexes was then irradiated with UV and placed horizontally on a denaturing SDS/polyacrylamide slabgel. Photocrosslinkedproteinswerethus elutedfromthe nativegelandfractionatedaccordingtotheir molecular sizes inthe second dimension(16) (Fig. 6Lower). As the photo- crosslinked protein monomer-DNAcomplexes resolved in the seconddimension havebeen identifiedpreviously (Fig.

5),the spots in thetwo-dimensionalpattern (Fig. 6Lower) could beinterpreted.Thistechnique,whilenotunequivocally abletoresolve allof the 10possibleLAPandC/EBPhomo- andheterodimers, isagreeable with the existence of most of them. Eachof the fourmajorD-site-bindingproteins, iden- tified in the second dimension (SDS gel)as a monomer, is associated with dimericcomplexes with different mobilities



C/EBP30 0





... ..- :..In1



LIP - 4

FIG. 6. Complexityofhomo-and heterodimericC/EBPandLAP complexes in isolated nuclei. (Upper) In vitroformationof recom- binant (LAP-LIP) heterodimers under various conditions (stock concentration forthesetwo recombinantproteins is -50 J,M), as monitored byelectromobility-shiftassay with a D-siteoligonucleo- tide probe.Lanes: 1, LAP alone(125nM); 2,LIP alone(125nM);

3-9, LAP and LIP(125 nMeach).Sampleswerepreincubatedat0°C (lanes1-3and6), 20°C(lanes4and7),and37°C (lanes5and8).The DNAprobe (75 nM)waseither included inthepreincubation (lanes 6-8) or added after the preincubation (lanes 1-5). All binding reactions(20


contained 400 ngofsonicated salmonspermDNA.

Complexes were resolved on 6.4% nondenaturating gel. (Lower) Albumin Dsite-binding proteinswereextracted fromisolatednuclei by their directtransfer to theazido-dUTP-substituted D-siteprobe at 0°C. After removal of the extracted nucleiby sedimentation, the protein-DNA complexes in the supernatant werefractionated by nondenaturating gel electrophoresis (first dimension). After UV irradiation, the polyacrylamide strip containing the photo- crosslinked complexes was placedhorizontally on adenaturating SDS/10%o polyacrylamide gel, and covalent radiolabeledprotein- DNA complexes were separatedby size (second dimension). An aliquot ofthe proteins used in the first dimension was directly photo-crosslinked,and theresultingcomplexeswereincludedassize markers in the second dimension (Lower leftlane).

in the first dimension (nativegel). For example, C/EBP-42 comigrates in the first dimension with dimeric complexes containingC/EBP-30, LAP, and LIP. Likewise, LIPcomi- gratesin thefirst dimensionwith dimersincludingC/EBP-42, C/EBP-30, and LAP. The existence ofin vivo-assembled


heterodimers has also been supported by immunoprecipitation experiments (ref. 17, and our unpub- lisheddata).


The intronless


genespecifies asinglemRNAbut two major protein species. This is accomplishedby


Proc. Natl.Acad Sci. USA 90



use oftwo AUG initiation codons within the same open readingframe. C/EBP-42, thelarger of thetwoproteins, is initiated at the first AUG. Another AUG is located 42 nt downstream ofthis initiation codon. In immunoblot analysis, aminorprotein speciescan be detected thatmay be initiated at this AUG. However, a second major C/EBP species, C/EBP-30is issuedfromanAUGlocated 351ntdownstream of the firstone.Three additional in-frame AUGs exist within the mRNAsequenceslocated even further downstream. One of these may,indeed, giverise toanadditional minorC/EBP form, C/EBP-20.The translation ofmultiple C/EBP proteins from a single mRNA is best explained by a ribosome- scanningmechanism. When the first AUG isplacedinto the perfect Kozak consensus sequenceCCACCAUGG, there- sulting mutant mRNA is only translated into C/EBP-42.

Apparently, most if not all ribosomes scanningthe mRNA sequence in a 5' -+ 3' direction initiate translation at this AUG. In contrast, the imperfect Kozak sequence CCCCCAUGG surrounding the first in-phase AUG within theC/EBP wild-typesequenceisapparently ignored bysome ribosomes. This results intranslationinitiation atalternative downstream AUGs,leading to theaccumulationofN-termi- nally truncatedproteins C/EBP-30 and, to alesser extent, C/EBP-20.

C/EBP-42andC/EBP-30differdramaticallyintheir tran- scriptional-activation potential. When overexpressed in HepG2 cells, C/EBP-42, but not C/EBP-30, efficiently ac- tivatestranscription fromamouseserumalbumin promoter.

As demonstrated by Friedman andMcKnight (7), the most potent C/EBP activation domain is, indeed, encoded by sequencessituated between thetwoAUGs used for transla- tioninitiationofC/EBP-42andC/EBP-30, respectively.

Thelap gene,aclose relativeofc/ebp,alsoproduces two proteins, LAP and LIP,by the same mechanism. In cotrans- fectionexperiments,LIPneutralizestheaction ofLAP, even atsubstoichiometric levels, atwhichmostof LIP would be expectedtobe in a(LAP/LIP) heterodimer.In contrast to the invariant ratio of (C/EBP-42/C/EBP-30) seen throughout postnatal liver development, the LAP/LIP ratio increases -5-foldduringthisperiod (5). Our protein-DNA crosslinking studiessuggestthatinconcert,thefour proteinsC/EBP-42,


LAP, andLIP, accountformostbinding activi- ties recognized in liver nuclear extracts by the albumin promoter element D. At least in vitro, these four related proteins contribute about equally to DNA binding and may form as many as 10distinct homo- and heterodimers. Our two-dimensional separation of photo-crosslinked protein- DNAcomplexesis compatible with the existence of most, if notall, of these dimeric forms in liver nuclei.

Althoughin vitrothe monomersexchange extremely rap- idlyat 37°C (ref. 18, this paper), in vivo, yet-undiscovered dimer-stabilizationmechanisms forcoiledcoilsmayexist. In this context, Williams etal. (17) and S. L. McKnight (per- sonalcommunication) noticedthat LAP andC/EBPcontain acysteineresidueatthe ultimate andpenultimate position, respectively. Oxidation of these C-terminal cysteines to intermolecular disulfide bridges would, of course, account forastabilization of dimers. Allofourattempts to demon- strate the presence of such C/EBPor LAPdimers in liver nuclear extractsfailed, even in conditions in which exoge- nously added in vitrooxidized dimerssurvived the homog- enization andseparation procedures (data not shown). How-

ever, theC-terminal cysteines could stabilize protein dimers through noncovalent interactions-e.g., by participating in chelate complexes with coordinated divalent cations such as Zn2+ or Cu2+.

The production of N-terminally truncated proteins is not only conserved between different members of the C/EBP family but also between the same members in different vertebrate species. Thus, the C/EBP-encoding genes of Xenopus, chicken, rat, mouse, and human all produce an N-terminally truncated species. Moreover, LIP homologs have been observed in mouse and human (P.D. and U.S., unpublished observations). This evolutionary conservation may reflect aphysiological significance of the shorter spe- cies.

Transcription factor antagonists operating byanumber of diverse mechanismshave been described forawidevariety of transactivators(forreview, seeref. 19). At first sight, the purposeofproducingsuch inhibitors may appearenigmatic.

However,their existence extends therepertoire ofregulatory mechanisms implicated in thefine-tuning ofgeneexpression because theactivityandaccumulationcanbemodulatednot onlyforthe transactivators but also fortheirantagonists.

We are grateful to Steven McKnightfor his generous gifts of C/EBPrecombinantplasmidsandC/EBP antisera,aswellasfor his valuable comments on the manuscript. We thank Uli Laemmli for helpful discussions,OttoHagenbuchleand HansTurlerforproviding N3-dUTP andCOScells, respectively, Edward Schmidt and Dan Lavery for their critical reading of the manuscript, and Nicolas Roggli for preparing theillustrations.This research was supported by the Swiss National Science Foundation and theState of Geneva.

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