Regulated Expression of Three C/EBP Lsoforms During Adipose Conversion of 3T3-L1 Cells

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Regulated expression of three C/EBP lsoforms during adipose conversion of 3T3-L1 cells

Zhaodan Cao, Robert M. Umek, and Steven L. McKnight 1 Department of Embryology, Howard Hughes Research Laboratories, Carnegie Institution of Washington, Baltimore, Maryland 21210 USA

In an effort to identify protein factors that play a regulatory role in the differentiation of adipocytes, we have isolated two genes that encode polypeptides related to CCAAT/enhancer-binding protein (C/EBP; hereafter termed C/EBPa). The proteins encoded by these C/EBP-related genes, termed C/EBPI~ and C/EBP~, exhibit similar DNA-binding specificities and affinities compared with C/EBPot. Furthermore, C/EBPI~ and C/EBP8 readily form heterodimers with one another as well as with C/EBPa. The transcriptional activating capacity of these two newly identified C/EBP isoforms was demonstrated by transient transfection experiments in which expression vectors encoding C/EBPI3 and C/EBP~ were observed to induce transcription from the promoter of the serum albumin gene in cultured hepatoma cells. The mRNAs encoding C/EBP~ and C/EBP~ were detected in a number of tissues, most of which corresponded to sites of expression of C/EBP~. The expression pattern of C/EBP[3 and C/EBP8 during adipose conversion of 3T3-L1 cells was examined by Western and Northern blotting assays. In contrast to the expression profile of the gene encoding C/EBP% whose product is not detectable until the late phase of adipocyte differentiation, the c/ebp[3 and c/ebp8 genes were actively expressed very early during adipocyte differentiation. Moreover, transcription of the c/ebp[3 and c/ebp8 genes was observed to be induced directly by adipogenic hormones. The accumulation of C/EBPI3 and C/EBP8 reached a maximal level during the first 2 days of differentiation and declined sharply before the onset of C/EBPot accumulation. The temporal pattern of expression of these three C/EBP isoforms during adipocyte differentiation may reflect the underpinnings of a regulatory cascade that controls the process of terminal cell differentiation. [Key Words: Transcription factors; cell differentiation; bZIP proteins] Received May 6, 1991; revised version accepted June 14, 1991.

CCAAT/enhancer-binding protein (C/EBP) is a transcrip- growth and acquire morphological and enzymatic prop- tion factor expressed primarily in liver, fat, and intesti- erties characteristic of adipocytes (Green and Kehinde nal tissues (Birkenmeier et al. 1989). C/EBP expression 1975; Russell and Ho 1976; Rubin et al. 1977; Student et is restricted to terminally differentiated cells, and evi- al. 1980). Several lines of evidence have indicated that dence has been presented indicating that it can stimulate C/EBP may participate in the terminal differentiation of the transcription of several genes that mark the differ- 3T3-L1 cells. First, the temporal profile of C/EBP expres- entiated state (Christy et al. 1989; Friedman et al. 1989; sion correlates with the progress of adipocyte differenti- Kaestner et al. 1990; Park et al. 1990). These observa- ation. The protein is undetectable in proliferating adipo- tions led to the speculation that C/EBP may play an im- blasts but accumulates to a substantial level in differen- portant role in establishing and/or maintaining the state tiated adipocytes (Birkenmeier et al. 1989). Furthermore, of terminal cell differentiation. premature expression of C/EBP impedes cell prolifera- The in vitro conversion of 3T3-L1 adipoblasts to adi- tion and accentuates differentiation in the presence of pocytes has provided a useful system to study the puta- adipogenic hormones (Umek et al. 1991 ). Second, C/EBP tive role of C/EBP in cell differentiation. The 3T3-L1 cell is capable of trans-activating genes encoding several ad- line was originally isolated from mouse fibroblasts ipose-specific proteins, including a fatty acid-binding (Green and Kehinde 1974). These cells exhibit a fibro- protein variously termed 422 or aP2, stearoyl coenzyme blast-like morphology and proliferate continuously A (CoA} desaturase, and the insulin-responsive glucose when maintained in growth medium. Upon appropriate transporter (Christy et al. 1989; Kaestner et al. 1990). hormonal stimulation, 3T3-L1 cells terminate mitotic Despite these observations, premature expression of C/EBP~ is not sufficient to convert adipoblasts to adipo- ~Corresponding author. cytes (Umek et al. 1991). This later observation indicates

1538 GENES& DEVELOPMENT 5:1538-1552 © 1991 by Cold Spring Harbor Laboratory ISSN 0890-9369/91 $3.00 Downloaded from genesdev.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press

that adipogenesis probably requires the function of un- rived from two different genes. One class contained 11 characterized regulatory proteins in addition to C/EBP. clones with the longest insert covering 1 kb, and the C/EBP belongs to a class of DNA-binding proteins other contained a single clone with an insert covering 0.6 termed bZIP proteins (Vinson et al. 1989). The bZIP do- kb. DNA sequence analysis of the inserts from the two main contains a region rich in basic amino acids linked classes revealed that both encoded bZIP proteins highly to a dimer-forming region called the leucine zipper similar to C/EBPoL (Fig. 1A, B). To simplify the nomen- (Landschulz et al. 1988b, 1989; Vinson et al. 1989). bZIP clature for this and future studies, the C/EBP-related pro- proteins are capable of forming homotypic and hetero- teins have been termed according to their chronological typic dimers by their leucine zippers (reviewed by P. order of isolation. According to this nomenclature, the Lamb and S.L. McKnight, in prep.). A different class of original C/EBP is now called C/EBPoL, the group that con- DNA-binding proteins, termed helix-loop-helix (HLH) tained 11 clones is called C/EBP[3, and the single clone is proteins, also mix as homotypic and heterotypic dimers referred to as C/EBPB (see Discussion). (Murre et al. 1989a, b). Certain members of the HLH fam- A somewhat unusual feature of the c/ebpa gene is that ily of transcription factors have been shown to play im- it is free of intervening sequences (Landschulz et al. portant roles in cell determination and differentiation 1988a). Judging from the close sequence relatedness (for reviews, see Olson 1990; Barinaga 1991). In light of among c/ebp[3, c/ebp3, and c/ebpa, we reasoned that the these relationships, we reasoned that the uncharacter- newly isolated genes might also be intron free. Genomic ized regulatory proteins required for adipogenesis might DNA fragments corresponding to c/ebp[3 and c/ebp3 be C/EBP related. Guided by this notion, we searched for were isolated and sequenced. Large open reading frames, C/EBP-related genes expressed in differentiating adipo- which contained the bZIP domain, were found in the cytes. genomic fragments corresponding to c/ebp~ and c/ebp3 Here we report the isolation and characterization of The open reading frame of the gene encoding C/EBP[3 two genes, termed c/ebp~ and c/ebp3, whose expression was observed to encode a hypothetical protein of 296 is regulated in a temporal manner during adipogenesis. amino acids with a predicted molecular mass of 31.3 kD. c/ebp[3 has been described previously and variously The c/ebp6 gene encoded a hypothetical protein of 268 termed NF-I16, LAP, I16-DBP, and AGP/EBP (Akira et al. amino acids with a predicted molecular mass of 28.6 kD. 1990; Chang et al. 1990; Descombes et al. 1990; Poli et The nucleotide sequences and conceptually translated al. 1990). To our knowledge, c/ebp3 has not been de- amino acid sequences of both C/EBP[3 and C/EBP~ are scribed previously. Both genes encode bZIP proteins that shown in Figure 1. TATAAA homologies were observed bind to DNA in a manner highly similar to C/EBP. More- within 100 nucleotides upstream from the putative over, C/EBP[3 and C/EBP8 can cross-dimerize with one translation initiation codon of both proteins, suggesting another as well as with C/EBP, hereafter termed C/EBPa. that each gene might be transcribed into a simple, in- Surprisingly, transcription of the genes encoding C/EBP[3 tron-free mRNA. To test this possibility we identified and C/EBP~ is directly responsive to adipogenic hor- the precise points of transcription initiation on each mones, which is reflected in the accumulation of these gene. Oligonucleotides complementary to sequences lo- proteins during the early stage of adipocyte differentia- cated just 3' to the putative translation initiation codon tion. The expression profiles of C/EBP[3 and C/EBP8 dur- of both C/EBP isoforms were synthesized (Fig. 1A, B), ing adipogenesis raise the possibility that these tran- radiolabeled, and used in primer extension assays scription factors perform important regulatory functions {McKnight et al. 1981) with poly(A) + RNA isolated from early during the process of terminal differentiation. differentiating 3T3-L1 cells. The lengths of the primer extension products shown in Figure 1C, are consistent with initiation of c/ebp[3 gene at a cluster of sites 54-58 Results bp upstream of the putative translation initiation codon and initiation of c/ebp6 gene at 38 bp upstream from the Isolation of c/ebp[3 and c/ebp8 genes putative translation start site. In both cases, the inferred To search for genes related to that which encodes initiation sites are located downstream of TATAAA ho- C/EBP{x, we constructed a cDNA library using mRNA mologies. isolated from 3T3-L1 cells midway through the differen- The deduced amino acid sequences of C/EBP[3 and tiation program. The library was screened with 150-bp C/EBP8 were compared with that of C/EBPoL with a com- DNA probe encoding the bZIP domain of C/EBP(x. The puter program that identifies regions of amino acid se- screen was conducted at moderate hybridization strin- quence similarity (Corpet 1988). As shown in Figure 2, gency so as to allow cDNA clones bearing similar se- the carboxy-terminal regions of the three proteins, quences to hybridize to the probe. Thirty hybridization- which contain the bZIP domain, are highly conserved. positive bacteriophage were isolated after screening of The basic regions of the three proteins share 21 of 24 3 x l0 s plaques. Eighteen of the phage contained se- identical amino acids (enclosed by a solid line in Fig. 2). quences complementary to the 3'-untranslated region of The amino acid sequence similarity between C/EBP[3 C/EBPcx cDNA when screened at high stringency. Rea- and C/EBPoL extended farther toward their respective soning that these phage contained C/EBPa-encoding in- amino termini, consisting of 8 identical residues within serts, we discarded them. The remaining 12 clones fell the next 11 positions. In contrast, the sequence of into two classes and were subsequently found to be de- C/EBP8 diverged in this region and included 2 proline

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cI~8

~2CC~TT~J~=C~C~CTTATRJL%CCTeCC~TC~C~CG~C~GCCGAOTCC~A 60 GAGCTCGATCCCTGTTCCGCCTT TGCTATGTCT~TCC TGCTT TGCGCGTGTCGG 60

~CC~CACCC~AC~.C~2AC, C~CCCCGCC~CCCC~GTTC£TCCA~C~C?~ 120 GGCC.JULATCCAC, ATTTTCATTTCGCT~TGGACGGCTAAGTAGG 120 M H R L 4 TCGT TCCCAGC~ 180 TC,~,~T(~G£~2AD,~AT~TCCCC~,CC~C~CC~CGCCTTTAQ~CCAT~AA~TGG 180 L A W B A A C L P P P P A A r R P M g V 24 CCCJLGTCCCTCOCCGCTCCGTCTCCGACCC2~'T GGGGC~GTGCGCGTCAGC 240

CX~TTC TACT&CGAD, CCC~ T~CTGC, CCT~C~_,CCAA~GGCCCC, CGccGCGC 240 TGGGGCTAC~I_%~r91~CGGCGGTCCGGCCCGGCGAGGTGACAC, C~TGGACGC~T 300 A N r Y Y E P D C L A Y C A ~( A A R A A 44 ~CGCGC~ 360 CGC ~CGCCCCC~CCGCcGAC,~CC, C~CAT T(k~2~ J~AC~,CCA TCGAc T T CkC,C C 300 M S A A L • S L D S P V R G T 15 P R A P A A E P A t G E H • R A Z D F S 64 CCCT~CTTCT~~CGGCCGA 420 CCTACCTG~C~,~CC(~(~TTC~C~CC~-=CGCCCGCGCACCACG 360 P W P T • P A A • Y • P G R V D K P G R 35 P Y L • I~ L & P & & D F A A P A P a H H 84 ~ TGGGC TCCACGACTCCTGCCATGTACGACGAC 480 ACT TCCTC TCCGAC~'TCT T~C~,kC~ACT kC~kAC, CCC, AF-,CAAGAAGCCC, C~CG 420 G P E P G • L G • L G S T T P A M Y D D 55 D F L S D L F k D D Y G A K P S K K P A 104 ~CATCGACT ~CTACATTGAC TCCAT GGCCGCCGTC, CCCACCCTAGAG 540 ACT~GGT TACGTC~CT~C,~C~GC~C~G~CGCGCCG~CC~C TGC TTcc 480 • S A I D • S A Y I D S M A A V P T L E 75 D Y G Y V ~ L G R A ~ A K A A ~ ~ k C F 124 CTGTGCC.ACC, ACGAACTC TTCGCCGACC TCT TC~C~G 600 C~CCGCCGCCTCC~(~,CT~~CGGC, CTTCGAA~C~TGCAAGC 540 L C B D • L F A D L • N S N H K A A G A 95 P P P P ~ A A L K A • P G F • P A D C K 144 GGCGGCCTGGAGC T~CTACGCGACCCCCGGGTGTGGGGTCTGTCGCT 660 GCGCGGA~C~GCCC~,~CAT~G~cCC~TT TCCCGTTC~CCT~C TACCTGG 600 G G L • L L Q G G P T R P P G V G S V A 115 R A D D A P k M A A • r P F A L R A Y L 164 AGGGGGCC GCTCJL%C~GCGAACCCGACTGGGGC~TC CCTGC TG 720 GCTAC~GACGCCGA~AGCAGCGGCAGCCTGTCCACGT~GTC~TC~TCCAGCC 660 R G P L K R • P D W G D G D A P G S L L 135 G Y Q a T P S ~ S S G S L S T S S S S S 184 ~TGGCGGTG TGCGCGCAGACAGTGGTGAGCTTGGCGGCCGCGGC TCAC, C CC 78 0 CGCCCGGCACGCCGAGCCCC~CCGAC~C~CGCGCCCG~CGCCTGCTTCC, CGC, C.GC 720 P A Q V A V C A Q T V V S L A A A A Q P 155 P P G T P S P A D A K A A P A A ~ i~ A G 204 JLCT CCACCCACTT ~ TCCTC~ GGGGCCGAC-CCTCGC GCCCGGC 840 CGCCGGCCGCGCCCGCCAAC, GCCAAC, C,C~GGTGC, A~AAC, CTG~GACGAGT 780 T P P T S P E P P R G S P G P S L A P G 175 P P A A P A K A K A K K T V D )( L S D E 224 AD-AGTCCG~ ~T (~ CCGGAGTACCGG 900 ACAAGATGC GGC GCGAG~ GCAACAACA T C GC GG TGCGC AAGAGCCGC GACAAGGC CAAGA 840 T V R • K G A G K R G P D R G S P E Y R 195 Y K M R R E R N N I A V R K S R D K A K 244 ~GAGC~T CGC TGTGCGCAAGAGCCGCGACAAGGCCAAGCGCC GC 960 TGCGCAACCTGGAGACGCAC, CACAAGGT GCTGGAC, CTGACGGC GGAGAACGAGCGGCTGC 900 Q R R E R N N I A V R K S R D K A K R R 215 M R N L E T Q H K V L E L T A E N E R L 264 AAC~GA~AAGCTGGTGGAGTTGTCGGCCGAC, AACGAGAAGCTGCATCAG 1020 ~AAGGTGGAGCAGC TGTCGCGAGAGCTCAC, C.ACCCTGCGGAACT TGTTCAAGCAGC 960 N Q • M Q Q K L V E L S A E N E K L H Q 235 Q K K V E Q L S R E L S T L R N L F K Q 284 CGCG~TCACCCGGGACCTGGCTGGCCTCCGGCAGT TCTTCAAAAAACTGCCC 1080 TGCCCGAGCCGCTGCTGGCCTCGGC~ACT GCTAGCGCGGCGCGGTGGCGT~ 1020 R V • Q L T R D L A G L R Q • F K K L P 255 L P g P L L A $ A G H C * 296 AC,CCCC, CCTTTCCTGCCGCCCACCGGCGCCGACTGCCGGTAACGCGCGGCGTC, GGCCT TT 1140 CC,CCGCGGCCACC GTGCGCCCTGCCCCGCGCGC TCCGGCCCCGCGCGCGCGCCCGGACCA 1080 S P P F L P P T G A D C R * 268

CCGTGCGTGCCC T GCGCGCACCTGCACC TGCACC~CCGC~ACCGC GG 1140 GAGACTCTGAACGACCTATACCTCAGACCCCGACAGCGGGGAC, CAGAC GCCC.CCCGAATC 1200

GCACGCGCGGCGCACGCACCTGCAC.AC, C GCACC GGGT T TCGGGAC T TGATGCAATCC GGA 1200 GCTAGTTTCTTTGGGACC TGCGAGCGACAGGAAGCTGCAGCTTGGGCACTGGACTGCGAG 1260

TCAAACGTGGCTGAGCGCGTGTGGACACGGGAC TACC, C AACACACGTG TAACTGTCTAGC i~60 AC,AAC.CTATATTAATCTT TCCCCTTAAATTATTTTTTATAATGGTAGCATTTTCTACGTC 1320

CGGGCCCTGAGTAATCACCTTAAAGATGTTCCTGCGCGGTTGT TGATG T TTTTGGT T T TG 1320 TTATTACCATTGCAGC TAAGGTACAT T TGTAGAAAAGACAT T T CCGACAGAC T T T TGT AG 1380

TTTTTGTTTTTTGTTTTGTTTTGTTTTTTTTTTTGGTCTTATTATTTTTTTTGTATTATA 1380 ATAAGAGGAAF=AGACTGCGCATGCTTTT TATAT TCATT TTTACAGTAT TTGTAAGAAT AA 1440

GAATAAGAATAAAGAAC, CATTTAAATC~ 1478 TAAAAAAGTTCTATTTCTATGAGAAAA~AC, GCGT ATGT ATATT TGAGAACC T T TTCCG T T 1440

TCGAGCAT TAAAGTGAAGACATT TTAATAAACT TTTT T GGGAGAATGT TT~CAAA 1500 Figure 1. Nucleotide sequences and conceptually translated // protein sequences o~ C/EBPI3 and C/EBPb. The nucleotide sequence of gene is shown in A, and that of is c/ebpfl c/ebp,5 C 123 nt elm al shown in B. TATAAA homologies, putative polyadenylation 110 nt am a~ signals, and sequences complementary to the oligonucleotides used for primer extension analyses {CI are underlined. The deduced amino acid sequences are shown in the single-letter code 90 .t ..I ..Q beneath the corresponding codons of the nucleotide sequence. Arrows indicate the sites of transcription initiation as deduced 76 nt 811 am by primer extension (CI. [CI An autoradiographic exposure of a 67 nt all, polyacrylamide gel used to size primer extension products. The locations of primer complementarity are shown in A and B. Poly(AI + RNA isolated from differentiating 3T3-L1 cells was annealed to synthetic oligonucleotides that had been end-labeled with [>-3~p]ATP and polynucleotide kinase. Annealed oligonucleotides served as primers for cDNA synthesis by reverse transcriptase. Lengths of extension products were determined 34 nt by their mobility relative to radiolabeled MspI fragments derived from pBR322 {indicated in nucleotides to the le[t of the autoradiograml. Sequence of C/EBPg has been submitted to GenBank.

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and 4 glycine residues that probably interrupt the a-he- hydrophobic dimerization interface (Landschulz et al. lical structure predicted to occur in this region of 1988b). With one exception, all three C/EBP isoforms C/EBPoL and C/EBPf~. This observation may be notewor- contain identical amino acids at the i + 4 positions of thy, as previous experiments demonstrated that muta- each repeat. The single exception to this i + 4 identity tions in the corresponding region of C/EBPoL resulted in a occurs in the most amino-terminal heptad repeat of notable decrease in DNA-binding affinity (Landschulz et C/EBP~, which contains a leucine residue rather than the al. 1989). valine present in C/EBP~ and C/EBP[3 (Fig. 2). Leucine is All three C/EBP-related proteins contain a heptad re- a reasonably conservative substitution for valine and is peat of leucines in perfect register relative to the location unlikely to alter substantially the amphipathic, a-helical of the highly conserved basic region (Fig. 2). The heptad property of this region. array of leucine residues is diagnostic of a dimerization The amino acid sequence similarities shared by interface termed the leucine zipper. In addition to the C/EBPc~, C/EBPf~, and C/EBP~ provide strong clues as to leucine repeats and their invariant location with respect the functions of the two newly identified proteins. They to the basic region, the three proteins contain highly are probably transcription factors that bind to DNA as related residues at the i + 4 position relative to each dimers. However, as Figure 2 clearly shows, the amino- leucine. When projected onto an idealized oL-helix, the terminal two-thirds of the proteins are largely unrelated amino acids at this i + 4 position are aligned on the at the level of primary amino acid sequence. This raises same helical face with the leucine residues forming a the possibility that each C/EBP isoform might be capable of mediating different regulatory processes.

C/EBP~ MHRLLAWDAACLPPPPAAF.RPMEVANFYYEPDCLAYGAKAARAAPRAPAAEPAIGEHER * , ** ** , ** , * , *** Comparison of DNA-binding properties C/EBP~ MESADFYEAEPRPPMSSHLQRPPHAPSNARLWLSPGRGPRAAPSPTCRPGAAGRICEHET , * , * , * ***** , among C/EBP isoforms C/EBP6 MSAALFSLDSPVRGTPWPT.EPAAFYEPGRV.DKPGRGPEPGELGE.LGSTTPAMYDDES Given the exceptional relatedness in the basic region of C/EBPe,, C/EBP[3, and C/EBPS, it was anticipated that C/EBP~ AIDFSPYLEPLAPAADFAAP..APAHHDFLSDLFADDYGAKP .... SKKPADYGYVSLGR ** , * , * , , , , ** , they might bind similar DNA substrates. To test this C/EBPd SIDISAYIDPAAFNDEFLADLFQHSRQQEKAKAAAGP~DFDYPGAPAGPGGAVMS. possibility each protein was purified and studied in ** ***** , , ** **

C/EBP6 AIDFSAYIDSMAAVPTL ..... ELCHDELFADLFNSNH ...... KAAGAGGLELL. DNA-binding assays. DNA fragments that encoded the bZIP domain of C/EBPf~ and C/EBP~ were cloned into T7 expression vectors (Studier and Moffatt 1986; Rosenberg C/EBP~ AGAKAAPA..C .... FPPPPPAAL...KAEPGFEPADCK...RADDAP...AMAAGFPF *** ** * * * * * * * * * ** et al. 1987). Each polypeptide was overexpressed in Esch- C/EBPd AGAHGPPPGYGCAAAGYLDGRMEPLYERVGAPAMRPLVIKQEPREEDEAKQLALAGLFPY erichia coli and purified to homogeneity according to * *** ** * , ,

C/EBP~ QGGPTRPPG ...... VGSVARGPL...KREPD ...... WGDGDA ..... PGSL.. methods described previously (Shuman et al. 1990). The purified proteins were tested for their ability to bind to a high-affinity C/EBPc~ target sequence in gel-shift assays CIEBP~ ALRA ...... YLGYQATPSGSSG.SLSTSSSSSPPGT.PSPADAKAAPA * * * * ** *** * * * (Fried and Crothers 1981). Both C/EBP~ and C/EBP~ C/EBP~ QPPPPPPPPHPHASPAHLAAPHLQFQIAHCGQTTMHLQPGHPTPPPTPVPSPHPAPAMGA were capable of shifting the mobility of the radioactively * * * * ** * *** ** *

C/EBP8 ...... LPAQVAVCAQTVVSLAAAAQPTPPT...SPEPPRGSPG labeled DNA probe in the presence of an excess amount of nonspecific competitor DNA (data not shown). To assess and compare DNA-binding affinities of the C/EBP~ ACFAGPPAA ...... PAKA~/~KTVDKLSDEYKMRP~RNNIAVRKS * ** **** *** * ** ************ three C/EBP proteins, we performed saturating binding C/EBPd AGLPGPGGSLKGLAGPHPDLRTGGGGGGGAG~SVDKNSNEYRVRP, ERNNIAVRKS assays in which a constant amount of protein was ti- * ** * * * * I** ************

C/EBP~ PSLA PGTV ...... REKGAGKRGPDRGSP YRQRRERNNIAVRKS trated with increasing amounts of a radiolabeled oligonucleotide containing an optimal C/EBP-binding sequence (Vinson et al. 1989). After incubation at 24°C for 20 min, the binding mixtures were subjected to polyacrylamide gel electrophoresis to separate protein-bound oligonucleotides from free ones. An autoradiographic C/EBP~ .RDKAKRR~E~QK~4E~K~QR~Q~RD~G~QF~ S pp F Lp p TGADCR image was used to locate the bound and free oligonucle- Figure 2. Amino acid sequence comparison of C/EBP~, otides {Fig. 3, top ). The corresponding gel segments were C/EBP[3, and C/EBPS. Amino acid sequences are displayed such then excised and measured for radioactivity by scintilla- that C/EBP[3 (top) and C/EBP8 (bottom) are compared with tion spectroscopy. Such assays revealed that with vari' C/EBPe~ (middle). Positions of identity are marked with an as- able rates, the amount of bound probe approached satu- terisk( *). The basic region of each protein, which is thought to ration within the range of input in all three sets of mediate direct contact with DNA, is enclosed by a solid line. assays, using polypeptides corresponding to C/EBPe~, Leucines organized in a heptad repeat are identified by dark shading. The i + 4 positions (relative to the heptad repeat of C/EBPI3, and C/EBP8 (Fig. 3, middle). The data were sub- leucines), which are thought to contribute to the hydrophobic jected to Scatchard analysis (Scatchard 1949), generating dimer interface, are indicated by light shading. The sequences the plots shown in the bottom panel of Figure 3. Such were aligned to reveal regions of similarity using a multiple analyses allowed calculation of dissociation constants sequence alignment program (Corpet 1988). (Kd) for each protein and revealed a hierarchy of binding

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affinities for the synthetic oligonucleotide wherein relatively high affinity to the same DNA sequence as C/EBP~ > C/EBPR > C/EBP8 (Fig. 3). Similar results C/EBP~ prompted us to test whether these two proteins were obtained in repeated experiments. Possible expla- might be capable of activating transcription. Previous nations for the observed differences in DNA-binding af- studies have demonstrated that C/EBPa can trans-acti- finity are outlined in the Discussion. vate the promoter of the serum albumin gene in transiently transfected hepatoma cells (Friedman et al. 1989). Gene-activating properties of the three C/EBP isoforms This same assay was used to test the potential of C/EBP~3 and C/EBP~ to activate transcription. The coding se- The confirmation that C/EBP~ and C/EBP8 bind with quences of both genes were cloned into a plasmid vector clwmw clvJ~f~ C/EBP8

Probe Pr~ Prol~

3, .j

/ /

• , . , • , . , . , . , . lo 20 30 40 50 60 70 • ,g " g " ~ " .~ " ~ " ~ " ~o °o i g " ,00

Total [nM] Total [riM] Total [nM]

1.2

y - 0.965-0.299x y - 2,834-X.153x |.0 .N4. " ...... R 2 - 0.936 • R 2- 0.866

kd - 3.344 nM • • kd - O.867 ,M O.3 • kd - 8.850 nH 0.8 ! 2, ! k 0.6 o.2 • lb •

~ o.4 2 i.

o.1 o.2

o.o ...... ,-~, . o 0 1 2 3 1 2 1 2

[Bound] nM [Bound] rd4 [Bound] nM

Figure 3. Measurements of relative DNA-binding affinities of C/EBP isoforms. Dissociation constant measurements for each C/EBP isoform were determined from quantitative evaluation of gel-shift assays visualized in the top panel of each column. [Top) Autora- diographic images identifying positions of bound and free probe. From left to right, each panel shows multiple lanes corresponding to binding reactions that contained a constant amount of purified polypeptide and increasing amounts of radiolabeled oligonucleotide containing a high-affinity C/EBPa-binding site (Shuman et al. 1990). Gel slices corresponding to the positions of bound and free forms of probe were excised and quantitated for radioactivity by scintillation spectroscopy. The amount of bound probe was plotted as a function of total input, as shown in each of the three middle panels. In the lower input range, the amount of bound probe increased rapidly with increased probe input. At higher input levels, however, the increase in bound probe was observed to reach a plateau level, apparently reflecting binding saturation. This apparent saturation was more evident in assays of C/EBPe~ and C/EBPB than of C/EBPS. (Bottom) Scatchard plots (Scatchard 1949) of saturation curves shown in middle panels. When plotted by the method of Scatchard (ratio of bound and free vs. bound) a linear correlation was observed between the two variables, allowing calculation of dissociation constants (Kd) wherein Kd = - 1/slope.

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in which expression of the particular c/ebp gene was Because the three isoforms of C/EBP are capable of under the control of the murine sarcoma virus long ter- binding to the same DNA sequence (see Fig. 3), it was minal repeat (Graves et al. 1985). Such expression vec- assumed that the observed transcriptional activation of tors were transfected into HepG2 cells along with a re- the albumin promoter occurred through the same cis- porter construct containing the promoter of the mouse regulatory sequence. To test this hypothesis, the reporter albumin gene linked to the herpes simplex virus (HSV) plasmid containing the native albumin promoter was re- thymidine kinase (tk)gene (Zaret et al. 1988). The ability placed with one containing a site-directed mutation in of the albumin promoter to direct transcription of the tk the C/EBPa-binding site (Friedman et al. 1989). The mu- gene in the absence or presence of the various C/EBP tated reporter plasmid was not expressed at a significant isoforms was measured by a primer extension assay with level in response to any of the three C/EBP isoforms. The RNA isolated from transiently transfected cells. Primer residual level of activation of the mutant plasmid might extension products were separated by gel electrophoresis result from protein bound to low-affinity sites within the and visualized by autoradiography. The results presented albumin promoter (Lichtsteiner et al. 1987). in Figure 4A show that hepatoma cells transfected with Western blots were conducted on protein extracts pre- the reporter construct alone failed to express the tk pared from transiently transfected cells to monitor the mRNA. In contrast, transcription of the tk gene became level of expression of the various C/EBP isoforms. Cell evident when the reporter plasmid was cotransfected cultures were transfected in duplicate with those used with expression vectors encoding each of the three for the primer extension assays described above. After C/EBP isoforms. According to this admittedly crude as- SDS-gel separation, proteins were transferred to nitro- say, the trans-activation potentials of the three C/EBP cellulose and probed with specific antisera raised against proteins differed less than threefold. synthetic peptides unique to the individual C/EBP iso-

43.0 kd -

29.0 kd -

18. 4 kd~ - i"

cl~ - ~ c/~ cI~6

Figure 4. Trans-activation of the promoter of the serum albumin gene by three isoforms of C/EBP. The gene-activating potentials of C/EBP[3 and C/EBP~ were compared with C/EBPoL with a transient transfection assay described previously (Friedman et al. 1989). Duplicate cultures of HepG2 cells were exposed to calcium phosphate precipitates containing 6 ~g of an albumin/tk reporter plasmid (pAT2) and 12 ~tg of one of three MSV-C/EBP expression plasmids (see Materials and methods). Two days after transfection one culture plate from each experiment was harvested for extraction of RNA and one plate was harvested for solubilization of total cell protein. Identical experiments were performed with a reporter plasmid (pAT2A) that was mutated at the high-affinity C/EBP-binding site within the albumin promoter (Friedman et al. 1989). (A) Autoradiographic exposures of sequencing gels that were used to size primer extension products. (Left) Products obtained with the pAT2 reporter plasmid; (right) products obtained with pAT2A. Locations of tk primer and extension products are designated. Left-most lanes of each experiment show primer extension reactions resulting from RNA prepared from HepG2 cells that had been transfected with the reporter plasmid alone. (B) Autoradiographic exposures of Western blots used to monitor expression of the three isoforms of C/EBP in transiently transfected HepG2 cells. Antisera used for Western blots are indicated below each autoradiographic exposure. Numbers shown to the left indicate positions of migration of molecular mass markers.

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forms (see Materials and methodsl. Multiple immunoreactive bands were visible on each filter. By the criteria of molecular size and transfection dependency, however, most bands could be identified as nonspecific, cross-re- active material. Transfection-dependent bands were readily detectable in the gel lanes containing proteins from cells transfected with C/EBP-encoding expression - 28S vectors. The molecular masses of these proteins, suggested by their rates of migration relative to molecular C/EBPu mass marker proteins, agreed well with the polypeptide - 18S sizes predicated by conceptual translation of the cloned genes (see Fig. 1). The results outlined in this section argue that the products of c/ebp[3 and c/ebp8 genes, like C/EBP~, can function as gene-activating proteins. - 28S c/~ Formation of heterodimers between C/EBP isoforms -~ 18S The observation that the c/ebp~ and c/ebp6 genes encode transcription factors closely related to C/EBPa raised the possibility that these factors might form mixed dimers. Substantial evidence has accumulated in- - 28S dicating that C/EBPa binds to DNA as an obligate dimer and that dimerization is mediated through the leucine C/E~8 - 18S zipper. The high degree of amino acid sequence similarity among the three C/EBP isoforms in their respective R a~ ~ leucine zippers, particularly along the hydrophobic dimer interface, strongly indicated that they would be Figure 5. Northern blot assays of mRNA accumulation of capable of forming heterodimers. Moreover, the fact that C/EBP isoforms in various mouse tissues. Total RNA was ex- cDNAs encoding all three isoforms were isolated from a tracted from indicated tissues of adult mice. Fifteen micrograms single cell type (adipocytesl suggested that all isoforms of RNA was loaded in each lane of a denaturing agarose gel. Ethidium bromide staining was used to ensure that each gel might be present simultaneously in a single cell. In an lane was loaded with an equivalent concentration of intact attempt to determine the generality of this observation, RNA (not shown). Sized RNAs were transferred to a nitrocellu- we examined the mRNA expression profile of the iso- lose membrane that was successively probed with radioactive forms in various mouse tissues. DNA corresponding to C/EBP~, C/EBPI3, and C/EBPK Autora- The results of Northern blot assays, shown in Figure 5, diographic images were obtained after each hybridization. Probe demonstrated that the three genes are expressed in a was removed between successive hybridization reactions by ex- common set of tissues (although the expression levels posing the filter to distilled water at 100°C for 5 min. The po- vary significantly). Similar to previous findings (Birken- sitions of 28S and 18S rRNA are indicated at right. meier et al. 1989), C/EBPa mRNA was found at abundant levels in liver and fat, and at low but detectable levels in intestinal and lung tissues. C/EBP[3 was de- pressed in E. coli and purified to homogeneity (see Ma- tected in all tissues examined with the exception of terials and methods). Two forms of C/EBPa, a 20- and a brain and testes. Relative to C/EBPa, C/EBP[3 mRNA 10-kD form, were prepared and used as a positive control levels were found to be quite high in intestine and lung. for dimer formation. Equal molar amounts of the 20- and C/EBP8 mRNA was most abundant in lung, intestinal, 10-kD polypeptides were incubated with a radiolabeled and adipose tissues. Such observations indicate that if a DNA fragment containing a high-affinity C/EBPet-bind- tissue expresses one type of C/EBP mRNA at a consid- ing site. After allowing protein-DNA complexes to erable level, it is likely to express at least one other type. form, the samples were subject to electrophoresis and Likewise, several tissues, such as brain and testes, fail to the positions of migration of the DNA probe were visu- express any of the three mRNA types at a considerable alized by autoradiography. Figure 6 shows that the 20- level. Assuming that mRNA levels reflect protein ex- and 10-kD C/EBPR polypeptides formed three complexes pression levels, it can be anticipated that the various that migrated slower than the free DNA. The fastest mi- isoforms are coexpressed in several different cell types. grating complex comigrated with the complex formed The results outlined in the previous paragraph when probe was mixed with the 10-kD species alone. prompted experiments that would test for the formation The slowest migrating complex comigrated with that of mixed dimers between the various C/EBP isoforms. formed with the 20-kD species. A third complex was Using the strategy developed by Hope and Struhl (1987), observed between the inferred 20- and 10-kD homodimer we performed gel-shift assays to test for heterotypic mix- complexes. The complex of intermediate mobility was ing of C/EBP isoform. Polypeptides of unique molecular interpreted to represent a heterodimeric complex com- size, corresponding to each C/EBP isoform, were exposed of one 20- and one 10-kD subunit.

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C/EBP~ 20kd - + + - + + - + + might help to regulate adipose conversion. As an initial C/EBP~ 10kd + + C/EBP~ 13kd + + + + goal we set out to determine the temporal pattems of C/EBP~ 9kd + + - + expression of C/EBP[3 and C/EBP8 during adipocyte differentiation. 3T3-L1 adipoblasts were induced to differentiate by using previously established methods (Stu- I bound dent et al. 1980). Cells were grown to confluence in growth medium containing calf serum and shifted to differentiation medium containing fetal calf serum, insulin, dexmethasone, and methylisobutylxanthine. Two free days later, dexmethasone and methylisobutylxanthine were removed. The progress of differentiation was mon- Figure 6. Assay for heterodimer formation between C/EBP iso- itored by measuring the accumulation of RNAs encoding forms. Purified polypeptides corresponding to each C/EBP iso- adipocyte-specific proteins (Bemlohr et al. 1984; Dobson form, and mixtures thereof, were exposed to a radiolabeled et al. 1987). Total RNA was harvested from cells imme- DNA fragment containing a high-affinity C/EBP-binding site. diately before and at daily intervals after the switch to Protein-DNA mixes were electrophoresed on a nondenaturing differentiation medium. The Northern blot images de- polyacrylamide gel (see Materials and methods). After electrophoresis the gel was dried and exposed to x-ray film. Bound and picted in Figure 7 show that expression of the mRNA free forms of the radiolabeled probe DNA are indicated. Poly- encoding fatty acid-binding protein 422 was first detect- peptides included in each binding reaction are indicated (+) able on day 3 and rose to a plateau level 7 days after the above each gel lane. switch to differentiation medium. Transcription of the gene encoding glycerol-3-phosphate dehydrogenase (GPDH) was initially detected at day 5 and reached a plateau level by the seventh day of the differentiation The ratio of the three C/EBPe~ complexes was program. The expression of these adipocyte-specific 1 : 2 : 1 corresponding, presumably, to homodimer 20 genes was accompanied by overt changes in cell mor- kD, heterodimer 20 kD/10 kD, and homodimer 10 kD. phology, including the accumulation of fat droplets (not The observed ratio of 1 : 2 : 1 is precisely that expected if shown). heterodimers form as readily as homodimers and have The RNA samples collected before and during differ- equivalent binding affinity for the target DNA. This re- entiation were also probed with radioactively labeled sult was anticipated as the two C/EBPa molecules bear cDNA fragments corresponding to each of the C/EBP- identical DNA-contact regions and dimerization inter- related genes. Consistent with earlier studies (Birken- faces. Figure 6 also shows the results obtained when the meier et al. 1989), the mRNA encoding for C/EBPa was radiolabeled DNA probe was incubated with C/EBPf~ or first detectable on day 2, increased gradually through day C/EBP8 polypeptides alone, in combination with one an- 6, and remained at an unchanged level thereafter. The other, or in combination with the 20-kD form of accumulation profiles of the C/EBP[3 and C/EBP~ C/EBPoL. When tested alone, each of the polypeptides mRNAs differed significantly from those of C/EBPa formed a complex of uniqtle mobility. When any combi- mRNA. The mRNAs encoding C/EBPB and C/EBP8 were nation of two isoforms was mixed, three complexes were detectable in undifferentiated adipoblasts. Furthermore, detected. Two of the three complexes corresponded to the mRNA levels of these two genes increased slightly the sizes of the parental homodimers, and one complex during the hormone-rich period early during the differ- exhibited an intermediate size. The size difference be- entiation program. Expression of C/EBPf~ mRNA was re- tween C/EBP[3 and C/EBP8 peptides was too small to duced during the latter half of the differentiation pro- allow a clear resolution of the three complexes in the gram, yet remained readily detectable. C/EBP~ mRNA autoradiogram that is shown in Figure 6. However, care- accumulation, on the other hand, was observed to be ful examination of a lighter autoradiographic exposure reduced dramatically during the final days of adipocyte revealed two species in the thick lower band. differentiation. In each reaction where different polypeptides were To test whether changes in mRNA levels of C/EBP mixed, the ratio of the complexes appeared to be isoforms were reflected by changes in the levels of their 1 : 2 : 1. However, one exception to this trend was ob- encoded proteins, SDS-solubilized extracts were pre- served. The presumed C/EBPoJC/EBP~ heterodimer pared from 3T3-L1 cells that had been differentiated in complex appeared to constitute a considerably higher parallel with those used for Northern blot analysis. Sol- proportion than expected. Possible explanations for this ubilized proteins were separated by SDS-PAGE gel elec- observation are addressed in the Discussion. trophoresis and analyzed by Western blot assays with antisera specific to each of the C/EBP isoforms {see Ma- terials and methods). The results of such studies, shown Regulation of C/EBP[3 and C/EBP3 expression during in Figure 8, indicated that the pattern of C/EBPa expres- adipocyte differentiation sion was similar to that of its encoding mRNA. The Having established an initial characterization of C/EBPB C/EBP~ polypeptide was undetectable in undifferenti- and C/EBPS, we undertook experiments designed to ask ated cells, first appeared at the fourth day of the program, whether these transcription factors, as with C/EBPoL, and increased in abundance throughout the remaining

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DAY.... : 0 ~ i: ,:2 ~ ....4 : :, 5 6 7 8 • sion precede the onset of C/EBP~ expression as well as the accumulation of adipocyte-specific mRNAs. This i c/r.sv~ temporal correlation, combined with previous studies showing that activation of C/EBP(x alone is insufficient to drive adipose conversion (Umek et al. 1991), raised the possibility that C/EBP~ and C/EBP8 regulate early events required for adipocyte differentiation. c/zm,13 The expression profiles of C/EBP~ and C/EBP8 also appeared to correlate with the presentation and with- drawal of adipogenic hormones, suggesting that these transcription factors might play a role in mediating cellular responses to such hormones. Following this lead we tested whether transcription of the c/ebp~ and c/ebp~5 C/ZBP~ genes might be regulated directly by adipogenic hormones (insulin, dexmethasone, and methylisobutylxanthine). 3T3-L1 adipoblasts were treated individually with insulin, dexmethasone, or methylisobutylxanthine m-qlllgl ,. in the presence of cycloheximide. Cycloheximide blocks protein synthesis and can therefore be used to discrimi- nate between direct and indirect action of hormones on the process of gene expression. After 4 hr of hormone treatment, RNA was prepared and analyzed by Northern lira ¢~oH blotting to determine the expression pattern of each C/EBP-related gene. The results shown in Figure 10 revealed an increase in C/EBP[3 mRNA in response to Figure 7. Measurements of accumulation of specific mRNAs methylisobutylxanthine and an increase in C/EBP~ during adipocyte differentiation. Total RNA was harvested mRNA in response to dexmethasone. The expression of from 3T3-L1 cells before (day 0) and at daily intervals after (days the C/EBP~ gene was undetectable in the presence of any 1-8) the switch from growth medium to differentiation medium (see Materials and methods). Ten-microgram aliquots of each of the three hormones, consistent with previous results RNA were size separated on five equivalent agarose electrophoresis gels. RNA was transferred to nitrocellulose and probed under stringent hybridization conditions with radioactive DNAs corresponding to the three C/EBP isoforms (C/EBP~, 0 1 2 3 4 5 6 7 8 C/EBPB, and C/EBPS) and two genes whose products are expressed in terminally differentiated adipocytes (fatty acid-binding protein 422 and glycerol-3-phosphate dehydrogenase). Each - C/EBPu mRNA migrated with an apparent size (not shown) consistent with that of previous reports (Bemlohr et al. 1984; Dobson et al. 0 1 2 3 4 5 6 7 8 1987; Landschulz et al. 1988a; Chang et al. 1990; see also Fig. 5).

days of differentiation. The expression profiles of the C/EBP~ and C/EBP~ polypeptides differed somewhat 0 1 2 3 4 5 6 7 8 from the profiles of their encoding mRNA. Specifically, both C/EBP[3 and C/EBP8 exhibited accentuated accu- ...... • CRM mulation upon hormone treatment relative to their ~,,~- - ClEBP8 mRNAs. That is, although both mRNAs were observed to increase slightly in abundance between days 0 and 1, the protein levels increased much more substantially Figure 8. Measurements of accumulation of various C/EBP- during this period. Such findings raise the possibility related polypeptides during adipocyte differentiation. Cultures that the expression of C/EBP[3 and C/EBP~ proteins is of 3T3-L1 cells were programmed to differentiate in parallel regulated at both transcriptional and post-transcriptional with those evaluated in Figure 7. Total cellular protein was levels. solubilized before (day OI and at daily intervals after (days 1-8) The expression profiles of the three C/EBP-related pro- the switch from growth medium to differentiation medium (see teins are plotted as a function of adipocyte differentia- Materials and methods). Proteins were electrophoresed on a denaturing polyacrylamide gel, transferred to nitrocellulose, and tion in Figure 9 (top). The times of addition and removal probed by Western blotting with antibodies specific to each of adipogenic hormones are also indicated. In Figure 9 C/EBP isoform. The positions of migration of each isoform are (bottom), the expression profiles of the adipocyte-spe- indicated at the right of each autoradiographic exposure. Cross- cific mRNA are similarly displayed. Such analyses show reacting materials (CRM) observed in samples probed with an- that concerted changes in C/EBP[3 and C/EBP8 expres- tibodies specific to C/EBPa and C/EBP~ are indicated.

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ADD: REMOVE: rich in basic amino acids to a region containing a heptad DEX DEX MIX MIX repeat of hydrophobic amino acids (often leucines). The INSULIN two regions have been proposed to occur in a contiguous c~-helical conformation. The (x-helix of the leucine repeat region is believed to intertwine with the analogous re- 100 gion of a second bZIP protein, forming a dimer. The basic regions of the paired subunits are believed to interact u a with each half of a dyad-symmetric DNA-binding site, |I ll I iI II I forming a "scissors grip" around the DNA substrate i| II I |I II I i I l1 I (Landschulz et al. 1988b, 1989; O'Shea et al. 1989; Vin- i I II l i f |l I son et al. 1989). 50 ,' I k Inspection of the primary amino acid sequences of bZIP proteins has shown that certain members of this

I I % I" family of proteins are more closely related than others. I~ %~ I For example, groupings of Fos-related proteins (Van Bev- ~ • , k'" "'~--• C/EBP6 eren et al. 1983; Cohen and Curran 1988; Zerial et al. ,4 L__~__~__~ ~ 1989; Nishina et al. 1990), Jun-related proteins (Maki et al. 1987; Ryder and Nathans 1988; Hirai et al. 1989; Ry- 0 1 2 3 4 5 6 7 8 der et al. 1989), and ATF/CREB-related proteins (Hoeffler et al. 1988; Lin and Green 1988; Hai et al. 1989; Berko- ADD: REMOVE: witz and Gilman 1990; Ivashkiv et al. 1990) have been described. Differences in amino acid sequence in the 100 bZIP region of these proteins influence two of their GPDH known functions--DNA-binding specificity and dimer- I:/ ization specificity. For example, the basic regions of ! C/EBP~ and GCN4, a bZIP protein that regulates genes i encoding enzymes necessary for de novo amino acid bio- synthesis in budding yeast (Struhl 1989), have been 50

! I I ! I ,-----.,;:I--Z. ...-llc. - .li I

0 1 2 3 4 5 6 7 8

DAYS OF DIFFERENTIATION Figure 9. Graphic representation of the temporal expression profiles of C/EBP isoforms and adipocyte-specific mRNAs during terminal differentiation. (Top)Accumulation of C/EBP isoforms as measured by Western blotting. The graph plots the percent maximal concentration of each protein (ordinate) as a function of the temporal order of differentiation [abscissa). {Bot- tom} A similarly arranged graph plotting the accumulation of 422 and GPDH mRNA. Data points were obtained by densito- metric scanning of the autoradiograms shown in Figs. 7 and 8. The times of addition (day 0) and removal [day 2) of adipogenic hormones [dexamethasone (DEX), methylisobutylxanthine {MIX), Figure 10. Evidence that genes encoding C/EBPI3 and C/EBP~ insulin] are indicated at top. are primary targets of adipogenic hormones. Undifferentiated adipoblast cells were preincubated with cycloheximide for 1 hr before challenge with individual hormones. Cells were incubated for 4 hr after addition of hormones. Total RNA was ex- demonstrating that C/EBP~ mRNA is not expressed in tracted from cells exposed to cycloheximide alone {CHX), or in proliferating adipoblasts. combination with individual adipogenic hormones; insulin (CHX + INSI, methylisobutylxanthine {CHX+ MIX), and dexamethasone (CHX+DEX). RNA samples were loaded in tripli- Discussion cate onto a denaturing electrophoresis gel, size-separated, transferred to nitrocellulose, and probed with radiolabeled DNA frag- C/EBPe~ is one of many proteins that contains a bZIP ments corresponding to C/EBP~ (top), C/EBP~ (midclle), and DNA-binding domain. DNA-binding proteins of this C/EBPc~ (bottom). Sizes of hybridizing RNA bands (not shown) class are characterized by the juxtaposition of a region were consistent with prior measurements (see Fig. 5).

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shown to dictate DNA-binding specificity (Agre et al. First, cDNAs encoding three forms of C/EBP have been 1989). Likewise, the leucine repeat segment of bZIP pro- isolated from the same cell type (differentiating adipo- teins has been found to dictate dimerization specificity. cytes). Second, a positive correlation was observed in the Fos-related proteins dimerize preferentially with Jun-re- distribution of tissues that express the three C/EBP isolated proteins (Halazonetis et al. 1988; Kouzarides and forms characterized in this study. Thus, we anticipate Ziff 1988; Rauscher et al. 1988; Sassone-Corsi et al. that certain cell types will simultaneously express more 1988; Smeal et al. 1989), yet neither Fos- nor Jun-related than one form of C/EBP, thereby offering the opportunity proteins will cross-dimerize with C/EBPot (Y. Nakabeppu for the formation of heterodimeric complexes. and W. Landschulz, unpubl.). Given the expectation that different C/EBPs will Several genes encoding proteins bearing a high degree cross-dimerize in vivo, we have referred to the variant of amino acid sequence similarity to the bZIP region of proteins as "isoforms." This term emphasizes the simi- C/EBPa have recently been isolated and characterized. larity between C/EBP variants. It is important, however, The first C/EBP-related gene to be characterized encodes to point out significant differences in the C/EBP iso- a protein termed NF-IL6 (Akira et al. 1990). The gene forms characterized to date. First, they are encoded by encoding NF-IL6 was cloned by DNA ligand-mediated different genes. Indeed, C/EBPet, C/EBPI3, and C/EBP8 are screening of an expression library prepared from human located on entirely different mouse chromosomes (E. glioblastoma cDNA. Recombinant DNA clones corre- Birkenmeier, Z. Cao, and S. McKnight, unpubl.). Second, sponding to the rat and mouse homologs of NF-IL6 were the amino acid sequences of the amino-terminal two- subsequently isolated. The protein products of these thirds of each of the C/EBP isoforms are not detectably genes have been variously termed LAP, IL-6DBP, and related (see Fig. 2; Roman et al. 1990). The amino-termi- AGP/EBP according to the perceived function of the pro- nal two-thirds of C/EBPa contains functional determi- tein (Chang et al. 1990; Descombes et al. 1990; Poli et al. nants important for its role in gene activation (Friedman 1990). Another closely related gene, encoding a protein and McKnight 1990; Pei and Shih 1991) and in mitotic termed Ig/EBP-1, has been isolated by DNA ligand-me- growth arrest (Umek et al. 1991). We anticipate that the diated screening of cDNAs prepared from cultured amino-terminal regions of C/EBP~3 and C/EBP8 will also mouse fibroblasts (Roman et al. 1990). Ig/EBP-1 binds perform important regulatory functions, perhaps distinct avidly to the E site of the immunoglobulin heavy-chain from that of C/EBPoL. For example, C/EBPI3 (NF-IL6) has gene enhancer. NF-IL6 (as well the homologous proteins been implicated in cellular response to interleukin 6 from rat and mouse) and Ig/EBP-1 bind DNA with a spec- (Poli et al. 1990). It is possible that the amino-terminal ificity similar to C/EBPe~ (Akira et al. 1990; Chang et al. region of C/EBP~3 is responsible for mediating the regu- 1990; Descombes et al. 1990; Poli et al. 1990; Roman et latory effects of interleukin 6. al. 1990), and both proteins have been shown to be ca- The heterodimerization between different C/EBP iso- pable of cross-dimerization with C/EBPa (Descombes et forms provides an additional level of complexity in ad- al. 1990; Poli et al. 1990; Roman et al. 1990). dressing the functional properties of this group of tran- Here we report the isolation of genes encoding two scription factors. As yet, we have not developed assays C/EBP-related proteins. One of the genes we have stud- for the function of C/EBP heterodimers. Each of the three ied, c/ebpfl, is the mouse homolog of NF-IL6. The other protein forms characterized in this study is capable of gene characterized in this report, c/ebp6, is distinct from avid interaction with DNA, and heteromeric dimers also other known C/EBP-related genes. Judging by the num- bind DNA avidly. When C/EBP~ and C/EBP~ were ber of C/EBP-related genes isolated in the past year, the mixed, the presumed heterodimeric complex was ob- size of the family can be expected to expand. In an at- served at a higher than expected ratio relative to the two tempt to clarify the confusing nomenclature that has homomeric complexes [see Fig. 6). We have not resolved evolved for proteins encoded by C/EBP-related genes, we the molecular basis for this somewhat unexpected ob- propose that proteins capable of cross-dimerization with servation. However, if bona fide, this observation might C/EBP be designated by C/EBP followed by a Greek letter indicate that a cell harboring equal amounts of both iso- indicative of their chronological order of characteriza- forms would contain relatively small amounts of ho- tion. According to this system, the first protein to be modimeric C/EBPa or C/EBP& Taking such speculation characterized by study of its encoding gene, originally one step further, one might imagine that certain func- termed C/EBP, will now be called C/EBPa. Similarly, tions of C/EBPa might require the protein in a ho- NF-IL6 will be termed C/EBPI3, Ig/EBP-1 will be termed modimeric form (e.g., during the late phase of adipocyte C/EBP% and the new family member characterized in differentiation}. If so, constitutive expression of C/EBP~ this study will be termed C/EBP& might impede the formation of C/EBPa homodimers and The critical criterion for assigning a bZIP protein to thereby subvert their hypothetical function. the C/EBP family is dimerization specificity. If proteins Quantitative DNA-binding assays of the various cross-dimerize readily in vitro, they may also cross- C/EBP isoforms revealed a hierarchy of avidities wherein dimerize in vivo. If so, their functional properties may be C/EBPI3 bound DNA more tightly than C/EBPa, which, integrated into a restricted "pod" of interacting proteins. in turn, bound DNA more tightly than C/EBP& We do Although in vivo cross-mixing of C/EBP isoforms has yet not know whether these differences in binding affinity to be conclusively demonstrated, several observations are meaningful, particularly given that they were ob- outlined in this report may be relevant to this issue. served with fragments derived from the various C/EBP

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isoforms. However, it is perhaps understandable that tory proteins other than those characterized in this C/EBP~ binds DNA less avidly than the other two iso- study. We do, however, find the temporal expression pat- forms. C/EBP8 lacks a stretch of basic amino acids com- terns of the three C/EBP isoforms sufficiently compel- mon to C/EBP~ and C/EBPB (see Fig. 2). A variant form ling to warrant further study and anticipate that efforts of C/EBP~ lacking this amino-terminal extension of the to discern and assign unique properties to each isoform "basic region" has been found to bind DNA severalfold will help elucidate potential roles for these transcription less avidly than the native polypeptide (Landschulz et al. factors during terminal cell differentiation. 1989). We hope that expanded knowledge of the specific roles played by different C/EBP-related proteins will eventu- Materials and methods ally provide a means of addressing the putative functions Cloning of c/ebp~3 and c/ebp8 genes of heterodimeric complexes. In this regard we pay par- Poly(A) + RNA was selected from total RNA extracted from ticular interest to the temporally regulated patterns of differentiating 3T3-L1 cells. A cDNA library was constructed C/EBP isoform expression during adipogenesis. Observa- using the ?~ZAP unidirectional cloning kit (Stratagene). The li- tions made thus far point to an important role for brary was screened at reduced hybridization stringency (Lee C/EBP~ in terminal differentiation of adipocytes. The 1990) with a 150-bp DNA probe corresponding to the bZIP do- temporal pattern of C/EBPc~ expression correlates with main of C/EBPa. Thirty bacteriophage, obtained from a screen the induction of adipocyte-specific gene expression and of 3 x l0 s plaques, survived tertiary screening. Eighteen of the the cessation of mitotic growth (Birkenmeier et al. 1989). recombinants tested positive when probed with a 1-kb HindIII- Premature expression of C/EBPc~ causes adipoblasts to EcoRI DNA fragment corresponding to the 3'-untranslated re- arrest mitotic growth, yet does not lead directly to adi- gion of the C/EBPc, mRNA (Landschulz et al. 1988a). These clones were judged to contain cDNA copies of the C/EBPc~ pocyte differentiation (Umek et al. 1991). The latter ob- mRNA and were discarded. Bluescript plasmids containing servation suggests that C/EBPc~ may not be the immedi- cDNA inserts were excised from the remaining 12 bacterio- ate target regulated by adipogenic hormones. Consistent phage isolates following recommended procedures (Stratagene). with this interpretation, the onset of C/EBP~ expression Eleven of these plasmids yielded a positive hybridization signal is delayed relative to the time of hormonal challenge when challenged at high stringency with the cDNA insert de- (Figs. 7 and 8; Birkenmeier et al. 1989), and hormonal rived from clone 5. The clone 5 cDNA insert was the largest of stimulation does not activate transcription from the the 12 and was later designated c/ebpfl. Clone 18, which did not gene encoding C/EBP~ in the presence of a protein syn- cross-hybridize to the clone 5 insert, was later designated thesis inhibitor (Fig. 10). c/ebpS. Both c/ebp[3 and c/ebpfl were sequenced by the dideox- In contrast to C/EBP~, the regulated patterns of ex- ynucleotide chain-termination method with a commercial sequencing kit (Pharmacia). pression of C/EBPI3 and C/EBP8 might implicate these Genomic DNA clones corresponding to c/ebpfl and c/ebp3 isoforms as mediators of adipoblast response to adipo- were also isolated and sequenced. The genomic clones were genic hormones. The expression levels of both C/EBPB found to contain DNA sequences colinear with their respective and C/EBP8 are increased dramatically during the time partial cDNAs. Sequence analysis of the genomic clones also of hormonal stimulation (see Fig. 8). Furthermore, the revealed that each contained an intron-free open reading frame. C/EBPI3- and C/EBP~-encoding genes are activated di- Furthermore, TATAAA homologies were found in close prox- rectly by adipogenic hormones (Fig. 10). Finally, the lev- imity 5' to the presumptive translation initiation codon, and els of these two proteins decline after the removal of putative polyadenylation sites were found in close proximity 3' hormones, before the overt onset of C/EBPa expression. to the translation termination codons. The sequential expression of C/EBP isoforms raises the possibility that C/EBP~3 and C/EBP~ are responsible Overexpression and purification of C/EBP[3 and C/EBP~ for the induction of C/EBPtx. Consistent with this pos- polypeptides sibility, Lane and co-workers have discovered an avid Polypeptides corresponding to the bZIP domains of C/EBPf3 and C/EBP-binding site in the promoter of the gene encoding C/EBP8 were overexpressed in E. coli with an expression system C/EBPe~ (Christy et al. 1991). Moreover, these investiga- directed by bacteriophage T7 RNA polymerase (Studier and tors have found that extracts prepared from differentiat- Moffatt 1986). Partial cDNA sequences of c/ebp~ and c/ebp8 ing adipocytes contain proteins, in addition to C/EBPc~, were cloned into a pET3 vector {Rosenberg et al. 1987) with the that bind to the C/EBP-binding site located within the correct open reading frame. The resulting plasmids express a C/EBPe~ promoter. Even if C/EBP~ and C/EBP~ do serve 13-kD C/EBPf3 polypeptide and a 9-kD C/EBP8 polypeptide. to induce expression of C/EBPa, such a simple regulatory Each polypeptide contains the entire DNA-binding domain. cascade cannot fully explain the process of adipocyte The C/EBP~ and C/EBP~ polypeptides were expressed in the differentiation. If the only role of the "early" C/EBP BL21 strain of E. coli and purified to homogeneity with column chromatographic techniques as described earlier (Shuman et al. isoforms (C/EBPB and C/EBPS) was to activate the ex- 1990). pression of the "late" form (C/EBPa), then premature expression of C/EBPa should lead directly to terminal differentiation--which it does not (Umek et al. 1991). Trans-activation of the albumin promoter by C/EBP[3 We have provided no direct evidence indicative of a and C/EBP3 functional role for C/EBPI3 and C/EBP8 during adipogen- The vector used to express full length of C/EBP[3 and C/EBP8 in esis. We also do not exclude the involvement of regula- eukaryotic cells was the same as was used to express C/EBPa

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(Friedman et al. 1989). Protein-encoding genomic DNA se- cording to the procedures of Chomczynski and Sacchi (1987). quences of c/ebp[3 and c/ebp8 were cloned into the vector be- Fifteen-microgram amounts of each RNA sample were electro- tween the Moloney routine sarcoma virus long terminal repeat phoresed through denaturing formaldehyde-agarose gels (Leh- (Graves et al. 1985) and the transcription termination/polyade- rach et al. 1977), transferred to GeneScreen membranes (NEN), nylation signals of the HSV tk gene (McKnight 1980). and probed with radiolabeled DNA fragments corresponding to Transfection of hepatoma cells was performed according to the three C/EBP isoforms. the calcium phosphate method (Graham and Van der Eb 1973). The reporter plasmid used to test for trans-activation by the various C/EBP isoforms was pAT2 (Zaret et al. 1988), which Preparation of rabbit antisera specific to C/EBP[3 and C/EBP~ consists of the promoter of the mouse serum albumin gene Synthetic peptides corresponding to the carboxy-terminal se- fused to the mRNA-coding segment of the HSV tk gene. Proce- quences of C/EBP~ (NH2-LRNLFKQLPEPLLASAGHC-COOH) dures for cell culture, transient transfection, RNA preparation, and C/EBP8 {NH2-LRQFFKKLPSPPFLPPTGADCR-COOH) were and primer extension have been described previously (Friedman prepared on an Applied Biosystems 430A peptide synthesizer. et al. 1989). Peptides were purified by high-performance liquid chromatog- raphy {HPLC), covalently coupled to keyhole limpet hemocya- Gel mobility-shift assays nin, and used to immunize rabbits as described previously The DNA probe used in gel mobility-shift assays (Fried and (Landschulz et al. 1988a). Antiserum specific to a 14-amino-acid Crothers 1981) was a 136-bp BglII-EcoRI fragment of the HSV tk peptide of C/EBPa has been described previously (Landschulz et gene modified to contain a high-affinity C/EBPa-binding site al. 1988a}. The specificity of the antiserum preparations was (Vinson et al. 1989). The probe DNA was radiolabeled with tested with bacterially expressed polypeptides corresponding to [etazP]dCTP and [~32p]dATP with a fill-in reaction. Polypeptides the bZIP domains of each C/EBP isoform. No cross-reactivity corresponding to the 20- and 10-kD forms of C/EBPa (Shuman was observed between the various antisera and polypeptides, et al. 1990), the 13-kD form of C/EBPI3, and the 9-kD form of consistent with the fact that the sequences of the synthetic C/EBP8 were added to binding reactions either alone or as mix- antigens were unrelated. tures prepared at a final concentration of 0.5 fM. Binding reactions (20 ~1) consisted of 10 mM Tris-HC1 (pH 7.5), 2 mM DTT, Western blot analysis 50 mM KC1, 10% (vol/vol) glycerol, 0.5 ~tg of poly[d(I-C)] (Sigma), and 1 mg/ml of bovine serum albumin (Boehringer Mannheim HepG2 cells transfected with plasmids expressing different Biochemicals). Before addition of probe DNA, binding reactions C/EBP isoforms were washed with phosphate-buffered saline were incubated at room temperature for 10 min to allow equil- {PBS) and scraped off of the tissue culture dish in 1 ml of PBS. ibration of polypeptide dimers. Probe DNA was added to a final Cells were pelleted in a microcentrifuge tube, lysed by the ad- concentration of 1-2 fM, and reactions were incubated for 20 dition of 100 ~1 of SDS-gel sample buffer, and heated to 100°C rain at room temperature before being loaded onto a 5% poly- for 5 min. Twenty-five-microliter aliquots of each sample were acrylamide gel containing 0.5x TBE buffer and 5% glycerol. electrophoresed through a 10% SDS-polyacrylamide gel {Laem- Gels were electrophoresed at a constant rate of 300 V at 4°C for mli 1970}. Electrophoresis proteins were then transferred to ni- 5 hr, dried, and exposed to x-ray film without an intensifying trocellulose filters and probed with appropriate antiserum prep- screen. arations {Towbin et al. 1979). Immunoreactive polypeptides Saturation-binding assays were carried out under the condi- were visualized by the ECL detection system (Amersham), fol- tions described above with a double-stranded oligonucleotide lowing procedures recommended by the supplier. To monitor probe containing a high-affinity C/EBP~-binding site (Shuman the expression patterns of C/EBP isoforms as a function of 3T3- et al. 1990). The DNA probe consisted of the self-complimen- L1 cell differentiation, protein extracts were prepared and used tary, dyad-symmetric sequence 5'-TGCAGATTGCGCAATC- for Western blot analyses as described previously (Friedman et TGCA-3' and was labeled at the 5' termini with [~32p]ATP and al. 19891. polynucleotide kinase. After gel electrophoresis and autoradiography, radioactive bands corresponding to free and protein- bound DNA probe were excised and quantitated by counting in Acknowledgments a scintillation spectrophotometer. We thank Dr. Ion Shuman for providing purified polypeptides corresponding to C/EBPa and for his extensive help in preparing Induction of 3T3-LI cell differentiation and purifying bacterially expressed polypeptides corresponding Culture and differentiation induction of 3T3-L1 cells was per- to C/EBPB and C/EBPS. We acknowledge the extensive help of formed as described previously (Student et al. 1980). Briefly, Brien Byers for the preparation and purification of synthetic cells were propagated to confluency in 90-mm plastic plates in peptides, Robert Kingsbury and Elizabeth Ryan for expert tech- Dulbecco's modified Eagle medium {DMEM) containing 10% nical assistance, and Ed Birkenmeier for communicating unpub- fetal calf serum. On differentiation day 0, cells were fed with lished observations regarding the genetic mapping and tissue DMEM supplemented with 10% fetal calf serum, 0.5 mM meth- distributions of expression of C/EBPB and C/EBPK Finally, we ylisobutylxanthine, 10 ~g/ml of insulin, and 10 -6 M dexameth- acknowledge contributions made by Peter Johnson, who first asone. Cells were changed to culture medium lacking methyl- discovered that a radiolabeled DNA fragment corresponding to isobutylxanthine and dexamethasone 2 days later (differentia- the DNA-binding domain of C/EBP was capable of hybridizing tion day 2) and harvested at daily intervals for analysis of pro- to multiple restriction fragments of mammalian DNA. During tein and mRNA accumulation. the course of these studies, Johnson isolated genomic copies of genes encoding three C/EBPa-related proteins This work was Northern blot analysis supported by funds provided to S.L.M. by the Howard Hughes Medical Institute and the Carnegie Institution of Washington. Total RNA was extracted from tissues of adult mice (C57BL6; R.M.U. was supported by a postdoctoral fellowship from the Jackson Laboratories} or from differentiating 3T3-L1 cells ac- Leukemia Society of America, and Z.C. was supported by a

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postdoctoral fellowship from the Howard Hughes Medical In- polypeptide segments of CCAAT/enhancer-binding protein stitute. required for transcriptional activation of the serum albumin The publication costs of this article were defrayed in part by gene. Genes & Dev. 4" 1416-1426. payment of page charges. This article must therefore be hereby Friedman, A.D., W.H. Landschulz, and S.L. McKnight. 1989. marked "advertisement" in accordance with 18 USC section CCAAT/enhancer binding protein activates the promoter of 1734 solely to indicate this fact. the serum albumin gene in cultured hepatoma cells• Genes & Dev. 3: 1314-1322. Graham, F.L. and A.J. Van der Eb. 1973. A new technique for the References assay of infectivity of human adenovirus 5 DNA. Virology 52: 456--457. Agre, P., P.F. Johnson, and S.L. McKnight. 1989. Cognate DNA Graves, B.J., R.N. Eisenman, and S.L. McKnight. 1985. 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Induction of protooncogene c-jun by serum growth factors. Proc. Natl. Acad. Sci.

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Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells.

Z Cao, R M Umek and S L McKnight

Genes Dev. 1991, 5: Access the most recent version at doi:10.1101/gad.5.9.1538

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