Proc. Natl. Acad. Sci. USA
Vol. 85, pp. 7857-7861, November 1988
Biochemistry

A gene activated in mouse 3T3 cells by serum growth factors
encodes a protein with “zinc finger” sequences

(transcription factors /serum response element)

BARBARA A. CHRISTY, LESTER F. LAu*, AND DANIEL NATHANS
Howard Hughes Medical Institute Laboratory and the Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine,

Baltimore, MD 21205

Contributed by Daniel Nathans, June 21, 1988

ABSTRACT We have recently identified by cDNA cloning
a set of genes that are rapidly activated in mouse 3T3 cells by
serum or purified growth factors. Here we report that the
cDNA (clone 268) derived from one of these immediate early
genes (zif/268) encodes a protein with three tandem “zinc
finger’’ sequences typical of a class of eukaryotic transcription
factors. The mRNA of zif/268 is present in many organs and
tissues of the mouse and is especially abundant in the brain and
thymus tissue. The 5’ genomic flanking sequence of zif/268 has
sequences related to binding sites for known regulatory pro-
teins, including four sequences that resemble the core of the
serum response elements (SREs) upstream of the c-fos and actin
genes. The SRE-like sequences could be responsible for the
coordinate activation of zif/268 and fos after serum stimulation
of 3T3 cells.

 

Growth factors and other extracellular ligands induce se-
quential changes in gene expression in target cells (1-4). The
initial changes are thought to occur via receptor-mediated
activation of messenger systems that modify preexisting
transcriptional regulators. Subsequent changes in gene
expression are presumably mediated by proteins encoded by
genes that are activated in the initial response. We and others
have been characterizing genes that are rapidly and tran-
siently activated after stimulation of resting murine 3T3
fibroblastic cells in culture with serum, platelet-derived
growth factor (PDGF), or fibroblast growth factor (FGF) (1,
3-7) in the expectation that some of these genes encode
transcriptional regulators. Among the ‘‘immediate early”
genes so far identified that appear to encode such proteins are
c-fos (8), junB (9), c-jun (ref. 10; K. Ryder and D.N.,
unpublished results), Krox 20 (11), and fra-/ (12). Here we
report that one of the rapidly activated genes previously
identified by cDNA cloning (clone 268, ref. 4) encodes a
protein with three ‘‘zinc finger’’ sequences (13) characteristic
of a class of eukaryotic transcription factors, including
TFINA (14), Spl (15), and SW15 (16). This mouse gene,
which we call zif/268, is homologous to a gene activated in rat
pheochromocytoma cells by nerve growth factor (17). The
same gene has also been identified recently by Sukhatme er

al, (18).

MATERIALS AND METHODS

Cell Culture. BALB/c 3T3 cells were maintained and
stimulated with serum as previously described (3). Transfec-
tion of NIH 3T3 cells was carried out by using the procedure
of van der Eb and Graham (19) followed by glycerol shock
(20). Chloramphenicol acetyltransferase (CAT) enzyme ac-
tivity was assayed as described (21). For measuring serum
induction of the zif/CAT constructs, duplicate cultures were

 

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7857

transfected in medium containing 10% fetal bovine serum and
then washed three times with medium containing 0.5% fetal
bovine serum. After glycerol shock and Hepes-buffered
saline rinse, cells were incubated in medium containing 0.5%
fetal bovine serum. At appropriate times prior to harvest (48
hr after transfection), fetal bovine serum was added directly
to the medium to a final concentration of 20%.

DNA Sequencing. zif/268 cDNA isolates were cloned into
Bluescript plasmids (Stratagene), and sets of 5’ and 3’
deletions were prepared by using exonuclease ITI [kindly
provided by B. Weiss (Johns Hopkins University) or pur-
chased from Promega Biotec, Madison, WI]. The resulting
subclones were sequenced by the dideoxynucleotide chain-
termination method (22), using deoxyadenosine 5’-[a-
[(?>S]thio]triphosphate and Sequenase (United States Bio-
chemical, Cleveland).t

Primer Extension and S1 Nuclease Assay. These procedures
were carried out as described (9). The primer used was a
32P.labeled oligonucleotide complementary to nucleotides
62-84 of the zif/268 RNA (Fig. 1).

In Vitro Transcription and Translation. In vitro transcrip-
tion and translation were carried out as described (23), except
that T7 RNA polymerase was used for transcription. The
cDNA template was the longest cDNA clone (Fig. 1) or a
subcloned Bel II restriction fragment containing nucleotides
338-1994.

RNA Biot Hybridization. This procedure was performed as
described (2) with 15 yg of total cellular RNA and randomly
primed (?*P]DNA probes.

In Situ Hybridization. Adult male BALB/c mice were killed
by asphyxiation and the brains were fixed and processed for
in situ hybridization as described (24), except that 5% acetic
acid/4% formaldehyde/85% ethanol (vol/vol) was used as a
fixative. Hybridization of tissue sections with *°S-labeled
sense or antisense RNA probe was carried out as described
(25) with slight modification.

RESULTS

Nucleotide Sequence of zif/268 cDNA. zif/268 mRNA
belongs to a class of mRNAs that we have called immediate
early mRNAs (4). It appears within minutes of treatment of
quiescent BALB/c 3T3 cells with serum, PDGF, or FGF and
is superinduced in the presence of a protein synthesis
inhibitor. A clone representing the 3’ end of zif/268 (clone 268
of ref. 4) was used to isolate a nearly full length CDNA clone
from a library prepared from poly(A)* RNA of BALB/c 3T3

 

Abbreviations: PDGF, platelet-derived growth factor; FGF, fibro-
blast growth factor; SRE, serum response element; CAT, chloram-
phenicol acetyitransferase.

*Present address: Department of Molecular Biology, Northwestern
University Medical School, 303 East Chicago Avenue, Chicago, IL
60611.

tThe sequence reported in this paper is being deposited in the
EMBL/GenBank data base (accession no. J04089).
7858 Biochemistry: Christy et al. Proc. Natl. Acad. Sci. USA 85 (1988)

cells 3 hr after stimulation by serum in the presence of cyclo- The 5’ end of the mRNA was identified by two methods,
heximide. By gel electrophoresis, the size of the zif/268 mRNA reverse transcription of the RNA using an oligonucleotide
was estimated to be about 3.3 kilobases (kb). The sequence primer and S1 nuclease analysis using a probe made with the
obtained from cDNAs of 2.8 and 3.2 kb is shown in Fig. 1. same primer. Both procedures resulted in products with

CGAGAGATCCCAGCGCGCAGAACT TGGGGAGCCGCCGCCGCGATTCGCCGCCGCCGCCAGCT TCCGCCGCCGCAAGATCGGCCCCTGCCCCAG
CCCTGCCCCAGCCTCCGCGGCAGCCCTGCGTCCACCACGGGCCGCGGCTACCGCCAGCCT GGGGGCCCACCTACACTCCCCGCAGT GT GCCCCTGCACC
CCGCATGTAACCCGGCCAACCCCCGGCGAGTGTGCCCTCAGTAGCTT CGGCCCCGGGCTGCGCCCACCACCCAACATCAGTICTICCAGCTCGCTGGTCC

292 GGG ATG GCA GCG GCC AAG GCC GAG ATG CAA TTG ATG TCT CCG CTG CAG ATC TCT GAC CCG TTC GGC TCC TTT CcT 24
MET Ala Ala Ala Lys Ala Glu MET Gln Leu MET Ser Pro Lev Gin Ile Ser Asp Pro Phe Gly Ser Phe Pro

367 CAC TCA CCC ACC ATG GAC AAC TAC CCC AAA CTG GAG GAG ATG ATG CTG CTG AGC AAC GGG GCT CCC CAG TTC CTC 49
His Ser Pro Thr MET Asp Asn Tyr Pro Lys Leu Glu Glu MET MET Lev Leu Ser Asn Gly Ala Pro Gln Phe Leu

442 GGT GCT GCC GGA ACC CCA GAG GGC AGC GGC GGT AAT AGC AGC AGC AGC ACC AGC AGC GGG GGC GGT GGT GGG GGC 74
Gly Ala Ala Gly Thr Pro Glu Gly Ser Gly Gly Asn Ser Ser Ser Ser Thr Ser Ser Gly Gly Gly Gly Gly Gly

517 GGC AGC AAC AGC GGC AGC AGC GCC TTC AAT CCT CAA GGG GAG CCG AGC GAA CAA CCC TAT GAG CAC CTG ACC ACA 99
Gly Ser Asn Ser Gly Ser Ser Ala Phe Asn Pro Gln Gly Glu Pro Ser Glu Gln Pro Tyr Glu His Leu Thr Thr

592 GAG TCC TTT TCT GAC ATC GCT CTG AAT AAT GAG AAG GCG ATG GTG GAG ACG AGT TAT CCC AGC CAA ACG ACT CGG 124
Glu Ser Phe Ser Asp Ile Ala Leu Asn Asn Glu Lys Ala MET Val Glu Thr Ser Tyr Pro Ser Gln Thr Thr Arg

667 TTG CCT CCC ATC ACC TAT ACT GGC CGC TTC TCC CTG GAG CCC GCA CCC AAC AGT GGC AAC ACT TTG TGG CCT GAA 149
Leu Pro Pro Ile Thr Tyr Thr Gly Arg Phe Ser Leu Glu Pro Ala Pro Asn Ser Gly Asn Thr Leu Trp Pro Glu

742 CCC CTT TTC AGC CTA GTC AGT GGC CTC GTG AGC ATG ACC AAT CCT CCG ACC TCT TCA TCC TCG GCG CCT TCT CCA 174
Pro Leu Phe Ser Leu Val Ser Gly Lev Val Ser MET Thr Asn Pro Pro Thr Ser Ser Ser Ser Ala Pro Ser Pro

817 GCT GCT TCA TCG TCT TCC TCT GCC TCC CAG AGC CCG CCC CTG AGC TGT GCC GTG CCG TCC AAC GAC AGC AGT CCC 199
Ala Ala Ser Ser Ser Ser Ser Ala Ser Glin Ser Pro Pro Leu Ser Cys Ala Val Pro Ser Asn Asp Ser Ser Pro

@92 ATC TAC TCG GCT GCG CCC ACC TTT CCT ACT CCC AAC ACT GAC ATT TIT CCT GAG CCC CAA AGC CAG GCC TTT CCT 224
Ile Tyr Ser Ala Ala Pro Thr Phe Pro Thr Pro Asn Thr Asp Ile Phe Pro Glu Pro Gln Ser Gln Ala Phe Pro

967 GGC TCG GCA GGC ACA GCC TTG CAG TAC CCG CCT CCT GCC TAC CCT GCC ACC AAA GGT GGT TTC CAG GIT CCC ATG 249
Gly Ser Ala Gly Thr Ala Leu Gln Tyr Pro Pro Pro Ala Tyr Pro Ala Thr Lys Gly Gly Phe Gln Val Pro MET

1042 ATC CCT GAC TAT CTG TTT CCA CAA CAA CAG GGA GAC CTG AGC CTG GGC ACC CCA GAC CAG AAG CCC TTC CAG GGT 274
Ile Pro Asp Tyr Leu Phe Pro Gln Gln Gln Gly Asp Leu Ser Leu Gly Thr Pro Asp Gln Lys Pro Phe Gin Gly

1117 CTG GAG AAC CGT ACC CAG CAG CCT TCG CTC ACT CCA CTA TCC ACT ATT AAA GCC TTC GCC ACT CAG TCG GGC TCC 299
Leu Glu Asn Arg Thr Gin Gln Pro Ser Leu Thr Pro Leu Ser Thr Ile Lys Ala Phe Ala Thr Gln Ser Gly Ser

1192 CAG GAC TTA AAG GCT CTT AAT ACC ACC TAC CAA TCC CAG CTC ATC AAA CCC AGC CGC ATG CGC AAG TAC CCC AAC 324
Gln Asp Leu Lys Ala Leu Asn Thr Thr Tyr Gln Ser Gln Leu Tle Lys Pro Ser Arg MET Arg Lys Tyr Pro Asn

1267 CGG CCC AGC AAG ACA CCC CCC CAT GAA CGC CCA TAT GCT TGC CCT GTC GAG TCC TGC GAT CGC CGC TTT TCT CGC 349
Arg Pro Ser Lys Thr Pro Pro His Glu Arg Pro Tyr Ala Cys Pro Val Glu Ser Cys Asp Arg Arg Phe Ser Arg

1342 TCG GAT GAG CTT ACC CGC CAT ATC CGC ATC CAC ACA GGC CAG AAG CCC TTC CAG TGT CGA ATC TGC ATG CGT AAC 374
Ser Asn Glu Leu Thr Arg His Tle Ara Tle His Thr Gly Gln Iys Pro Phe Gln Cys Arg Ile Cys MET Arg Asn

1417 TTC AGT CGT AGT GAC CAC CIT ACC ACC CAC ATC CGC ACC CAC ACA GGC GAG AAG CCT TIT GCC TGT GAC ATT TGT 399
Phe Ser Arg Ser Asp His Leu Thr Thr His Ile Arg Thr His. Thr Gly Glu lvs Pro Phe Ala Cys Aso Jle Cys

1492 GGG AGG AAG TTT GCC AGG AGT GAT GAA CGC AAG AGG CAT ACC AAA ATC CAT TTA AGA CAG AAG GAC AAG AAA GCA 424
Gly Ara Lys Phe Ala Arg Ser Aso Glu Aro Ivs Aro His Thr Lys Ile Hig Leu Arg Gln Lys Asp Lys Lys Ala

1567 GAC AAA AGT GTG GTG GCC TCC CCG GCT GCC TCT TCA CTC TCT TCT TAC CCA TCC CCA GTG GCT ACC TCC TAC CCA 449
Asp Lys Ser Val Val Ala Ser Pro Ala Ala Ser Ser Leu Ser Ser Tyr Pro Ser Pro Val Ala Thr Ser Tyr Pro

1642 TCC CCT GCC ACC ACC TCA TTC CCA TCC CCT GTG CCC ACT TCC TAC TCC TCT CCT GGC TCC TCC ACC TAC CCA TCT 474
Ser Pro Ala Thr Thr Ser Phe Pro Ser Pro Val Pro Thr Ser Tyr Ser Ser Pro Gly Ser Ser Thr Tyr Pro Ser

1717 CCT GCG CAC AGT GGC TTC CCG TCG CCG TCA GTG GCC ACC ACC TTT GCC TCC GTT CCA CCT GcT TIC ccc ACC CAG 499
Pro Ala His Ser Gly Phe Pro Ser Pro Ser Val Ala Thr Thr Phe Ala Ser Val Pro Pro Ala Phe Pro Thr Gln

1792 GTC AGC AGC TTC CCG TCT GCG GGC GTC AGC AGC TCC TTC AGC ACC TCA ACT GGT CTT TCA GAC ATG ACA GCG ACC 524
Val Ser Ser Phe Pro Ser Ala Gly Val Ser Ser Ser Phe Ser Thr Ser Thr Gly Leu Ser Asp MET Thr Ala Thr

1867 TTT TCT CCC AGG ACA ATT GAA ATT TGC TAA AGGGAATAAAAGAAAGCAAA AGGAAAGACATAAAAGCACAGGAGGGAAGA 533
Phe Ser Pro Arg Thr Ile Glu Ile Cys *
GATGGCCGCAAGAGGGGCCACCTCTTAGGTCAGATGGAAGATCTCAGAGCCAAGTCCTTCTACTCACGAGT AGAAGGACCGTTGGCCAACAGCCCTTTC
CACTTACCATCCCTGCCTCCCCCGTCCTGTTCCCCTTTTGACTTCAGCTGCCTGAAACAGCCATGTCCAAGTICTTCACCTCTATCCAAAGGACTIGAT
TIGCATGGTATTGGATAAATCATTTCAGTATCCTCTCCATCACATGCCTGGCCCTIGCTCCCT TCAGCGCTAGACCATCAAGT TGGCATAAAGAAAAAA
AAATGGGTTTGGGCCCTCAGAACCCTGCCCTGCATCTTTGTACAGCATCTGTGCCATGGATITTIGTITTCCTIGGGGTATTCTTGATGTGAAGATAATT
TGCATACTCTATTGTATTATTTGGAGTTAAATCCTCACTTTGGGGGAGGGGGGAGCAAAGCCAAGCAAACCAATGATGATCCTCTATITTGTGATGACT
CTGCTGTGACAT TAGGTTTGAAGCATTTITTTTTTTCAAGCAGCAGTCCTAGGTATTAACTGGAGCATGTGTCAGAGTGTTGTTCCGTTAATTTIGTAAA
TACTGCTCGACTGTAACTCTCACATGTGACAAAGTATGGTTTGTTIGGTTGGGTTTTGTTTITGAGAATITITI TGCCCGTCCCTTTGGTTTCAAAAGT
TICACGTCTTGGTGCCTTTTGTGTGACACGCCTTGCCGATGGCTT GACATGCGCAGATGTGAGGGACACGCT CACCT TAGCCT TAAGGGGGTAGGAGTG
ATGTTTTGGGGGAGGCT TTGAGAGCAAAAACGAGGAAGAGGGCTGAGCTGAGCTTTCGGTCICCAGAATGTAAGAAGAAAAAA TT TAAACAAAAATCTG
AACTCTCAAAAGTCTATTTTTCTAAACTGAAAATGTAAATT TATACATCTATTCAGGAGT TGGAGTGT TGT GGT TACCTACTGAGTAGGCTGCAGTITT
TGTATGTTATGAACATGAAGTTCATTATITIGTGGTTITATTTTACTTTGTACTTGTGTTTGCTTAAACAAAGTAACCTGTTIGGCTTATAAACACATT
GAATGCGCTCTATTGCCCATGGGATATGTGGTGTGTATCCTTCAGAAAAAT TAAAAGGAAAAAT AAAGAAAAAAAAAAAAAAARA

Fic. 1. The cDNA sequence and inferred amino acid sequence of zif/268. Numbers appearing in the left margin refer to the first nucleotide
listed on that line. Numbers appearing in the right margin refer to the last amino acid encoded on that line. The start of the mRNA is indicated
by v. Nucleotides 1-12 were determined by sequencing a genomic clone. The portion of the DNA that encodes three potential zinc-finger
structures is underlined.
Biochemistry: Christy ef al.

similar mobility, indicating that the 5’ end of the mRNA is
about 11 nucleotides upstream of the longest cDNA clone.
This site is 25 nucleotides downstream from a TATA-like
element, AAATAGA (see below).

There are 5 ATG codons within the first 400 nucleotides of
cDNA sequence, at positions 197, 295, 316, 325, and 379. The
first ATG, at position 197, is followed immediately by a
termination codon. The 2nd, 3rd, 4th, and Sth ATG codons
are all in the same long open reading frame; there are no
in-frame termination codons upstream of these ATGs. Al-
though the ATG codon at position 379 is in the most favorable
context for initiation (26), in vitro translation of zif/268
transcripts indicates that one of the preceding AUGs is the
preferred initiation codon in a reticulocyte extract. As shown
in Fig. 2, a nearly full-length transcript of zif/268 cDNA
directs the synthesis of a protein of electrophoretic mobility
lower than that directed by a truncated transcript missing the
three upstream AUGs. The first of the upstream in-frame
ATGs (at position 295) is followed by an open reading frame
of 1599 nucleotides, a termination codon, 1223 nucleotides of
nontranslated 3’ sequence, anda 3’ poly(A) tail. Polyadenylyl-
ation signal sequences (27, 28) are found at positions 1901,
3094, and 3107. Between these polyadenylylation signals are
two ATTTA sequences; multiple corresponding sequences of
this type in the 3’ untranslated region of a mRNA have been
shown to be related to message instability (29), which is a
general property of immediate early mRNAs (4).

Predicted Protein Sequence Derived from the zif/268 cDNA.
Fig. 1 shows the amino acid sequence of the protein encoded
by the long open reading frame of the zif/268 cDNA. If
translation begins at ATG 295, the predicted protein would be
533 amino acids in length. The most notable feature of the
amino acid sequences is the presence of spaced cysteine and
histidine residues near the carboxy! end of the protein that
correspond to three tandem copies of the consensus sequence
for a postulated DNA- and metal-binding domain, the zinc
finger structure (13). Compared to published sequences of
putative zinc finger-containing proteins, the zif/268 product
is nearly identical to NGFI-A, the product of a gene activated

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Fic. 2. In vitro translation of zif/268 transcripts. RNA tran-
scripts from a nearly full-length cDNA clone or from a truncated
clone containing a Bgl 11 cDNA fragment (nucleotides 338-1994)
were translated in a rabbit reticulocyte extract in the presence of
(S]methionine. The products were analyzed by electrophoresis in
a sodium dodecyl! sulfate/10% acrylamide gel. Lane M, molecular
weight markers (given x 10~+); lane 1, products of large transcript;
lane 2, products of truncated transcript; lane 3, products of both
transcripts cotranslated; lane 4, products 1 and 2 mixed; lane 5, no
transcript. Note that zif/268 product has an anomalously low
electrophoretic mobility.

Proc. Natl. Acad. Sci. USA 85 (1988) 7859

by nerve growth factor in rat PC12 pheochromocytoma cells
(17). The zif/268 product has 18 amino acid substitutions or
deletions relative to NGFI-A.

Serum Responsiveness of a zif/CAT Plasmid. To determine
whether nucleotide sequences upstream of the zif/268 gene
confer serum responsiveness on its promoter, as found for
c-fos (30, 31), a cloned fragment of zif/268 genomic DNA
containing 338 nucleotides of 5’ cDNA sequence and about
2.5 kb of contiguous upstream sequence was ligated to the
coding region of the bacterial CAT gene (21). This test
plasmid (zif/CAT) was transfected into NIH/3T3 cells and
the time course of expression of CAT activity was measured
after serum starvation and stimulation of the cells with 20%
fetal bovine serum. As shown in Fig. 3, the zif/CAT construct
was induced by serum. The overall level of induction was
about 8.3-fold, and the peak of enzyme induction was at
about 2-4 hr. The zif/268 promoter had no detectable activity
when the genomic fragment was linked to the CAT gene in the
opposite orientation. When a zif/CAT construct containing
only 1 kb of 5‘ genomic sequence was used, serum respon-
siveness was also observed. We conclude that the zif/268
promoter and sequences that confer serum responsiveness
are contained within 1 kb of the start site of transcription.

Nucleotide Sequence of the zif/268 Upstream Sequences.
The sequence of the zif/268 promoter region sufficient to
confer serum responsiveness is shown in Fig. 4. The previ-
ously noted sequence AAATAGA at ~25 relative to the
transcription start site presumably functions as a TATA-like
element. Also present are two CCAAT sequences (36) at
positions —227 and 336 and core Spl sequences (37).
Upstream of the TATA-like element of zif/268 are four
repeats of a sequence resembling or identical to the core of
the serum response element (SRE) found 5’ of the c-fos gene
and shown to mediate serum responsiveness (30, 38). How-
ever, none of the four zif/268 sequences shows the degree of
symmetry outside the core sequence seen in the c-fos or
Xenopus actin SREs (39). It remains to be seen whether one
or more of the zif/268 sequences actually functions as an
SRE.

Presence of zif/268 mRNA in Mouse Cell Lines and Tissues.
To determine how widely expressed the zif/268 gene is in
mouse cell lines and tissues, total RNA from various prolif-
erating lines and from adult tissues were analyzed by blot
hybridization analysis for the presence of zif/268 RNA. The
RNA was detected in many cell lines, including C127 cells,
1-7 cells, Friend leukemia cells, NB41 neuroblastoma cells,
and various teratocarcinoma cell lines. Of the tissues exam-

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Fic. 3. Serum responsiveness of the zif/268 promoter in NIH
3T3 cells. Cells were transfected in duplicate with the zif/CAT
plasmid and treated as described in the text. The figure is an
autoradiogram of ('C)chloramphenicol and its acetylated products
after chromatography. Numbers at the bottom of the figure are
average percent conversion (per 100 xg of total protein) of chlor-
amphenicol to acetylated form in 1 hr; lane SV, cells transfected with
simian virus 40-CAT plasmid, pSV2-CAT (21); lane M, mock-
transfected; numbers at the top, hr of stimulation with 20% serum
prior to harvest. CAT activity of transfected cells in the absence of
added serum varied by less than 1.5-fold during the 24-hr incubation.
7860 Biochemistry: Christy ef al.

Proc. Natl. Acad. Sci. USA 85 (1988)

AACAGATCCT GGCGGGGACT TAGGACTGAC CTAGAACAAT CAGGGTTCCG CAATCCAGGT CCCAAAGGTG GGATCCTCAA CCGCAGGACG GAGGGAATAG CCTTTCGATT CTGGGTGGTG

-1023 AP2-like

CATTGGAAGC CCCAGGQTCT AAAACCCCCA ACCTACTGAC TGGTGGCCGA GTATGCACCC GACTGCTAGC TAGGCAGTGT CCCAAGAACC AGTAGCCAAA TGTCTTGGCC TCAGTTTTCC

-903

CGGTGACACC TGGAAAGTGA CCCTGCCATT AGTAGAGGCT CAGGTCAGGG CCCCGCCTCT CCTGGGCGGC CTCTGCCCTA GCCCGCCCTG CCGCTCCTICC TCTCCGCAGG CTCGCTCCCA

-783 APl-like

CGGTCCCCGA GGTGGGCGGG TGAGCCCAGG ATGACGGCTG TAGAACCCCG GCCIGACTCG] CCCTCGCCCC CGCGCCGGGC CIGGGCTTCC CTAGCCCAGC TCGCACCCGG GGGCCGTCGG

-663

AGCCGCCGCG CGCCCAGCTC TACCGGCCTG GCGCCCTCCC CACGCGGGCG TCCCCGACTC CCGCGCGCGC TCAGGCTCCC AGTTGGGAAC CAAGGAGGGG GAGGATGGGG GGGGGGGTGT

-$43 SRE-lLike

SRE-like

GCGCCGACCC GGAAACGECA TATAAGGAGC AGGAAGGATC CCCCGCCGGA ACAGGCCTTA TTTCCCCAGC GECTTATATG GAGTGGCCCA ATATGGCCCT GCCGCTTCCG GCTCTGGGAG

-423

GAGGGGCGAG CGGGGGTTGG_ GGCGGGGGCA AGCTGGGAAC TCCAGGCGCC TGGCCCGGGA GGCCACTGCT GCTGTTCCAA TACTAGGCTT TCCAGGAGCC TGAGCGCTCG CGATGCCGGA

-303 CRE-like

SRE~iike SRE-like

GCGGGTCGCA GGGTGGAGGT GCCCACCACT CTTGGATGGG AGGGCTICAC GTCACTCCGG GICCTCCCGG CCGGTCcT CATATTAGGG CTTCCTGCTT CECATATATG GCCATGTACG

-183

TCACGGCGGA GGCGGGCCCG TGCTGTTCCA GACCCTTGAA ATAGAGGCCG ATTCGGGGAG TCGCGAGAGA TCCCAGCGCG CAGAA

-63

+1

Fic. 4. Nucleotide sequence of the zif/268 5’ flanking region. Numbers on the left side refer to the first nucleotide listed on the line directly
above, with mRNA start site designated as +1. The TATA-like element, CCAAT sequences, and GC boxes (which contain the core sequences
of Sp1 binding sites) are underlined. Boxed sequences: a sequence identical to an activator protein 2 (AP-2) site found in the human growth
hormone promoter (32) at position — 887 to — 894; a sequence resembling an AP-1 binding site (33, 34) at position — 604 to —610; a sequence
resembling a cAMP-responsive element (CRE) (35) at position —130 to —137; four sequences that resemble the core sequences of the
serum-response elements (SREs) of the c-fos and actin genes at positions —397 to — 406, —343 to —352, —95 to —104, and —73 to —82.
Additional sequence (to nucleotide — 1383) is included in the sequence data being deposited in the EMBL/GenBank data base.

ined, brain and thymus had a high level of zif/268 mRNA,
while lung and heart had moderate levels (Fig. 5). Low but
detectable levels were found in almost all other tissues tested,
with the exception of liver. (However, zif/268 mRNA was
found in regenerating liver within 1 hr after partial hepatec-
tomy.) The RNA was also present in mouse embryos and in
placenta. In those tissues in which zif/268 mRNA is most
abundant, lesser amounts of smaller hybridizing RNAs were
found, including an RNA of about 1.6 kb, a size consistent
with utilization of the poly(A) signal at nucleotide 1901 (Fig.
1). To localize zif/268 mRNA within the brain, we analyzed
its distribution in brain sections by in situ hybridization (Fig.
6). The RNA is most readily detected in the cerebral cortex
and hippocampus.

DISCUSSION

The main conclusion of this report is that the immediate early
growth response gene (clone 268), previously identified by
cDNA cloning from mRNA of BALB/c 3T3 cells stimulated
with serum (4), encodes a protein of 533 amino acids with
three typical zinc finger sequences characteristic of a class of
eukaryotic transcription factors (40). zif/268 is homologous

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Fic. 5. Blot hybridization analysis of mouse tissue RNA. Total
RNA (15 yg) from various mouse tissues was fractionated by
electrophoresis and blotted onto nitrocellulose, and the filter was
probed with a nearly full-length cDNA clone for zif/268. The lane
marked 3CH contains 1 yg of total RNA from BALB/c 3T3 cells
stimulated with serum for 3 hr in the presence of cycloheximide.
Other abbreviations: BR, brain; LI, liver; K, kidney; I, intestine; M,
muscle; S, spleen; TH, thymus; TE, testes; H, heart; and LU, lung.

to a gene (NGFI-A) activated in rat pheochromocytoma cells
by nerve growth factor (17); the same gene has been inde-
pendently identified by Sukahtme er al. (18) and designated
Egr-1. Within the zinc finger region zif/268 is closely related
to the recently described Krox gene family (47) and more
distantly related to Sp/ (15). Outside the zinc finger se-
quences, however, the sequence of zif/268 is quite different
from the sequences of Krox 20 [the one member of the Krox
family whose complete cDNA sequence has been reported
(11)] and Sp/ (15).

In addition to its expression in mouse cell lines stimulated
with serum or growth factors, zif/268 is expressed in a
number of mouse organs and tissues, notably brain, thymus,
lung, and heart (Fig. 5 and ref. 18). In mouse brain zif/268
mRNA is especially prominent in the cortex and hippocam-
pus, as visualized by in situ hybridization (Fig. 6). In more
extensive studies of the distribution of zif/268 mRNA in rat
brain, Saffen et al. (41) have found that administration of
convulsants causes a prompt increase in the mRNA in
neurons of the hippocampus and the entorhinal cortex,
suggesting that zif/268 plays a role in regulating the genomic
response to stimulation of nerve cells.

The activation of the zif/268 gene in 3T3 cells by serum,
PDGF, or FGF is coordinate with that of c-fos (4, 5). The
inducibility of c-fos expression by serum is mediated by a
22-base-pair sequence with dyad symmetry 5’ to the fos

 

Fic. 6. Localization of zif/268 mRNA in mouse brain by in situ
hybridization. Adult mouse brain was fixed and sectioned sagitally.
Hybridizations were performed as described in the text, using either
the antisense probe (Righ#) or the sense probe (Left) for the zif/268
cDNA. Photographs shown are negative images of film exposed on
the slides for 3 days. The regions displaying the highest levels of
zif/268 hybridization were the hippocampus and the cerebral cortex.
Biochemistry: Christy er al.

TATA signal (30, 31). This SRE is the binding site of a cellular
protein thought to serve as a transcriptional activator of fos
(38, 39, 42) and perhaps other genes coordinately regulated
with fos (39). It is therefore of interest that upstream of the
zif/268 TATA-like sequence there are four possible SREs.
All four share the common core motif CC(A or T),GG, one
of which is identical in sequence to that of fos, and another
is identical to an artificial SRE (42), but they all lack the
extensive flanking sequence symmetry seen in the c-fos and
other active SREs (39, 43). Whether one or more of these
putative SREs is functional remains to be determined. If
zif/268 does have multiple functional SREs, this could
explain the brisk response of this gene to serum and growth
factors (4, 5, 17). In addition to potential SREs zif/268 has
other possible transcription factor binding sites, namely core
Sp1-like sites (37), CCAAT sequences (36), an AP1-like site
(33, 34), an AP-2 site (32, 44), and a site similar to the
consensus sequence for cAMP-regulated promoters (35) (Fig.
4).

The emerging picture of the early genomic response to
growth factors is one of considerable complexity. A large
number of genes are rapidly activated in 3T3 cells as part of
the direct response to receptor-mediated second messenger
systems. Several of these immediate early genes encode
known or probable transcription factors: c-fos (8), junB (9),
c-jun (AP1) (33, 34), Krox 20 (11), and zif/268 (Egr-/,
NGFI-A). Others encode secreted proteins (7) or membrane
proteins (S. Hartzell, K. Ryder, and D.N., unpublished
results). A possible role of immediate early transcription
factors is the repression of immediate early genes. Another
possible role is the activation of genes expressed later in the
growth response, including those whose products regulate
DNA replication. At either site of action, the transcription
factors may act independently or as part of a multicomponent
transcription complex (10, 45, 46). The first step in elucidat-
ing the possible role of the zif/268-encoded protein in gene
regulation is to find genes with binding sites for this protein.

Note Added in Proof. The nucleotide sequence of a cDNA essentially
identical to that of zif/268 has recently been reported by Lemaire et
al. (48).

We thank Se-Jin Lee for the embryonic, placental, and cell line
RNAs; Stephen Desiderio and Keith Peden for critically reviewing
the manuscript; Laura Sanders and Michael McLane for technical
assistance; Clark Riley, Lawrence Cisek, and Jeffry Corden for
computer assistance; and Lily Mitchell for preparing the manuscript.
This research was supported in part by Grant 5 P01 CA16519 from
the National Cancer Institute. L.F.L. was a Postdoctoral Fellow of
the Helen Hay Whitney Foundation.

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