MOLECULAR AND CELLULAR BIOLOGY, June 1996, p. 2802–2813
Copyright ? 1996, American Society for Microbiology
Vol. 16, No. 6
An Exceptionally Conserved Transcriptional Repressor, CTCF,
Employs Different Combinations of Zinc Fingers To
Bind Diverged Promoter Sequences of Avian
and Mammalian c-myc Oncogenes
GALINA N. FILIPPOVA,1SARA FAGERLIE,1ELENA M. KLENOVA,2† CENA MYERS,1
YVONNE DEHNER,1GRAHAM GOODWIN,2PAUL E. NEIMAN,1
STEVE J. COLLINS,1AND VICTOR V. LOBANENKOV1*
Fred Hutchinson Cancer Research Center, Seattle, Washington 98104,1and Chester Beatty Laboratories,
Institute of Cancer Research, London SW3 6JB, United Kingdom2
Received 19 September 1995/Returned for modification 20 December 1995/Accepted 6 March 1996
We have isolated and analyzed human CTCF cDNA clones and show here that the ubiquitously expressed
11-zinc-finger factor CTCF is an exceptionally highly conserved protein displaying 93% identity between avian
and human amino acid sequences. It binds specifically to regulatory sequences in the promoter-proximal
regions of chicken, mouse, and human c-myc oncogenes. CTCF contains two transcription repressor domains
transferable to a heterologous DNA binding domain. One CTCF binding site, conserved in mouse and human
c-myc genes, is found immediately downstream of the major P2 promoter at a sequence which maps precisely
within the region of RNA polymerase II pausing and release. Gel shift assays of nuclear extracts from mouse
and human cells show that CTCF is the predominant factor binding to this sequence. Mutational analysis of
the P2-proximal CTCF binding site and transient-cotransfection experiments demonstrate that CTCF is a
transcriptional repressor of the human c-myc gene. Although there is 100% sequence identity in the DNA
binding domains of the avian and human CTCF proteins, the regulatory sequences recognized by CTCF in
chicken and human c-myc promoters are clearly diverged. Mutating the contact nucleotides confirms that
CTCF binding to the human c-myc P2 promoter requires a number of unique contact DNA bases that are
absent in the chicken c-myc CTCF binding site. Moreover, proteolytic-protection assays indicate that several
more CTCF Zn fingers are involved in contacting the human CTCF binding site than the chicken site. Gel shift
assays utilizing successively deleted Zn finger domains indicate that CTCF Zn fingers 2 to 7 are involved in
binding to the chicken c-myc promoter, while fingers 3 to 11 mediate CTCF binding to the human promoter.
This flexibility in Zn finger usage reveals CTCF to be a unique ‘‘multivalent’’ transcriptional factor and
provides the first feasible explanation of how certain homologous genes (i.e., c-myc) of different vertebrate
species are regulated by the same factor and maintain similar expression patterns despite significant promoter
The c-myc proto-oncogene encodes a nuclear phosphopro-
tein with leucine zipper and helix-loop-helix structural motifs
which is involved in regulating important cellular functions,
including cell cycle progression, differentiation, and apoptosis
(27, 31, 40). In several types of human and animal cancers, myc
is deregulated. Maintenance of the level of the c-myc mRNA is
achieved by regulation of both transcription initiation and
transcriptional elongation (for reviews, see references 27 and
40). A wide variety of signals can influence both initiation and
elongation of the c-myc mRNA. Most of these signals work
through cis-acting regulatory DNA sequences near or within
the c-myc gene, and some of these sequences bind specifically
a number of nuclear factors (for details, see reference 27).
Identification and study of such factors may provide insight
into molecular mechanisms of normal and aberrant c-myc ex-
Analysis of factors binding to the 5?-flanking noncoding
DNA sequences of the chicken c-myc gene (25) resulted in
identification (22, 23), purification (24), and molecular cloning
of the chicken 11-Zn-finger transcription factor termed CCCTC-
binding factor (CTCF) (15).
We have now isolated and analyzed human CTCF cDNA
clones and show here that the ubiquitously expressed CTCF
factor is (i) an exceptionally highly conserved protein display-
ing 93% identity between avian and human amino acid se-
quences and (ii) a ‘‘multivalent’’ factor which utilizes different
combinations of individual Zn fingers to specifically bind to
diverged regulatory DNA sequences within the promoter-
proximal regions of chicken, mouse, and human c-myc genes.
We also demonstrate that CTCF contains two strong tran-
scriptional repressor domains transferable to the GAL4 DNA
binding domain and that it can repress transcription from re-
porter constructs containing the P2 promoter of the human
c-myc gene. The P2-proximal CTCF binding sequence maps
precisely within the important regulatory region where pausing
of polymerase II transcription complexes is regulated, and
CTCF is a predominant nuclear factor binding to this region.
We show that elimination of CTCF binding by specific muta-
tion of the P2 promoter-proximal sequence results in increased
transcription from stably transfected human c-myc reporter
constructs. Since chicken CTCF is a repressor for the chicken
* Corresponding author. Mailing address: Fred Hutchinson Cancer
Research Center, Suite C2-023, 1124 Columbia St., Seattle, WA 98104.
Phone: (206) 667-4419 or (206) 667-4850. Fax: (206) 667-6523. Elec-
tronic mail address: email@example.com.
† Present address: Department of Biochemistry, University of Ox-
ford, Oxford OX1 3QU, United Kingdom.
c-myc gene promoter (13), function of CTCF appears to be
also conserved. Therefore, taken together, our results indicate
that CTCF is a major, evolutionarily conserved, negative reg-
ulator of vertebrate c-myc genes.
MATERIALS AND METHODS
Isolation of human CTCF cDNAs. Two primers corresponding to DNA se-
quences at amino acids (aa) 1 to 6 and 7 to 13 of the chicken CTCF amino-
terminal peptide 1 (15) and three primers corresponding to aa 266 to 271, 276 to
282, and 283 to 288 of the first chicken CTCF Zn finger were used in six
combinations to PCR amplify a fragment(s) of human CTCF cDNA from puri-
fied, size-fractionated double-stranded human muscle cDNA (Quick-clone
cDNA; Clontech Laboratories, Inc.). One of six reactions produced three dis-
crete DNA bands of about 600, 800, and 1,100 bp. DNA from these bands was
isolated and ligated into the TA cloning vector (Invitrogen, San Diego, Calif.).
The insert sequences of 36 independent plasmids were determined by automated
sequencing with a Taq DyeDeoxy Terminator Cycle sequencing kit (Applied
Biosystems, Inc.) and run through the FASTA DNA sequence homology search
using the Wisconsin Genetics Computer Group package. Four inserts were
found to have about 82% homology with the chicken CTCF cDNA sequence.
One plasmid, p800-3, containing the human CTCF cDNA fragment was used to
screen a cDNA library in Uni-ZAP XR vector constructed from poly(A)?RNA
isolated from early-passage human myeloid cell line HL-60 (3) with a ZAP-
cDNA synthesis kit (Stratagene, La Jolla, Calif.). Fourteen positive clones were
helper-excised from lambda phage into the Bluescript plasmid. The seven longest
clones had identical sequences at the ends of about 4.5-kbp inserts. The three
longest clones, p7.1, p9.1, and p10.2, were sequenced on both strands with
identical consecutive sets of primers.
In vitro transcription-translation and nuclear extracts. Full-length human
CTCF and the DNA binding domain of CTCF were synthesized from the p7.1
CTCF cDNA (see Fig. 2A) subcloned into the Bluescript vector with the T3
promoter in the sense orientation and from the pCITE/CTCF1 template con-
taining the 11-Zn-finger domain of CTCF under control of the T7 promoter-
CITE leader (15), respectively, by using the TnT reticulocyte lysate coupled in
vitro transcription-translation system (Promega Co., Madison, Wis.) as described
in the manufacturer’s manual. Twelve plasmids for the TnT in vitro synthesis of
truncated zinc finger forms of the CTCF DNA binding domain were constructed
by cloning in frame into pCITE-4a(?) (Novagen, Madison, Wis.) the human
CTCF cDNA fragments PCR amplified with pairs of primers designed to cover
certain groups of CTCF Zn fingers as follows: (i) p4a-ZF(1-11), encoding the
full-length 11-Zn-finger domain from aa 236 to 622; (ii) amino-terminally trun-
cated forms, i.e., p4a-ZF(2-11), fingers 2 to 11 beginning at the middle of the Zn
finger 1 at position 275 and ending at aa 622; p4a-ZF(3-11), fingers 3 to 11, aa
307 to 622; p4a-ZF(4-11), fingers 4 to 11, aa 332 to 622; p4a-ZF(5-11), fingers 5
to 11, aa 367 to 622; and p4a-ZF(6-11), fingers 6 to 11, aa 388 to 622; and (iii)
carboxy-terminally truncated forms, i.e., p4a-ZF(1-10), fingers 1 to 10, aa 236 to
549; p4a-ZF(1-9), fingers 1 to 9, aa 236 to 520; p4a-ZF(1-8), fingers 1 to 8, aa 236
to 492; p4a-ZF(1-7), fingers 1 to 7, aa 236 to 463; p4a-ZF(1-6), fingers 1 to 6, aa
236 to 433; and p4a-ZF(1-5), fingers 1 to 5, aa 236 to 404. Translation products
synthesized in the presence of [35S]Met were visualized on sodium dodecyl
sulfate (SDS) gels as described previously (36). Nuclear protein extracts were
prepared from isolated cell nuclei by using NUN solution containing 0.3 M NaCl,
1 M urea, and 1% nonionic detergent Nonidet P-40 as described elsewhere (19)
and protease and phosphatase inhibitors as described previously for purification
of chicken CTCF by sequence-specific chromatography (24).
EMSA, methylation interference, and missing-contact analyses. Four consec-
utive fragments of human c-myc DNA (A, positions ?56 to ?111 relative to ?1
at the P2 initiation site; B, positions ?225 to ?38; C, positions ?353 to ?166;
and D, positions ?489 to ?329) and four consecutive mouse c-myc DNA frag-
ments (?, positions ?237 to ?87; ?, positions ?157 to ?18; ?, positions ?49 to
?113; and ?, positions ?85 to ?254), one by one covering partially overlapping
promoter DNA sequences of interest, were PCR amplified and simultaneously
end labelled on either strand by using pairs of 15- to 22-bp-long primers, one of
which was 5? end labelled with [?-32P]ATP and T4 polynucleotide kinase. A
positive-control DNA fragment bearing CTCF binding sequence footprint V of
the chicken c-myc gene was amplified from the pFpV plasmid (23, 24). These
fragments were gel purified by Elutip-D (Schleicher & Schuell, Keene, N.H.)
minicolumn chromatography, and basically equal amounts of each fragment were
utilized for both electrophoretic mobility shift assay (EMSA) and methylation
interference and missing-contact analyses. Each of four DNA fragments con-
taining CTCF binding sites revealed by the EMSA experiments, 5? end labelled
on either the top (coding) strand or the bottom (anticoding) strand, was then
either partially methylated at guanines with dimethyl sulfate or modified at
pyrimidine bases with hydrozine by the C?T reaction of Maxam and Gilbert (28)
and incubated with the in vitro-translated DNA binding domain of CTCF. Free
DNA probe was separated from the CTCF-bound probe by preparative EMSA,
and DNA was isolated from the gel, cleaved at modified bases with piperidine,
and analyzed on a sequencing gel as described in detail previously (15, 24). For
EMSA with each DNA probe, 1 to 10 ?l of the in vitro translation product or
nuclear extract was used for reactions in the presence of cold, double-stranded
competitor DNAs [poly(dI-dC) plus poly(dG)-poly(dC) plus oligonucleotide
containing strong binding sites for both Sp1 and Egr1 proteins] in a phosphate-
buffered saline (PBS)-based buffer containing standard PBS with 5 mM MgCl2,
0.1 mM ZnSO4, 1 mM dithiothreitol, 0.1% Nonidet P-40, and 10% glycerol.
Reaction mixtures were incubated for 30 min at room temperature and then
analyzed on 5% polyacrylamide gels run in 0.5? Tris-borate-EDTA buffer.
Proteolytic-protection analyses. Two DNA fragments of identical lengths,
harboring either the chicken c-myc site V sequence or the human c-myc site A
sequence, were PCR amplified and simultaneously end labelled as described
above. These DNA probes were incubated for 30 min at room temperature with
5 ?l of the in vitro-translated 11-Zn-finger DNA binding domain of CTCF
(CTCF1) in 20 ?l of the PBS-based EMSA buffer; then proteinase K (Merck)
was added to final concentrations of 0, 0.01, 0.1, 1, 3, 5, and 10 mg/ml, and the
samples were incubated for an additional 20 min and loaded on the EMSA gel.
Reporter and expression constructs and stable and transient transfections.
Expression constructs pGal-CTCF-N and pGal-CTCF-C, containing N- and C-
terminal amino acid sequences of chicken CTCF fused in frame to the GAL4
DNA binding domain, respectively, were prepared by ligating the SmaI-HindIII
(positions 113 to 830) and MscI-XbaI (positions 1908 to 2850) fragments of the
chicken CTCF cDNA (15) into the pSG424 vector (35). The luciferase reporter
construct p5xUAS/TK-Luc contains five GAL4 binding sites (5xUAS ) in-
serted upstream of the minimal herpes simplex virus (HSV) thymidine kinase
(TK) promoter of plasmid pTK-Luc. A total of 106QT6 quail fibroblasts growing
at about 50% confluence were transfected by the calcium phosphate method (36)
with 2.5 ?g of the reporter, 2.5 ?g of the expression vector, 0.4 ?g of the
transfection efficiency control plasmid pSV/?-gal, and 5 ?g of the pUC18 DNA
as a carrier. Cell extracts were prepared 48 h posttransfection, and activity of the
reporter constructs was measured by the Luciferase Assay System as described by
the manufacturer (Promega). Expression of the GAL-CTCF fusion proteins was
monitored by Western blot (immunoblot) analysis using monoclonal antibodies
against GAL4 (1-147) protein (a gift from P. Chambon, Institut de Ge ´ne ´tique et
de Biologie Mole ´culaire et Cellulaire). Reporter plasmid pAPwtCAT was con-
structed by ligating the ApaI-PvuII fragment of the human c-myc 5? noncoding
region from positions ?121 to ?352 relative to the P2 site into the pBLCAT3
promoterless chloramphenicol acetyltransferase (CAT) construct (26). To obtain
the pAPacaCAT plasmid, the ACA mutation of the CTCF binding site shown in
Fig. 5A was introduced by two-step PCR amplification (with two mutant primers
and two flanking normal primers) and religation. The mutated sequence was
verified by sequencing. The pCI/CTCF expression construct was made by ligating
the insert from the p7.1 full-length human CTCF cDNA plasmid into the pCI
expression vector (Promega) under the control of the cytomegalovirus (CMV)
immediate-early enhancer-promoter. Each of the two reporter plasmids was
cotransfected with the pCMV/?-gal plasmid and with the pSV2neo plasmid into
mouse NIH 3T3 fibroblasts by the lipofection method. The molar ratio of CAT
reporter plasmid to ?-galactosidase (?-gal)-expressing plasmid and neo-express-
ing plasmid was about 10:1:1. Polyclonal stably transfected cell lines were estab-
lished by pooling all G418-resistant clones from each transfection. Transient-
cotransfection experiments were performed with human embryonic kidney 293
cells by using pHIV-LTR/?-gal for normalizing transfection efficiency, the pCI/
CTCF expression vector as an effector, and pAPwtCAT and pAPacaCAT as
reporter constructs. A number of cotransfection experiments and EMSAs have
been initially carried out to ensure that (i) in 293 cells, the pCI/CTCF expression
vector is able to produce CTCF, detectable by Western immunoblotting, at levels
proportional to the amount of transfected plasmid; (ii) transient transfection into
the 293 cell line reproducibly resulted in sufficient signal from our short CAT
constructs containing only the P2-proximal c-myc promoter region; and (iii) the
human immunodeficiency virus (HIV) long terminal repeat (LTR)-driven ?-gal
construct employed as an internal control for cell transfection efficiency itself
neither binds nor responds to CTCF. In both stable-transfection and transient-
cotransfection experiments, CAT activity, normalized to the internal copy num-
ber of stably integrated reporter constructs or control ?-gal activity, was assayed
in cell extracts prepared from equal numbers of transfected cells as described
Western, Northern (RNA), and Southern blots. The Ab1 affinity-purified poly-
clonal antibody against the N-terminal epitope of CTCF conserved in vertebrates
(15) was used at a 1:100 dilution to probe protein gel blots as described by the
manufacturer of the enhanced chemiluminescence (ECL) detection kit (Amer-
sham International plc). Northern (RNA) and Southern (DNA) blots were
probed with the human CTCF cDNA probe labelled with [?-32P]dCTP by nick
translation and washed in 0.1? SSC (1? SSC is 0.15 M NaCl plus 0.015 M
sodium citrate) at 65?C (36).
Nucleotide sequence accession numbers. The GenBank/EMBL database ac-
cession numbers for the human and mouse CTCF cDNA sequences are U25435
and U51037, respectively.
Isolation of the human CTCF cDNA. The chicken CTCF
gene, which encodes a factor binding to the chicken c-myc
promoter, was cloned previously (15, 22–25). We based our
strategy for cloning human CTCF on evidence that at least two
VOL. 16, 1996CONSERVED ‘‘MULTIVALENT’’ REPRESSOR OF c-myc PROMOTERS2803
domains of CTCF were highly conserved. Polyclonal antibod-
ies against an amino-terminal peptide of the chicken CTCF
detected nuclear CTCF protein in cells of a variety of verte-
brate species, from frogs to humans (Fig. 1A) (15). In addition,
EMSAs detected the presence of CTCF DNA binding activity in
nuclear extracts from cells of a variety of vertebrates tested
(Fig. 1B and data not shown). These observations indicate
conserved domains of CTCF polypeptide in both amino-ter-
minal and DNA binding regions. Therefore, primers corre-
sponding to DNA sequences at these regions were used to
amplify and subclone a fragment of human CTCF cDNA that
we then used to isolate and analyze several lambda clones of
human CTCF cDNA as described in Materials and Methods.
Figure 2A shows the complete human CTCF cDNA sequence
and its longest open reading frame (ORF). In comparing chick-
en and human coding sequences, we observed about 20% di-
vergence, primarily at the third DNA base pair of CTCF codons
(6). In addition to highly conserved coding regions, the 5? non-
coding regions of chicken, mouse, and human CTCF cDNAs and
their 1.2-kbp-long 3? untranslated regions have multiple domains
of 100% homology (6), indicating putative important conserved
sequences that might be involved in control of CTCF mRNA
turnover, cellular compartmentalization, or translation efficiency.
Chicken and human CTCF proteins are strictly conserved.
Comparison of human and chicken CTCF amino acid se-
quences (Fig. 2B) shows that the two proteins are practically
identical, with homology extending well outside the completely
conserved 11-Zn-finger domain. Analysis of the human CTCF
sequence reveals the same structural domains previously noted
in the amino acid sequence of chicken CTCF (15), namely, 10
Zn fingers of the C2H2 type and 1 Zn finger of the C2HC class,
two highly positive domains flanking the 11-Zn-finger domain,
three acidic regions in the carboxy-terminal part of the se-
quence, and putative serine phosphorylation sites adjacent to a
potential nuclear localization signal. Unlike most of the Kru ¨p-
pel-GLI class of factors (12), not all of CTCF’s 11 Zn fingers
are separated by the highly conserved 7-aa linkers (H-C links)
of the form (T/S)GE(K/R)P(F/Y)X (5), suggesting that the
CTCF DNA binding domain may be formed by discrete groups
Probing a ‘‘zoo’’ DNA blot with labelled human CTCF
cDNA fragments displays single-copy CTCF genes in frog,
chicken, mouse, and human genomes (6). CTCF expression is
not restricted to a particular cell type, since Northern blot
analysis of total RNA from a variety of chicken, mouse, and
human cell lines and tissues detects comparable levels of ex-
pression of ?4-kb CTCF mRNA in all cells tested (6, 14).
It was previously observed that the apparent mobility in SDS
gels of CTCF protein purified from chicken cells by sequence-
specific chromatography suggested a protein of 130 or 160 kDa
(23, 24). Moreover, the major CTCF form detected by Western
immunoblotting is also about 160 kDa (Fig. 1A). However,
the practically identical ORFs of chicken and human CTCF
cDNAs (Fig. 2B) predict a protein of 82 kDa. We therefore
screened different chicken, mouse, and human cDNA libraries
but could not isolate any CTCF cDNAs containing a longer
ORF (6, 13). However, when the human cDNA was tran-
scribed and translated in vitro, we noted a single protein with
a mobility of about 160 kDa in SDS gels (Fig. 1C, lane 1). Such
anomalous electrophoretic migration of proteins is uncommon
but has been observed with other translation products (2, 34),
including zinc finger proteins (7). It appears that the amino
acid sequence responsible for the aberrant migration of CTCF
is located outside the DNA binding region, since the in vitro-
translated 11-Zn-finger domain of CTCF migrates in accord
with its predicted size of about 40 kDa (Fig. 1C, lane 3). The
in vitro-translated CTCF product and endogenous CTCF from
nuclear extracts comigrate when loaded on the same gel and
assayed by immunoblotting (13) and also generate in EMSA
retarded complexes with similar mobilities (Fig. 1B). There-
fore, both chicken (15) and human (this report) ?4-kb cDNAs
represent full-length copies of the mature polyadenylated
CTCF mRNA and encode a protein identical to the endoge-
CTCF protein binds specifically to the promoter-proximal
regions of avian, mouse, and human c-myc genes. Using the
in vitro-translated DNA binding domain of CTCF for gel shift
experiments and methylation interference and missing-con-
tact assays, we have determined CTCF binding sequences in
the promoter-proximal region of mouse and human c-myc
genes. Figure 3A demonstrates a schematic outline of our ap-
proach. Four consecutive DNA fragments representing DNA
sequences of the promoter-proximal regions of mouse c-myc
(fragments ?, ?, ?, and ?) and human c-myc (fragments A, B,
C, and D) were synthesized by PCR amplification with pairs of
primers (one of which was32P end labelled) in order to obtain
DNA probes suitable for both EMSA and methylation inter-
ference experiments. As a positive control for CTCF bind-
ing, a DNA fragment spanning the footprint V region of the
chicken c-myc promoter (24) was also included in these exper-
FIG. 1. Characterization of endogenous and cloned CTCF proteins. (A)
CTCF proteins are present in cells of different vertebrates. Results of Western
immunoblot analysis of Saccharomyces cerevisiae, chicken, mouse, and human
total cell lysates probed with CTCF antibody Ab1 (15) are shown. BM2 and HD3
are chicken erythroid and myeloid cell lines, respectively; HL-60 and K562 are
human myeloid and erythroid cell lines, respectively; and Rauscher MEL,
C2C12, and NIH 3T3 are mouse erythroleukemia, myoblast, and fibroblast cell
lines, respectively. The major endogenous CTCF protein (arrow), with an ap-
parent molecular mass of about 160 kDa, and positions of the Rainbow molec-
ular mass protein markers (on the left) are indicated. (B) CTCF protein, synthe-
sized from the cDNA template in vitro, and nuclear CTCF proteins of different
vertebrates form identical sequence-specific DNA-protein complexes. EMSAs using
in vitro-translated human CTCF (IVT CTCF) and nuclear extracts prepared
from chicken BM2, mouse NIH 3T3, and human K562 cell lines were performed
with32P-labelled DNA fragment A from the P2 promoter-proximal region of the
human c-myc gene (see also Fig. 3 and 4 legends for details). In the EMSA reac-
tion (lane ?), a control TnT reticulocyte lysate transcription-translation mixture
with no template was used. The positions of the unbound DNA probe and the
CTCF-DNA complexes are indicated (arrows). (C) The in vitro-synthesized
CTCF polypeptide with a predicted molecular mass of 82 kDa migrates as a
160-kDa protein in SDS-polyacrylamide gels. Full-length CTCF cDNA (lanes 1
and 2) and the 11-Zn-finger domain (lanes 3 and 4) were in vitro transcribed in
either the sense (T3 for p7.1/HuCTCF and T7 for pCITE/CTCF1) or the anti-
sense orientation and simultaneously translated in the TnT reticulocyte lysate
containing [35S]methionine. Positions of markers are shown on the left.
2804 FILIPPOVA ET AL.MOL. CELL. BIOL.
FIG. 2. The primary sequence of CTCF protein is conserved in vertebrates. (A) Nucleotide sequence of the human CTCF cDNA and the inferred CTCF protein
primary sequence (shown in single-letter code). Eleven-amino-acid sequences conforming to the C2H2 and C2HC class of zinc finger consensus motifs (reviewed in
reference 5) are identified (dotted underlines). (B) Alignment of human (HU) and chicken (CH) CTCF amino acid sequences. Identities (lines) and conservative
substitutions and amino acids with similar properties (colons and dots, respectively) are indicated. The two sequences have been aligned for maximal match by the
BestFit program of the Genetics Computer Group package.
VOL. 16, 1996 CONSERVED ‘‘MULTIVALENT’’ REPRESSOR OF c-myc PROMOTERS2805
FIG. 3. Sequence-specific CTCF binding to DNA fragments from the promoter-proximal regions of mouse and human c-myc genes. (A) Schematic outline of the
approach to screen the promoter regions of human and mouse c-myc genes for CTCF binding sites. We utilized the indicated DNA fragments from human c-myc and
mouse c-myc P1-P2 promoter regions in EMSA analysis. The ?30 region of polymerase II (Pol II) pausing and promoter melting (18) and summary of CTCF binding
sites in the human c-myc promoter are also shown. (B) EMSA analysis of CTCF binding to human c-myc DNA fragments. DNA fragments shown in panel A were tested
by EMSA for specific binding to the in vitro-translated 11-Zn-finger domain of CTCF (CTCF1). DNA fragment V harboring the previously defined CTCF binding site
of the chicken c-myc promoter (24) was also included in these experiments as a positive control. With each DNA probe, EMSA analysis was carried out with either
5 ?l of control (no template) TnT reticulocyte lysate or 5 ?l of the lysate containing the CTCF1 DNA binding domain synthesized from the pCITE/CTCF1 template
(15). To challenge the specificity of CTCF binding to the human c-myc fragments, cross-competition EMSA reactions were also performed by including in the EMSA
incubation mixture a 500-fold excess of unlabelled DNA fragments A, D, and V. Note that in these assays, the relatively different positions of shifted bands in EMSA
with CTCF1 and different DNA fragments (e.g., with fragments A and V) are due to the different lengths of the DNA probes employed. If fragment V is synthesized
to match precisely the length of fragment A, then the positions of CTCF-shifted bands are identical with the two probes (6). (C) EMSA reactions with four mouse c-myc
promoter DNA fragments with increasing amounts (0 to 5 ?l) of in vitro-translated CTCF1 were carried out as described above (but excluding cross-competition
2806 FILIPPOVA ET AL.MOL. CELL. BIOL.
iments. Figure 3B and C show that in addition to control
fragment V, three of eight DNA fragments efficiently bind the
11-Zn-finger domain of CTCF protein, namely, DNA frag-
ments A and B from the human c-myc gene and fragment ?
from the mouse gene. Comparison of the proportions of each
DNA probe bound by an equal amount of CTCF indicated that
binding to fragments A, B, and ? is comparable to that for
chicken fragment V. Binding to fragment C was weaker and
not characterized further. Unlabelled DNA fragments A, V,
and D were also used as competitors in a cross-competition
EMSA experiment (Fig. 3B). Fragment A efficiently competed
for CTCF binding to itself and to fragments V and B and
fragment V competed for binding to itself and to fragments A
and B, whereas fragment D, which did not bind CTCF, did not
compete for CTCF binding. Seven other 120- to 220-bp-long
GC-rich DNA fragments containing multiple CCTC motifs
(from the HIV LTR and from the chicken c-myc promoter-
proximal regions upstream and downstream of site V) were
also tested for CTCF binding and found to be negative (6).
To determine which nucleotides are recognized by CTCF in
human and mouse fragments A, B, and ? and to compare them
with the recognition sequence in chicken fragment V, we car-
ried out missing-contact analysis (for C plus T bases) and
methylation interference (for G bases) assays for both strands
of each DNA fragment. DNA bases which on removal or
modification reduced binding of CTCF resulted in sequencing
gel bands of decreased intensity in lanes of CTCF-bound DNA
(Fig. 4, lanes B) compared with the free-DNA lanes (lanes F).
Inspection of bases required for CTCF binding to four DNA
sequences (Fig. 4) reveals the following. (i) CTCF binds to a
DNA sequence from positions ?5 to ?45 immediately down-
stream of the P2 initiation site of both human (fragment A)
and mouse (fragment ?) c-myc promoters. (ii) This P2-proxi-
mal CTCF binding sequence is well conserved in the two mam-
malian c-myc genes; moreover, most of the CTCF-contacting
nucleotides within the human and the mouse sites are identi-
cal. (iii) CTCF also binds to a different GC-rich sequence (in
fragment B) immediately downstream of the P1 initiation site
of the human c-myc promoter. (iv) The P2-proximal CTCF-
binding sequence shared by the human and mouse c-myc genes
(Fig. 4A and C) and the P1-proximal CTCF binding sequence
of the human gene (Fig. 4B) are significantly different from
one another and from CTCF binding sequence V in the
chicken c-myc gene (Fig. 4D).
Different combinations of CTCF Zn fingers bind to diver-
gent sequences in the chicken and human c-myc promoters. As
noted above, the amino acid sequence of the CTCF 11-Zn-
finger DNA binding domain is 100% conserved between chick-
en and human CTCF proteins (Fig. 2B), yet visual inspection
of the nucleotide contact points of CTCF in the human (frag-
ment A; Fig. 4A) and chicken (fragment V; Fig. 4D) c-myc
promoters indicates that these CTCF target sequences are
clearly divergent. How do the identical CTCF 11-Zn-finger
DNA binding domains contact clearly divergent DNA regula-
tory sequences? A pairwise comparison of CTCF contact points
in the chicken and human fragments (Fig. 5A) indicates that
CTCF contacts bases within a GC-rich core common to the
chicken and human fragments (Fig. 5A, subregion A2). How-
ever, in human fragment A, CTCF also contacts sequences at
least 12 nucleotides upstream of this GC-rich core (Fig. 5A,
subregion A1), and such contact points are absent in chicken
fragment V. To determine whether these more upstream se-
quences are critical for CTCF binding to human fragment A,
we selectively mutated three nucleotides within this region by
changing TGT to ACA, as noted in Fig. 5A. EMSA with the in
vitro-translated DNA binding domain shows that this ACA
mutation knocks out CTCF binding (Fig. 5B). Therefore, the
contact bases critical for recognition by CTCF are clearly dif-
ferent in human fragment A and chicken fragment V.
Since human fragment A harbors more CTCF binding nu-
cleotides than chicken fragment V (Fig. 5A), one would predict
that more CTCF Zn fingers may be involved in binding to
fragment A than to fragment V. To confirm this hypothesis, we
first performed proteolytic-protection assays with CTCF target
fragment complexes. Specific regions of DNA-binding proteins
that make direct contact with the corresponding DNA target
fragments are selectively protected from proteolytic degrada-
tion (1, 38). We treated the in vitro-synthesized 11-zinc-finger
CTCF domain prebound to either DNA fragment A or DNA
fragment V with increasing amounts of proteinase K and an-
alyzed the resulting DNA-protein complexes by gel shift assays.
Figure 6 shows that the proteinase-resistant complexes formed
with both DNA fragments migrated faster than the complexes
formed with the untreated, full-length 11-Zn-finger domain.
Moreover, proteinase-treated complexes formed with frag-
ment A (Fig. 6B) migrated more slowly than complexes formed
with fragment V (Fig. 6A). Since the two DNA fragments were
exactly the same length, this result indicates that not all 11
fingers are absolutely required for binding to both fragments
and that the site A DNA sequence protects a significantly
larger part of the 11-zinc-finger domain than the site V se-
quence does. The difference in relative mobility was about
20%, suggesting that at least two fingers more are involved in
binding to human site A than to chicken site V.
To directly determine which CTCF Zn fingers might be
involved in binding to human fragment A versus chicken frag-
ment V, we utilized these fragments as probes in gel shift
assays together with serially truncated in vitro-translated
CTCF products. As detailed in Materials and Methods, we
engineered five amino-terminally (Fig. 7D) and six carboxy-
terminally (Fig. 7A) truncated in vitro-translated products of
the 11-Zn-finger domain of CTCF. Gel shift assays of these
different forms of the CTCF DNA binding domain using the
two DNA fragments containing either chicken c-myc CTCF
binding site V (Fig. 7B and E) or human c-myc CTCF binding
site A (Fig. 7C and F) demonstrate that N-terminal fingers 1
and 2 are dispensable for binding to site A (Fig. 7F) but that
finger 2 is required for binding to the site V sequence (Fig. 7E).
On the other hand, C-terminal fingers, including finger 11, are
absolutely required for binding to the P2-proximal site A of
human c-myc (Fig. 7C), but fingers 11 to 8 are dispensable for
binding to site V of chicken c-myc (Fig. 7B).
Taken together, these data indicate that the group of six zinc
fingers, fingers 2 to 7, is sufficient for CTCF binding to chicken
c-myc site V, while another group of nine fingers, from 3 to 11,
mediates CTCF binding to human c-myc site A. Because of its
ability to recognize and bind to different DNA sequences by
employing different groups of Zn fingers, we propose to call
CTCF a multivalent factor.
CTCF contains two transcriptional repressor domains and
negatively regulates the human c-myc P2 promoter. Taken
together, the strict evolutionary conservation of CTCF and its
unusual ability to bind specifically to a number of diverged
DNA sequences in the promoter-proximal regions of human,
mouse, and chicken c-myc genes suggest that CTCF plays an
important role in regulation of c-myc genes in vertebrate spe-
cies. To determine whether CTCF might be a positive or neg-
ative transcriptional regulator, both amino- and carboxy-ter-
minal CTCF protein domains flanking the 11-Zn-finger region
were individually fused to the GAL4 DNA binding domain to
produce the pGal-CTCF-N and pGal-CTCF-C expression vec-
tors, respectively, and cotransfected with the reporter pro-
VOL. 16, 1996CONSERVED ‘‘MULTIVALENT’’ REPRESSOR OF c-myc PROMOTERS 2807
FIG. 4. Identification of variant DNA sequences specifically recognized by CTCF in the promoter-proximal regions of human, mouse, and chicken c-myc genes. The
results of experiments to determine all DNA bases required for recognition of vertebrate c-myc promoters by CTCF are shown. Each of four DNA fragments containing
CTCF binding sites revealed by the EMSA experiments (Fig. 3) was subjected to methylation interference analysis (with DNA probes partially methylated at guanines
with dimethyl sulfate [DMS]) or missing-nucleoside analysis (with DNA probes modified at pyrimidine bases with hydrozine [HZ]). Lanes F, free DNA probes separated
from the CTCF1-bound probes (lanes B). DNA bases which, when missing from the labelled strand or modified, reduce binding of CTCF (bars) and particular
methylated G residues preferentially found in CTCF-bound DNA molecules (circles) are indicated. In each panel, the G ladder and the C?T ladder lanes show
sequencing reactions run in parallel to facilitate reading of the nucleotide sequence within each CTCF binding site. For each DNA fragment, both coding and noncoding
DNA strands (except for mouse fragment ?, which is homologous to human fragment A) were analyzed by both methylation interference and missing-contact analyses,
resulting in the summary of guanine and pyrimidine residues required for sequence recognition by CTCF shown below each panel. DNA bases which are different within
CTCF binding sites in mouse and human P2-proximal sequences are underlined in panel C.
moter containing GAL4 binding sites. The reporter gene con-
sisted of the luciferase gene with either five GAL4 binding
sites (11) upstream of a minimal HSV TK promoter (p5xUAS/
TK-Luc) or just a TK promoter (pTK-Luc). When cotrans-
fected with the pSG424 vector expressing only the GAL4
DNA binding domain, the two reporter constructs had simi-
lar levels of basal transcription (Fig. 8A). Cotransfection with
the Gal-CTCF fusion expression vectors, pGal-CTCF-N and
pGal-CTCF-C, results in 20- and ?100-fold repression of the
p5xUAS/TK-Luc reporter activity, respectively, while activity
of the pTK-Luc reporter with no binding sites for the fusion
proteins is not inhibited (Fig. 8A). Western immunoblot anal-
ysis of total transfected-cell lysates with anti-GAL4 monoclo-
nal antibodies showed production of approximately equal
amounts of two fusion proteins (14). Therefore, transcriptional
repression was specifically mediated by binding of the Gal-
CTCF fusion proteins to the reporter promoter. Figure 8A also
indicates that in QT6 fibroblasts, the C-terminal CTCF domain
appears to be a stronger repressor than the N-terminal do-
main. These results indicate that CTCF harbors at least two
transcriptional repressor domains.
CTCF is the major protein in nuclear extracts binding to the
P2-proximal DNA sequence A of the human c-myc gene under
our EMSA conditions (Fig. 1B). To determine whether CTCF
might act as a transcriptional repressor when bound to the hu-
man c-myc promoter, we analyzed the functional contributions
of both endogenous and exogenous CTCF binding to the P2-
proximal site of the human c-myc gene. The site is situated
within the region between 121 bp upstream (ApaI site) and
352 bp downstream (PvuII site) of the P2 promoter (Fig. 8B),
which excludes the P1 upstream sequences. This 473-bp se-
quence around the P2 promoter is sufficient to correctly initi-
ate RNA transcription from stably transfected constructs (21)
and is fully responsible for the suppression of c-myc transcrip-
tion upon induced cell differentiation (10). We prepared CAT
reporter constructs harboring this P2-proximal human c-myc
DNA sequence (Fig. 8B), and in one of these constructs we
engineered the ACA mutation that specifically eliminates CTCF
binding to this P2-proximal sequence (Fig. 5). To generate
stable transfectants with these two reporter constructs, we co-
transfected them with the pSV/neo plasmid along with the
pCMV/?-gal plasmid into mouse NIH 3T3 fibroblasts. G418-
resistant clones from each transfection were pooled, and CAT
FIG. 5. Selective mutation of nucleotides which distinguish the human c-myc P2-proximal CTCF binding sequence from the chicken c-myc promoter CTCF binding
site V eliminates specific recognition. (A) Comparison of the primary sequence and DNA bases required for CTCF binding to human P2-proximal site A and to chicken
promoter site V. CTCF-contacting purine (filled circles) and pyrimidine (open circles) bases determined in two sequences by methylation interference and missing-
contact assays (Fig. 4) are indicated. CCCTC motifs formerly implicated in CTCF binding (24) (underline arrows) are indicated. The TGT-to-ACA substitution within
subregion A1 of human c-myc CTCF binding site A is also shown. (B) Two DNA fragments of identical length harboring the P2-proximal DNA sequence of human
c-myc, with and without the ACA mutation shown in panel A, were synthesized and end labelled by PCR amplification and used for EMSA with 0, 1, and 5 ?l of the
in vitro-translated DNA binding domain of CTCF (CTCF1). Free DNA probes (F) and the presence of an endogenous reticulocyte lysate activity (e.a.) binding outside
the CTCF binding sequence are also indicated.
FIG. 6. Two different CTCF binding DNA sequences protect different num-
bers of zinc fingers from proteolytic attack. Two DNA fragments of identical
length, harboring either chicken c-myc site V sequence (A) or human c-myc site
A sequence (B), were preincubated with the in vitro-translated 11-Zn-finger
DNA binding domain of CTCF (CTCF1), then treated with increasing amounts
of proteinase K as described in Materials and Methods, and analyzed by EMSA
on the same gel. The identical mobilities of two free DNA probes and of two
untreated DNA-CTCF1 complexes (lower and upper dashed lines, respectively)
and the positions of complexes retaining DNA-protein binding during proteinase
treatment (arrows) are indicated.
VOL. 16, 1996 CONSERVED ‘‘MULTIVALENT’’ REPRESSOR OF c-myc PROMOTERS2809
activity, normalized to the reporter copy number or ?-gal ac-
tivity, was assayed in extracts from equal numbers of these
cells. We measured CAT activity in cells grown under three
different conditions: normal growth, when cells were passaged
every third day and did not reach confluence; growth arrest,
when confluent cells were kept in serum-deprived medium for
2.5 days; and serum response, when confluent cells were serum
starved for 2 days and then transferred to a fresh serum-con-
taining medium for 12 h prior to being harvested. Under all
three cell growth conditions, the ACA mutation results in a
three- to sixfold increase in reporter gene activity (Fig. 8C).
Moreover, the repressing effect of CTCF binding to the P2-
proximal site was most profound in growth-arrested cells, i.e.,
under conditions in which transcription from the c-myc pro-
moter has been reported to be inhibited (see reference 27 for
a review). Thus, mutational analysis of the P2-proximal CTCF-
binding site strongly suggests that CTCF is a repressor of tran-
scription from the major human c-myc gene promoter.
To examine the ability of exogenously supplied CTCF to
repress the c-myc P2 promoter, we performed transient-co-
transfection experiments with a CMV promoter-driven CTCF
expression vector and the two c-myc promoter-CAT reporter
constructs described above (Fig. 8B). These transient-cotrans-
fection experiments are potentially complicated by endoge-
nous CTCF present in target cells which might repress reporter
constructs and mask any effect of the exogenous CTCF. There-
fore, to assess any effect of exogenous CTCF produced by the
transfected expression vector, conditions in which endogenous
CTCF was limiting with respect to the transfected target con-
structs were established (i.e., binding of endogenous CTCF
was saturated). Under such conditions, an excess of target
constructs free of bound endogenous CTCF should respond to
exogenous CTCF produced by the cotransfected expression
vector. Figure 8D (bars for 0 ?g of CTCF) shows that with an
input of 1 ?g of c-myc promoter-CAT constructs per transfec-
tion, the target constructs appeared to be in excess, since there
was little difference in CAT reporter activity between the wild-
type and mutated constructs. Under these conditions, intro-
duction of as little as 0.2 ?g of CTCF expression vector re-
sulted in repression of the wild-type but not the ACA-mutated
promoter, indicating that the sequence-specific interaction of
exogenously expressed CTCF with the P2-proximal DNA re-
gion can specifically repress the promoter. At a higher input of
exogenous CTCF (2.0 and 10 ?g of expression vector), a stron-
ger repression was achieved. However, some of this stronger
repressing effect does not require binding of CTCF to the P2-
proximal site because the ACA-mutated promoter also be-
comes repressed (Fig. 8D, two rightmost bars). This finding
indicates that at a high input level, CTCF can either bind to
low-affinity sites in the mutated promoter or interact with other
transcription factors involved in transcription from the P2 pro-
moter of the human c-myc gene. This may be quite specific for
the P2 c-myc promoter, since in cotransfection experiments
with several other promoters, including HIV LTR, murine
leukemia virus LTR, simian virus 40, and HSV TK, we noted
no promoter suppression by even high levels of exogenously
expressed CTCF (data not shown).
Taken together, the presence of two strong CTCF repressor
domains (Fig. 8A), our observation that mutation of a CTCF
binding site within the c-myc promoter results in increased
reporter gene activity (Fig. 8C), and the suppression of the
c-myc promoter activity by exogenous CTCF (Fig. 8D) indicate
that CTCF is a major physiological repressor of the human
c-myc P2 promoter.
We have cloned the human c-myc promoter-binding protein
CTCF and noted ?93% identity with the chicken CTCF pro-
tein. While more than 90% amino acid identity between avian
and mammalian nuclear proteins has been described for some
structural DNA- and RNA-binding factors (such as histones
and SR proteins), it is not common for sequence-specific
DNA-binding transcription factors. Among them, only a few
examples of such extreme conservation have been found: oct-1
(33), gata-3 (16), ets-1 (44), and max (39). The fact that chicken
and human CTCF amino acid sequences did not noticeably
diverge (Fig. 2B) during the estimated 200 to 300 million years
of evolution (20) is suggestive of a vital CTCF function con-
served in all vertebrates. Moreover, no significant amino acid
sequence alterations, either inside or outside the CTCF DNA
binding domain, were tolerated to maintain this conserved
function. Here, we present data showing that at least one such
conserved function of CTCF involves binding and repressing
c-myc gene promoters.
We found that CTCF is able to bind specifically to a number
of diverged DNA sequences in the promoter-proximal regions
of chicken, mouse, and human c-myc genes (Fig. 3 and 4).
FIG. 7. Different combinations of CTCF zinc fingers are required to bind
human and chicken c-myc promoters. Each lane is labelled by two numbers
indicating the first and the last zinc finger of each truncated form of the 11-finger
CTCF DNA binding domain. Full-length 11-Zn-finger polypeptide (panel A,
lane 1-11) and six carboxy-terminal deletion (panel A, lanes 1-10 to 1-5) and five
amino-terminal deletion (panel D, lanes 2-11 to 6-11) forms of the DNA binding
domain were synthesized in vitro as described in Materials and Methods, and
1-?l aliquots of each translation product were analyzed by SDS gel electrophore-
sis. (A and D) Basically equal amounts of each truncated form were synthesized.
Positions of the molecular mass markers are indicated on the left. (B and C)
EMSA analysis of C-terminally truncated forms binding to chicken c-myc frag-
ment V and human c-myc fragment A (Fig. 3), respectively. (E and F) Similar
analysis for N-terminally truncated forms. Gel shift reactions included equal
amounts (5 ?l) of each in vitro translation product with the control reticulocyte
lysate mixture (lanes ?) and with no protein (lanes No prot.).
2810FILIPPOVA ET AL.MOL. CELL. BIOL.
Although there is absolute (100%) identity between the
chicken and human CTCF proteins within the 11-zinc-finger
domain (Fig. 2B), the specific sequences to which CTCF binds
in the respective c-myc promoters are clearly divergent (Fig. 4
and 5A). How do identical CTCF DNA binding domains con-
tact these different DNA regulatory sequences? We have now
determined that CTCF utilizes different combinations of Zn
fingers to bind to these diverged chicken and human c-myc
promoters. First, proteinase protection assays indicated that
more CTCF Zn fingers are involved in binding to the P2-
proximal human promoter sequence (site A) than in binding to
chicken site V (Fig. 6). Second, a gel shift analysis using serially
truncated Zn fingers reveals that fingers 2 to 7 are involved in
binding to the chicken c-myc promoter (site V), while fingers 3
to 11 appear to be required for binding to the human c-myc
promoter (site A) (Fig. 7). Therefore, in binding to the chicken
c-myc promoter, only 6 of the 11 CTCF Zn fingers are utilized,
while human CTCF utilizes no more than 9 fingers in binding
to the diverged human c-myc promoter. In so concluding, how-
ever, we acknowledge certain limitations of our gel shift DNA
binding studies with terminally deleted CTCF forms possibly
arising from an apparent lack of rigorous quantitative analyses
of binding affinities of different groups of CTCF Zn fingers to
different DNA sequences and also from possible complex in-
terdependence of individual Zn fingers and other regions of
the full-length CTCF protein. A definitive demonstration of
exactly how different individual CTCF Zn fingers recognize
different nucleotide sequences will perhaps require a crystal
structure analysis of CTCF complexes with a number of its
DNA binding sites. However, such analysis may be a problem
because of the size of the components involved. At present, it
is worthwhile to note that the two different base-specific con-
tact patterns in chicken and human c-myc promoters deter-
mined by the methylation interference experiments (Fig. 4) are
consistent with DNA subsite sequences predicted for CTCF
fingers 2 to 7 and for fingers 3 to 11 by the set of rules proposed
for the Zn finger DNA recognition code based on the cocrystal
structure of several Zn finger domains and their cognate DNA
binding sites (43).
There appears to be considerable evolutionary conservation
in the patterns of c-myc expression, with myc expression en-
hanced in mitogen-stimulated cells and repressed during
terminal differentiation in multiple vertebrate species. It is per-
haps not surprising that CTCF, which appears to be an impor-
tant transcriptional repressor of c-myc, also displays marked
evolutionary conservation. What is quite surprising, however,
is the considerable evolutionary divergence of the CTCF target
sequences in the human and chicken c-myc promoters that
requires different combinations of CTCF Zn fingers to bind to
these regulatory sequences. Despite this strict evolutionary
conservation, there appears to be considerable flexibility inher-
ent in the CTCF DNA binding domain to enable it to bind to
divergent sequences within the c-myc promoters of different
Two other relatively large Zn finger proteins, Evi-1 and
MZF1, which can bind different DNA sequences have been
previously reported. MZF1 protein is a 13-Zn-finger protein
which appears to harbor two independent DNA binding do-
mains, a nine-Zn-finger domain separated from an additional
four-Zn-finger domain by a glycine-proline-rich region (9).
The Evi-1 oncogene protein also contains two domains of Zn
fingers, an amino-terminal domain of seven fingers and a car-
boxy-terminal domain of three fingers (29). In both MZF1 (30)
and the Evi-1 protein (8, 32), each of these domains binds
independently to distinct, diverged target DNA sequences. In
contrast, CTCF also binds to diverged sequences but does so
FIG. 8. CTCF contains two transcriptional repressor domains and negatively
regulates the P2 promoter of the human c-myc gene. (A) Expression vectors
producing the GAL4 (1-147) DNA binding domain alone (pSG424) or fused to
the C-terminal (pGal-CTCF-C) or N-terminal (pGal-CTCF-N) CTCF domain
flanking the 11-Zn-finger region were cotransfected into QT6 fibroblasts along
with the TK promoter-based reporter plasmids containing five (p5xUAS/TK-
Luc) or no (pTK-Luc) GAL4 binding sites, and the activity of the reporter,
normalized to the expression from the cotransfected pSV/?-gal construct, was
measured. Representative results from one of five independent experiments are
shown. The standard deviation calculated from five experiments was ?10%. (B)
Scheme for the reporter c-myc–CAT constructs. See text for details. (C) Tran-
scriptional activities of the wild-type P2 promoter-CAT construct (pAPwtCAT)
and of the CTCF binding site-mutated construct (pAPacaCAT) stably trans-
fected into NIH 3T3 fibroblasts and assayed under three different cell growth
conditions as described in the text. Standard error was calculated by measuring
normalized CAT activity in four separate plates of each stably transfected mass
culture. Results of these experiments were identical when CAT activity was
normalized to either ?-gal activity from cotransfected pCMV/?-gal plasmid or
the copy number of stably integrated reporter constructs estimated by Southern
blotting. (D) Transcriptional repression assayed by measuring CAT activity from
the pAPwtCAT and pAPacaCAT reporter constructs transiently cotransfected
along with increasing amounts of the pCI/CTCF expression vector into 293 cells.
Error bars represent the standard deviations of the means of four transfections
for each combination of the reporter and effector constructs.
VOL. 16, 1996CONSERVED ‘‘MULTIVALENT’’ REPRESSOR OF c-myc PROMOTERS2811
by utilizing different combinations of Zn fingers within a single
DNA binding domain. In this respect, we propose to define
CTCF as a multivalent factor.
We have accumulated several lines of evidence indicating
that CTCF is a negative regulator of the human c-myc pro-
moter. Transient-transfection assays utilizing chimeric GAL4
DNA binding domain-CTCF fusion products indicate that
CTCF harbors at least two transcriptional repressor domains
(Fig. 8A). Moreover, in stable-transfection experiments utiliz-
ing c-myc promoter-CAT constructs, we have observed that
selectively mutating the CTCF binding site within the human
P2-proximal promoter region (site A, Fig. 5A) results in in-
creased reporter gene activity (Fig. 8C). Finally, we have ob-
served in transient-cotransfection assays that CTCF specifically
represses human c-myc promoter activity through both DNA
binding-dependent and -independent pathways (Fig. 8D).
Taken together, these observations provide strong evidence
that CTCF is a negative regulator of c-myc transcription.
Several previous observations indicate that the P2-proximal
region to which CTCF binds and which it negatively regulates
appears to be critical for c-myc transcriptional regulation: (i)
the level of mRNAs initiated at the P2 promoter is usually
more than 80% of steady-state c-myc RNA levels in normal
cells (40); (ii) activity of transcription from the P2 promoter is
regulated by the rate of pausing and release of polymerase II
at the sequence immediately downstream of P2 (4, 41, 42); (iii)
pausing of polymerase II and promoter melting demonstrated
by in vivo footprinting analysis occur at around position ?30
(18), at a sequence that maps precisely within the CTCF bind-
ing site (Fig. 4A); and (iv) transcription of P2-initiated RNA is
blocked at the same site when c-myc is down-regulated in cells
induced to differentiate (17). Note that EMSA analysis of both
mouse and human nuclear extracts with human fragment A
containing these P2-proximal sequences indicates that CTCF is
the predominant nuclear protein interacting with the DNA
region downstream of the P2 initiation site (Fig. 1B). Thus,
CTCF binding to the P2-proximal site likely plays a critical role
in the complex regulatory events mediated through this site.
Since we did not attempt to distinguish the effect of CTCF on
initiation versus elongation, the precise molecular mechanism
of this repression remains to be determined.
Taken together, the marked evolutionary conservation of
CTCF repressor domains and the ability to bind promoters of
avian and mammalian c-myc genes suggest that the c-myc-
repressing function of CTCF may be conserved also in verte-
brates. Although previous mutational analysis of the CTCF
binding site in the chicken c-myc promoter indicated that the
Nsi mutation designed to specifically knock out CTCF binding
results in a decrease of transcription (15), we have recently
found that besides CTCF, this mutation also eliminates an
overlapping strong binding site for the Egr1 family of tran-
scription activators (6). We have also found that CTCF and
two Sp1-like and Egr1 proteins bind to the site V sequence of
the chicken c-myc promoter in a pairwise mutually exclusive
fashion and demonstrated by cotransfection experiments that
chicken CTCF represses the chicken c-myc promoter (13). This
suggests that chicken CTCF might actively repress the chicken
c-myc promoter by a combination of two mechanisms, by dis-
placing positive Sp1 and Egr1 family factors from the site V
sequence and bringing its own transcription repressor domains
to the promoter.
How can CTCF be a negative regulator of endogenous c-myc
gene transcription if it is ubiquitously expressed in different
cells, including proliferating cells with active c-myc expression?
Perhaps there is a specific posttranslational modification of
CTCF that regulates this repressor activity. Indeed, we have
recently documented (14) that CTCF is phosphorylated in vivo
and that the negative effect of CTCF on transcription strongly
depends on the site-specific reversible phosphorylation of its
C-terminal trans-repressor domain. Moreover, in comparison
with the wild-type CTCF protein, transient expression of the
constitutively hypophosphorylated form of CTCF resulted in
much stronger repression of target promoters (14). An addi-
tional reason to believe that the repression of c-myc genes by
CTCF is regulated at the level of specific posttranslational
modifications comes from our recent observation that in rap-
idly growing erythroid precursor HD3 cells CTCF is highly
phosphorylated. However, upon induction of terminal differ-
entiation of the cells, both c-myc expression and CTCF phos-
phorylation are extinguished (14).
In conclusion, we again note the 100% evolutionary conser-
vation within the entire CTCF 11-Zn-finger domain, including
those fingers that are not directly involved in binding to the
c-myc promoters. This suggests that these fingers are involved
in another conserved biological function(s), which might in-
clude zinc finger protein-protein or protein-RNA interactions
or interactions with regulatory elements of other specific target
genes. It is therefore likely that the strict conservation of CTCF
is driven by another preserved function(s) beyond binding to
c-myc promoters. We are now attempting to identify such a
We are grateful to Paul Goodwin and Tim Knight for assistance with
image analysis and help in preparing figures; to Michael Parker for
providing help with DNA sequence assembly and analysis; to Mary
Kay Dolejsi for automated DNA sequencing and oligonucleotide syn-
thesis; and to LeMoyne Mueller, Gilbert Loring, and Sandra Jo
Thomas for technical assistance. We thank V. KewalRamani for the
293 cell line, M. Linial for the QT6 cell line, Anton Krumm and Mary
Peretz for mouse and human c-myc gene plasmids, and P. Chambon
for anti-GAL4 antibodies. Michael Emerman, Philippe Soriano, Mark
Groudine, and Stephen Tapscott are thanked for discussions and re-
view of the manuscript.
Cena Myers was supported by a 1994 summer graduate student
fellowship. This work was funded by NIH/NCI grants RO1 CA20068 to
P. E. Neiman and RO1 CA55397 to S. J. Collins, by the NIH RO3
TW00057 Fogarty Award and by American Cancer Society grant DB54
to P. Neiman and V. Lobanenkov, by a Human Frontier Science
Program (HFSP) Long-Term Fellowship to E. M. Klenova, by Cancer
Research Campaign grants to G. H. Goodwin, and by Pilot Study
Seattle Breast Cancer Research Program grant NCI P20 CA66186-01
to V. Lobanenkov.
1. Bogenhagen, D. F. 1993. Proteolytic footprinting of transcription factor
TFIIIA reveals different tightly binding sites for 5S RNA and 5S DNA. Mol.
Cell. Biol. 13:5149–5158.
2. Casaregola, S., A. Jacq, D. Laoudj, G. McGurk, S. Margarson, M. Tempete,
V. Norris, and I. B. Holland. 1992. Cloning and analysis of the entire
Escherichia coli ams gene: ams is identical to hmp1 and encodes a 114 kDa
protein that migrates as a 180 kDa protein. J. Mol. Biol. 228:30–40.
3. Collins, S. J., R. C. Gallo, and R. E. Gallagher. 1977. Continuous growth and
differentiation of human myeloid leukemia cells in suspension culture. Na-
ture (London) 270:347–349.
4. Eick, D., F. Kohlhuber, D. A. Wolf, and L. J. Strobl. 1994. Activation of
pausing RNA polymerases by nuclear run-on experiments. Anal. Biochem.
5. El-Baradi, T., and P. Tomas. 1991. Zinc finger proteins: what we know and
what we would like to know. Mech. Dev. 35:155–169.
6. Filippova, G. N., P. E. Neiman, S. J. Collins, and V. V. Lobanenkov. Un-
7. Franklin, A. J., T. L. Jetton, K. D. Shelton, and M. A. Magnuson. 1994. BZP,
a novel serum-responsive zinc finger protein that inhibits gene transcription.
Mol. Cell. Biol. 14:6773–6788.
8. Funabiki, T., B. L. Kreider, and J. N. Ihle. 1994. The carboxyl domain of zinc
fingers of the Evi-1 myeloid transforming gene binds a consensus sequence
of GAAGATGAG. Oncogene 9:1575–1581.
2812FILIPPOVA ET AL.MOL. CELL. BIOL.
9. Hromas, R., S. J. Collins, D. Hickstein, W. Raskind, L. L. Deaven, P. Download full-text
O’Hara, F. S. Hagen, and K. Kaushansky. 1991. A retinoic acid-responsive
human zinc finger gene, MZF1, preferentially expressed in myeloid cells. J.
Biol. Chem. 266:14183–14187.
10. Ishida, S., K. Shudo, S. Takada, and K. Koike. 1994. Transcription from the
P2 promoter of human protooncogene myc is suppressed by retinoic acid
through an interaction between the E2F element and its binding proteins.
Cell Growth Differ. 5:287–294.
11. Kakidani, H., and M. Ptashne. 1988. Gal4 activates gene expression in
mammalian cells. Cell 52:161–167.
12. Kinzler, K. W., J. M. Ruppert, S. H. Bigner, and B. Vogelstein. 1988. The
GLI gene is a member of the Kruppel family of zinc finger proteins. Nature
13. Klenova, E. M., G. N. Filippova, C. Meyers, S. Fagerlie, P. E. Neiman, G. H.
Goodwin, and V. V. Lobanenkov. Unpublished data.
14. Klenova, E. M., G. N. Filippova, P. E. Neiman, G. H. Goodwin, and V. V.
Lobanenkov. Functional regulation of the chicken c-myc transcriptional re-
pressor CTCF by phosphorylation. Submitted for publication.
15. Klenova, E. M., R. H. Nicolas, H. F. Paterson, A. F. Carne, C. M. Heath,
G. H. Goodwin, P. E. Neiman, and V. V. Lobanenkov. 1993. CTCF, a con-
served nuclear factor required for optimal transcriptional activity of the
chicken c-myc gene, is an 11-Zn-finger protein differentially expressed in
multiple forms. Mol. Cell. Biol. 13:7612–7624.
16. Ko, L. J., M. Yamamoto, M. W. Leonard, K. M. George, P. Ting, and J. D.
Engel. 1991. Murine and human T-lymphocyte GATA-3 factors mediate
transcription through a cis-regulatory element within the human T-cell re-
ceptor ? gene enhancer. Mol. Cell. Biol. 11:2778–2784.
17. Kohlhuber, F., L. J. Strobl, and D. Eick. 1993. Early down-regulation of
c-myc in dimethylsulfoxide-induced mouse erythroleukemia (MEL) cells is
mediated at the P1/P2promoters. Oncogene 8:1099–1102.
18. Krumm, A., T. Meulia, M. Brunvand, and M. Groudine. 1992. The block to
transcriptional elongation within the c-myc gene is determined in the pro-
moter-proximal region. Genes Dev. 6:2201–2213.
19. Lavery, D. J., and U. Schibler. 1993. Circadian transcription of the choles-
terol 7a hydroxylase gene may involve the liver-enriched bZIP protein DBP.
Genes Dev. 7:1871–1884.
20. Lewin, B. 1990. Genes IV, p. 504–506. Oxford University Press, New York.
21. Lipp, M., R. Schilling, S. Wiest, G. Laux, and G. W. Bornkamm. 1987. Target
sequences for cis-acting regulation within the dual promoter of the human
c-myc gene. Mol. Cell. Biol. 7:1393–1400.
22. Lobanenkov, V., and G. Goodwin. 1989. CCCTC-binding protein: a new
nuclear protein factor which interaction with 5?-flanking sequence of chicken
c-myc oncogene correlates with repression of the gene. Proc. Acad. USSR
23. Lobanenkov, V. V., V. V. Adler, E. M. Klenova, R. H. Nicolas, and G. H.
Goodwin. 1989. CCCTC-binding factor (CTCF): a novel sequence-specific
DNA binding protein which interacts with the 5?-flanking sequence of the
chicken c-myc gene, p. 45–68. In T. S. Papas (ed.), Gene regulation and
AIDS: transcriptional activation, retroviruses and pathogens. Portfolio Pub-
lishing Corp., Woodlands, Tex.
24. Lobanenkov, V. V., R. H. Nicolas, V. V. Adler, H. Paterson, E. M. Klenova,
A. V. Polotskaja, and G. H. Goodwin. 1990. A novel sequence-specific DNA
binding protein which interacts with three regularly spaced direct repeats of
the CCCTC-motif in the 5? flanking sequence of the chicken c-myc gene.
25. Lobanenkov, V. V., R. H. Nicolas, M. A. Plumb, C. A. Wright, and G. H.
Goodwin. 1986. Sequence-specific DNA-binding proteins which interact with
(G?C)-rich sequences flanking the chicken c-myc gene. Eur. J. Biochem.
26. Luckow, B., and G. Schutz. 1987. CAT constructions with multiple unique
restriction sites for the functional analysis of eukaryotic promoters and
regulatory elements. Nucleic Acids Res. 15:5490.
27. Marcu, K. B., S. A. Bossone, and A. J. Pate. 1992. Myc function and regu-
lation. Annu. Rev. Biochem. 61:809–860.
28. Maxam, A. M., and W. Gilbert. 1980. Sequencing end-labeled DNA with
base-specific chemical cleavages. Methods Enzymol. 65:499–560.
29. Morishita, K., D. S. Parker, M. L. Mucenski, N. A. Jenkins, N. G. Copeland,
and J. N. Ihle. 1988. Retroviral activation of a novel gene encoding a
zinc-finger protein in IL-3-dependent myeloid leukemia cell lines. Cell 54:
30. Morris, J. F., R. Hromas, and F. J. Rauscher III. 1994. Characterization of
the DNA-binding properties of the myeloid zinc finger protein MZF1: two
independent DNA-binding domains recognize two DNA consensus se-
quences with a common G-rich core. Mol. Cell. Biol. 14:1786–1795.
31. Packham, G., and J. L. Cleveland. 1995. c-Myc and apoptosis. Biochim.
Biophys. Acta 1242:11–28.
32. Perkins, A. S., R. Fishel, N. A. Jenkins, and N. G. Copeland. 1991. Evi-1, a
murine zinc finger proto-oncogene, encodes a sequence-specific DNA-bind-
ing protein. Mol. Cell. Biol. 11:2665–2674.
33. Petryniak, B., L. M. Staudt, C. E. Postema, W. T. McCormack, and C. B.
Thompson. 1990. Characterization of chicken octamer-binding proteins
demonstrates that POU domain-containing homeobox transcription factors
have been highly conserved during vertebrate evolution. Proc. Natl. Acad.
Sci. USA 87:1099–1103.
34. Query, C. C., R. C. Bentley, and J. D. Keene. 1989. A common RNA
recognition motif identified within a defined U1 RNA binding domain of the
70K U1 snRNP protein. Cell 57:89–101.
35. Sadowski, I., and M. Ptashne. 1989. A vector for expressing Gal (1-147)
fusions in mammalian cells. Nucleic Acids Res. 17:7539.
36. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a
laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y.
37. Seed, B., and J.-Y. Sheen. 1988. A simple phase-extraction assay for chlor-
amphenicol acetyltransferase activity. Gene 67:271–277.
38. Shuman, J. D., C. R. Vinson, and S. L. McKnight. 1990. Evidence of changes
in protease sensitivity and subunit exchange rate on DNA binding by C/EBP.
39. Sollenberger, K., T. Kao, and E. Taparowsky. 1994. Structural analysis of the
chicken max gene. Oncogene 9:661–664.
40. Spencer, C. A., and M. Groudine. 1991. Control of c-myc regulation in
normal and neoplastic cells. Adv. Cancer Res. 56:1–48.
41. Strobl, L. J., and D. Eick. 1992. Hold back of RNA polymerase II at the
transcription start site mediates down-regulation of c-myc in vivo. EMBO J.
42. Strobl, L. J., F. Kohlhuber, J. Mautner, A. Polack, and D. Eick. 1993.
Absence of a paused transcription complex from the c-myc P2promoter of
the translocation chromosome in Burkitt’s lymphoma cells: implication for
the c-myc P1/P2promoter shift. Oncogene 8:1437–1447.
43. Suzuki, M., M. Gerstein, and N. Yagi. 1994. Stereochemical basis of DNA
recognition by Zn fingers. Nucleic Acids Res. 22:3397–3405.
44. Watson, D. K., M. J. McWilliams, P. Lapis, J. A. Lautenberger, C. W.
Schweinfest, and T. S. Papas. 1988. Mammalian ets-1 and ets-2 genes encode
highly conserved proteins. Proc. Natl. Acad. Sci. USA 85:7862–7866.
VOL. 16, 1996 CONSERVED ‘‘MULTIVALENT’’ REPRESSOR OF c-myc PROMOTERS2813