THE JOURNAL OF BIOLOGICAL CHEMISTRY© 1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 272, No. 50, Issue of December 12, pp. 31777–31784, 1997
Printed in U.S.A.

A Transcription Activator with Restricted Tissue Distribution
Regulates Cell-specific Expression of a1(XI) Collagen*(Received for publication, August 5, 1997)

Akihiro Kinoshita‡, Patricia Greenwel‡, Shizuko Tanaka‡, Maurizio Di Liberto§,
Hidekatus Yoshioka¶, and Francesco Ramirez‡i
From the ‡Brookdale Center for Developmental and Molecular Biology, Mt. Sinai School of Medicine, New York,New York 10029, the §Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New
York, New York 10021, and the ¶Department of Molecular Biology and Biochemistry, Okayama University Medical
School, Okayama 565, Japan

Different regulatory programs are likely to control
expression of the a1(XI) collagen (COL11A1) gene in cartilaginous and non-cartilaginous tissues and in coordination with different collagen genes. Here, we report
the identification of a cis-acting element that is requiredfor constitutive and tissue-specific activity of the proximal COL11A1 promoter. The element binds an apparently novel activator whose expression is restricted
mostly, but not exclusively, to cells of mesenchymal origin. Transient transfection experiments using wildtype and mutant constructs demonstrated the criticalcontribution of a 45-base pair upstream element (FP9) topromoter activity. The same functional tests and DNA
binding assays narrowed down the critical portion of
FP9 to a 20-base pair sequence, which consists of animperfect palindrome with strong homology to the
GATA consensus motif. Despite being able to bind GATA
proteins in vitro, FP9 is actually recognized by a distinct;100-kDa polypeptide (FP9C) probably belonging to thezinc-finger family of transcription factors. FP9C binding
was mostly identified in nuclei from cells of mesenchymal origin, including those actively engaged in COL11A1
transcription. A positive correlation was also established between the level of FP9C binding and the degreeof cell differentiation in vitro. Thus, FP9C represents anunusual example of tissue-specific and differentiationrelated transcription factor with overlapping expression in hard and soft connective tissues.

Proper expression of collagen genes during embryogenesis
and in the adult organism are required for the correct assembly
and the physiological maintenance of the extracellular matrix(ECM)1 (1). Conversely, deregulated production of collagenmolecules is the hallmark of a variety of connective tissue
disorders (2). During the past few years, substantial effort has* This work was supported by Grant R37-38648 from the NIAMS,
National Institutes of Health. This is article 244 from the Brookdale
Center for Developmental and Molecular Biology. The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. .
i To whom correspondence should be addressed: Brookdale Center for
Developmental and Molecular Biology, Mount Sinai School of Medicine,
One Gustave Levy Place, New York, NY 10029. Tel.: 212-241-7984;
E-mail: ramirez@msvax.mssm.edu.
1
The abbreviations used are: ECM, extracellular matrix; bp, basepair(s); CAT, chloramphenicol acetyltransferase; COL5A2, the genecoding for the a2 chain of type V collagen; COL11A1, the gene coding forthe a1 chain of type XI collagen; EMSA, electrophoretic mobility shift
assay; HUVEC, human umbilical vein endothelial cells; MELC, mouseerythroleukemia cells; OPA, 1,10-phenanthroline; smc, smooth muscle
cell(s).
This paper is available on line at http://www.jbc.org

been directed toward understanding the transcription of collagen genes, including the identification of factors that confer
spatiotemporal specificity. By and large, most of the progresshas been made with the genes coding for the subunits of types
I and II collagen. This body of work has identified cis-acting
elements and trans-acting factors that restrict expression ofthese collagens to distinct mesenchymal cell lineages. Several
reports have demonstrated that transcription of the a1(I) collagen gene in osteoblasts is under the control of an upstream
element that contains multiple binding sites for nuclear proteins (4 –7). This modularly arranged cis-acting element is recognized by both ubiquitous and osteoblast-specific factors (6, 7).
There is evidence suggesting that the latter includes an apparently new member of the homeodomain proteins (7). Similarly,
an 18-bp sequence within the first intron of the a1(II) collagengene has been shown to control promoter expression in cartilaginous tissues by binding a transcriptional complex that includes the SOX9 activator (8, 9). Finally, expression of theproximal promoter of a2(I) collagen in dermis, tendon, and thefibrous layers of many internal organs has been reported to besignificantly augmented by a far-upstream enhancer that contains fibroblast-specific DNase-hypersensitive sites (10).
We are interested in the control of the genes coding for thesubunits of the so-called minor fibrillar collagens (11). The two
members of this subgroup of collagens, types V and XI, play a
critical role in regulating the formation of the types I and II
fibrillar networks in cartilaginous and non-cartilaginous tissues, respectively (12, 13). They can also give rise to cross-type
trimers consisting of a2(V) and a1(XI) chains in vascular muscles and in the developing bone, among other tissues (11).
Although the precise function of this cross-type molecule remains a mystery, its tissue distribution implies that the a2(V)and a1(XI) collagen (COL5A2 and COL11A1) genes are more
broadly expressed than those coding for the subunits of theparental trimers, as well as the co-expressed types I and II
collagen genes. This in turn endows COL11A1 with the distinction of being the sole collagen gene to be transcribed in both
cartilaginous and non-cartilaginous tissues. We have previously reported the characterization of the proximal upstream
sequence of COL11A1 (14). DNA sequencing has revealed thatthe promoter contains GC-rich boxes in place of the TATA
motif. DNA transfections have established that cis-acting elements located between nucleotides 2541 and 2199 drive constitutive transcription from the proximal promoter in a1(XI)
collagen-producing cells. The latter tests have employed therhabdomyosarcoma A-204 line and vascular smooth muscle
cells (smc), as the COL11A1-positive cells, and the fibrosarcoma HT-1080 line, as the COL11A1-negative cell. Finally,
DNase I footprinting assays have mapped nine areas of interaction with nuclear proteins (FP1 to FP9) within the proximal

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31778Cell Type-specific Transcription Factor

promoter (Fig. 1). Based on sequence homologies, most of themwere thought to represent binding sites for ubiquitous activators, like AP2 and Sp1 (14). An interesting correlation was also
noted with the proximal promoter of the coordinately expressed
COL5A2 gene (15). The two collagens were in fact found toshare an upstream element, called FPB in COL5A2 and FP7 in
COL11A1, that binds the same ubiquitously expressed transacting factor (14).
The present study was designed to further characterize thecis-acting elements of the 2541 to 2199 segment and implicitly, the trans-acting factors involved in COL11A1 regulation.
The results indicate that constitutive and cell type-specifictranscription of the 2541COL11A1 promoter is under the control of FP9, an element farther upstream of FP7. More importantly, the experimental evidence suggests that the transcription factor binding to FP9 is an apparently new member of thezinc-finger family of nuclear proteins. The factor (FP9C) ismostly expressed in cells of mesenchymal origin and always inthose actively transcribing COL11A1. Interestingly, we also
found a positive correlation between the level of FP9C binding
and the stage of differentiation of osteoblastic and skeletal
muscle cell lines.

medium supplemented with 10% fetal calf serum. MC3T3-E1 and Jurkat cells were cultured in 10% fetal calf serum supplemented witha-minimal essential medium and RPMI medium, respectively; melanocytes and keratinocytes were cultured in Medium 154 supplemented
with HMGS and HKGS, respectively; HUVEC were grown in Medium
200 supplemented with LSGS. C2C12 myoblasts and the myogenic P2line were induced to differentiate by placing confluent cells in 2% horse
serum (16). Short term cultures of mouse costal chondrocytes wereprepared as described (17). RNA was purified from cultured cells andsubjected to Northern hybridization to probes for COL11A1 and glyceraldehyde-3-phosphate dehydrogenase according to standard protocols(18). Progressive 59 deletion of the COL11A1 promoter and internal
substitutions of the 45-bp and 20-bp FP9 with unrelated sequenceswere engineered using specific oligonucleotide primers and the polymerase chain reaction technique and were all verified by DNA sequencing(18). The mutant sequences were subcloned 59 of the chloramphenicolacetyltransferase (CAT) reporter gene in the vector pBLCAT3 (19).

MATERIALS AND METHODS

Cells and Other Reagents—Human cell lines included primary embryonic dermal fibroblasts (CF 37), adult keratinocytes, neonatal melanocytes, umbilical vein endothelial cells (HUVEC), and the rhabdomyosarcoma A-204 and T-lymphocyte Jurkat lines. Keratinocytes,
melanocytes and HUVEC were purchased from Cascade Biologics, Inc.
(Portland, OR). Rat cells included primary vascular smooth muscle cells(smc) and hepatocytes, the neurogenic PC12 line, the osteosarcoma
lines ROS 17/2.8 and ROS 25, and the chondrosarcoma line RCS. Mousecells included primary costal chondrocytes, NIH3T3 fibroblasts, themultipotential mesodermal line C3H10T1/2, C2C12 myoblasts, theC3H10T1/2-derived P2 myogenic line, MC3T3-E1 osteoblast-like calvarial cells, and erythroleukemia cells (MELC). Consensus recognition
sequences for and antibodies against various transcription factors werepurchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell Cultures, RNA Analysis, and DNA Transfection Experiments—
Most cells were grown and maintained in Dulbecco’s modified Eagle’s

FIG. 2. Functional analysis of the342-bp promoter segment in A-204
cells. A, schematic map of the proximal
promoter, which includes the nucleotide
position of the 59 ends of the deletion mutants, the location of the footprinted areas, and the CAT reporter gene represented by the gray rectangle. B and C
show the functional assays of the 59 deletion mutants (2541 to 2199) and of theconstructs with nucleotide substitutions(2541/45 and 2541/20) or internally deleted (2541/D167). Histograms indicate
the percentage of CAT activity normalized for the co-transfected control and expressed relatively to pSVCAT activity.
The data represent an average of three tofive independent tests 6 S.D.

FIG. 1. COL11A1 proximal promoter. Top, schematic representation of the promoter showing the relative positions of the footprinted
areas including those six that lay between nucleotides 2541 and 2199
(14). The nucleotide composition of the 45-bp long FP9 is shown below
with the 20-bp core highlighted by the brackets; the inverted GATA-like
repeats are underlined; and the major and minor contact points of thenuclear factors are indicated by closed and open circles, respectively (seeFig. 3). Below the sequence are the nucleotide substitutions in the 20-bp
core used in the competition assays shown in Fig. 3A.

Cell Type-specific Transcription Factor

31779

FIG. 3. Binding specificity of A-204
nuclear proteins to FP9. A, EMSA was
performed with the 45-bp probe and binding was competed with 20- and 50-foldmolar excess of the same unlabeled probe(45) or the wild-type (20) and mutant versions (M1–M4) of the 20-bp core. The composition of the 45-bp and 20-bp oligonucleotides are shown in Fig. 1. B, EMSAs
were performed using the indicatedprobes and nuclear extracts without (2)and with preincubation with antibodies
against GATA-1 (a1), GATA-3 (a3), orSp1 (aS). C, EMSAs were performed using the indicated probes and A-204 nuclear extract without (2) and with unlabeled competitors corresponding to the
GATA (G) and FP9 (F9) sequences. In the
EMSA on the left, competitors were added
at 100-fold excess; in the EMSA on theright, the GATA competitor was added at
20-, 50-, 100-, and 300-fold excess and theFP9 competitor at 20- and 50-fold excess.
In all panels, the arrow and the closed
circle identify FP9C and GATA-1,
respectively.

Purified plasmid DNA was introduced into the cells by the calcium
phosphate method according to the published protocol along with thenormalizing standard pSVLUC, a plasmid containing the luciferase
reporter gene under the transcriptional control of the SV40 promoter(20). About 48 h after transfection, cells were harvested and assayed for
CAT and luciferase activities. Values were normalized for the co-transfected plasmid and expressed relatively to parallel transfections of thepSVCAT vector. Cell transfections were performed multiple times andthe resulting data were evaluated using Student’s t test.
In Vitro DNA Binding Assays—Crude nuclear extracts were purified
according to our published protocol (15). Unless otherwise specified, 5
mg of nuclear proteins were used in the electrophoretic mobility shift
assay (EMSA) together with approximately 10,000 dpm of labeled oligonucleotide probe as described previously (15). Unlabeled competitors
were added in the amounts indicated in the figure legends, or otherwise
at 100-fold molar excess. In the zinc-dependence assay, 0.5 ml of 1,10phenanthroline (OPA) resuspended in ethanol at the concentration of 1M, 0.2 M, or 0.1 M was added to 47.5 ml of buffer solution containing
nuclear proteins and dI-dC and was then incubated on ice for 5.5 h. Thiswas followed by addition of the labeled probe and incubation on ice foran additional 309 prior to EMSA. Conditions for DNase I footprinting
and DNA methylation interference assays were essentially identical tothe published protocols (15).
Gel Filtration Chromatography—To estimate the molecular weightof denatured FP9C, nuclear extracts from A-204 cells (500 mg) weredissolved in 500 ml of denaturing buffer containing 6 M guanidine HCl,
50 mM Tris-HCl, pH 8.0, 0.1 mM dithiothreitol, 0.1 mM of phenylmethylsulfonyl fluoride and left on ice for 30 min. After clearing it bycentrifugation, the solution was applied to a Sephacryl S-300 column(50 cm 3 1 cm, inner diameter) equilibrated in the same denaturing
buffer. Fractions (0.75 ml) were collected and analyzed by EMSA after
microdialysis of 100-ml aliquots against 20% glycerol, 20 mM Hepes, pH
7.9, 0.1 mM dithiothreitol, 0.1 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride. Marker proteins ranging from 14 to 100 kDa were frac-tionated through the same column under identical conditions and analyzed by SDS-polyacrylamide gel electrophoresis.
RESULTSA previous study has delineated the shortest segment of theproximal COL11A1 promoter capable of directing constitutive
and cell type-specific expression of the reporter CAT gene intransient transfection experiments (14). The segment extends
from nucleotide 2541 to nucleotide 2199 with respect to themajor start site of transcription of this tissue-specific TATAless gene. Within it, DNase I footprinting assays identified six
distinct areas of protection from nuclease digestion by nuclear
proteins, which were termed FP9 to FP4 (Fig. 1). In the present
study, the same combination of DNA transfection and DNAbinding assays was used to further characterize the 342-bp
upstream segment of COL11A1.
FP9 Is Required for 2541COL11A1 Promoter Activity—To
dissect the functional properties of the 342-bp segment, theactivity of mutant constructs harboring progressive 59 promoter deletions, from FP9 to FP4, were compared after transfection into A-204 cells. The analysis showed that all mutant
plasmids drive expression of the CAT gene to levels comparable
to the basal activity of the 2199COL11A1 promoter (Fig. 2A).
The results therefore suggested that the 45-bp-long FP9 may
be the principle contributor to transcription from the 2541
promoter. This postulate was corroborated by the finding thatthe 2541 promoter construct containing an unrelated sequencein place of the 45-bp FP9 (construct 2541/45) displayed thesame activity as the basal 2199COL11A1 plasmid (Fig. 2C). To

31780Cell Type-specific Transcription Factor

FIG. 4. Methylation interference analyses. A, methylation interference analysis of the single complex formed between the FP9 probe and theA-204 nuclear extract. Major and minor methylated G are indicated by closed and open circles, respectively. B, methylation interference analysisof the B1 and B2 complexes formed between the FP9 probe and smc nuclear extract (see EMSA on the left). The amount of smc nuclear extract usedin the EMSA was 5, 10, and 20 mg. Contact points are indicated by the open and closed circles in B1 and by the asterisk in B2. The pattern of B1
should be compared with that in A, whereas the pattern of B2 should be compared with that in C. C, methylation inference analysis of the single
complex formed between the GATA consensus sequence and the MELC. The contact point is indicated by the asterisk. In all panels, the results ofthe sense and antisense strand probes are shown along with the sequence around the contact points. Lane F, free DNA; lane B, bound DNA
fragment.

establish whether other elements in the 342-bp segment might
be required to support FP9 activity and indirectly to confirm
the above results, another mutant construct was tested bytransient transfection. The mutation was engineered using
BstXI restriction sites conveniently located at nucleotides 2482
and 2316 and resulted in the internal loss of the segment
spanning from FP8 to FP6 (Fig. 2A). The mutant plasmid(construct 2541/D167) exhibited slightly higher activity than
the wild-type construct (Fig. 2C). Although a similar test was
not performed for the sequence encompassing FP4 and FP5,
the results strongly suggested that FP9 is the major contributor to constitutive and cell type-specific transcription from theproximal COL11A1 promoter. Incidentally, wild-type and mutant constructs transfected into the HT-1080 line were transcriptionally inactive (data not shown).
The FP9 Binding Site Contains a GATA Palindrome—The
EMSA was initially employed to assess the complexity of theFP9 binding pattern, as well as its specificity. To this end,
nuclear proteins purified from A-204 cells were incubated withthe radiolabeled 45-bp-long FP9 probe without and with increasing amount of the same unlabeled sequence. The results
revealed the formation of a single predominant complex which
migrates toward the top of the gel and is specifically competedby the same 45-bp-long sequence (Fig. 3A). Occasionally, weobserved a lesser intense and faster moving complex (see Fig.
3C) whose identity was not investigated in the present study.
Next, the methylation interference assay was performed tofurther characterize the trans-acting factor that binds to FP9.
This delineated the main area of DNA-protein interaction byidentifying the G residues that are contacted by the nuclear
protein. Three major contact points and two minor ones werelocated within the 20-bp core of FP9 (Fig. 4A). This sequence ischaracterized by an imperfect palindrome with a 2-bp interruption and strong homology to the binding site for GATA proteins(Fig. 1) (21). The EMSA reiterated the importance of the 20-bp
core by documenting the ability of this sequence to compete forbinding to FP9 almost as effectively as the 45-bp long oligonucleotide (Fig. 3A). It also documented the specificity of theinteraction, in that both the 45-bp and 20-bp oligonucleotides
competed formation of the complex with comparable efficiency(Fig. 3A). Furthermore, it showed that the competitiveness ofthe core sequence was somewhat diminished by substitutions
outside of the GATA motifs (M1 and M4, Fig. 3A) and wascompletely abolished by those introduced within them (M2 andM3, Fig. 3A). Altogether, the results of the in vitro binding tests
implicated the imperfect GATA palindrome as the major recognition site of the FP9 complex (FP9C). Consistent with thisconclusion, substitution of the 20-bp core of FP9 with an unrelated sequence rendered the 2541 promoter construct (2541/
20) as inactive as the plasmid without the entire FP9 or theconstruct with the mutant version of the 45 bp (Fis. 2, B and C).
FP9C Is a Novel Zinc-finger Protein—The GATA represents
a family of six related zinc-finger transcription factors, whichbind to the same (A/T)GATA(AG/GC) consensus sequence andare differently expressed in various tissues, including vascular
muscles (21). To test whether or not the imperfect FP9 palindrome could be recognized in vitro by GATA proteins, bindingof MELC nuclear proteins to FP9 and the GATA consensus
sequence were compared. The erythroleukemia cells were chosen for the test because they produce large amounts of GATA-1,
the prototype of the GATA family (22). The assay documented
the ability of FP9 to bind in vitro the same protein of the MELC
nuclear extract as the GATA consensus sequence (Fig. 3B).
Aside from the similar migration, identity between the twocomplexes was supported by the finding that they were bothsupershifted by anti-GATA-1 antisera but not by anti-GATA-3
or anti-Sp1 antibodies (Fig. 3B). Based on the above results, wetested whether A-204 nuclei contain a GATA-like binding ac-

Cell Type-specific Transcription Factor
tivity and found that the GATA consensus sequence failed toyield a retarded band in the EMSA (Fig. 3C). To confirm theabsence of GATA-like proteins in this a1(XI) collagen-producing tumor line, two additional EMSAs were performed. The
first revealed that the A-204 nuclear factors that binds to the
FP9 sequence is not recognized by the anti-GATA antibodies orby the control anti-Sp1 antisera (Fig. 3B). In the second EMSA,
formation of the complex between A-204 nuclear proteins andthe FP9 sequence was challenged with molar excess of the
GATA consensus sequence or the 45-bp oligonucleotide. As
expected, 20-fold excess of the latter abrogated FP9C binding;by contrast, competition with the GATA consensus sequence
had no significant effect on FP9C formation even when theoligonucleotide was added in vast molar excess (Fig. 3C).
Hence, a transcription factor other than GATA binds to theimperfect GATA palindrome of FP9 in the COL11A1-positive
rhabdomyosarcoma cells.
OPA is a potent chelator of zinc and an effective inhibitor of
DNA binding by zinc-finger proteins (23). Consistent with thisproperty, formation of GATA-1 between the MELC extract and
the GATA consensus sequence was gradually blocked by increasing amounts of OPA (Fig. 5A). The same result was obtained with the FP9 probe and the A-204 nuclear extract, thus
implying that the factor recognizing the dyad of symmetry in
the COL11A1 promoter is most certainly a zinc-finger protein.
Assuming a globular conformation for both proteins, the slower
migration of FP9C compared with GATA-1 could be interpreted
as suggesting that the former complex may be larger than the
average GATA protein (;50 kDa) (21). To test this hypothesis,
we estimated the molecular mass of the denatured protein by
gel filtration chromatography. To this end, A-204 nuclear extract was denatured before being applied to the column equilibrated in the same denaturing buffer and the resulting fractions were renatured prior to be tested by the EMSA.
Maximum FP9C binding activity was observed around a molecular range (80 –120 kDa) significantly greater than the one
expected for a GATA protein (Fig. 5B). A similar estimate wasobtained for the native protein using glycerol gradient sedimentation analysis (data not shown). In sum, the above experiments suggested that an apparently novel ;100-kDa zincfinger protein binds to a critical upstream regulator of theproximal COL11A1 promoter.
FP9C Expression Is Cell Type-restricted—The absence of
FP9C binding activity in MELC nuclei raised the possibility
that the COL11A1 activator may only be present in a restricted
number of cells. To test this hypothesis, we undertook a systematic EMSA screen of nuclear extracts from normal andtransformed cell lines. In addition to the rhabdomyosarcoma
A-204, substantial amount of FP9C was observed in cells of
mesenchymal origins, such as chondrocytes and smc, and in
keratinocytes and vascular endothelial cells (Fig. 6A). By contrast, little or no FP9C binding activity was seen with nuclear
extracts from fibroblasts, T-lymphocytes, neurogenic cells,
hepatocytes, and melanocytes (Fig. 6A). Binding of the ubiquitous NF-1 protein to its cognate site was nearly the same infibroblasts compared to A-204 nuclear extracts, thus indirectly
validating the significant difference in FP9C content between
the cell lines (Fig. 6B). Finally, Northern blot hybridizations
established a positive correlation between FP9C binding activity and COL11A1 gene expression (Fig. 6C). Altogether, theresults strongly suggested that FP9C may be a regulator of cell
type-specific transcription, in addition to controlling COL11A1
gene expression.
Aside from FP9C, smc nuclear extracts yielded a second andless evident complex whose relative migration appeared similar to that of GATA-1 (Fig. 6A). This raised the possibility that

31781

FIG. 5. Characterization of FP9C. A, EMSAs were performed with
the indicated probes and nuclear extracts, and under increasing concentrations of OPA (in mM) for 5.5 h. Aside from the sample without
OPA (2), another control included addition of ethanol to the binding
reaction at the same final concentration as in the OPA-treated samples.
B, gel filtration chromatography of denatured A-204 nuclear proteins.
Aliquots (10 ml) from fractions were examined for FP9C binding activity
by EMSA following renaturation. The migration of size markers (inkDa) is indicated above each autoradiography. In all panels, the arrow
and the closed circle identify FP9C and GATA-1, respectively.

the second complex could either correspond to an smc-specific
GATA protein or represent a product of FP9C degradation. A
methylation interference test was therefore performed on eachof the smc retarded bands to discriminate between these alternatives. As expected, the slower migrating EMSA band of thesmc sample (B1) yielded the same contact points as the single
band of the A-204 sample (Fig. 4, compare A and B). By contrast, the faster migrating band of the smc sample (B2) yieldeda pattern different from FP9C but identical to GATA-1 (Fig. 4,
compare B and C). Furthermore, competition with GATA consensus sequence eliminated the faster but not the slower migrating complex of the smc sample (data not shown). Recent
expression data support the idea that the smc complex boundby FP9 is likely to correspond to GATA-6 (24). Aside fromestablishing the contact points of GATA-6, the methylationinterference assay reiterated the specificity of FP9C binding
and the identity of the FP9C protein in different cell lines. The
competition experiments described in the previous section
strongly suggested that FP9C binds to the 45-bp probe withsignificantly higher affinity than GATA-1 (Fig. 3C). Consistent
with this postulate, addition of increasing amounts of smc
nuclear extract to the FP9 binding reaction translated into
substantially higher intensification of the B1 complex relatively to B2 (Fig. 4B). This last result was interpreted asindicating that GATA-6 binding in vitro may have no physiological relevance to COL11A1 gene regulation.

31782Cell Type-specific Transcription Factor

FIG. 6. Screening cells for FP9C binding activity and COL11A1 expression. A, EMSAs were performed with the same amount of FP9probe (10,000 dpm) and the same amount of nuclear extracts (5 mg) purified from A-204 (Rh), smc, chondrocytes (ch), MELC (Er), lymphocytes (T),NIH3T3 (mf) and CF37 (hf) fibroblasts, keratinocytes (kc), melanocytes (mc), hepatocytes (hp), and neurogenic cells (nc). The closed circle denotes
the complex in common between smc and MELC extracts (see Fig. 3B), whereas the arrow points to FP9C. B, comparison of the binding intensityof F9PC and NF-1 proteins in A-204 versus NIH3T3 nuclear extracts. Equal amounts (5 mg) of nuclear extracts and radiolabeled probes (10,000dpm) were added in each binding assay. C, Northern analysis of RNA from the above cells hybridized to the COL11A1 and glyceraldehyde-3phosphate dehydrogenase probes.

The screen for FP9C binding activity included myogenic celllines that can be readily differentiated in vitro and osteoblastic
representatives of different stages of differentiation. Together,
they provided the experimental means to examine the possible
relationship between FP9C expression and differentiation ofskeletal muscle and bone cells. In the osteoblastic model, thetransition from early to late stages of differentiation is represented by MC3T3-E1, ROS 25, and ROS 17/2.8 cells, in thatorder (25). EMSA revealed that FP9C binding increases gradually with differentiation (Fig. 7A). Comparable changes in
FP9C expression were observed in the myogenic differentiation
model (Fig. 7A). This is exemplified by undifferentiated and invitro differentiated C2C12 myoblasts, and by undifferentiated
C3H10T1/2 cells and the clonally derived P2 line differentiatedin vitro (16). As a control, the same EMSAs were repeatedusing the binding site of a transcription factor that remains
constant during skeletal muscle and bone cell differentiation(Fig. 7B). An additional control included the Northern analysis

of COL11A1 gene expression in the same panel of cell lines(Fig. 7C). Within the experimental limitations of the two models, the results were therefore consistent with the notion that
FP9C may be implicated in terminal differentiation of celllineages that share a common embryological origin.
DISCUSSION

The list of nuclear factors that control transcription in specific cell types is growing at an increasing pace, thanks to theimplementation of new screening strategies and the continued
progress in the study of tissue-specific gene regulation. Although more tedious and time-consuming, the latter approachhas the virtue of identifying cell type-specific factors throughthe cognate cis-acting elements and thus, of readily relatingthem with the expression and function of the target genes.
Examples of nuclear factors identified in this way include theerythroid GATA-1 protein and the B lymphocyte activator
Oct-2, among several others (22, 26). Very recently, the study ofCell Type-specific Transcription Factor

31783

FIG. 7. FP9C binding and cell differentiation. A and B, EMSAs were performed using the same amount of labeledprobe (10,000 dpm) and the same amountof nuclear proteins (5 mg) purified from
MC3T3-E1 (MC), Ros 25 (R25), and Ros
17/2.8 (R17) cells; from undifferentiated(Cu) and in vitro differentiated (Cd)C2C12 myoblasts; and from C3H1OT1/2(C3) and in vitro differentiated P2 cells(P2). The probes were the FP9 sequence(A) and the high affinity Sp1 binding site(B); the arrow in A points to FP9C. C,
Northern analysis of RNA from the above
cells hybridized to the COL11A1 and glyceraldehyde-3-phosphate dehydrogenaseprobes.

the osteocalcin gene has led to the demonstration that Osf2/
Cbfa1, the mammalian homologue of the Drosophila Runt protein, is a major determinant of osteoblasts differentiation and a
regulator of several ECM-coding genes in bone (27–29). However, the Osf2/Cbfa1 case represents the exception rather than
the rule among ECM-coding genes. Indeed, the genetic determinants responsible for orchestrating the assembly and remodeling of tissue-specific ECMs remain virtually unknown. By
analogy to other systems, it is safe to predict that the diversification of matrix-coding gene expression in distinct mesenchymal cell lineages is probably achieved through combinatorialinteractions of ubiquitous and cell type-specific factors. It isalso reasonable to argue that expression of ECM-coding genes
requires significantly more complicated networks of interactions than other tissue-specific gene families, both in terms oftrans-acting factors and cis-acting elements. The collagengenes are a case in point. Current evidence excludes the existence of shared regulatory mechanisms involving common cisacting elements. Although fibroblast-specific elements havebeen identified in the coordinately regulated type I collagengenes, they differ in composition and interact with distinct
nuclear proteins (4, 5, 30 –34). A SOX9 binding activity has
recently been reported to be necessary for cartilage-specificproduction of type II collagen (8, 9). By contrast, there is noevidence of SOX9 participation in the exquisite specificity ofa2(XI) collagen gene expression in restricted domains of thedeveloping limbs (35). Relevant to the present study, there isalso no apparent similarity between the cis-acting elements of
the a2(XI) collagen gene and of the one coding for the a1(XI)
partner. We believe the results described in this report areconsistent with having identified a cell type-specific regulatorof collagen gene expression.
Although solely based on transient transfection assays, FP9adheres to the two experimental criteria that are normallyused to define a tissue-specific element. First, FP9 is required
for transcription from the proximal 2541COL11A1 promoter.
This was demonstrated in functional assays that utilized progressively shorter promoter segments, internal deletions, andsequence substitutions of and within FP9. Particularly convincing was the finding that transcription from the 2541 promoter
depends on the 20-bp core sequence of FP9, the element that isprincipally involved in the binding of the cognate factor. To oursurprise, however, the effects of more subtle mutations within
the 20-bp core were not as drastic on transcription as they wereon in vitro binding (data not shown). One of the problems withthe functional evaluation of the promoter is the intrinsic weakness of the COL11A1 promoter. Work with transgenic mice iscurrently searching for upstream and downstream elementsthat may enhance promoter expression. The problem of promoter strength notwithstanding, FP9 conforms to probably themost important criterion for tissue specificity. It in fact binds atranscription factor, FP9C, which is produced in significant
amounts only by a restricted number of cells. Albeit far frombeing exhaustive, the screen nevertheless suggests that FP9C
is predominantly, but not exclusively, a mesenchymal geneproduct. Relevant to the main scope of the study, FP9C isconsistently found in cells actively engaged in the synthesis ofa1(XI) collagen. As already mentioned, the binding specificity
of FP9C is dictated by a sequence that consists of two GATAlike motifs arranged in a palindromic configuration interrupted
by the GG dinucleotide. Like the remaining of the 2541 promoter, this element is remarkably conserved in the mouse gene(data not shown). In point of fact, there are only two nucleotide
substitutions in the 20-bp core sequence of the two promoters.
The substitutions result in a GG 3 AA transition in the mousegene at the 2-bp interruption of the GATA palindrome; this isthe very same mutation (M1) that was found inconsequential
for FP9C binding (Fig. 3A). FP9 can bind GATA-1 in vitro
nearly as efficiently as the GATA consensus sequence. However, failure of the GATA consensus sequence to interfere withformation of FP9C clearly demonstrated that the 20-bp core isbound by the natural complex with very strong affinity, certainly higher than GATA-1. Along this line, incremental addition of smc nuclear extract augmented binding of FP9C significantly more than GATA-6. The sensitivity of FP9C to OPA
treatment is widely regarded as a strong indication that thefactor probably belongs to the zinc-finger family of proteins.
Additional analyses suggested (but did not prove) that FP9C
consists of a single polypeptide of about 100 kDa. Altogether
the size of the nuclear factor, the composition of the cognatesite and the functional effects of the FP9 mutations indicatethat FP9C is probably a novel zinc-finger activator.
By extrapolating from the in vitro data, we suggest thatorgans actively involved in FP9C expression may include the31784Cell Type-specific Transcription Factor

vascular system, skeletal muscle, bone, cartilage, and probably
skin. In this respect, FP9C joins the list of transcription factors
with restricted tissue distribution. One of such examples isMHox, whose expression in skeletal muscles and chondrocytes
resembles that of FP9C (36). Relevant to ECM-coding genes,
FP9C represents the first regulator that is expressed in both
hard and soft connective tissue. In at least two of the available
systems, myoblasts and osteoblasts, FP9C levels seem to increase in concert with cell differentiation. Within the limitationof the models employed, we propose that FP9C is not only atissue-specific regulator but also a determinant of cell differentiation. The cloning of the FP9C gene will provide the means toeventually test this hypothesis and elucidate the mechanisms
underlying FP9C regulation of COL11A1 transcription. Moregenerally, it may also shed new light on the pathways thatgovern cell commitment and differentiation in embryologicallyrelated but phenotypically distinct lineages.
Acknowledgments—We thank Drs. B. de Crombrugghe, M. Goldfarb,
G. Karsenty, J. H. Kimura, V. Lefebvre, D. Sassoon, and M. Takigawa
for helpful suggestions and invaluable reagents. We also thank KarenFrith for typing the manuscript and Cindy Else for excellent technicalassistance.
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