From the
Cartilage matrix protein (CMP) is synthesized by chondrocytes in
a developmentally regulated manner. Here we have dissected promoter
upstream elements involved in its transcriptional regulation. We show
that although the 79-base pair CMP minimal promoter is promiscuous,
1137 base pairs of 5`-flanking region are capable of directing tissue-
and developmental stage-specific transcription when fused to a reporter
gene. This results from two positive control regions which, in
proliferating chondrocytes, relieve the repression mediated by two
non-tissue-specific negative control regions. Characterization of the
promoter proximal silencer by DNase I footprinting and gel shifts
revealed the presence of two elements, SI and SII, which bound
mesenchymal cell proteins. Methylation interference analysis indicated
a gapped palindromic binding site similar to nuclear factor I (NF-I)
family proteins within SI, but only a half-site within SII. Gel shift
assays with specific NF-I and mutated SI competitors, binding of
recombinant NF-I, as well as supershift analysis with NF-I-specific
antiserum verified the binding of NF-I family proteins to the SI
element. Double-stranded SI and SII oligonucleotides inserted in single
copy in either orientation were found to repress both homologous and
heterologous promoters upon transfection into mesenchymal cells.
Transcriptional repression also occurred when a consensus NF-I site
itself was fused to the CMP minimal promoter. We conclude that
NF-I-related protein(s) can mediate transcriptional repression in cells
of mesenchymal origin.
Selective activation of eukaryotic genes during development or
in response to extracellular signals is mediated at the transcription
initiation level by the regulated assembly of stereospecific
nucleoprotein complexes on promoters and enhancers (reviewed in Refs.
1, 2). Additionally, however, an increasing body of evidence indicates
the importance of negative control regions, such as silencers, in
restricting the expression of genes to certain tissues or developmental
stages
(3, 4) . Some data suggest that, like promoters
and enhancers, transcriptional silencers may also have a modular
structure
(5) and can function via a complex interplay between
trans-acting factors and cis-acting elements
(3, 4) . Analysis of additional negative control regions
and transcription factors binding to them is required for a better
understanding of the role and mechanisms of transcriptional repression
in eukaryotes.
Cartilage is a unique tissue which provides a
challenging model system for studying both tissue- and developmental
stage-specific gene regulation. During endochondral bone formation,
committed mesenchymal cells differentiate through a series of events to
proliferating chondrocytes, which further differentiate to hypertrophic
chondrocytes
(6, 7) . Chondrogenesis is concomitant with
marked changes in gene expression. The condensed mesenchymal cells stop
synthesizing type I collagen and switch to the synthesis of
cartilage-specific gene products (reviewed in Refs. 7, 8), including
collagens type II, IX, and XI, aggrecan, and other non-collagenous
proteins such as link protein and cartilage matrix protein
(CMP).
CMP is an
extracellular glycoprotein synthesized by chondrocytes in a
developmentally regulated manner. It is a homotrimer of 54-kDa subunits
assembled via a coiled-coil
Previously,
we have identified the promoter of the chicken CMP gene and located two
transcription start sites 31 and 39 bp downstream of the TATA box
(11, 15) . Preliminary characterization of the control
region in a transient expression system indicated the presence of a
silencer between 334 and 15 bp upstream of the TATA motif and the
occurrence of a fragment with enhancer-like properties in the first
intron
(11) . In this study, we report a more precise mapping
and tissue-specific activity of the promoter upstream regulatory
regions using deletion series. Among the two positive and two negative
control regions defined, here we describe the identification and
functional analysis of two negative elements, which are located within
the silencer region adjacent to the minimal promoter. We also provide
evidence that both elements bind to NF-I-like proteins and that these,
as well as a consensus NF-I site, can repress the CMP minimal promoter.
The filled-in HinfI fragment
(
The 1.2-kb CMP promoter fragment cleaved
with HinfI and AvaII was subcloned into M13 and
pBS(+) vectors, and the nucleotide sequences were determined on
both DNA strands by the standard dideoxy chain termination method. The
structures of all CAT fusion constructs were confirmed by
double-stranded sequencing using Sequenase version 2.0 (U. S.
Biochemicals Corp.), according to the manufacturer's protocol.
When more upstream
sequences were incorporated and tested in CEC cultures, the activity
dropped 24-30-fold in constructs N(-334) and N(-669), was
restored in N(-799), followed by a 46-fold decrease in N(-878), and a
complete restoration of the minimal promoter activity in construct N
(Fig. 1). This suggests that the moderately high level of minimal
promoter activity was modulated by two negative and two positive
control regions. The first negative control region covered the silencer
(NR1) identified previously
(11) between
When longer promoter fragments were tested in HDM and CEF
cultures, no tissue-specific differences were observed in the
activities of NR1 and NR2, which decreased the promoter activity to the
same very low level (
To sum up, although the CMP minimal promoter showed a
promiscuous character, the 1.2-kb promoter fragment displayed the
tissue specificity that conformed with the in vivo expression
of the gene. Apparently, this was due to the tissue- and developmental
stage-specific function of the upstream positive cis elements,
which abrogated the general effect of the negative regulatory regions,
allowing complete restoration of the full transcriptional activity only
in CEC, the fully CMP-expressing cell type.
Insertion of a single copy SI element
into pTK
To gain insight into the transcriptional control mechanisms
of the chicken CMP gene, we have started mapping the promoter upstream
regulatory regions involved in the tissue- and developmental
stage-specific expression of the gene. Based on transient expression
studies of deletion constructs, we have localized two negative control
regions (NR1 and NR2) and two tissue- and developmental stage-specific
positive regions (PR1 and PR2) alternating upstream of a 79-bp minimal
promoter. The promoter adjacent negative region includes the silencer
identified previously
(11) , which, based on data presented
here, extends from
Data presented here indicate that the CMP minimal promoter has a
moderately high activity in mesenchymal cells, irrespective of CMP mRNA
expression, and is not influenced by polylinker sequences present at
the vector-insert border. Furthermore, this promoter (
We performed in vitro analysis of
the NR1 silencer elements to understand the general principles of how
such elements can function in several cell types. Contact point
analysis defined a binding site similar to the consensus CTF/NF-I
recognition sequence
(24) for SI and a half-site for SII.
Further evidence that NF-I-family proteins can bind to SI was obtained
by three independent approaches, including competition EMSAs with a
high affinity NF-I-binding site
(24) , supershift analyses with
a CTF-2-specific antiserum, and binding of a recombinant NF-I
(42) . In mesenchymal cells, both SI and the high affinity NF-I
site repressed the CMP minimal promoter. The SI element was also
capable of mediating transcriptional repression to the heterologous
thymidine kinase promoter, but its left half-site mutant (MLH)
abolished this repression. Accordingly, this mutant did not displace
the SI
Comparison of SI and SII with other
known eukaryotic silencing elements, or repressor binding sites, shows
only limited sequence homology. Despite the partial homology of SI to
the purine-rich 5` half of the IF-1 binding element
(44) , the
IF-1-specific oligonucleotide was not capable of competing with SI for
binding CEF nuclear proteins (data not shown). Based on available data,
the SI element is most similar to that reported for the rat growth
hormone silencer, which has recently been shown
(45) to bind to
a protein from rat liver that is antigenically related to NF1-L, a
truncated variant of NF-I previously isolated and cloned from the same
tissue
(46) .
The involvement of NF-I half- or palindromic
sites in transcriptional activation and viral replication is well known
(47, 48) . Although some reports have implied
(45, 49, 50) , the involvement of NF-I proteins
in negative transcriptional regulation has not been clearly
demonstrated to date, and it is poorly understood how they can mediate
negative regulation in general. The NF-I proteins originate from a
multigene protein family spread along the vertebrate phylum
(46, 51, 52, 53) , and splice variants
have also been reported to exist for most of them. In fact, chicken
tissues contain NF-I proteins that are products of four separate genes,
each of which generate a number of spliced forms, resulting in a total
of at least 12 isoforms in chicken
(53) . Although all of them
share a highly homologous N-terminal DNA-binding domain, their
C-terminal activation domains differ significantly. Since the various
NF-I proteins are also known to bind to their cognate half-sites either
as stable homo- or heterodimers
(54) , this increases further
the diversity of this protein family. The various homo- and
heterodimers may differ in their tissue distribution and also in terms
of their transcriptional activation properties.
It is thus possible
that an isoform exists which lacks a functional activation domain, or
contains a repressor domain, and this isoform plays a role in mediating
repression by the SI element. In fact, our data suggest that CEF
nuclear proteins interacting with SI are similar, but not identical to
vCTF-1, as judged by their mobilities and immune reactivities when
bound to either the SI or NF-I probes in gel shift assays. A plausible
explanation can be that some of the SI-binding proteins are products of
the chicken NF-I-C gene
(53) , which is the closest relative of
the human CTF isoforms, but that other CTF/NF-I proteins, acting as
repressors, may be more abundant in mesenchymal cells. On this premise,
heterodimers formed between the truncated and full-length form of CTF
could be envisaged to bind to SI and mediate repression.
Repression
mediated by SI may not only involve previously unidentified isoforms of
NF-I but also a tissue-specific cofactor/corepressor that functions
with NF-I. As it has been shown recently, CTF-1, one of the best
characterized activator of the NF-I family works via the antirepression
mechanism, utilizing cofactors to antagonize the histone-mediated
repression of the basal transcription machinery
(42) .
Similarly, nucleosomal structure has been implicated in the regulation
of NF-I-activated promoters
(55, 56) . Here we showed
that not only the SI and SII elements, but also a high affinity,
artificial NF-I-binding site was capable of conferring inhibition to
the CMP minimal promoter, when placed 18 bp upstream of the TATA motif,
that is very close to the natural position of SI (
Purification and further
characterization of the SI- and SII-binding protein(s) can contribute
to the identification of new NF-I isoforms and to an understanding of
their role in transcriptional repression in particular cell-types or on
particular promoters.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank/EMBL Data Bank with accession number(s) M97497.
We thank N. Tanese and N. Mermod for critical reading
of the manuscript. We are grateful to I. W. Mattaj and J. Bernues for
introducing P. Szabó into footprint analysis. We are
particularly thankful to R. Tjian and N. Tanese for providing us
several antisera raised against CTF-1 and 2, A. Udvardy for
Drosophila histone H1-specific antiserum, and N. Mermod for
the vCTF-1, respectively. We also thank Cs. Bachrati for doing the
transcription factor data base search, I. Fekete and A. Simon for their
excellent technical assistance, and A. Borka, M. Tóth and P.
Kós for the artwork.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
Hypertrophic chondrocytes, however,
synthesize predominantly type X collagen as a specific product. The
early and later events of chondrogenic differentiation can be studied
in high density mesenchyme cultures undergoing chondrogenesis in
vitro and in various chondrogenic primary culture systems,
respectively (reviewed in Refs. 6-8). Genes for several cartilage
proteins have been isolated
(7, 8) , but only the
control regions of type II collagen gene have been characterized
(9, 10) . In an attempt to obtain further information
about the common and distinct molecular mechanisms involved in the
transcriptional control of cartilage-specific genes, we have reported
previously a preliminary analysis of the regulation of chicken CMP gene
in a transient expression system
(11) .
-helix and stabilized by disulfide
bridges
(12, 13, 14) . Electron microscopy of
the native protein shows three compact ellipsoids connected at one end
(14) . In addition to the C-terminal trimerization domain, the
monomer is composed of two large repeated domains separated by an
epidermal growth factor module
(13, 15) . The repeats
show sequence homology to the type A domains of von Willebrand factor
and other members of this superfamily
(15, 16) .
Copurification of CMP with cartilage proteoglycans
(12, 13) and its binding to type II collagen fibrils
(17) suggest a role in the assembly of the cartilaginous matrix.
The CMP transcript is first detectable in limb buds of stage 26
(18) chicken embryos, and its amount increases sharply
concomitant with chondrogenesis
(13, 19) .
Immunohistochemical studies in normal
(20) , as well as
transgenic mice
(21) , and in situ hybridizations
(22) indicate the highest level of CMP expression in the
proliferative and upper hypertrophic zones, while its expression is
more restricted as compared to type II collagen and aggrecan at the
early stage of chondrogenesis
(19, 22) .
Plasmid Constructions and DNA Sequencing
As in
our previous work
(11) , all positions are given in bp from the
first T in the TATA motif of the CMP gene, unless noted otherwise.
Conventional recombinant DNA procedures were performed according to
standard protocols
(23) . Construct N, which carries the CMP
promoter fragment between positions 1137 and +64 fused to
the CAT reporter gene, has been described (pCMP
CAT
N, Ref.
11). Unidirectional 5`-deletion mutants were made by linearizing
pgCMP107/112
(11) with BglII at
1137 within the
CMP 5`-flanking region and digesting with Bal 31 exonuclease. The
shortened fragments were blunted, then cleaved with AvaI and
ligated to the 4.4-kb SalI (filled-in)- AvaI fragment
isolated from construct N. Deletion of nucleotides between
948
and
334 from construct N resulted in derivative N(-B). The
HindIII- PvuII fragment carrying 5`-flanking CMP
sequences between
878 and
809 was isolated from
derivative N(-878) and inserted into the HindIII/blunted
PstI site of N(-15).
220 to +127) of pgCMP107/112 was subcloned into the
HincII site of pBS(+) (Stratagene) in both orientations
to produce T54/11 and T54/13, respectively. The blunted
SacI- AvaI fragment from position
332 to
12 (NR1 silencer) was inserted in reverse orientation into the
filled-in SalI site of pTK
CAT, 118 bp upstream of the
transcription start site of the thymidine kinase promoter
(pTK
CAT
(C)
S4(
), Ref. 11). Double-stranded (ds)
CMP SI oligonucleotide and its mutated derivative MLH, as listed below,
were also ligated into the same site of pTK
CAT in both
orientations. SI, SII, NF-I
(24) ds oligonucleotides, and an
intronic 25-mer were also inserted in both direct and reverse
orientations into the blunted PstI site of N(-15), 18 bp
upstream of the TATA motif.
Oligonucleotides
The following oligonucleotides
were synthesized on a Millipore Cyclone DNA synthesizer, purified, and
used in double-stranded form. ( A)Oligonucleotides
carrying binding sites of known transcription factors: Sp1,
5`-AGCTGATCGGGGCGGGGCAGCT-3`
(25) ; AP-2,
5`-GAACTGACCGCCCGCGGCCCGT-3`
(26) ; HNF-4,
5`-CAGGTGACCTTTGCCCAGCGC-3`
(27) ; the EGR-1 site,
5`-TCGACGCCCTCGCCCCCGCGCCGG-3`
(28) ; a high affinity
NF-I-binding site 5`-TTTTGGCTTGAAGCCAATAT-3`
(24) .
( B)Oligonucleotides car-rying CMP cis elements: CMP SI, 5`-CAGGGGCTGGCCCCATGCCTCCCCGA-3`; CMP SII,
5`-GAGGGGCTGGCAGGGCTGGACGTCCA-3`; an intronic 25-mer
5`-ACTATGCTGAGTCAGAATGTCACCC-3`. ( C)Mutated SI
derivatives (mutated nucleotides are underlined): G29,
5`-CAGGGGCTAGCCCCATGCCTCCCCGA-3`; P1d,
5`-CAGGGGCTAGCCCCATGCTTCCCCGA-3`; P1-2d,
5`-CAGGGGCTAACCCCATGTT-TCCCCGA-3`; MLH,
5`-CAGGGGCTAATCCCATGCCTCCCCGA-3`.
Cell Culture
Primary cultures were made mostly
from chicken embryos of Rhode Island White homozygotes and partly from
a hybrid termed Ross and a specific pathogen-free White Leghorn
homozygotes. The hybrid Arbor Acres Hybro used in the previous work
(11) has been replaced since persistent infection by herpes
virus had been noted in the population. Primary chicken embryo
chondrocytes (CEC) were obtained from sterna of day 14 embryos using
0.1% collagenase digestion (Sigma type I) at 37 °C, and the cells
were cultivated in 60-mm plates in DMEM containing 10% fetal calf serum
(Life Technologies, Inc., Flow, or Jacques-Boyd). Chicken embryo
fibroblasts (CEF) were prepared from day 8 to day 10 chicken embryos by
standard procedures
(29) , maintained in DMEM containing 5%
fetal calf serum, and transfected after two passages. Limb buds of
stage 23-24 chicken embryos were used to make high density
mesenchyme (HDM) cultures undergoing chondrogenesis in vitro (30) , by cultivating 5 10
cells in
35-mm plates in Ham's F12/DMEM 6:4 medium
(31) supplemented with 10% fetal calf serum.
Transient Expression Assays
CEC and CEF cultures
were transfected with 10 µg of DNA by the calcium phosphate
coprecipitation method, and assayed for CAT activity essentially as
described
(11) , but with some modifications
(32) . The
precipitate remained on the cells for 18 h, and the cells were
harvested 44 h after transfection. The HDM cultures were transfected in
a similar way, but the cells were harvested after 96 h of in vitro chondrogenesis. Duplicate or triplicate plates containing an equal
number of cells were made for each DNA, and the reference or control
plasmid, such as N(-15) or pTKCAT, was also transfected parallely.
To correct for transfection efficiency, but to avoid the interference
observed between the CMP control region and the cotransfected internal
control,
(
)
15-µl aliquots from the cell
suspensions were subjected to dot-blot analysis and hybridization with
P-labeled CAT fragment as described
(33) . CAT
activities were normalized to both the amount of protein and DNA used
in the assay and were expressed as percentage CAT activity, relative to
the level directed by the control or reference plasmid adjusted at 100%
(relative CAT activity). Each transfection experiment was repeated
5-15 times with at least two independent plasmid preparations
purified by two cycles of CsCl-ethidium-bromide gradient
centrifugation. Data are presented as means ± S.E. Significant
differences among the activities of reference and test plasmids were
discerned by paired t tests
(34) .
Isolation of Nuclei
Cells from CEF, CEC, and HDM
cultures were lysed as described
(35) , with the modification
that DTT, phenylmethylsufonyl fluoride, and benzamidine at 0.5
mM each, and leupeptin and pepstatin at 5 µg/ml were also
added to the lysis buffer. Crude nuclear pellets were suspended in 1.0
M sucrose, 10% glycerol, 10 mM HEPES, pH 7.6,
supplemented with proteolytic inhibitors as above, layered over a
cushion of 1.2 M sucrose buffered similarly, and spun in a
swinging-bucket rotor at 75,000 g for 45 min. Pure
nuclei were suspended in 25 mM HEPES, pH 7.6, 25% glycerol, 3
mM MgCl
, 0.1 mM Na
EDTA, 1
mM EGTA, 5 mM DTT, frozen in liquid N
,
and stored at
80 °C.
Preparation of Nuclear Extracts
Frozen nuclei were
quick-thawed and extracted at 4 °C with buffer B containing 530
mM NaCl
(36) . Nuclear extracts fractionated by
precipitation with ammonium sulfate to 70% saturation were dialyzed
overnight against 2 250 ml of D buffer (10 mM HEPES,
pH 7.6, 0.1 mM Na
EDTA, 20% glycerol, 50
mM KCl, and 3.5 mM DTT) and centrifuged at 105,000
g for 15 min. The protein concentration of the
supernatant was measured by Bradford microassay
(37) and stored
in small aliquots as described above for nuclei. For DNase I
footprinting, 10 mg of ammonium sulfate-precipitated CEF nuclear
proteins were dialyzed overnight against a modified D buffer,
containing 10% glycerol, 0.1% Triton X-100, 100 mM KCl, and
proteolytic inhibitors, centrifuged, and applied to a 5-ml
heparin-agarose (Sigma) column equilibrated in the same buffer. The
column was eluted with a 50-ml linear gradient of 0.1-1.0
M KCl in the modified D buffer. The collected fractions (1 ml)
were dialyzed against D buffer, concentrated by Centricon-10 (Amicon)
to one-tenth of the original volume, aliquoted, and tested by
electrophoretic mobility shift assay (EMSA).
EMSA
Crude or partially purified nuclear extracts
were incubated with 0.2-2.0 µg of poly(dI-dC) and various
cold competitors in a binding buffer (20 mM Tris-HCl, pH 7.9,
50 mM KCl, 3.5 mM DTT, 0.2 mM EDTA, 4
mM MgCl, and 2% Ficoll) for 10 min on ice.
20-90 fmol (30,000 cpm) of 5`-end-labeled oligonucleotides were
then gently added, and following 20 min of incubation at 30 °C,
were electrophoresed on prerun 5% polyacrylamide gels at 10 V/cm in
22.5 mM Tris borate, 0.25 mM EDTA, pH 8.0. Gels were
dried and autoradiographed using intensifying screens
(23) .
DNase I Footprinting
20-50 fmol of DNA
fragments (30,000 cpm) end-labeled at EcoRI or NcoI
sites were incubated for 20 min at 22 °C in the presence or absence
of heparin-agarose purified DNA-binding proteins in 20-µl EMSA
binding buffer containing 10% glycerol instead of Ficoll. 1.5 µl
(1.3-2.3 ng) of previously titrated DNase I (Sigma Type I) was
added and the incubation continued for 2 min further. Digestion was
stopped by addition of 200 µl of stop solution (50 mM
Tris-HCl, pH 7.9, 0.1 M NaCl, 50 µg/ml tRNA, 0.5% SDS) and
200 µl of phenol/chloroform (1:1). Samples were further processed,
electrophoresed, and autoradiographed according to standard protocols
(38) .
Methylation Interference Analysis
CMP SI and SII
oligonucleotides labeled strand specifically at the 5`-ends were used
for methylation interference analysis as described
(39) . The
same experiments were also performed after cloning the SI and SII
oligonucleotides into the HincII site of pBS(+) in single
copy and using the fragments end-labeled at the EcoRI or
HindIII sites of the polylinker. Bound and free DNA were
eluted from preparative scale EMSA gels by passive diffusion and
batch-purified with DE52 equilibrated in 10 mM Tris-HCl, pH
8.0, 100 mM NaCl, and 1 mM EDTA. Equal counts/minute
of piperidine-cleaved DNA were loaded on 8 or 15% sequencing gels to
visualize interfered guanine residues.
Supershift Analyses
Supershifts with
trans-acting factor-specific antisera were performed
essentially as described
(40) . Polyclonal antisera raised
against CTF-2 and a peptide present at the C terminus of CTF-1
(41) was a kind gift of N. Tanese and R. Tjian. vCTF-1
(42) , the protein produced by HeLa cells, infected with a
vaccinia virus construct carrying the entire coding region of CTF-1,
was a kind gift of N. Mermod.
Negative and Tissue-specific Positive Control Regions
Restrict the Expression of the CMP Gene to Chondrocytes
In an
attempt to map cis-acting elements involved in the regulation
of the chicken CMP gene, we fused a series of 5`-deleted promoter
fragments to the CAT reporter gene (Fig. 1). To test for the
tissue specificity of the control regions, transient expression of the
constructs was studied in three cell types of mesenchymal origin. Among
these, CEF does not express CMP, and HDM undergoing chondrogenesis in
in vitro culture expresses the gene at lower level than the
fully expressing CEC.(
)
Figure 1:
Transient
expression analysis of the CMP promoter 5`-deletion mutants. The
left panel shows the structures of constructs generated by
progressive 5`-deletions using Bal31 exonuclease or by introducing
internal deletions at restriction cleavage sites indicated. Positions
for the 5` ends of the shortened promoter fragments ( thick
lines), as well as both end points of the internal deletions
( dotted lines) are given relative to the TATA, as previously
(11). Relative CAT activities measured in CEC, HDM, and CEF cultures
obtained from Rhode Island White chicken embryos, are depicted in the
right panel by solid, open, and striated
bars, respectively. CAT activities are expressed in percentage of
that of pTKCAT control. Data represent means of 5-15
independent experiments ± S.E. NR1 and NR2,
negative control regions; PR1 and PR2, positive
control regions; nd, not
determined.
The CMP minimal
promoter construct, N(-15), carrying 15 bp upstream and 64 bp
downstream of the TATA motif, yielded 29.4 ± 2.3 and 36.9
± 2.9% relative CAT activities in CEC and HDM cultures,
respectively, as compared to pTKCAT (Fig. 1), and only a
somewhat lower value (26.1 ± 3.8%) was measured in the
non-CMP-expressing CEF. Deletion of 4- or 8-bp polylinker sequence at
the upstream vector/insert border of construct N(-15) did not alter the
minimal promoter activity significantly ( p > 0.05), nor
affect its promiscuity. Similar activity was detected when the
15 to +50 CMP fragment was fused to the CAT reporter gene
using different cloning sites (data not shown).
334 and the
minimal promoter, while the second one was located between positions
878 and
799 (NR2). Inclusion of NR2 sequences from
878 to
809 also repressed the minimal promoter activity
5-fold in construct N(-15)NR2. In CEC cultures, the repression was
relieved by positive control elements located from
799 to
669 (PR1) and from
1137 to
942 (PR2). In construct
N(-B), PR2 was capable of abrogating the effect of the NR1 silencer and
increasing the CAT activity to a level higher than that of the minimal
promoter.
1.6%) in N(-334), N(-669), and N(-878) in all
mesenchymal cells, irrespective of whether the cell type expressed the
CMP gene or not (Fig. 1). In contrast, the positive upstream
elements in construct N and N(-799) displayed 2-3-fold lower
activities in HDM cultures undergoing chondrogenesis in vitro than in CEC cultures, while their activities were even more
diminished in the non-expressing CEF cultures (5.1 ± 1.3% for
construct N). This, as well as the high activity of construct N(-B) in
CEC, indicated that the positive elements in PR2 functioned
6-8-fold more efficiently in CEC as compared to CEF, and
therefore conferred specific activation only in the CMP-expressing cell
type.
Identification of the SI and SII cis Elements
within the NR1 Silencer by Footprint Analysis
Nucleotide
sequence of the 5`-flanking region up to the BglII cleavage
site was determined and compared with consensus binding sites for known
transcription factors listed in the NIH Transcription Factor Data base
(43) . Among 145 putative sites, a consensus PEA-3 site and two
copies of the glucocorticoid response element were found within the
minimal promoter and PR2, respectively (data not shown). In order to
identify cis control elements involved in the transcriptional
regulation of the CMP gene, we first focused our attention on the NR1
silencer. This silencer was interesting in that it inhibited both
homologous and heterologous promoters in all mesenchymal cell types
tested (11, and present work), suggesting common mechanisms of
repression. To address this question, we prepared nuclear extracts from
both CEC and CEF, and in preliminary EMSAs found that the 347-bp
HinfI fragment (220 to +127) covering the major
part of NR1 as well as the TATA motif, mediated specific interactions
with these extracts (data not shown). To delineate relevant cis elements within this fragment, we subjected the CEF nuclear
extract to heparin-agarose chromatography and tested the fractions
eluted at various KCl concentrations in DNase I footprint assays.
Several protected regions were observed with these fractions
(Fig. 2). Among them, an extended footprint for fractions 21 and
22 and a shorter one for fractions 23-30 were located in the NR1
silencer. We analyzed first the latter cis element mapped
close to the TATA motif and determined the area of protection at the 3`
end of NR1 on both strands of the DNA using fractions enriched for this
activity.
Figure 2:
DNase I footprint assay with fractionated
CEF nuclear extracts. The coding strand of the insert from T54/11,
covering the CMP promoter sequence from 220 to +127, was
labeled at the EcoRI end, and incubated with partially
purified CEF nuclear proteins. Lane minus, no protein added;
lanes 16- 26 and 30 represent fractions eluted at 115,
165, 254, 307, 367, 406, 462, 491, 525, 587, and 692 mM KCl
concentrations, respectively. Thick lines indicate the various
protected regions. Positions are given on the right. NE,
nuclear extract; HepA, heparin-agarose
fractions.
As shown in Fig. 3, the protected regions extended
from 36 to
13 (SI) and at higher protein concentration,
from
144 to
121 (SII). Element SI was capable of stable
binding of nuclear factor(s) at 300-fold molar excess of poly(dI-dC)
(Fig. 3 A, lane 8) and remained intact even at
700-1000-fold molar excess of the bulk competitor (data not shown).
Binding to element SII, however, was less stable and competed by
300-fold excess of bulk competitor (Fig. 3 A, lane
8). Both footprints were abolished by 50-fold excess of cold NR1
fragment (data not shown), as well as 10-fold molar excess of the ds SI
oligonucleotide (
37 to
12) (Fig. 3 B,
lane 5). Some protection at the TATA motif was also observed
even in the presence of 1200-fold excess of poly(dI-dC)
(Fig. 3 B, lane 4).
Figure 3:
Identification of the SI and SII elements
by competition DNase I footprinting. A, the noncoding strand
of the insert (220 to + 127) from T54/13 was labeled at the
EcoRI end and used in footprint analysis with a
heparin-agarose fraction of CEF nuclear extract eluted at 525
mM KCl (fraction 25; Fig. 2). Lanes 1, 6, and 9 did
not contain nuclear proteins. Lanes 2-5 contained 1, 3,
5, and 8 µl of the fraction, respectively. 5 µl of the same
fraction was tested in the presence of 150- and 300-fold molar excess
of poly(dI-dC) in lanes 7 and 8, respectively.
AG, A+G sequence ladder of the same DNA. Numbers on either side indicate the positions and the TATA motif is also
marked. Open boxes at the left side of the figure, mark the
protected regions SI and SII, respectively. B, the coding
strand of the CMP fragment (
220 to +68) from T54/11,
labeled at the NcoI site at +68 was used in footprint
analysis in the absence ( lanes 1 and 6) and presence
of 2 µl ( lane 2) and 6 µl ( lanes 3-5)
of fraction 25 as in A. DNase I protection was performed in
the presence of 1200-fold molar excess of poly(dI-dC) ( lane 4)
and 10-fold molar excess of SI ds oligonucleotide ( lane 5).
Other symbols as in A.
Based on the facts that
SII protection was competed both by an overlapping fragment as well as
the synthetic SI site, but was easily abolished by poly(dI-dC), we
concluded that the two sites bound to protein species of similar site
preference, but with different affinities. This conclusion was also
supported by the observation that the 5`-half of both footprints is
composed of the same 10-bp sequence (AGGGGCTGGC), including a putative
AP-2 recognition site (GGGCTGGC). An imperfect repeat of the same
sequence occurred in inverted or in tandem copies in SI and SII,
respectively. SI also carried a putative HNF-4 recognition sequence
(TGGCCCC).
Binding of Nuclear Proteins to the SI Element in
EMSA
Incubation of radiolabeled ds SI oligonucleotide with
nuclear proteins extracted from mesenchymal cell types resulted in the
formation of a single shifted complex of similar mobility in EMSA,
irrespective of whether the extract originated from expressing or
non-expressing cell-type (Fig. 4 A). The broad band
produced by the CEC extract ( lane 3) was due to contaminating
proteoglycans, which were extracted along with DNA-binding proteins
from partially purified CEC nuclei. In some CEF extract preparations,
however, more than one band could be occasionally seen (see for example
Fig. 4B). SII also formed a major DNAprotein
complex of similar mobility as SI in EMSAs, but it was easily
displacable by increasing concentration of poly(dI-dC) (data not
shown), indicating a weaker binding in accordance with the footprint
assays (Fig. 3 A).
Figure 4:
EMSA of the SI nucleoprotein
complex. All lanes contained 90 fmol (30,000 cpm) of 5`-end-labeled SI
probe and 500 ng of poly(dI-dC) as nonspecific competitor. Lanes
F, no protein added. A, lanes 1-3 contained 10 µg of CEF, 10 µg of HDM, and 2 µg of CEC
nuclear extracts, respectively. Band C,
lanes 1-11 contained 2.6 µg of CEF nuclear extract,
and in addition, lanes 2-11 included specific ds
competitors as bracketed on top. The exact molar excesses of
these competitors over the labeled probe are indicated on top of each
lane.
To obtain an insight into the
nature of the nuclear proteins that bind to the SI element, competitor
ds oligonucleotides were designed for proteins having putative binding
sites within SI (AP-2 and HNF-4) as well as for others which are known
to bind to GC-rich regulatory regions (Sp1 and WT-1). Careful titration
experiments in EMSAs showed that while the CEF nuclear proteins bound
to SI could be completely displaced by a 50-fold molar excess of
homologous competitor, it required 500- and 4000-fold molar excesses of
the SII- and HNF-4-specific oligonucleotides, respectively, to do so
(Fig. 4, B and C). On the other hand, the EGR-1
oligonucleotide (Wilms tumor protein-binding site)
(Fig. 4 C), as well as the Sp1 and AP-2 oligonucleotides
(data not shown) did not compete even at 4000-fold molar excess. These
experiments further confirmed the sequence specificity of SI element
binding and a similar, but weaker binding for the SII element.
Nucleotides in Tight Contact with Proteins within the SI
Element Demarcate a ``Gapped'' Palindrome
Methylation
interference analysis was performed to determine the guanine residues
of SI and SII in direct contact with CEF nuclear protein (Figs. 5 and
6). This analysis showed that methylating 3 guanine residues on either
the coding or the noncoding strand excluded the SI oligonucleotide from
complex formation, while methylating other guanine residues on either
strand had little effect (Fig. 5). As summarized in Fig. 7,
a three-nucleotide stretch 5`-GGC-3` on either strand was strongly
contacted, and the site encompassed is 5`-GCTGGCCCCATGCCTC-3` (top
strand indicated, contacted nucleotides underlined). The G-interference
pattern is similar to the NTTGGCNGCCAAN consensus sequence
(24) identified for the NF-I family of transcription factors.
Figure 5:
Methylation interference analysis of the
SI element. The ds SI oligonucleotide, cloned into the HincII
site of pBS(+), was 3`-end-labeled in the polylinker region at the
HindIII site ( A, coding strand), or at the
EcoRI site ( B, noncoding strand). The
probes were partially methylated at the G residues, incubated with CEF
nuclear proteins, and separated by EMSA as described under
``Materials and Methods.'' Large arrowheads represent methylated guanines which interfere strongly with
protein binding; small arrowheads indicate weaker
interactions. AG, CT, and G show Maxam-Gilbert
sequence ladders of input DNA. Lanes B and F, bound
and unbound DNA, respectively.
Figure 7:
Summary of
DNase I protection and methylation interference analyses. Solid
bars depict the SI and SII footprints determined in several
experiments and shown in Fig. 3; weakly protected nucleotides are
bordered with dotted lines. Horizontal arrows delimit the
imperfect inverted and tandem repeats. DNase I hypersensitive
nucleotides are marked with arrowheads. G residues interacting
with CEF nuclear proteins are also shown. Strong and weak contacts,
determined in several methylation interference assays, appear as
closed and open circles, respectively. The top strand
of the NF-I consensus recognition sequence (24) is also shown for
comparison.
A similar pattern of interference (5`-GGC-3`) was also observed for
the SII oligonucleotide (Fig. 6) but only on the coding strand,
when the SI type shifted complex was analyzed. Although the same
three-nucleotide motif recurred both upstream and downstream of the
contacted site in this strand, these were either not involved or
contacted imperfectly, probably due to the lack of another half site in
inverted copy separated by a spacer of N. The interference
pattern of SII therefore suggests that the protein(s) binds to the GGC
stretch as a half-site.
Figure 6:
Methylation interference analysis of the
SII element. A, coding strand or B, noncoding
strand of SII oligonucleotide was labeled at the 5`-end and annealed to
the complementary strand. Partially methylated probes were incubated
with CEF nuclear proteins and the interfered G residues were visualized
as described (39). For explanation of symbols, see legend to Fig.
5.
Site Competition Assays with a Consensus NF-I
Oligonucleotide and SI Mutants
To obtain a direct evidence that
a NF-I-like recognition sequence is involved in protein binding at the
SI site, site competition assays were performed, using first a high
affinity NF-I sequence characterized by Chodosh et al.
(24) (Fig. 8). This experiment showed that the consensus
NF-I recognition sequence competed equally well as the homologous
competitor. In further site competition assays, SI derivatives were
used where the NF-I-like strong contact points determined in
methylation interference experiments were systematically mutated
(Fig. 8). These experiments clearly showed that not only point
mutations in both half-sites, but even a single point mutation in the
left half-site at 29 (G29) was sufficient to destroy the ability
of the competitors to displace the SI
nucleoprotein complex. These
experiments supported the observation that a NF-I recognition site is
indeed involved in protein binding to the SI element.
Figure 8:
EMSA of the SI nucleoprotein
complex with NF-I and mutated SI competitors. Each lane contained 90
fmol (30,000 cpm) of the CMP SI probe and 1 µg of poly(dI-dC).
Except for lane F, all other lanes included 1.2 µg of CEF
nuclear extract. Lanes 2-17 contained specific
competitors as bracketed on top. The exact molar excesses of
the cold ds competitors over the labeled probe are indicated on top of
each lane. The sequences of the mutated SI competitors are aligned with
SI underneath the figure. The NF-I-like contact points of SI are
boldfaced, while the mutated nucleotides are
underlined.
Supershift Analysis Confirms the Binding of NF-I Family
Protein(s) to SI
We wished to ascertain if the protein(s) that
bound to the SI element were antigenically related to the NF-I/CTF
family of transcription factors. For this purpose, we performed
supershift analysis using rabbit polyclonal antiserum directed against
the entire CTF-2, but reacting mostly with epitopes clustered in the
C-terminal half (from residues 221 to 499) of the molecule.(
)
This experiment (Fig. 9 A, lane 4)
clearly demonstrated that the SI
nucleoprotein complex could be
specifically recognized by the CTF-2-specific antiserum, while the
preimmune serum or a histone H1-specific antiserum did not generate
similarly supershifted bands (Fig. 9 A, lanes 2 and 6).
Figure 9:
Supershift analysis of the
SInucleoprotein complex. Each lane contained 90 fmol (30,000 cpm)
of 5`-end-labeled ds oligonucleotide as a probe and 1 µg of
poly(dI-dC). A, the CMP SI probe was used and incubated
with 1.2 µg of CEF nuclear proteins in lanes 1, 2, 4,
and 6. Lanes 2-6 contained preimmune,
CTF-2-specific ( Anti- NF-I) or histone H1-specific
( Anti- H1) antisera as indicated on top. Solid
arrow and arrowhead indicate the shifted and supershifted
complexes, respectively. B, the SI and NF-I probes used
are bracketed on top. Lanes F, no protein added.
Lanes 1-12, the type and amount of proteins and antisera
used are given over each lane. Anti-M, the same CTF-2-specific
antiserum as Anti- NF-I in A;
Anti- C-pep, antiserum directed against a C-terminal
peptide of CTF-1 (41). Solid and open arrows mark the
positions of shifted complexes produced by CEF proteins and vCTF-1,
respectively. Solid arrowheads depict positions of
probe-protein-antibody ternary complexes formed by CTF-2-specific
antiserum ( M), anti-C-pep antiserum ( C), or
wellshifts ( W), respectively.
Further experiments were carried out to
compare the mobility and supershifting pattern of CEF nuclear proteins
and vCTF-1 when bound to SI and NF-I probes (Fig. 9 B).
Although the CEF nucleoprotein complexes migrated faster than the
vCTF-1 counterparts, they yielded supershifts of similar mobilities
with the CTF-2-specific antiserum ( M, lanes 2,
5, 8, and 11). Additionally, the same
antiserum formed a wellshift with vCTF-1 complexes ( W,
lanes 5 and 11). An antiserum specific for a
C-terminal peptide of CTF-1
(41) supershifted vCTF-1 bound to
either probes ( C, lanes 6 and 12) but did
not produce detectable supershifts with CEF nucleoprotein complexes
( lanes 3 and 9). These data suggest that CEF nuclear
proteins bound to SI have antigenic similarity to CTF-2, but do not
carry the C-terminal peptide of CTF-1.
Functional Analysis of the SI, SII, and NF-I
Elements
NF-I proteins are well known as transcriptional
activators. To test whether the SI element which binds a NF-I-related
protein can be involved in transcriptional repression, we performed
transient expression studies. SI-specific ds oligonucleotide was
inserted into the construct N(-15) at a position very close to its
natural context, and the CAT activities were measured in CEC and CEF
cultures (). In direct orientation, a single copy of SI
element was capable of exerting 6-9-fold repression on the
homologous promoter activity. These values are not far from the
14-16-fold repression observed for the NR1 silencer, which
contains both SI and SII elements. The inhibition in reverse
orientation was only 3-4-fold, but still significant. Even a
single copy of the SII oligonucleotide, which formed low stability
nucleoprotein complex, was also capable of inhibiting the CMP promoter
in transient expression studies. Interestingly it caused larger
repression in reverse (9-fold), than in direct orientation
(
3-fold). Finally, an orientation independent repression between
4-6-fold was observed in both CEC and CEF cultures, when a high
affinity NF-I-binding site
(24) was inserted in a single copy
at the same position into the CMP minimal promoter construct
(). Insertion of an unrelated oligonucleotide at the same
position, however, did not cause significant changes in the CMP minimal
promoter activity (intronic 25-mer; ), indicating that the
repression caused by the other oligonucleotides was specific and not
due to some artifact, such as the disruption of a binding site created
at the vector-insert border.
CAT, 92 bp upstream of the thymidine kinase promoter,
exerted 3-5-fold inhibition on the heterologous promoter
activity, as compared to the 6-8-fold repression mediated by the
NR1 silencer (). Mutation of the NF-I contact points at
the left half-site of SI (MLH), however, abolished the repression of
the heterologous promoter. These data suggest that the SI and SII
elements of the CMP gene, as well as an artificial NF-I-binding site,
are indeed capable of mediating transcriptional repression of the CMP
gene, both in CEC and CEF.
334 to
12 (NR1) and functions in
mesenchymal cells. Here, we have characterized the SI
cis-control element (
36 to
12) and to some
extent, the related SII element (
144 to
121), both of
which were located within the NR1 silencer by DNase I footprinting, and
were capable of conferring negative regulation in functional assays.
15 to
+50) inserted into a different vector (pBS+), and surrounded
by completely different sequences, functions at a level similar to the
mouse albumin promoter in an in vitro transcription
system.
(
)
These, as well as the repeatedly
decreasing and increasing CAT activity profile of the deletion
constructs, make it unlikely that the transient expression data depend
upon the presence of cryptic activation binding sites in the vector or
at the vector/insert border. Our data are more consistent with the
model that tissue- and developmental stage-specific regulation is
conferred to the promiscuous minimal promoter by more upstream positive
elements located in PR1 (
799 to
669) and PR2 (
1137
to
942), which relieve the general inhibitory role of NR1 and
NR2 only in CMP-expressing cells. Contrary to this, the collagen II
gene has high promoter activity in HDM cultures, utilizes promoter
upstream silencers functional only in non-expressing cells, and its
identified silencer elements are not similar
(9, 10) .
This suggests that the two cartilage protein genes employ different
control mechanisms, even though their expression pattern is only
slightly different in cartilage
(20, 21, 22) .
We had previously found that the constructs N and N(-B) exhibited no
chondrocyte-specific expression
(11) . As shown here, tissue
specificity of N and N(-B) expression could indeed be observed with
statistical confidence, based on numerous transient transfections of
the basic constructs using three independent chicken strains. As the
previous studies were conducted with cells from a chicken strain
infected with herpes virus, we believe that the data presented here
represent the actual regulatory properties of the various CMP promoter
fragments in chondrocytes. This implies that, due to the presence of
PR1 and PR2, the 1.2-kb CMP promoter fragment carries all the
information necessary to direct efficient and selective transcription
of a reporter gene in tissue culture cells, a result that is congruent
with the in vivo expression of the CMP gene
(19, 20, 21, 22) . In fact, the low
activity of PR1 and PR2 in HDM cultures representing the early stage of
chondrogenesis is also in keeping with the later onset of the CMP gene
as compared to that of the collagen II gene during in vivo chondrogenesis, as well as with the low activity of both the
endogenous gene and the chicken CMP transgene in the zone of youngest
chondrocytes in transgenic mice
(19, 20, 21, 22) . It requires further
studies, however, to define the tissue- and developmental
stage-specific cis elements in PR1 and PR2, and reveal if they
have enhancer activity.
nucleoprotein complex in EMSA either (data not shown), in
common with other SI derivatives mutated in the NF-I contact points
(Fig. 8). Taken together, our data indicate that binding of
protein(s) related to the CTF/NF-I family of ubiquitous transcription
factors is involved in the transriptional repression by the SI element
in cells of mesenchymal origin.
12).
Therefore, it is possible that in close proximity to the RNA polymerase
II assembly site and in the absence of an upstream activating element,
NF-I could suppress transcription by ( a) simple steric
interference with TBP; ( b) failing to relieve repression of
basal transcription mediated by histones or by TFIID inhibitory
components such as NC1, NC2, Dr
, and Dr
(reviewed in Ref. 57), or ( c) by utilizing its own
negative-acting cofactors to actually promote repression. However, the
observation that the SI element can repress the heterologous thymidine
kinase promoter from a distance of 92 bp, suggests a more specific
effect than directly occluding TBP, perhaps utilizing negative-acting
cofactors. Some credence has led to this possibility from the
observation that NF-I/CTF binding to the eH element of the serum
albumin enhancer, inhibited the transcriptional activation by HNF3
via a specific mechanism which could not be explained by simple steric
interference with TBP
(50) .
Table:
The effect of various CMP control
elements and a high affinity NF-I site on the activity of the CMP
minimal promoter-CAT construct in expressing and non-expressing cell
types
Table:
Effect of the CMP SI element and its mutated
derivative on the heterologous thymidine kinase promoter activity in
expressing and non-expressing cell types
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.