©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a Nuclear Factor-I Family Protein-binding Site in the Silencer Region of the Cartilage Matrix Protein Gene (*)

Piroska Szabó(§)(¶) , Jaideep Moitra (¶) , Altanchimeg Rencendorj , Gábor Rákhely (**) , Tibor Rauch , Ibolya Kiss (§§)

From the (1) Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, P. O. Box 521, H-6701 Szeged, Hungary

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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).() 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) .

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 -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) .

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.


MATERIALS AND METHODS

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 (pCMPCATN, 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).

The filled-in HinfI fragment (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 pTKCAT, 118 bp upstream of the transcription start site of the thymidine kinase promoter (pTKCAT(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 pTKCAT 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.

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.

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 10cells 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 NaEDTA, 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 NaEDTA, 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.


RESULTS

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).

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 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.

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 (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.

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.

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 SInucleoprotein 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 SInucleoprotein 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.

Insertion of a single copy SI element into pTKCAT, 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.


DISCUSSION

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 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.

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 (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.

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 SInucleoprotein 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.

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 (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) .

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.

  
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



FOOTNOTES

*
This work was supported by grants OTKA T896, B11 113, and C038 from the Hungarian National Scientific Research Foundation, Grant PHARE ACCORD H9112-0145 from the National Committee for Technological Development, and Grant AKA 1-300-2-92-0-829 from the Research Foundation of the Hungarian Academy of Sciences (to I. K.) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) M97497.

§
Present address: Division of Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010.

These authors should be considered as equal first authors.

**
Recipient of a Science Foundation fellowship from the Hungarian Credit Bank Ltd.

§§
To whom correspondence should be addressed. Tel.: 36-62-432-232; Fax: 36-62-433-506.

The abbreviations used are: CMP, cartilage matrix protein; bp, base pair(s); CAT, chloramphenicol acetyl transferase; ds, double stranded; CEC, chicken embryo chondrocyte; CEF, chicken embryo fibroblast; HDM, high density mesenchyme; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; NF-I, nuclear factor I; cpm, counts/min; kb, kilobase(s).

I. Kiss, unpublished observation.

S. Muratoglu, F. Deák, and I. Kiss, unpublished observations.

N. Tanese, personal communication.

J. Moitra and I. Kiss, unpublished observation.


ACKNOWLEDGEMENTS

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.


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