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Originally published In Press as doi:10.1074/jbc.M502681200 on August 8, 2005

J. Biol. Chem., Vol. 280, Issue 42, 35417-35423, October 21, 2005
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Identification of a Repressor in the First Intron of the Human {alpha}2(I) Collagen Gene (COL1A2)*

Taras T. Antoniv{ddagger}1, Shizuko Tanaka{ddagger}1, Bayan Sudan{ddagger}, Sarah De Val§, Ke Liu¶, Lu Wang{ddagger}, Dominic J. Wells¶, George Bou-Gharios§, and Francesco Ramirez{ddagger}||2

From the {ddagger}Laboratory of Genetics and Organogenesis, Research Division of the Hospital for Special Surgery, and Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, New York 10021, the Gene Targeting Unit, Division of Neuroscience and Mental Health, Imperial College School of Medicine, Charing Cross Hospital, London W6 8RP, United Kingdom, §Renal Medicine, Imperial College School of Medicine, Hammersmith Campus, London W12 ONN, United Kingdom, and ||CEINGE-Biotecnologie Avanzate, 80131 Naples, Italy

Received for publication, March 10, 2005 , and in revised form, June 23, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The human and mouse genes that code for the {alpha}2 chain of collagen I (COL1A2 and Col1a2, respectively) share a common chromatin structure and nearly identical proximal promoter and far upstream enhancer sequences. Despite these homologies, species-specific differences have been reported regarding the function of individual cis-acting elements, such as the first intron sequence. In the present study, we have investigated the transcriptional contribution of the unique open chromatin site in the first intron of COL1A2 using a transgenic mouse model. DNase I footprinting identified a cluster of three distinct areas of nuclease protection (FI1-3) that span from nucleotides +647 to +760, relative to the transcription start site, and which contain consensus sequences for GATA and interferon regulatory factor (IRF) transcription factors. Gel mobility shift and chromatin immunoprecipitation assays corroborated this last finding by documenting binding of GATA-4 and IRF-1 and IRF-2 to the first intron sequence. Moreover, a short sequence encompassing the three footprints was found to inhibit expression of transgenic constructs containing the COL1A2 proximal promoter and far upstream enhancer in a position-independent manner. Mutations inserted into each of the footprints restored transgenic expression to different extents. These results therefore indicated that the unique open chromatin site of COL1A2 corresponds to a repressor, the activity of which seems to be mediated by the concerted action of GATA and IRF proteins. More generally, the study reiterated the existence of species-specific difference in the regulatory networks of the mammalian {alpha}2(I) collagen coding genes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The collagens represent a large family of extracellular proteins that impart specific physical properties to tissues, in addition to playing important roles during morphogenesis and growth and in tissue homeostasis and repair (1). Collagen I is the most abundant and most widely distributed collagen type, with high prevalence in bone, skin, teeth, ligaments, and tendons (2). It consists of two {alpha}1(I) chains and one {alpha}2(I) chain that are produced by two fairly large genes that reside on different chromosomes in both the human and the mouse genome (3). Structural mutations in the collagen I chains give rise to manifestations that affect the integrity of multiple organ systems in patients with osteogenesis imperfecta and Ehlers-Danlos syndrome (3). Likewise, excessive deposition of collagen I resulting from dysregulated expression of the corresponding genes (COL1A1 and COL1A2)3 is the hallmark of many fibrotic disorders that impair the function of affected organs (4-6). Expression of the collagen I genes is tightly controlled during development and in a discrete subset of mesenchymal cell types. That collagen I transcripts are found in the same 2:1 ratio as the corresponding chains has been interpreted to suggest that common regulatory programs coordinate expression of the two genes (7). However, multiple studies have failed to reveal a common organization of cis-acting elements and cognate trans-acting factors that would be consistent with the notion of shared regulatory programs between the collagen I genes (6). In point of fact, transgenic studies have revealed that the regulatory networks of the mammalian collagen I genes are organized very differently. On the one hand, tissue-specific production of {alpha}1(I) collagen chains is under the control of distinct and separate DNA elements scattered throughout the 3.2 kb immediately upstream of the start site of transcription (8-11). On the other hand, proper {alpha}2(I) collagen synthesis is the result of combinatorial interactions among nuclear factors that bind to overlapping DNA motifs clustered within the proximal promoter, as well as between them and those binding to a far upstream enhancer (12-15).

Species-specific differences have also emerged with respect to the organization of the regulatory network of the human COL1A2 and mouse Col1a2 gene (12-15). Chromatin analyses have shown that COL1A2 and Col1a2 share five DNase I hypersensitive sites (HS) within nearly identical sequences of the proximal promoter (HS1), 2.3 kb (HS2) and 20 kb upstream of the transcription start site (HSs 3-5) (12, 13, 16). Furthermore, studies in transgenic mice have demonstrated that high and tissue-specific expression of both COL1A2 and Col1a2 proximal promoters requires interaction with the upstream sequence containing HSs 3-5, also known as the far upstream enhancer (12, 13). Deletion experiments have, however, shown that the region around HS5 is dispensable in the mouse but absolutely required in the human transgene (13, 14). Additional analyses have documented that the proximal promoter or the far upstream enhancer of the human, but not of the mouse gene, can by itself drive transgenic expression in osteoblasts (15).

The transcriptional contribution of the first intron sequence is another potential difference between the two species. First, we have identified an open chromatin site, HS(In), that is unique to the first intron of the human gene (13). Second, earlier cell transfection experiments had assigned an enhancing activity to the first intron of Col1a2 and an inhibitory role to the COL1A2 counterpart (17, 18). As part of our effort to delineate the full anatomy of the COL1A2 regulatory network, we have revisited these early studies using the transgenic mouse model in conjunction with DNA binding assays and guided by the knowledge that the intronic sequence contains an open chromatin site (13). The results indicate that the sequence harboring HS(In) acts as a strong inhibitor of COL1A2 transcription, thus supporting the earlier contention of Sherwood et al. (17). Our investigations also mapped relevant cis-acting elements within the HS(In) sequence, identified the cognate trans-acting factors, and demonstrated that full HS(In) repressing activity requires the concerted action of GATA and IRF transcription factors. This study therefore extended the characterization of the major functional elements of the COL1A2 regulatory network, in addition to identifying another species-specific difference between the human and mouse genes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
DNA Binding Assays—Nuclear extracts were purified from cultured WI-38 human lung fibroblasts, NIH-3T3 mouse fibroblasts, or Jurkat T cells according to the previously published protocol (19). For DNase I footprinting assay, a plasmid DNA containing the intron 1 sequence that spans from nucleotides +524 to +895 was cleaved internally with HindIII, end-labeled by filling in 3'-recessed ends with the Klenow enzyme, excised from the plasmid backbone with EcoRI, and incubated with nuclear extracts with or without the addition of DNase I as described previously (19, 20). Likewise, the electrophoretic mobility shift assay (EMSA) conditions for oligonucleotide end-labeling and incubation with nuclear extracts were essentially the same as described previously (19, 20). In some experiments, nuclear extracts were preincubated with commercial antibodies against GATA or IRF proteins (Santa Cruz Biotechnology, Santa Cruz, CA), or molar excesses of unlabeled mutant or wild-type oligonucleotides were added to the nuclear extract incubation. Footprinted sequences or DNA-protein complexes were resolved by polyacrylamide gel electrophoresis and visualized by autoradiography. The chromatin immunoprecipitation (ChIP) assay was performed on WI-38 cells using a commercial kit (Upstate, Lake Placid, NY) and according to the published protocol (21). Oligonucleotide primers corresponding to +680/+705 and +1074/+1047 (intron-specific) or corresponding to -2472/-2450 and -2358/-2339 (negative control) were employed to PCR-amplify sequence potentially bound by various nuclear proteins. The PCR reaction was performed for a total of 38 cycles after initial denaturation at 93 °C for 3 min; amplification conditions included denaturation at 93 °C for 45 s, annealing at 55 °C (intron-specific) or 47 °C negative control)for 1 min, and elongation at 72 °C for 2 min. One-seventh of the immunoprecipitated genomic fragment was used as a template for amplification, except for the input sample in which 0.01% of the total DNA was used. Results of the ChIP analysis were visualized by Southern blot hybridization to the intron or upstream sequences of the amplification products separated on a 2% agarose gel. Intron sequences from different vertebrate organisms were derived from the Ensemble data base and aligned using the program GeneDoc.

Transgenic Constructs—The control LacZ reporter constructs harboring the far upstream enhancer and proximal promoter of COL1A2 have been already described (13). Mutant constructs were engineered using PCR amplification to insert single nucleotide substitutions into the various nuclear protein-binding sites of the intronic sequence. Preparation of linearized plasmid DNA for microinjection was according to the standard protocol (22).

Generation and Analysis of Transgenic Embryos—Transgenic embryos were produced by the standard pronuclear injection of DNA into fertilized C57Bl/10 x CBA/Ca F1 eggs (22). Plasmid DNA was digested with appropriate enzymes, purified from agarose gel, and microinjected at a concentration of 2-4 ng/ml in 10 mM Tris (pH 7.4) and 0.1 mM EDTA. Embryos were collected from the recipient females mainly at 15.5 days postcoitum (embryonic day 15.5) for whole mount fixation and staining. This stage was chosen because it is characterized by high Col1a2 activity and to avoid decreased skin permeability due to increased keratinization (12). Southern blot hybridization and/or PCR amplification of placental DNA were used to assess transgene integration as described previously (12). After cutting open the thorax and abdomen, embryos were placed in cold phosphate-buffered saline and fixed for 45-60 min in 0.2% glutaraldehyde, 2% formalin in 0.1 M phosphate buffer, pH 7.3, containing 2 mM MgCl2 and EGTA. After three washes of 1 h each in the same buffer supplemented with 0.1% sodium deoxycholate and 0.2% Nonidet P-40, embryos were stained overnight at room temperature in 1 mg/ml 5-bromo-4-chloro-3-indolyo-{beta}-D-galactosidase (X-gal) solution containing 5 mM potassium ferrocyanide and 5 mM ferricyanide. For histology, X-gal-positive embryos were dehydrated and wax-embedded, and 6-µm tissue sections were prepared, dewaxed, and counterstained with eosin. The data presented are from embryos with comparable numbers of transgenic inserts, 2-5.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Early cell transfection experiments have shown that the first intron of Col1a2 and COL1A2 stimulates and inhibits transcription, respectively (17, 18). These studies were, however, performed using long intronic sequences (1.2-1.7 kb) and without prior knowledge of the precise location of relevant cis-acting element(s). Subsequent analyses of chromatin structure located a unique DNase-sensitive site, termed HS(In), at about +730 in the first intron of COL1A2 and within an evolutionarily divergent sequence (13, 17). The present study was designed to characterize this putative regulatory element of COL1A2 using transgenic mice in combination with DNA binding assays. Accordingly, the DNase I footprinting assays were first performed on a genomic fragment spanning from nucleotides +524 to +801 to map sites of nuclear protein interaction around HS(In). The analysis located a cluster of three clearly distinct footprinted areas within the HS(In) encompassing region, which were designated FI1 (+647/+676), FI2 (+696/+734), and FI3 (+746/+760) (Fig. 1A). Radiolabeled oligonucleotides containing each of the footprinted areas were then used in the EMSA to confirm binding of nuclear proteins. The EMSA revealed formation of specific retarded bands with each of the three probes (Fig. 2). A computer-aided inspection of the footprinted areas identified potential binding sites for transcription factors GATA (FI1 and FI2) and IRF (FI3) (Fig. 1B). Wild-type and mutant forms of these recognition sites were therefore tested in competition assays against the respective labeled probes. The resulting EMSAs documented the ability of the wild-type oligonucleotides and the inability of the mutant sequences to affect complex formation (Fig. 2). Cross-species sequence alignment of the relevant intron elements revealed only a modest level of sequence homology and loss of most of the GATA- and IRF-binding sites identified in the human gene (Fig. 1C).

Additional EMSAs using specific antibodies confirmed that GATA and IRF proteins indeed bind to the FI1 and FI2 sequences and to the FI3 sequence, respectively. Specifically, the assays showed that the FI1 and FI2 probes bind GATA-4 and not GATA-1, whereas probe FI3 recognized both IRF-2 and IRF-1 (Fig. 2). That the IRF-1 antibodies reduced FI3 complex formation without yielding a supershift could be accounted for by unspecific antibody interference. However, lack of IRF-1 antibody interference with Jurkat nuclear extracts excluded this possibility (Fig. 3A). Consistent with the differential distribution of the two GATA proteins (23), the specificity of the FI2 complex was indirectly corroborated by the finding that GATA-2 and GATA-3 or GATA-4 bind to FI2 in Jurkat T-cells and fibroblasts, respectively (Fig. 3A). The same results were obtained with the IF1 probe (data not shown). These in vitro binding assays were confirmed in vivo by ChIP analysis of human lung fibroblasts. Sequence-specific PCR amplification of genomic DNA immunoprecipitated with antibodies against GATA-4, IRF-1, or IRF-2, but not with unspecific antibodies, yielded reproducibly positive signals when hybridized to the HS(In) probe (Fig. 3B). Furthermore, specificity of in vivo binding was independently confirmed by lack of positive signals in a parallel control sample in which the ChIP assay was performed with an upstream sequence of COL1A2 (Fig. 3B). Preliminary evidence also suggested that a potential CREB/AP1 recognition sequence in FI2 binds c-Jun (data not shown).



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  		FIGURE 1.
DNase I footprinting of the COL1A2 intron. A, an end-labeled genomic fragment spanning from nucleotides +542 to +801 was incubated with increasing amounts of DNase I in the presence (+) or absence (-) of WI-38 nuclear extracts. The numbers on the side of the protected areas correspond to the footprinted sequences (FI1-3) shown in B, where the locations of the GATA and IRF consensus sequences are also indicated along with the nucleotide substitutions (underlined) of the mutant sequences used in the transgenic experiments and EMSAs. C, cross-species sequence alignment of the relevant intronic elements shown in B and analyzed in Fig. 2.

 
Having determined the precise location of the HS(In) element and the identity of the interacting nuclear factors, the next experiments examined its functional contribution to COL1A2 transcription. Transient transfections using a 372-bp intronic sequence (+524 to +895) inclusive of the HS(In) element showed a slight down-regulation of the -378 promoter activity, consistent with the earlier notion of a repressing activity of the COL1A2 intron (data not shown) (17). Based on this preliminary evidence, the transgenic model was then employed to examine the HS(In) element within the in vivo context and in relationship to the interaction between the proximal promoter and far upstream enhancer. Transgenic constructs included the original 21.1/18.8pLAC plasmid, which contains the core sequence of the far upstream enhancer and the -378 proximal promoter (13) and a modification of 21.1/18.8pLAC in which the wild-type 372-bp segment containing the HS(In) element had been inserted downstream of the reporter gene (21.1/18.8pLAC-In) or between the far upstream enhancer and proximal promoter (21.1/18.8(In)pLAC) (Fig. 4). Several 21.1/18.8pLAC-In founders were generated, and all showed lower {beta}-galactosidase staining in most tissues when compared with embryos harboring the intronless 21.1/18.8pLAC transgene (Fig. 5, A and B). Histological sections demonstrated that {beta}-galactosidase staining in different transgenics, albeit variable in intensity, was always confined to collagen I-producing cells (Fig. 5, D-I). Overall, the intensity and distribution of X-gal staining in the 21.1/18.8pLAC-In transgene was reminiscent of the pattern observed previously with the proximal promoter transgene without the far upstream enhancer (13). Limited staining was seen in some ossification centers of intramembranous bones (Fig. 5, H and I) and in patches of skin fascia and tendon (Fig. 5G), as well as in a few internal organs, such as the forming kidney and spleen (Fig. 5, D and F). By contrast, no staining was detected in the lung, heart, gut, and blood vessels (Fig. 5E). Similar results were obtained with 21.1/18.8(In)pLAC, the construct in which the intronic sequence had been inserted between the enhancer and the promoter (Fig. 5C). Together, these findings demonstrated the inhibitory effect of the HS(In) sequence on COL1A2 transcription.



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  		FIGURE 2.
Gel mobility shift assays of the HS(In) footprints. Labeled oligonucleotides corresponding to the sequences of FI1, FI2, and FI3 were incubated with nuclear extracts purified from NIH-3T3 with or without molar excesses (50-100-fold) of unlabeled oligonucleotides corresponding to wild-type (wt) and mutant (mt) versions of the original probes and the wild-type or mutant GATA (Gwt and Gmt) and IRF (Iwt and Imt) consensus sequences, as well as preincubation with antibodies ({alpha}) against the indicated transcription factors. Symbols indicate retarded (arrow) or supershifted (arrowhead) complexes and nonspecific bands (asterisk).

 



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  		FIGURE 3.
GATA and IRF proteins binding to HS(In) elements. A, antibody interference of FI2 and FI3 complex formation using the indicated antisera ({alpha}) and nuclear extracts purified from fibroblasts (F) or Jurkat cells (T). B, ChIP analysis of chromatin from WI-38 cells before (lane 1 and 2) and after immunoprecipitation (in duplicate) with antibodies against IRF-1 (lanes 3 and 4), IRF-2 (lanes 5 and 6), and GATA-4 (lanes 7 and 8). Other controls include IgG-treated sample (lane 9) and input DNA (lane 10). Top (Intron), PCR-amplified products of the +680 to 1074 intron segment bearing the GATA- and IRF-binding sites. Bottom (Control), PCR-amplified products of the -2472 to -2339 upstream sequence of COL1A2. Amplified products were electrophoresed in a 2% agarose gel and visualized by Southern blot hybridization to the original intron or upstream probes.

 



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  		FIGURE 4.
Transgenic constructs. A schematic representation of the LacZ reporter constructs employed in the transgenic analyses, and containing the intronic elements shown in Fig. 1B, is illustrated here.

 



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  		FIGURE 5.
Functional analysis of HS(In) in transgenic mice. Whole mount X-gal staining of illustrative embryonic day 15.5 embryos harboring the 21.8/18.8pLAC (A), 21.1/18.8pLAC-In (B), or 21.1/18.8(In)pLAC transgenes (C) is shown. X-gal staining of tissue sections from 21.1/18.8pLAC-In transgenic embryos showing {beta}-galactosidase activity in a few collagen I-producing cells, such as those in the splenic primordium (D, s) and around the vertebra but not in blood vessels (E, s), is shown. Positive mesenchymal cells of the developing kidney and tendons are shown in F and G, respectively. A few osteoblasts are positively stained (arrows) in the parietal bone (H) and maxillary gland (I).

 
Based on the above findings, we assessed the contribution of individual nuclear protein-binding sites to HS(in) inhibition of 21.1/18.8pLAC expression by examining {beta}-galactosidase activity in transgenic mouse embryos harboring mutated versions of FI1, FI2, or FI3. The mutations included nucleotide substitutions in the binding sites of each footprint (21.1/18.8pLAC-Inm1, 21.1/18.8pLAC-Inm2, and 21.1/18.8pLAC-Inm3) and in the GATA-binding sites of both FI1 and FI2 (21.1/18.8pLAC-Inm1,2) (Fig. 4). X-gal staining revealed comparable {beta}-galactosidase levels that were similar to that of the intronless 21.1/18.8pLAC transgene (Fig. 6, A-D). They also identified a few interesting differences among the four mutant transgenes. First, X-gal staining in the skin and other tissues of the 21.1/18.8pLAC-Inm1 and 21.1/18.8pLAC-Inm2 transgene was lower than in 21.1/18.8pLAC-Inm3 embryos (Fig. 6, A, B, and D). The sole exception was the unusually strong {beta}-galactosidase activity in 21.1/18.8pLAC-Inm1 and 21.1/18.8pLAC-Inm2 bones (Fig. 6, K and L). In point of fact, this was the strongest X-gal staining ever recorded in bone with any of the COL1A2 constructs examined in this and previous studies (13, 15). Second, LacZ gene expression in internal organs of the 21.1/18.8pLAC-Inm2 transgene was consistently higher than it was for the 21.1/18.8pLAC-Inm1 construct (data not shown). Third, the combination of both GATA mutations (21.1/18.8pLAC-Inm1,2) yielded the same level of X-gal staining as the mutation of only the IRF-binding site (21.1/18.8pLAC-Inm3) and was the closest to that of the intronless control transgene (Fig. 6, C and D). Several founders harboring transgenes with m1, m2, and m3 mutations were sectioned and examined histologically. This revealed intense {beta}-galactosidase activity in all type I collagen-producing cells, such as skin fascia fibroblasts (Fig. 6E), skeletal muscle cells, and tendon and blood vessels (Fig. 6H). High level staining was also noted in gut, lung, pancreas, and splenic primordium (Fig. 6, F-I). Collectively, these results indicated that full repressor activity requires the participation of all nuclear protein-binding sites and that each of them may play a more prominent role within individual cellular contexts.

In summary, the present study has demonstrated for the first time that the HS(In) element is a strong repressor of COL1A2 transcription in vivo. This conclusion was based on the ability of a short HS(In) containing sequence to inhibit the activity of an experimental model that closely replicates the expression pattern of COL1A2 in transgenic mice. Within the limitations of this in vivo model, our result supports Sherwood et al. (17) cell transfection data and reiterates the existence of functional differences in the organization of the regulatory network of the Col1a2 and COL1A2 genes. As such, it underscored the peril of extrapolating functional conclusions from one mammalian species to another.

The EMSA and the ChIP assay have correlated the inhibitory activity of the HS(In) sequence with the specific binding of GATA and IRF proteins to three nearly juxtaposed elements. Moreover, transgenic experiments have demonstrated that inhibition requires the full complement of nuclear protein-binding sites. They have also raised the possibility that each of the cis-acting HS(In) elements imparts slightly different properties to the inhibitory protein complex. GATA proteins represent a small family of zinc finger transcriptional regulators that are expressed in hematopoietic stem cells (GATAs 1-3) and in a variety of mesoderm- and endoderm-derived tissues (GATAs 4-6) (23). Consistent with the tissue distribution of GATA family members, we observed binding of GATA-4 and GATAs 2 and 3 with nuclear extracts from NIH-3T3 and Jurkat cells, respectively. GATA proteins have been reported to modulate tissue-specific gene expression across various cell types by interacting with a large array of transcriptional activators and repressors (23). Along these lines, we recently found that the HS2 element of COL1A2 is another GATA-binding site that represses transcription from the -378 promoter (24). It is therefore conceivable to argue that combinatorial interactions among GATA proteins and co-factors at different COL1A2 sites may orchestrate expression of this mesenchyme-specific gene within different cellular contexts.



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  		FIGURE 6.
Effects of HS(In) mutations on transgene activity. Whole mount X-gal staining of illustrative embryonic day 15.5 embryos harboring the 21.1/18.8pLAC-Inm1 (A), 21.1/18.8pLAC-Inm2 (B), 21.1/18.8pLAC-Inm1,2 (C), and 21.1/18.8pLAC-Inm3(D) transgene is shown. Tissue sections from embryos representative of mutations m1, m2, and m3 (E-J)or m1 and m2 (K and L) showing {beta}-galactosidase activity in skin fibroblasts (E) but not keratinocytes (arrow), gut muscular layers (F, m), pancreas (G), blood vessels (H, arrow), tendon (H, t), lung fibroblasts (I), and splenic primordium (J, s) but not kidney (k) are shown. Intense staining was also seen in all osteoblasts, including those in growth plates (K, arrows) and ribs (L, arrows).

 
Originally identified as transcriptional repressors or activators of interferon-{beta} (IFN-{beta}) and of IFN-{gamma}-inducible genes, IRFs have later emerged as broader regulators of other biological processes, such as cell growth, in conjunction with other nuclear proteins (25). A case in point is IRF-2, which has been shown to repress IFN-{beta} activation by coactivator repulsion (26). In this novel regulatory mechanism, incorporation of IRF-2 into the enhanceosome prevents recruitment of the CREB-binding protein and RNA polymerase II holoenzyme complex through a specific protein domain. Similar to the relatively higher activity of 21.1/18.8pLAC-Inm3 when compared with the other mutant transgenes in most collagen I-producing tissues, inactivation of IRF-2 in mice has been shown to expand the number of cells that respond to viral infection by inducing IFN-{beta} gene transcription (26). Although our study did not address whether a similar mechanism may operate in COL1A2, it nonetheless suggested that other factors that normally counteract HS(In) repression in collagen I-producing cells are not present in the transgenic model used as our experimental readout. Among others, probable modulating factors include additional sequences and cognate trans-acting factors and/or appropriate spacing of the interacting domains in the regulatory network (27). Work in progress is characterizing the precise mechanism of the transcriptional repression by the intronic elements and the identity of the trans-acting factors involved in this process. It is also exploring the possibility that HS(In) may contain another interferon-responsive element of the COL1A2 gene.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant AR386481, the St. Giles Foundation, the James D. Farley Family, and the Arthritis Research Campaign. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Laboratory of Genetics and Organogenesis Hospital for Special Surgery, 535 East 70th St., New York, NY 10021. Tel.: 212-774-7554; Fax: 212-774-7864; E-mail: ramirezf{at}hss.edu.

3 The abbreviations used are: COL1A2 and Col1a2, human and mouse {alpha}2(I) collagen gene, respectively; EMSA, electrophoretic mobility shift assay; HS, DNase I hypersensitive site; HS(In), DNase I-hypersensitive site in the first intron of Col1A2; IRF, interferon regulatory factor; ChIP, chromatin immunoprecipitation; X-gal, 5-bromo-4-chloro-3-indolyo-{beta}-D-galactosidase; IFN, interferon; CREB, cAMP-response element-binding protein. Back


   ACKNOWLEDGMENTS
 
We thank Karen Johnson for organizing the manuscript and Graham Reed for the photographic reproductions. This investigation was conducted in a facility constructed with support from Research Facilities Improvement Program Grant Number C06-RR12538-01 from the National Center for Research Resources, National Institutes of Health.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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