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Published online before print
September 4, 2007, 10.1101/gr.6224007 Genome Res. 17:1438-1447, 2007 ©2007 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/07 $5.00
Letter Computational identification and functional validation of regulatory motifs in cartilage-expressed genes1 Department of Orthopaedic Surgery, Washington University School of Medicine, St. Louis, Missouri 63110, USA; 2 Department of Biomedical Engineering, Washington University, St. Louis, Missouri 63130, USA; 3 Department of Pediatrics, University of Texas Medical School at Houston, Houston, Texas 77030, USA; 4 Shriners Hospital for Children, Houston, Texas 77030, USA; 5 Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110, USA; 6 Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA
Chondrocyte gene regulation is important for the generation and maintenance of cartilage tissues. Several regulatory factors have been identified that play a role in chondrogenesis, including the positive transacting factors of the SOX family such as SOX9, SOX5, and SOX6, as well as negative transacting factors such as C/EBP and delta EF1. However, a complete understanding of the intricate regulatory network that governs the tissue-specific expression of cartilage genes is not yet available. We have taken a computational approach to identify cis-regulatory, transcription factor (TF) binding motifs in a set of cartilage characteristic genes to better define the transcriptional regulatory networks that regulate chondrogenesis. Our computational methods have identified several TFs, whose binding profiles are available in the TRANSFAC database, as important to chondrogenesis. In addition, a cartilage-specific SOX-binding profile was constructed and used to identify both known, and novel, functional paired SOX-binding motifs in chondrocyte genes. Using DNA pattern-recognition algorithms, we have also identified cis-regulatory elements for unknown TFs. We have validated our computational predictions through mutational analyses in cell transfection experiments. One novel regulatory motif, N1, found at high frequency in the COL2A1 promoter, was found to bind to chondrocyte nuclear proteins. Mutational analyses suggest that this motif binds a repressive factor that regulates basal levels of the COL2A1 promoter.
Gene expression and its regulation are important for the coordination of various activities of a cell. This complex regulatory circuit involves a multitude of transcription factors (TFs) and their corresponding cis-acting regulatory elements whose interaction in a variety of permutational and combinatorial events enable the function and maintenance of tissues. An understanding of the transcriptional regulatory network (Covert et al. 2004  ) can be attempted using a computational approach in which TF-binding sites can be modeled, searched, and identified in the non-coding sequence through the use of position weight matrices (PWMs) and DNA pattern recognition programs (Stormo 2000). The conservation of functional cis-acting elements among the non-coding sequences of orthologous genes denoted by "phylogenetic footprinting" has refined computational approaches to allow for a credible identification of cis-acting elements (Tagle et al. 1988; Wasserman et al. 2000; Blanchette and Tompa 2002).
The successful use of DNA pattern recognition programs in inferring regulatory circuits has been evident in the detection of novel regulatory elements involved in heat-shock response (GuhaThakurta et al. 2002a
Many genes such as collagen type II (COL2A1), collagen type IX (COL9A1), collagen type XI (COL11A2), and melanoma inhibitory activity (MIA) that are normally expressed predominantly and almost exclusively in cartilage are known to share common regulatory factors (Okazaki et al. 2002
Analyses of previously characterized TF-binding motifs in cartilage characteristic genes Our analyses included steps to identify known TF-binding motifs, novel binding motifs, and paired SOX-binding sites within cartilage enhancer sequences by examining the conserved sequence in 18 orthologous pairs of human and mouse genes. The results of the analysis of the characterized TF binding motifs from TRANSFAC are shown in Table 1A. Transcription factor-binding motifs were ranked by the log ratio of the probability scores, taking into account multiple predicted binding sites within a promoter, calculated using cartilage genes and background genes, respectively (see Methods). Based on this model, a higher log ratio value indicates a higher probability that the TF will bind to the promoter of these cartilage genes. The log ratios of the probability scores for TFs with documented roles in chondrogenesis are given in Table 1B.
Identification of paired SOX-binding motifs in cartilage characteristic genes One TF we expected to detect with a high log ratio in the analyses above is SOX9, a master regulator of chondrogenesis (Wagner et al. 1994 ; Wheatley et al. 1996; Lefebvre et al. 1997; Ng et al. 1997; Bi et al. 1999; Wegner 1999; Akiyama et al. 2002). However, the log ratio values for both the TRANSFAC SOX9 and SOX5 motifs in cartilage genes were relatively low compared to other motifs (0.842 for SOX9 and 1.351 for SOX5) given the known functional roles these factors play in vivo. SOX transcription factors bind to cis-regulatory sequences through a conserved HMG domain. The SOX subfamily of HMG proteins is remarkable as they appear to play distinct and essential roles in many different developmental processes, yet the DNA-binding sites they recognize are highly degenerate. Their trans-activating functions and specificities appear to be highly dependent on both the orientation of, and the spacing between, their binding sites and the binding sites of other cofactors (Wegner 1999; Peirano and Wegner 2000; Bridgewater et al. 2003). This suggests that the SOX motif present in TRANSFAC may not model functional SOX-binding sites present in chondrocyte genes precisely and accurately. Therefore, to better model the binding sites involved in cartilage gene regulation, we constructed a cartilage-specific SOX-binding profile from enhancer elements known to specify cartilage gene expression in vivo (Fig. 1A,B). We collected a set of 31 previously documented SOX-binding sites (Supplemental Table 1) from three cartilage-specific genes. The consensus sequence of this profile is CTTT GWW, which is slightly different from the TRANSFAC motifs for SOX5 (ATTGTT) or SOX9 (CYATTGTT). These three SOX-binding profiles are short and contain degenerate positions. Therefore, to reduce the false-positive discovery rate, we used evolutionary conservation to search for SOX sites conserved in human and mouse cartilage genes. This approach significantly decreased the number of predicted SOX sites in cartilage-expressed genes (data not shown).
To test the sensitivity of our SOX model, we used this motif to predict experimentally identified SOX-binding sites in our training set of cartilage regulatory regions of the Mia, Col2a1, and Col11a2 genes (Fig. 2A–C). Although using the SOX profile to identify the contributing SOX-binding sites seems circular, the purpose of this test is to identify (1) any SOX site in the training set that is significantly different from other SOX sites and (2) additional unknown SOX sites in the known cartilage regulatory regions. All of the sites used to train the motif were also predicted from our search algorithm with the exception of one in the 183-bp Mia gene promoter element, indicating that our search criteria were functioning appropriately. When the orientation and spacing of the predicted sites were examined, we found at least one tandem pair of sites with opposite orientations, with a short spacer sequence in between (3–8 bp), for each of the regulatory elements used to develop our model motifs (Fig. 2A–C).
Multiple sites were predicted in the 183-bp element, located at –2251 to –2058 of the Mia promoter, an essential part of the cartilage regulatory module (Okazaki et al. 2006 ). Indeed, it has been shown in previous studies that the E class of SOX factors, which includes SOX9, can exhibit homodimeric, cooperative DNA binding that is dependent on closely spaced and oppositely oriented binding sites (Peirano and Wegner 2000; Sock et al. 2003). Next, we searched the entire Mia gene promoter sequence to see if our algorithm could predict a known SOX9-binding site at –410 (Xie et al. 1999). In addition to predicting our previously identified SOX9 binding site, a novel, closely spaced, and oppositely orientated site was identified immediately downstream at –395 (Fig. 2D). We mutated both the –410 and –395 sites individually and analyzed the effects using luciferase reporter constructs in transient transfection experiments in RCS cells (Fig. 3). As previously reported, the full-length –2251-bp construct that is required for cartilage-specific expression exhibited strong activity (Xie et al. 1999). In contrast, the negative control construct truncated to –401 bp (and missing the –410 SOX9 site) had virtually no activity. Mutation of two core nucleotides in the newly identified motif at –395 bp similarly ablated activity of the full-length promoter construct (–2251 bp) despite the presence of the –410 SOX9-binding site that has previously been characterized (Fig. 3) (Xie et al. 1999). Thus, both the –410 and –395 sites appear to be equally essential to the full-length promoter activity.
Since experimental data suggested that pairing of SOX motifs is an important part of the chondrocyte regulatory module, we refined our search criteria to include not just the conservation between the mouse and human sequences, but also the close positioning of the sites that are 3–8 bp apart. A summary of all the conserved sites predicted in the promoters of all the searched cartilage genes is shown in Supplemental Table 2. As the table indicates, the conservation between human and mouse orthologous sequences significantly reduced the number of motifs that would be predicted to be functional. There were only three genes in our cartilage set (COL9A2, COMP, FGF18) that did not have any conserved pair of SOX sites. To test the specificity of our search criteria, we also collected the promoters of 13 previously reported liver-specific genes (Krivan and Wasserman 2001 ) and performed the exact same search. Only six of the 13 liver-specific genes have conserved paired SOX sites in their promoters (Supplemental Table 3).
In addition to SOX factors, our previous results have implicated the repressor C/EBP beta as a TF that may work in these regulatory sequences to restrict cartilage gene expression (Okazaki et al. 2006
Since the SOX-binding module seemed a predominant feature in our gene set, we then decided to test these predicted sites for functional activity in another well-characterized cartilage gene promoter, COL9A1, that is known to be activated by SOX9 in chondrocytes (Zhang et al. 2003
Identification of a novel motif N1 in cartilage-expressed genes Although the TRANSFAC database curates  500 vertebrate TF-binding profiles, there are estimated to be 2000 TF proteins in the human genome. Therefore, the binding profiles of many TFs are still not available, and the computational analysis described above using the TRANSFAC motifs may have missed other chondrocyte cis-regulatory elements. To circumvent this problem, we applied the DNA pattern recognition algorithm CONSENSUS (Stormo and Hartzell 1989; Hertz and Stormo 1999) to identify potential regulatory motifs in chondrocyte genes that have not been identified in TRANSFAC. The input data set for CONSENSUS is the conserved sequences of the 18 cartilage genes. We analyzed the human and mouse sequence sets separately and used the ALLR statistics (Wang and Stormo 2003) to find nonredundant candidate motifs that were conserved between human and mouse and over-represented in cartilage promoters. By this procedure, 87 conserved motifs were identified. Comparison and consolidation between similar motifs generated 23 nonredundant conserved motifs. The log ratio of the probability scores was calculated for each motif (Table 1C), and the sequence logo for the five top ranking motifs that are not present in the TRANSFAC database is shown in Figure 5. The distribution of all five novel motifs in cartilage-characteristic genes and their genomic location is shown in Supplemental Table 4.
The location of the N1 motif in the promoter regions of both the COL2A1 and COL11A2 genes made this motif a good candidate for experimental validation of its function. The consensus sequence determined for this motif is CCAGAGCCC. Significantly, evolutionarily conserved N1 motifs were detected in the proximal promoter region of 11 of the 18 cartilage genes analyzed in both human and mouse (Table 2). In some genes, multiple conserved N1 motifs were detected. For example, in the COL2A1 promoter, four conserved N1 motifs were identified within –1 kb (N-125/126, N-135, N-147, and N-991), and one conserved N1 motif was identified at the +16 (N+16) position relative to the transcriptional start site.
To assess the function of the N1 motif and to determine its relevance in the regulation of chondrogenesis, oligonucleotides (15 bp in length) were generated from the promoter sequences of the cartilage genes in which this motif was recognized (see Table 2) and used as probes in electromobility gel shift assays using nuclear extracts from the RCS cells to determine protein-binding capacity (Fig. 6). Significantly, a major DNA–protein complex with similar mobility was generated for all the 16 oligonucleotides tested (Fig. 6, O1–O16, arrowhead), suggesting that these protein–DNA complexes probably have an identical TF binding to the N1 motif. In all of these cases, the binding could be competed by the addition of nonradiolabeled oligonucleotides to the reaction, demonstrating the specific nature of this binding. For the oligonucleotides O6, O13, O14, and O16, an additional, faster migrating complex (double arrowhead) was also observed, suggesting additional TFs may bind to these sequences as well. Four conserved N1 sites were identified in the COL2A1 promoter within –1 kb from the transcription start site (O6, O7, O8, O9). The N1 site at –125/126 was found to be present on both strands of the COL2A1 promoter sequence at the indicated positions. Interestingly, this motif, CCCCGGAGCCC, partially overlaps a previously identified EGR1 site (with overlap shown in bold/underlined) that mediates repression of the COL2A1 gene by interleukin 1 beta (IL1B) (Tan et al. 2003 ).
To test the functional significance of the N1 motif in the COL2A1 gene and validate the computational search, a mutational analysis of the N1 motif was performed. To avoid complications in the analysis caused by the binding of other factors to the site at –125/126, the more distal N1 motif at –147 in a COL2A1 promoter-luciferase reporter construct was mutated to test its function in the RCS cell line in which strong COL2A1 expression is normally observed. This mutational analysis involved deletion of the four most conserved bases from the N1 motif (CCAGTGCCC to CCA----CC) at the –147 position. Deletion of these four core bases increased the basal COL2A1 promoter activity in our reporter construct as measured by relative luciferase activity (Fig. 7A), suggesting that this is probably a motif that binds to a repressor resulting in negative regulation. As IL1B is known to repress chondrogenesis, the response of the mutated COL2A1 promoter was also tested by measuring its response to IL1B (Fig. 7A). As expected, the wild-type COL2A1 promoter demonstrated a reduction in its basal activity (by 62%) in the presence of IL1B. The mutant promoter activity was also repressed (by 54%–63%), suggesting that the regulation to inflammatory mediators was still active. Interestingly, the repression of the mutant promoter in the presence of IL1B results in its activity being equal to the activity of the wild-type promoter in the absence of IL1B again, further suggesting that the –147 N1 motif probably binds to a negatively regulating TF.
The functional significance of the N1 motifs found in the promoter of an additional cartilage characteristic gene, COMP, was also analyzed. When oligonucleotides representing an N1 motif in the COMP promoter were tested by EMSA as above, a high-molecular-weight DNA–protein complex similar to that of the other tested oligonucleotides was observed. This DNA–protein complex was competed away with non-radiolabeled oligonucleotides representing the wild-type N1 motif, but not by oligonucleotides with mutation(s) of the core nucleotides within the N1 motif (data not shown; for details, see Supplemental Methods). Furthermore, to test the function of this motif in vitro, analogous mutations in the human COMP gene promoter-luciferase reporter construct were made, mutating the N1 sequence motif from CACCACAGCCC to CACTGTGGCCC to form N1Mut (mutational changes are underlined) (Fig. 7B). The RCS cell line transfected with this mutant construct also showed an increase in activity compared to the wild-type promoter, again demonstrating a negative transacting cis-regulatory function for this motif.
In this study, we have taken a computational approach to annotate and validate important cis-regulatory motifs in cartilage-specific genes. The use of standard molecular biology techniques such as systematic sequence deletions and other mutagenesis approaches to define cartilage-specific regulatory regions would have been time-consuming given the size of the promoter region and the number of genes that we have used in this study. To circumvent this problem, we used computational methods that utilized position weight matrices and phylogenetic footprinting to increase the sensitivity of our predictions. Using this approach, we screened our cartilage-specific gene set for previously characterized TF-binding motifs from the TRANSFAC database and novel TF-binding sites with customized binding motifs. Statistical analysis of motif enrichment was performed to predict those that are most likely to regulate the chondrocyte genes.
Several TRANSFAC motifs were highly represented in our gene set for factors that play critical roles in patterning required for skeletal development (Nissen et al. 2003
Using this information, we were able to refine our computational search and predict novel SOX-binding motifs for experimental validation more reliably. We found many high-scoring SOX-binding sites in our cartilage genes using the customized SOX-binding profile, but only some of them were present in a tandem pair with opposite orientations. We hypothesized that this is the preferred architecture of the chondrocyte regulatory module. Our analysis identified five novel SOX-binding motifs in the COL9A1 promoter in addition to one that is already published. Mutations in any of these SOX-binding motifs significantly decreased promoter activity in luciferase reporter assays, validating our computational approach. We also identified a novel SOX-binding motif in the cartilage-specific Mia gene and similarly validated its function by mutational analyses. Although we did not test it for functional activity, our model also predicted an additional SOX site (+130 bp), oppositely orientated and adjacent to, a documented site (+120 bp) in the HAPLN1 gene (also called cartilage linking protein 1 gene) (Kou and Ikegawa 2004
Another challenge to defining biologically functional SOX-binding sites through computational approaches is that in vivo, some SOX sites function only in the context of other cis-acting motifs and TFs (Kamachi et al. 2000
One drawback of performing computational promoter analysis based on the TRANSFAC database is that currently we do not have a complete collection of the binding profiles of all transcription factors. Although recent studies have proposed methods to computationally identify mammalian TF-binding profiles using comparative genomics approaches (Tan et al. 2005 ; Xie et al. 2005), the latest version of TRANSFAC has only the weight matrix models for about a quarter of the TFs in the human genome. Therefore, to identify enriched motifs in cartilage genes that have not been found in TRANSFAC, we also applied DNA pattern discovery algorithms on our cartilage gene set. Using this computational approach, we were able to identify and validate the function of one novel binding motif, N1, that appeared to participate in regulating the basal promoter activity of the COL2A gene. In addition to COL2A1, the N1 motif was also identified and validated in the promoter regions of other cartilage-expressed genes such as COMP and COL9A1 by mutational analyses (data not shown), supporting its role as a cis-regulatory motif. We do not know the identity of the factor that binds to N1, as it does not correspond to any known TF-binding profile in the TRANSFAC database or any cis-acting regulatory elements known to drive cartilage-specific gene expression. Several N1 motifs were close to or overlapping with previously identified binding sites for SP1, EGR1 (Tan et al. 2003), or Maf (Huang et al. 2002). However, we were not able to verify binding of any of these proteins to the N1 motif using specific antibodies in gel shift experiments (data not shown). Given that the mutation of the motif appeared to increase basal promoter activity of several cartilage genes, it is likely that there is a repressive factor in RCS cells that binds to this motif. The identity of the N1-binding protein may be uncovered when the TRANSFAC database is more complete.
In summary, there are several challenges in the use of computational approaches to identify TF-binding motifs in mammalian organisms. One major challenge is the fact that mammalian species have very long intergenic sequence, and it is very difficult to detect regulatory sequences in such a large search space. In our approach, we used phylogenetic footprinting methods (Wasserman et al. 2000
Definition of promoter sequences A set of 18 orthologous human and mouse genes with documented expression in cartilage either during development or maintenance of the tissue was selected for this analysis: ACAN, CILP, COL11A1, COL11A2, COL2A1, COL9A1, COL9A2, COL9A3, COMP, HAPLN1, CTGF, FGF18, IHH, MATN1, MATN3, MIA (Cdrap), PRG4, and SOX9. An additional set of 13 previously reported liver-specific genes was also collected for testing specificity. The promoter sequences from these gene sets were selected for the analyses as follows. For most genes, the promoter sequence was defined as the 10-kb upstream and the 5-kb downstream sequence according to the transcriptional start site. For some genes, the promoter sequence was truncated when an upstream gene was encountered (e.g., the COL11A2 gene) or when the translation start site was reached. Focusing on the 15-kb genomic sequence around the transcription start site for each gene in our analysis is keeping within the limit of current DNA recognition pattern programs. For each human gene, the mouse ortholog was determined by the NCBI’s HomoloGene database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=homologene). The mouse ortholog was further verified for the reciprocal best match of the protein sequences using WU-BLAST (http://blast.wustl.edu/). Promoter sequences of human and mouse genes were retrieved from the UCSC genome browser (http://www.genome.ucsc.edu). Repetitive elements in the promoter sequences were masked by the program RepeatMasker (http://www.repeatmasker.org) with the slow and sensitive mode.
Determination of conserved regions in promoters
Identification of evolutionarily conserved TF-binding motifs
Enrichment of binding motifs
)
; Hu et al. 2004).
Construction of a cartilage-specific SOX-binding profile
Identification of paired SOX-binding sites in cartilage genes
Identification of novel regulatory motifs in cartilage-characteristic genes
Cell culture
Electromobility gel shift assays (EMSA) and transient transfections
We thank Hua Yu for her technical assistance in preparation of mutant constructs. We also thank David Stokes (Thomas Jefferson University, Philadelphia) for providing us with the COL9A1 promoter construct (p846). This work was supported by NIH grants RO1 AR36994, R01 AR45550, and RO1 AR50847 to L.J.S. and NIH grants HG00249 and GM63340 to G.D.S.
7 Present address: Division of Oncology, Washington University School of Medicine, St. Louis, MO 63110, USA. 
E-mail sandelll{at}wudosis.wustl.edu; fax (314) 454-5900. [Supplemental material is available online at www.genome.org.] Article published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.6224007
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Abstract
Results
subunit is required for bone formation and hematopoietic maturation. Nat. Genet. 32: 645–649.
mediate the repression of gene transcription of cartilage-derived retinoic acid-sensitive protein induced by interleukin-1
and 
globin genes of a prosimian primate (Galago crassicaudatus). Nucleotide and amino acid sequences, developmental regulation and phylogenetic footprints. J. Mol. Biol. 203: 439–455.