Molecular Vision 2004; 10:758-772 <http://www.molvis.org/molvis/v10/a91> ©2004 Molecular Vision Received 12 August 2004 | Accepted 7 October 2004 | Published 7 October 2004 Analysis of transcriptional regulation of the small leucine rich proteoglycans Elena S. Tasheva,1 Bernward Klocke,2 Gary W. Conrad1 1Kansas State University, Division of Biology, Manhattan, KS; 2Genomatix Software GmbH, München, Germany Purpose: Small leucine rich proteoglycans (SLRPs) constitute a family of secreted proteoglycans that are important for collagen fibrillogenesis, cellular growth, differentiation, and migration. Ten of the 13 known members of the SLRP gene family are arranged in tandem clusters on human chromosomes 1, 9, and 12. Their syntenic equivalents are on mouse chromosomes 1, 13, and 10, and rat chromosomes 13, 17, and 7. The purpose of this study was to determine whether there is evidence for control elements, which could regulate the expression of these clusters coordinately. Methods: Promoters were identified using a comparative genomics approach and Genomatix software tools. For each gene a set of human, mouse, and rat orthologous promoters was extracted from genomic sequences. Transcription factor (TF) binding site analysis combined with a literature search was performed using MatInspector and Genomatix’ BiblioSphere. Inspection for the presence of interspecies conserved scaffold/matrix attachment regions (S/MARs) was performed using ElDorado annotation lists. DNAseI hypersensitivity assay, chromatin immunoprecipitation (ChIP), and transient transfection experiments were used to validate the results from bioinformatics analysis. Results: Transcription factor binding site analysis combined with a literature search revealed co-citations between several SLRPs and TFs Runx2 and IRF1, indicating that these TFs have potential roles in transcriptional regulation of the SLRP family members. We therefore inspected all of the SLRP promoter sets for matches to IRF factors and Runx factors. Positionally conserved binding sites for the Runt domain TFs were detected in the proximal promoters of chondroadherin (CHAD) and osteomodulin (OMD) genes. Two significant models (two or more transcription factor binding sites ar- ranged in a defined order and orientation within a defined distance range) were derived from these initial promoter sets, the HOX-Runx (homeodomain-Runt domain), and the ETS-FKHD-STAT (erythroblast transformation specific-forkhead- signal transducers and activators of transcription) models. These models were used to scan the genomic sequences of all 13 SLRP genes. The HOX-Runx model was found within the proximal promoter, exon 1, or intron 1 sequences of 11 of the 13 SLRP genes. The ETS-FKHD-STAT model was found in only 5 of these genes. Transient transfections of MG-63 cells and bovine corneal keratocytes with Runx2 isoforms confirmed the relevance of these TFs to expression of several SLRP genes. Distribution of the HOX-Runx and ETS-FKHD-STAT models within 200 kb of genomic sequence on human chromosome 9 and 500 kb sequence on chromosome 12 also were analyzed. Two regions with 3 HOX-Runx matches within a 1,000 bp window were identified on human chromosome 9; one located between OMD and osteoglycin (OGN)/ mimecan genes, and the second located upstream of the putative extracellular matrix protein 2 (ECM2) promoter. The intergenic region between OMD and mimecan was shown to coincide with different patterns of DNAse I hypersensitivity sites in MG-63 and U937 cells. ChiP analysis revealed that this region binds Runx2 in U937 cells (mimecan transcript note detectable), but binds Pitx3 in MG-63 cells (expressing high level of mimecan), thereby demonstrating its functional association with mimecan expression. Upon comparing the predictions of S/MARs on the relevant chromosomal context of human chromosomes 9 and 12 and their rodent equivalents, no convincing evidence was found that the tandemly arranged genes build a chromosomal loop. Conclusions: Twelve of 13 known SLRP genes have at least one HOX-Runx module match in their promoter, exon 1, intron 1, or intergenic region. Although these genes are located in different clusters on different chromosomes, the com- mon HOX-Runx module could be the basis for co-regulated expression. The process of transcription is the key element in gene nals for transcription factors and that promoters bear the his- expression and, as such, an attractive control point for regula- tone code: H3 hyperacetylation and methylation of lysine 4 tion of gene expression in cell and tissue specific manners. It [1]. Various models demonstrate how the two types of com- is not surprising that considerable research has been conducted plexes, the nucleosome remodeling complexes of the SWI/ on elucidating the mechanisms by which genes are regulated SNF (switch/sucrose non-fermentable) type, which use the [1-7]. Current views of transcriptional regulation incorporate energy of ATP-hydrolysis to alter histone-DNA contacts, and the histone code hypothesis which proposes that different com- the enzymatic complexes that modify histones by acetylation, binations of histone modifications function as recognition sig- methylation, phosphorylation, and ubiquitinylation participate in chromatin remodeling [1-7]. Models also explain how regu- Correspondence to: Elena S. Tasheva, Division of Biology, Ackert latory motifs act at a distance and involve looping to bring Hall, Kansas State University, Manhattan, KS, 66506-4901; Phone: regulatory elements in contact with distant promoters. Regu- (785) 532-6553; FAX: (785) 532-6653; email: [email protected] latory motifs are seen as binding sites for proteins that induce 758 Molecular Vision 2004; 10:758-772 <http://www.molvis.org/molvis/v10/a91> ©2004 Molecular Vision chemical modifications and structural alterations propagating ture and that regulatory effects of a control region depend on down the fiber [5]. In addition to studying regulation of tran- the specific combination of elements, as well as the order and scription through a variety of biological and biochemical ap- orientation in which they occur [8,10,16]. The ability of a given proaches, recently there has been much interest in the possi- sequence-specific transcription factor to interact with both co- bility of using bioinformatics approaches to identify gene regu- activators and co-repressors and/or to recruit histone-modify- latory elements [8-10]. Large scale cross-species DNA se- ing proteins is thought to provide a simple means for generat- quence comparisons reveal regions of highly conserved non- ing on-off switches in a tissue specific manner during the cell- coding sequences located upstream of transcription initiation cycle, and in development [1-4,7]. In addition to the order and sites (gene promoters), or in introns and intergenic regions orientation of units of transcription regulatory regions, the (enhancers, silencers, scaffold/matrix attachment region (S/ abundance of transcriptional cofactors in a certain cell type MARs), and locus control regions) [11-15]. Functional analy- also influences the ability of site specific factors to regulate ses of these conserved regions show that they represent cis- gene expression [17]. Thus, a combination of bioinformatics regulatory elements that control expression of nearby genes. and wet lab experiments has emerged as a successful method Data indicate that these regulatory regions have modular struc- for detecting cis-regulatory elements in many genomic loci, including those for homeodomain containing transcription factors (HOX), immunoglobulin, β-globin, and IL4/IL13/IL5- cytokine gene clusters [18-21]. The small lecine rich proteoglycans (SLRPs) are a well- known family of secreted proteoglycans present in many con- nective tissues. Crucial roles of these macromolecules in ma- trix assembly and modulation of cellular growth have been demonstrated extensively in the literature [22-25]. In the eye, SLRPs have been shown to be equally important for develop- ment and maintenance of corneal transparency and for pro- viding the structural link between the neural retina and retinal pigment epithelium [26,27]. Alterations in the balance of cor- neal SLPGs result in increased hydration and loss of corneal transparency. In vitro data demonstrate that these molecules regulate axon guidance and synapse formation during the de- velopment of nervous tissue and the vertebrate retina. Indi- vidual roles of several SLRPs have been demonstrated by pro- duction of gene knock-out animals. All single or double SLRP- null mice generated so far displayed abnormal collagen fibrillogenesis and developed a variety of diseases such as osteoporosis, osteoarthritis, muscular dystrophy, Ehlers-Danlos syndrome, and corneal pathology, e.g. diseases that appear to result from collagen defects [28-33]. Phenotypic changes in different SLRP-null mice are mild, indicating that these pro- teins can compensate for one another, as evidenced by an in- creased amount of lumican in fibromodulin-null mice [31]. Although the molecular bases for these compensatory mecha- nisms presently are unknown, it is likely that this might occur at the level of transcription. The notion of regulated expres- sion of the SLRPs at the level of transcription is supported by their genomic organization. Thus, 10 of the 13 members of the SLRP gene family are organized in clusters on three chro- mosomes: opticin (OPTC), proline arginine rich end leucine rich repeat protein (PRELP), and fibromodulin (FMOD) on human chromosome 1q3 (their