Analysis of transcriptional regulation of the small leucine richproteoglycans

[摘要] Purpose: Small leucine rich proteoglycans (SLRPs) constitute afamily of secreted proteoglycans that are important for collagenfibrillogenesis, cellular growth, differentiation, and migration. Ten ofthe 13 known members of the SLRP gene family are arranged in tandemclusters on human chromosomes 1, 9, and 12. Their syntenic equivalentsare on mouse chromosomes 1, 13, and 10, and rat chromosomes 13, 17, and7. The purpose of this study was to determine whether there is evidencefor control elements, which could regulate the expression of theseclusters coordinately.Methods: Promoters were identified using a comparative genomicsapproach and Genomatix software tools. For each gene a set of human,mouse, and rat orthologous promoters was extracted from genomicsequences. Transcription factor (TF) binding site analysis combined witha literature search was performed using MatInspector and Genomatix'BiblioSphere. Inspection for the presence of interspecies conservedscaffold/matrix attachment regions (S/MARs) was performed using ElDoradoannotation lists. DNAseI hypersensitivity assay, chromatinimmunoprecipitation (ChIP), and transient transfection experiments wereused to validate the results from bioinformatics analysis.Results: Transcription factor binding site analysis combined with aliterature search revealed co-citations between several SLRPs and TFsRunx2 and IRF1, indicating that these TFs have potential roles intranscriptional regulation of the SLRP family members. We thereforeinspected all of the SLRP promoter sets for matches to IRF factors andRunx factors. Positionally conserved binding sites for the Runt domainTFs were detected in the proximal promoters of chondroadherin (CHAD) andosteomodulin (OMD) genes. Two significant models (two or moretranscription factor binding sites arranged in a defined order andorientation within a defined distance range) were derived from theseinitial promoter sets, the HOX-Runx (homeodomain-Runt domain), and theETS-FKHD-STAT (erythroblast transformation specific-forkhead-signaltransducers and activators of transcription) models. These models wereused to scan the genomic sequences of all 13 SLRP genes. The HOX-Runxmodel was found within the proximal promoter, exon 1, or intron 1sequences of 11 of the 13 SLRP genes. The ETS-FKHD-STAT model was foundin only 5 of these genes. Transient transfections of MG-63 cells andbovine corneal keratocytes with Runx2 isoforms confirmed the relevanceof these TFs to expression of several SLRP genes. Distribution of theHOX-Runx and ETS-FKHD-STAT models within 200 kb of genomic sequence onhuman chromosome 9 and 500 kb sequence on chromosome 12 also wereanalyzed. Two regions with 3 HOX-Runx matches within a 1,000 bp windowwere identified on human chromosome 9; one located between OMD andosteoglycin (OGN)/mimecan genes, and the second located upstream of theputative extracellular matrix protein 2 (ECM2) promoter. The intergenicregion between OMD and mimecan was shown to coincide with differentpatterns of DNAse I hypersensitivity sites in MG-63 and U937 cells. ChiPanalysis revealed that this region binds Runx2 in U937 cells (mimecantranscript note detectable), but binds Pitx3 in MG-63 cells (expressinghigh level of mimecan), thereby demonstrating its functional associationwith mimecan expression. Upon comparing the predictions of S/MARs on therelevant chromosomal context of human chromosomes 9 and 12 and theirrodent equivalents, no convincing evidence was found that the tandemlyarranged genes build a chromosomal loop.Conclusions: Twelve of 13 known SLRP genes have at least oneHOX-Runx module match in their promoter, exon 1, intron 1, or intergenicregion. Although these genes are located in different clusters ondifferent chromosomes, the common HOX-Runx module could be the basis forco-regulated expression.

[学科分类] 生物化学/生物物理