Regulation of human COL9A1 gene expression. Activation of the proximal promoter region by SOX9.
ABSTRACT The COL9A1 gene contains two promoter regions, one driving expression of a long alpha1(IX) chain in cartilage (upstream) and one driving expression of a shorter chain in the cornea and vitreous (downstream). To determine how the chondrocyte-specific expression of the COL9A1 gene is regulated, we have begun to characterize the upstream chondrocyte-specific promoter region of the human COL9A1 gene. Transient-transfection analyses performed in rat chondrosarcoma (RCS) cells, human chondrosarcoma (HTB) cells, and NIH/3T3 cells showed that the COL9A1 promoter was active in RCS cells but not HTB or NIH/3T3 cells. Inclusion of the first intron had no effect on promoter activity. In transient-transfection analyses with promoter deletion constructs, it was found that full promoter activity in RCS cells depended on the region from -560 bp to +130 bp relative to the transcriptional start site (+1). Sequence analysis of the region from -890 bp to the transcriptional start predicted five putative SOX/Sry-binding sites. Mutation analysis revealed that two of three putative SOX/Sry binding sites within the -560 to +130 bp region are responsible for most of the COL9A1 promoter activity in RCS cells. Co-transfection experiments with a SOX9 expression plasmid revealed that a construct containing the five putative SOX/Sry-binding sites was transactivated 20- to 30-fold in both HTB and NIH/3T3 cells. Further co-transfection experiments showed that two of the SOX/Sry-binding sites located within the -560 to +130 bp region were required for full transactivation. However, mutation and deletion analyses indicated that a region from -560 to -357 bp, which does not contain any other conspicuous SOX9 sites, is also important for full promoter activity. DNA-protein binding assays and super-shift analysis revealed that SOX9 can form a specific complex with one of the SOX/Sry-binding sites with in the -560 to +130 region.
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ABSTRACT: Chronic articular cartilage defects are the most common disabling conditions of humans in the western world. The incidence for cartilage defects is increasing with age and the most prominent risk factors are overweight and sports associated overloading. Damage of articular cartilage frequently leads to osteoarthritis due to the aneural and avascular nature of articular cartilage, which impairs regeneration and repair. Hence, patients affected by cartilage defects will benefit from a cell-based transplantation strategy. Autologous chondrocytes, mesenchymal stem cells and embryonic stem cells are suitable donor cells for regeneration approaches and most recently the discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options. It is the aim of this review to summarize recent advances in the use of amniotic fluid stem cells as novel cell sources for the treatment of articular cartilage defects. Molecular aspects of articular cartilage formation as well as degeneration are summarized and the role of growth factor triggered signaling pathways, scaffolds, hypoxia and autophagy during the process of chondrogenic differentiation are discussed.Stem cell reviews 08/2012; · 5.08 Impact Factor
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ABSTRACT: Epigenetic modifications are heritable changes in gene expression without changes in DNA sequence. DNA methylation has been implicated in the control of several cellular processes including differentiation, gene regulation, development, genomic imprinting and X-chromosome inactivation. Methylated cytosine residues at CpG dinucleotides are commonly associated with gene repression; conversely, strategic loss of methylation during development could lead to activation of lineage-specific genes. Evidence is emerging that bone development and growth are programmed; although, interestingly, bone is constantly remodelled throughout life. Using human embryonic stem cells, human fetal bone cells (HFBCs), adult chondrocytes and STRO-1(+) marrow stromal cells from human bone marrow, we have examined a spectrum of developmental stages of femur development and the role of DNA methylation therein. Using pyrosequencing methodology we analysed the status of methylation of genes implicated in bone biology; furthermore, we correlated these methylation levels with gene expression levels using qRT-PCR and protein distribution during fetal development evaluated using immunohistochemistry. We found that during fetal femur development DNA methylation inversely correlates with expression of genes including iNOS (NOS2) and COL9A1, but not catabolic genes including MMP13 and IL1B. Furthermore, significant demethylation was evident in the osteocalcin promoter between the fetal and adult developmental stages. Increased TET1 expression and decreased expression of DNA (cytosine-5-)-methyltransferase 1 (DNMT1) in adult chondrocytes compared to HFBCs could contribute to the loss of methylation observed during fetal development. HFBC multipotency confirms these cells to be an ideal developmental system for investigation of DNA methylation regulation. In conclusion, these findings demonstrate the role of epigenetic regulation, specifically DNA methylation, in bone development, informing and opening new possibilities in development of strategies for bone repair/tissue engineering.PLoS ONE 01/2013; 8(1):e54957. · 3.73 Impact Factor
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ABSTRACT: Type XI collagen, a heterotrimer composed of α1(XI), α2(XI), and α3(XI), plays a critical role in cartilage formation and in skeletal morphogenesis. However, the transcriptional regulation of α1(XI) collagen gene (Col11a1) in chondrocyte is poorly characterized. In this study, we investigated the proximal promoter of mouse Col11a1 gene in chondrocytes. Major transcription start site was located at -299 bp upstream of the translation start site, and the proximal promoter lacks a TATA sequence but has a high guanine-cytosine (GC) content. Cell transfection experiments demonstrated that the segment from -116 to -256 is necessary for activation of the proximal Col11a1 promoter, and an electrophoretic mobility shift assay showed that a nuclear protein is bound to the segment from -116 to -176 in this promoter. Additional comparative and in silico analyses demonstrated that an ATTGG sequence, which is critical for binding to nuclear factor Y (NF-Y), is within the highly conserved region from -135 to -145. Interference assays using wild-type and mutant oligonucleotide or with specific antibody revealed that NF-Y protein is bound to this region. Cell transfection experiments with reporter constructs in the absence of NF-Y binding sequence exhibited the suppression of the promoter activity. Furthermore, chromatin immunoprecipitation assay demonstrated that NF-Y protein is directly bound to this region in vivo, and overexpression of dominant-negative NF-Y A mutant also inhibited the proximal promoter activity. Taken together, these results indicate that the transcription factor NF-Y regulates the proximal promoter activity of mouse Col11a1 gene in chondrocytes.In Vitro Cellular & Developmental Biology - Animal 10/2013; · 1.29 Impact Factor