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Noriyuki Tsumaki - One of the best experts on this subject based on the ideXlab platform.
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Direct induction of chondrogenic cells from human dermal fibroblast culture by defined factors.
PloS one, 2013Co-Authors: Hidetatsu Outani, Hideki Yoshikawa, Minoru Okada, Akihiro Yamashita, Kanako Nakagawa, Noriyuki TsumakiAbstract:The repair of large cartilage defects with hyaline cartilage continues to be a challenging clinical issue. We recently reported that the forced expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) can induce chondrogenic cells from mouse dermal fibroblast culture without going through a pluripotent state. We here generated induced chondrogenic (iChon) cells from human dermal fibroblast (HDF) culture with the same factors. We developed a chondrocyte-specific COL11A2 promoter/enhancer lentiviral reporter vector to select iChon cells. The human iChon cells expressed marker genes for chondrocytes but not fibroblasts, and were derived from non-chondrogenic COL11A2-negative cells. The human iChon cells formed cartilage but not tumors in nude mice. This approach could lead to the preparation of cartilage directly from skin in human, without going through pluripotent stem cells.
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Time-lapse observation of the dedifferentiation process in mouse chondrocytes using chondrocyte-specific reporters
Osteoarthritis and Cartilage, 2013Co-Authors: Y. Minegishi, Ko Hosokawa, Noriyuki TsumakiAbstract:Summary Objective When chondrocytes prepared from cartilage are expanded in monolayer culture, fibroblast-like cells gradually prevail. Although these prevailing fibroblast-like cells are believed to emerge because of the dedifferentiation of chondrocytes, the definite origin of the prevailing fibroblast-like cells has not been determined. We herein examined whether the prevailing non-chondrocytic cells observed after monolayer expansion culture arise from dedifferentiating chondrocytes or are the result of the overgrowth of fibroblasts that are present at the start of the culture. We also evaluated whether chondrocytes dedifferentiate because they proliferate or because they are cultured in monolayers. Methods Chondrocytes were prepared from COL11A2-EGFP transgenic mice and COL11A2-Cre; R26-stop flox -EYFP transgenic mice, which respectively express enhanced green fluorescent protein (EGFP) and Cre specifically in chondrocytes under the control of COL11A2 promoter/enhancer sequences. COL11A2-Cre; R26-stop flox -EYFP mice express enhanced yellow fluorescent protein (EYFP) only in cells which express or used to express Cre. We performed a time-lapse observation of the chondrocytes during monolayer expansion culture, and also observed the chondrocytes after treatment with mitomycin C. Results A time-lapse observation showed that COL11A2-EGFP chondrocytes underwent cell divisions, lost GFP fluorescence, increased cell numbers, and prevailed during the expansion culture. The observation of the COL11A2-Cre; R26-stop flox -EYFP chondrocytes confirmed that most of the cells after expansion in monolayer culture had been chondrocytes. Mitotically inactive chondrocytes generated by treatment with mitomycin C still underwent dedifferentiation, thus suggesting that chondrocyte dedifferentiation is not associated with cell division. Conclusion The non-chondrocytic cells that prevail after the monolayer expansion culture of chondrocytes originate from chondrocytes, and are not generated by the overgrowth of fibroblasts that are present at the start of the culture. Chondrocyte dedifferentiation does not appear to be associated with cell division.
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Insulation of the ubiquitous Rxrb promoter from the cartilage-specific adjacent gene, COL11A2.
The Journal of biological chemistry, 2008Co-Authors: Junko Murai, Daisuke Ikegami, Mina Okamoto, Hideki Yoshikawa, Noriyuki TsumakiAbstract:Abstract The retinoid X receptor β gene (Rxrb) is located just upstream of the α2(XI) collagen chain gene (COL11A2) in a head-to-tail manner. However, the domain structures of these genes are unknown. COL11A2 is specifically expressed in cartilage. In the present study, we found Rxrb expression in various tissues with low expression in the cartilage. COL11A2 1st intron enhancer directed cartilage specific expression when linked to the heterologous promoter in transgenic mice. These results suggest the presence of enhancer-blocking elements that insulate Rxrb promoter from the COL11A2 enhancer. So far, most of insulators examined in vertebrates contain a binding site for CTCF. We found two possible CTCF-binding sites: one (11P) in the intergenic region between Rxrb and COL11A2 by electrophoretic mobility shift assays, and the other in the 4th intron of RXRB by data base search. To examine the function of these elements, we prepared bacterial artificial chromosome (BAC) transgene constructs containing a 142-kb genomic DNA insert with RXRB and COL11A2 sequences in the middle. Mutation of 11P significantly decreased the RXRB promoter activity in muscular cells and significantly increased expression levels of RXRB in chondrosarcoma cells. In transgenic mouse assays, the wild-type BAC transgene partly recapitulated endogenous Rxrb expression patterns. A 507-bp deletion mutation including 11P enhanced the cartilage-specific activity of the RXRB promoter in BAC transgenic mice. Chromatin immunoprecipitation analysis showed that CTCF was associated with RX4, but not with 11P. Our results showed that the intergenic sequence including 11P insulates Rxrb promoter from COL11A2 enhancer, possibly associating with unknown factors that recognize a motif similar to CTCF.
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COL11A2 collagen gene transcription is differentially regulated by ews erg sarcoma fusion protein and wild type erg
Journal of Biological Chemistry, 2003Co-Authors: Yoshito Matsui, Liu Yang, Hideki Yoshikawa, Noriyuki Tsumaki, Howard A. Chansky, Akira Myoui, Fariba Barahmandpour, Anna Zielinskakwiatkowska, David R. EyreAbstract:Abstract A specific t(21;22) chromosomal translocation creates the chimeric EWS/ERG gene in some cases of Ewing's sarcoma. In the resultant EWS/ERG fusion protein, the N-terminal part of the ETS family protein ERG is replaced by the N terminus of the RNA-binding protein EWS. We found that both the EWS/ERG andCOL11A2 genes are expressed in the Ewing's sarcoma cell line, CADO-ES1. To investigate a potential role for EWS/ERG inCOL11A2 gene expression, we characterized theCOL11A2 promoter and tested the ability of wild-type ERG and EWS/ERG sarcoma fusion protein to transactivate COL11A2promoter using a luciferase assay. We found that expression of EWS/ERG, but not wild-type ERG, transactivated the COL11A2 promoter and that this transactivation required not only the N-terminal region of EWS but also an intact DNA-binding domain from ERG. Electrophoretic mobility shift assay using COL11A2 promoter sequence showed involvement of EWS/ERG in the formation of DNA-protein complexes, and chromatin immunoprecipitation assay revealed direct interaction betweenCOL11A2 promoter and EWS/ERG fusion protein in vivo. EWS/ERG, but not wild-type ERG, bound to RNA polymerase II. Treatment of cells with the histone deacetylase inhibitor trichostatin A enabled ERG to transactivate the COL11A2 promoter, therefore abolishing the differential effects of EWS/ERG and ERG. Taken together, these findings indicate that the COL11A2 gene is regulated both by potential ERG association with a histone deacetylase complex and by direct EWS/ERG recruitment of RNA polymerase II.
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COL11A2 Collagen Gene Transcription Is Differentially Regulated by EWS/ERG Sarcoma Fusion Protein and Wild-type ERG
The Journal of biological chemistry, 2003Co-Authors: Yoshito Matsui, Liu Yang, Hideki Yoshikawa, Noriyuki Tsumaki, Howard A. Chansky, Fariba Barahmand-pour, Anna Zielinska-kwiatkowska, Akira Myoui, David R. EyreAbstract:Abstract A specific t(21;22) chromosomal translocation creates the chimeric EWS/ERG gene in some cases of Ewing's sarcoma. In the resultant EWS/ERG fusion protein, the N-terminal part of the ETS family protein ERG is replaced by the N terminus of the RNA-binding protein EWS. We found that both the EWS/ERG andCOL11A2 genes are expressed in the Ewing's sarcoma cell line, CADO-ES1. To investigate a potential role for EWS/ERG inCOL11A2 gene expression, we characterized theCOL11A2 promoter and tested the ability of wild-type ERG and EWS/ERG sarcoma fusion protein to transactivate COL11A2promoter using a luciferase assay. We found that expression of EWS/ERG, but not wild-type ERG, transactivated the COL11A2 promoter and that this transactivation required not only the N-terminal region of EWS but also an intact DNA-binding domain from ERG. Electrophoretic mobility shift assay using COL11A2 promoter sequence showed involvement of EWS/ERG in the formation of DNA-protein complexes, and chromatin immunoprecipitation assay revealed direct interaction betweenCOL11A2 promoter and EWS/ERG fusion protein in vivo. EWS/ERG, but not wild-type ERG, bound to RNA polymerase II. Treatment of cells with the histone deacetylase inhibitor trichostatin A enabled ERG to transactivate the COL11A2 promoter, therefore abolishing the differential effects of EWS/ERG and ERG. Taken together, these findings indicate that the COL11A2 gene is regulated both by potential ERG association with a histone deacetylase complex and by direct EWS/ERG recruitment of RNA polymerase II.
Leena Ala-kokko - One of the best experts on this subject based on the ideXlab platform.
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Dominant and recessive forms of fibrochondrogenesis resulting from mutations at a second locus, COL11A2
American Journal of Medical Genetics Part A, 2012Co-Authors: Stuart W. Tompson, Leena Ala-kokko, Eissa Faqeih, Jacqueline T. Hecht, Rika Miki, Tara L. Funari, Vincent Funari, Lisette Nevarez, Deborah Krakow, Daniel H. CohnAbstract:Fibrochondrogenesis is a severe, recessively inherited skeletal dysplasia shown to result from mutations in the gene encoding the proα1(XI) chain of type XI collagen, COL11A1. The first of two cases reported here was the affected offspring of first cousins and sequence analysis excluded mutations in COL11A1. Consequently, whole-genome SNP genotyping was performed to identify blocks of homozygosity, identical-by-descent, wherein the disease locus would reside. COL11A1 was not within a region of homozygosity, further excluding it as the disease locus, but the gene encoding the proα2(XI) chain of type XI collagen, COL11A2, was located within a large region of homozygosity. Sequence analysis identified homozygosity for a splice donor mutation in intron 18. Exon trapping demonstrated that the mutation resulted in skipping of exon 18 and predicted deletion of 18 amino acids from the triple helical domain of the protein. In the second case, heterozygosity for a de novo 9 bp deletion in exon 40 of COL11A2 was identified, indicating that there are autosomal dominant forms of fibrochondrogenesis. These findings thus demonstrate that fibrochondrogenesis can result from either recessively- or dominantly-inherited mutations in COL11A2.
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A stop codon mutation in COL11A2 induces exon skipping and leads to non-ocular Stickler syndrome.
American journal of medical genetics. Part A, 2004Co-Authors: Mirka Marjanna Vuoristo, John Georgios Pappas, Valerie Jansen, Leena Ala-kokkoAbstract:Mutations in COL11A2 cause a spectrum of phenotypes affecting chondrogenic tissues. We analyzed this gene by conformation sensitive gel electrophoresis (CSGE) and sequencing in a family with non-ocular Stickler syndrome, and found a heterozygous C --> T mutation in exon 57 + 13 in affected members, resulting in Arg893Stop codon. Since heterozygous nonsense mutations in COL11A2 do not usually lead to any obvious phenotype, all exons and exon boundaries of COL11A2 in the sample of the propositus were sequenced. Because no disease-associated alterations were found, we performed RT-PCR analysis on the RNA. Analysis showed skipping of exon 57 in one allele, resulting in an inframe deletion of 54 bp or 18 amino acids, which would explain the phenotype observed in the family. Thus, the exon skipping resulted from a nonsense-associated altered splicing (NAS). This article contains supplementary material, which may be viewed at the American Journal of Medical Genetics website at http://www.interscience.wiley.com/jpages/0148-7299/suppmat/index.html.
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A stop codon mutation in COL11A2 induces exon skipping and leads to non-ocular Stickler syndrome.
American Journal of Medical Genetics Part A, 2004Co-Authors: Mirka M. Vuoristo, John Georgios Pappas, Valerie Jansen, Leena Ala-kokkoAbstract:Mutations in COL11A2 cause a spectrum of phenotypes affecting chondrogenic tissues. We analyzed this gene by conformation sensitive gel electrophoresis (CSGE) and sequencing in a family with non-ocular Stickler syndrome, and found a heterozygous C → T mutation in exon 57 + 13 in affected members, resulting in Arg893Stop codon. Since heterozygous nonsense mutations in COL11A2 do not usually lead to any obvious phenotype, all exons and exon boundaries of COL11A2 in the sample of the propositus were sequenced. Because no disease-associated alterations were found, we performed RT-PCR analysis on the RNA. Analysis showed skipping of exon 57 in one allele, resulting in an inframe deletion of 54 bp or 18 amino acids, which would explain the phenotype observed in the family. Thus, the exon skipping resulted from a nonsense-associated altered splicing (NAS). This article contains supplementary material, which may be viewed at the American Journal of Medical Genetics website at http://www.interscience.wiley.com/jpages/0148-7299/suppmat/index.html. © 2004 Wiley-Liss, Inc.
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Sequence variations in the collagen IX and XI genes are associated with degenerative lumbar spinal stenosis
Annals of the rheumatic diseases, 2003Co-Authors: N Noponen-hietala, Minna Männikkö, E Kyllönen, E Ilkko, Jaro Karppinen, Jurg Ott, Leena Ala-kokkoAbstract:Background: Degenerative lumbar spinal stenosis (LSS) is usually caused by disc herniation or degeneration. Several genetic factors have been implicated in disc disease. Tryptophan alleles in COL9A2 and COL9A3 have been shown to be associated with lumbar disc disease in the Finnish population, and polymorphisms in the vitamin D receptor gene (VDR) ( Fok I and Taq I), the matrix metalloproteinase-3 gene (MMP-3) and an aggrecan gene (AGC1) VNTR have been reported to be associated with disc degeneration. In addition, an IVS6-4 a>t polymorphism in COL11A2 has been found in connection with stenosis caused by ossification of the posterior longitudinal ligament in the Japanese population. Objective: To study the role of genetic factors in LSS. Methods: 29 Finnish probands were analysed for mutations in the genes coding for intervertebral disc matrix proteins, COL1A1, COL1A2, COL2A1, COL9A1, COL9A2, COL9A3, COL11A1, COL11A2, and AGC1. VDR and MMP-3 polymorphisms were also analysed. Sequence variations were tested in 56 Finnish controls. Results: Several disease associated alleles were identified. A splice site mutation in COL9A2 leading to a premature translation termination codon and the generation of a truncated protein was identified in one proband, another had the Trp2 allele, and four others the Trp3 allele. The frequency of the COL11A2 IVS6 −4 t allele was 93.1% in the probands and 72.3% in controls (p = 0.0016). The differences in genotype frequencies for this site were less significant (p = 0.0043). Conclusions: Genetic factors have an important role in the pathogenesis of LSS.
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Genomic organization of the human COL3A1 and COL5A2 genes: COL5A2 has evolved differently than the other minor fibrillar collagen genes.
Matrix Biology, 2001Co-Authors: Merja Välkkilä, Miia Melkoniemi, Laura Kvist, Helena Kuivaniemi, Gerard Tromp, Leena Ala-kokkoAbstract:We report here on the complete structure of the human COL3A1 and COL5A2 genes. Collagens III and V, together with collagens I, II and XI make up the group of fibrillar collagens, all of which share a similar structure and function; however, despite the similar size of the major triple-helical domain, the number of exons coding for the domain differs between the genes for the major fibrillar collagens characterized so far (I, II, and III) and the minor ones (V and XI). The main triple-helical domain being encoded by 49-50 exons, including the junction exons, in the COL5A1, COL11A1 and COL11A2 genes, but by 43-44 exons in the genes for the major fibrillar collagens. Characterization of the genomic structure of the COL3A1 gene confirmed its association with the major fibrillar collagen genes, but surprisingly, the genomic organization of the COL5A2 gene was found to be similar to that of the COL3A1 gene. We also confirmed that the two genes are located in tail-to-tail orientation with an intergenic distance of approximately 22 kb. Phylogenetic analysis suggested that they have evolved from a common ancestor gene. Analysis of the genomic sequences identified a novel single nucleotide polymorphism and a novel dinucleotide repeat. These polymorphisms should be useful for linkage analysis of the Ehlers-Danlos syndrome and related disorders.
Yoshito Matsui - One of the best experts on this subject based on the ideXlab platform.
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sp1 family of transcription factors regulates the human α2 xi collagen gene COL11A2 in saos 2 osteoblastic cells
Journal of Bone and Mineral Research, 2006Co-Authors: Tomohiro Goto, Yoshito Matsui, Russell J. Fernandes, Dennis A. Hanson, Takahiro Kubo, Kiminori Yukata, Toshimi Michigami, Toshihisa Komori, Takashi Fujita, Liu YangAbstract:Genes encoding type XI collagen, normally associated with chondrogenesis, are also expressed by osteoblasts. By studying Saos-2 cells, we showed that the transcription factors, Sp1, Sp3, and Sp7 (Osterix), regulate COL11A2 expression through its proximal promoter. The findings indicate both ubiquitous and osteoblast-specific mechanisms of collagen gene regulation. Introduction: Type XI collagen is essential for skeletal morphogenesis. Collagen XI gene regulation has been studied in chondrocytes but not in osteoblasts. Materials and Methods: We cultured Saos-2 cells, a human osteosarcoma-derived line of osteoblasts, and analyzed them for 2(XI) protein and COL11A2 regulatory mechanisms. Results and Conclusions: Although types I and V were the dominant collagens deposited by Saos-2 cells, they expressed COL11A2 mRNA, and 2(XI) chains were present in the extracellular matrix. The COL11A2 promoter region (from -149 to -40) containing three Sp1 binding sites was required for promoter activity in transient transfection assays. All three Sp1 sites were critical for binding by nuclear proteins in electrophoretic mobility shift assays. Further analysis using consensus oligonucleotides and specific antibodies as well as chromatin immunoprecipitation assay implicated Sp1 and Sp3 in binding to this promoter region. Overex- pressing Sp1 or Sp3 significantly increased COL11A2 promoter activity and endogenous COL11A2 gene expression, an effect that was suppressed by the Sp1-binding inhibitor mithramycin A. Further experiments showed that Sp1, Sp3, CREB-binding protein (CBP), p300, and histone deacetylase (HDAC) were physically associated and HDAC inhibitors (trichostatin A or NaB) upregulated COL11A2 promoter activity and endogenous gene expression. Another Sp1 family member, Sp7 (Osterix), was expressed in Saos-2 cells, but not in chondrocytes, and was shown by chromatin immunoprecipitation to occupy the COL11A2 promoter. Overexpressing Sp7 increased COL11A2 promoter activity and endogenous gene expression, an effect also blocked by mithramycin A. Using siRNA to knockdown Sp1, Sp3, or Sp7, it was shown that depression of any of them decreased COL11A2 promoter activity and endogenous gene expression. Finally, primary cultures of osteoblasts expressed COL11A2 and Sp7, upregulated COL11A2 promoter activity and endogenous gene expression when Sp1, Sp3, or Sp7 were overexpressed, and downregulated them when Sp1, Sp3, or Sp7 were selectively depressed. The results establish that Sp1 proteins regulate COL11A2 transcription by binding to its proximal promoter and directly interacting with CBP, p300, and HDAC.
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Sp1 Family of Transcription Factors Regulates the Human α2 (XI) Collagen Gene (COL11A2) in Saos‐2 Osteoblastic Cells
Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 2006Co-Authors: Tomohiro Goto, Yoshito Matsui, Russell J. Fernandes, Dennis A. Hanson, Takahiro Kubo, Kiminori Yukata, Toshimi Michigami, Toshihisa Komori, Takashi Fujita, Liu YangAbstract:Genes encoding type XI collagen, normally associated with chondrogenesis, are also expressed by osteoblasts. By studying Saos-2 cells, we showed that the transcription factors, Sp1, Sp3, and Sp7 (Osterix), regulate COL11A2 expression through its proximal promoter. The findings indicate both ubiquitous and osteoblast-specific mechanisms of collagen gene regulation. Introduction: Type XI collagen is essential for skeletal morphogenesis. Collagen XI gene regulation has been studied in chondrocytes but not in osteoblasts. Materials and Methods: We cultured Saos-2 cells, a human osteosarcoma-derived line of osteoblasts, and analyzed them for 2(XI) protein and COL11A2 regulatory mechanisms. Results and Conclusions: Although types I and V were the dominant collagens deposited by Saos-2 cells, they expressed COL11A2 mRNA, and 2(XI) chains were present in the extracellular matrix. The COL11A2 promoter region (from -149 to -40) containing three Sp1 binding sites was required for promoter activity in transient transfection assays. All three Sp1 sites were critical for binding by nuclear proteins in electrophoretic mobility shift assays. Further analysis using consensus oligonucleotides and specific antibodies as well as chromatin immunoprecipitation assay implicated Sp1 and Sp3 in binding to this promoter region. Overex- pressing Sp1 or Sp3 significantly increased COL11A2 promoter activity and endogenous COL11A2 gene expression, an effect that was suppressed by the Sp1-binding inhibitor mithramycin A. Further experiments showed that Sp1, Sp3, CREB-binding protein (CBP), p300, and histone deacetylase (HDAC) were physically associated and HDAC inhibitors (trichostatin A or NaB) upregulated COL11A2 promoter activity and endogenous gene expression. Another Sp1 family member, Sp7 (Osterix), was expressed in Saos-2 cells, but not in chondrocytes, and was shown by chromatin immunoprecipitation to occupy the COL11A2 promoter. Overexpressing Sp7 increased COL11A2 promoter activity and endogenous gene expression, an effect also blocked by mithramycin A. Using siRNA to knockdown Sp1, Sp3, or Sp7, it was shown that depression of any of them decreased COL11A2 promoter activity and endogenous gene expression. Finally, primary cultures of osteoblasts expressed COL11A2 and Sp7, upregulated COL11A2 promoter activity and endogenous gene expression when Sp1, Sp3, or Sp7 were overexpressed, and downregulated them when Sp1, Sp3, or Sp7 were selectively depressed. The results establish that Sp1 proteins regulate COL11A2 transcription by binding to its proximal promoter and directly interacting with CBP, p300, and HDAC.
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Overexpression of HMGA2-LPP fusion transcripts promotes expression of the α 2 type XI collagen gene
Biochemical and biophysical research communications, 2005Co-Authors: Takahiro Kubo, Tomohiro Goto, Yoshito Matsui, Kiminori Yukata, Natsuo YasuiAbstract:Abstract In a subset of human lipomas, a specific t (3; 12) chromosome translocation gives rise to HMGA2-LPP fusion protein, containing the amino (N)-terminal DNA binding domains of HMGA2 fused to the carboxyl (C)-terminal LIM domains of LPP. In addition to its role in adipogenesis, several observations suggest that HMGA2-LPP is linked to chondrogenesis. Here, we analyzed whether HMGA2-LPP promotes chondrogenic differentiation, a marker of which is transactivation of the α 2 type XI collagen gene ( COL11A2 ). Real-time PCR analysis showed that HMGA2-LPP and COL11A2 were co-expressed. Luciferase assay demonstrated that either of HMGA2-LPP, wild-type HMGA2 or the N-terminal HMGA2 transactivated the COL11A2 promoter in HeLa cells, while the C-terminal LPP did not. RT-PCR analysis revealed that HMGA2-LPP transcripts in lipomas with the fusion were 591-fold of full-length HMGA2 transcripts in lipomas without the fusion. These results indicate that in vivo overexpression of HMGA2-LPP promotes chondrogenesis by upregulating cartilage-specific collagen gene expression through the N-terminal DNA binding domains.
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COL11A2 collagen gene transcription is differentially regulated by ews erg sarcoma fusion protein and wild type erg
Journal of Biological Chemistry, 2003Co-Authors: Yoshito Matsui, Liu Yang, Hideki Yoshikawa, Noriyuki Tsumaki, Howard A. Chansky, Akira Myoui, Fariba Barahmandpour, Anna Zielinskakwiatkowska, David R. EyreAbstract:Abstract A specific t(21;22) chromosomal translocation creates the chimeric EWS/ERG gene in some cases of Ewing's sarcoma. In the resultant EWS/ERG fusion protein, the N-terminal part of the ETS family protein ERG is replaced by the N terminus of the RNA-binding protein EWS. We found that both the EWS/ERG andCOL11A2 genes are expressed in the Ewing's sarcoma cell line, CADO-ES1. To investigate a potential role for EWS/ERG inCOL11A2 gene expression, we characterized theCOL11A2 promoter and tested the ability of wild-type ERG and EWS/ERG sarcoma fusion protein to transactivate COL11A2promoter using a luciferase assay. We found that expression of EWS/ERG, but not wild-type ERG, transactivated the COL11A2 promoter and that this transactivation required not only the N-terminal region of EWS but also an intact DNA-binding domain from ERG. Electrophoretic mobility shift assay using COL11A2 promoter sequence showed involvement of EWS/ERG in the formation of DNA-protein complexes, and chromatin immunoprecipitation assay revealed direct interaction betweenCOL11A2 promoter and EWS/ERG fusion protein in vivo. EWS/ERG, but not wild-type ERG, bound to RNA polymerase II. Treatment of cells with the histone deacetylase inhibitor trichostatin A enabled ERG to transactivate the COL11A2 promoter, therefore abolishing the differential effects of EWS/ERG and ERG. Taken together, these findings indicate that the COL11A2 gene is regulated both by potential ERG association with a histone deacetylase complex and by direct EWS/ERG recruitment of RNA polymerase II.
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COL11A2 Collagen Gene Transcription Is Differentially Regulated by EWS/ERG Sarcoma Fusion Protein and Wild-type ERG
The Journal of biological chemistry, 2003Co-Authors: Yoshito Matsui, Liu Yang, Hideki Yoshikawa, Noriyuki Tsumaki, Howard A. Chansky, Fariba Barahmand-pour, Anna Zielinska-kwiatkowska, Akira Myoui, David R. EyreAbstract:Abstract A specific t(21;22) chromosomal translocation creates the chimeric EWS/ERG gene in some cases of Ewing's sarcoma. In the resultant EWS/ERG fusion protein, the N-terminal part of the ETS family protein ERG is replaced by the N terminus of the RNA-binding protein EWS. We found that both the EWS/ERG andCOL11A2 genes are expressed in the Ewing's sarcoma cell line, CADO-ES1. To investigate a potential role for EWS/ERG inCOL11A2 gene expression, we characterized theCOL11A2 promoter and tested the ability of wild-type ERG and EWS/ERG sarcoma fusion protein to transactivate COL11A2promoter using a luciferase assay. We found that expression of EWS/ERG, but not wild-type ERG, transactivated the COL11A2 promoter and that this transactivation required not only the N-terminal region of EWS but also an intact DNA-binding domain from ERG. Electrophoretic mobility shift assay using COL11A2 promoter sequence showed involvement of EWS/ERG in the formation of DNA-protein complexes, and chromatin immunoprecipitation assay revealed direct interaction betweenCOL11A2 promoter and EWS/ERG fusion protein in vivo. EWS/ERG, but not wild-type ERG, bound to RNA polymerase II. Treatment of cells with the histone deacetylase inhibitor trichostatin A enabled ERG to transactivate the COL11A2 promoter, therefore abolishing the differential effects of EWS/ERG and ERG. Taken together, these findings indicate that the COL11A2 gene is regulated both by potential ERG association with a histone deacetylase complex and by direct EWS/ERG recruitment of RNA polymerase II.
Martin P. Snead - One of the best experts on this subject based on the ideXlab platform.
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Stickler syndrome: further mutations in COL11A1 and evidence for additional locus heterogeneity.
European journal of human genetics : EJHG, 1999Co-Authors: Sam Martin, Allan J. Richards, John R.w. Yates, John D. Scott, Michael Pope, Martin P. SneadAbstract:Stickler syndrome (hereditary arthro-ophthalmopathy) is a dominantly inherited connective tissue disorder with ocular, oro-facial, auditory and skeletal manifestations. It is genetically and phenotypically heterogeneous with the majority of families having mutations in the gene encoding type II collagen (COL2A1) and exhibiting a characteristic ‘membranous’ or type 1 vitreous phenotype. More recently a novel mutation in the gene encoding the α1 chain of type XI collagen (COL11A1) was reported in a Stickler syndrome pedigree with a different ‘beaded’ or type 2 vitreous phenotype. In the present study five more families with the type 2 vitreous phenotype were examined for linkage to four candidate genes: COL2A1, COL5A2, COL11A1 and COL11A2. Two families were linked to COL11A1 and sequencing identified mutations resulting in shortened α1(XI) collagen chains, one via exon skipping and the other via a multiexon deletion. One of the families showed weak linkage to COL5A2 but sequencing the open reading frame failed to identify a mutation. In the remaining two families all four loci were excluded by linkage analysis. These data confirm that mutations in COL11A1 cause Stickler syndrome with the type 2 vitreous phenotype and also reveal further locus heterogeneity.
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a family with stickler syndrome type 2 has a mutation in the col11a1 gene resulting in the substitution of glycine 97 by valine in alpha 1 xi collagen
Human Molecular Genetics, 1996Co-Authors: A J Richards, John R.w. Yates, John D. Scott, Rebecca Williams, S J Payne, Michael F Pope, Martin P. SneadAbstract:Stickler syndrome (hereditary arthro-ophthalmopathy) is the commonest inherited cause of retinal detachment and one of the commonest autosomal dominant connective tissue dysplasias. There is clinical and locus heterogeneity with about two thirds of families linked to the gene encoding type II procollagen (COL2A1). Families with Sticklers syndrome type 1 have a characteristic congenital vitreous anomaly and are linked without recombination to markers at the COL2A1 locus. In contrast families with the type 2 variety have a different vitreo-retinal phenotype and are not linked to the COL2A1 gene. Type XI collagen is a quantitatively minor fibrillar collagen related to type V collagen and associated with the more abundant type II collagen fibrils. A mutation in COL11A2, the gene for alpha 2 (XI) procollagen, has recently been found in a family described as having Stickler syndrome, although there was no ocular involvement. Here we show for the first time that a family with the full Type 2 Stickler syndrome including vitreous and retinal abnormalities is linked to the COL11A1 gene and characterise the mutation as a Glycine to Valine substitution at position 97 of the triple helical domain caused by a single base G-->T mutation. These results are the first to provide confirmation that type XI collagen is an important structural component of human vitreous. They also support previous work suggesting that mutations in the genes encoding collagen XI can give rise to some manifestations of Stickler syndrome, but of these, only mutations in COL11A1 will give the full syndrome including the vitreo-retinal features.
Fransiska Malfait - One of the best experts on this subject based on the ideXlab platform.
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novel pathogenic col11a1 COL11A2 variants in stickler syndrome detected by targeted ngs and exome sequencing
Molecular Genetics and Metabolism, 2014Co-Authors: Frederic Acke, Fransiska Malfait, Olivier Vanakker, Wouter Steyaert, Kim De Leeneer, Geert Mortier, Ingeborg Dhooge, Anne De Paepe, Els De Leenheer, Paul CouckeAbstract:Abstract Introduction Stickler syndrome is caused by mutations in genes encoding type II and type XI collagens. About 85% of the pathogenic variants is found in COL2A1 (Stickler type 1), whereas a minority of mutations has been reported in COL11A1 (Stickler type 2) and COL11A2 (Stickler type 3). Beside the typical skeletal and orofacial manifestations, ocular anomalies are predominantly present in type 1 and type 2, while hearing loss is more pronounced in type 2 and type 3. Methods We performed COL11A1 mutation analysis for 40 type 2 Stickler patients and COL11A2 mutation analysis for five type 3 Stickler patients, previously all COL2A1 mutation-negative, using targeted next-generation sequencing (NGS) whereas whole-exome sequencing (WES) was performed in parallel for two patients. Three patients were analyzed for both genes due to unclear ocular findings. Results In total 14 COL11A1 and two COL11A2 mutations could be identified, seven of which are novel. Splice site alterations are the most frequent mutation type, followed by glycine substitutions. In addition, six variants of unknown significance (VUS) have been found. Identical mutations and variants were identified with both NGS techniques. Conclusion We expand the mutation spectrum of COL11A1 and COL11A2 in Stickler syndrome patients and show that targeted NGS is an efficient and cost-effective molecular tool in the genetic diagnosis of Stickler syndrome, whereas the more standardized WES might be an alternative approach.
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Novel pathogenic COL11A1/COL11A2 variants in Stickler syndrome detected by targeted NGS and exome sequencing
Molecular genetics and metabolism, 2014Co-Authors: Frederic Acke, Fransiska Malfait, Olivier Vanakker, Wouter Steyaert, Kim De Leeneer, Geert Mortier, Ingeborg Dhooge, Anne De Paepe, Els De Leenheer, Paul CouckeAbstract:Abstract Introduction Stickler syndrome is caused by mutations in genes encoding type II and type XI collagens. About 85% of the pathogenic variants is found in COL2A1 (Stickler type 1), whereas a minority of mutations has been reported in COL11A1 (Stickler type 2) and COL11A2 (Stickler type 3). Beside the typical skeletal and orofacial manifestations, ocular anomalies are predominantly present in type 1 and type 2, while hearing loss is more pronounced in type 2 and type 3. Methods We performed COL11A1 mutation analysis for 40 type 2 Stickler patients and COL11A2 mutation analysis for five type 3 Stickler patients, previously all COL2A1 mutation-negative, using targeted next-generation sequencing (NGS) whereas whole-exome sequencing (WES) was performed in parallel for two patients. Three patients were analyzed for both genes due to unclear ocular findings. Results In total 14 COL11A1 and two COL11A2 mutations could be identified, seven of which are novel. Splice site alterations are the most frequent mutation type, followed by glycine substitutions. In addition, six variants of unknown significance (VUS) have been found. Identical mutations and variants were identified with both NGS techniques. Conclusion We expand the mutation spectrum of COL11A1 and COL11A2 in Stickler syndrome patients and show that targeted NGS is an efficient and cost-effective molecular tool in the genetic diagnosis of Stickler syndrome, whereas the more standardized WES might be an alternative approach.
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Diagnostic criteria for Stickler syndrome based on comprehensive clinical and molecular analysis
2014Co-Authors: Frederic Acke, Olivier Vanakker, Ingeborg Dhooge, Anne De Paepe, Els De Leenheer, Paul Coucke, Kristien Hoornaert, Fransiska MalfaitAbstract:Stickler syndrome is a heterogeneous disorder variably affecting the ocular, orofacial, auditory and skeletal system. Mutations in COL2A1, COL11A1 and COL11A2 have been found to cause Stickler syndrome and result in slightly distinct phenotypes, referred to as type 1, type 2 and type 3 respectively. Due to the large phenotypic variability, no consensus about minimal clinical diagnostic criteria exists. Currently, diagnosis is mainly based on expert opinion and positive mutation analysis. The aim of this study is to better define the syndrome and its different types by creating clinically-based guidelines. Medical records of more than 250 probands with a clinical suspicion of Stickler syndrome were reviewed for relevant symptoms and molecular results. COL2A1 analysis was performed in all patients, and COL11A1 and COL11A2 were subsequently analyzed in the COL2A1-negative patients. In 90% of the probands, the disease-causing mutation was detected, of which 82% was located in the COL2A1 gene, 14% in COL11A1 and 4% in COL11A2. Most COL2A1 mutations lead to haploinsufficiency (nonsense mutations, out-of-frame deletions), whereas the majority of mutations in COL11A1/COL11A2 exhibit a dominant-negative effect (in-frame exon deletions, glycine substitutions). Symptoms that are more present in the mutation-positive patients and thus stronger direct towards Stickler syndrome, are high myopia, retinal detachment, cleft palate and a positive familial history suggesting autosomal dominant inheritance. Hearing loss, joint hypermobility and premature arthropathy are frequently found in Stickler syndrome, but are less specific. The main characteristics differentiating the three types of Stickler syndrome are appearance of the vitreous (membranous in type 1, beaded in type 2 and normal in type 3) and severity of hearing loss (mild high-frequency hearing loss in type 1, moderate pan-frequency hearing loss in type 2 and type 3), although this distinction is not absolute. Based on these clinical as well as molecular results, diagnostic criteria for the different types of Stickler syndrome using a point-scale of different symptoms are proposed. These novel criteria may guide clinicians to better diagnose Stickler syndrome and might help to select the correct molecular analysis.
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Hearing impairment in Stickler syndrome: a systematic review
Orphanet journal of rare diseases, 2012Co-Authors: Frederic Acke, Fransiska Malfait, Ingeborg Dhooge, Els De LeenheerAbstract:Background Stickler syndrome is a connective tissue disorder characterized by ocular, skeletal, orofacial and auditory defects. It is caused by mutations in different collagen genes, namely COL2A1, COL11A1 and COL11A2 (autosomal dominant inheritance), and COL9A1 and COL9A2 (autosomal recessive inheritance). The auditory phenotype in Stickler syndrome is inconsistently reported. Therefore we performed a systematic review of the literature to give an up-to-date overview of hearing loss in Stickler syndrome, and correlated it with the genotype.
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Hearing impairment in Stickler syndrome: a systematic review
Orphanet Journal of Rare Diseases, 2012Co-Authors: Frederic R E Acke, Fransiska Malfait, Ingeborg J M Dhooge, Els M R De LeenheerAbstract:Background Stickler syndrome is a connective tissue disorder characterized by ocular, skeletal, orofacial and auditory defects. It is caused by mutations in different collagen genes, namely COL2A1 , COL11A1 and COL11A2 (autosomal dominant inheritance), and COL9A1 and COL9A2 (autosomal recessive inheritance). The auditory phenotype in Stickler syndrome is inconsistently reported. Therefore we performed a systematic review of the literature to give an up-to-date overview of hearing loss in Stickler syndrome, and correlated it with the genotype. Methods English-language literature was reviewed through searches of PubMed and Web of Science, in order to find relevant articles describing auditory features in Stickler patients, along with genotype. Prevalences of hearing loss are calculated and correlated with the different affected genes and type of mutation. Results 313 patients (102 families) individually described in 46 articles were included. Hearing loss was found in 62.9%, mostly mild to moderate when reported. Hearing impairment was predominantly sensorineural (67.8%). Conductive (14.1%) and mixed (18.1%) hearing loss was primarily found in young patients or patients with a palatal defect. Overall, mutations in COL11A1 (82.5%) and COL11A2 (94.1%) seem to be more frequently associated with hearing impairment than mutations in COL2A1 (52.2%). Conclusions Hearing impairment in patients with Stickler syndrome is common. Sensorineural hearing loss predominates, but also conductive hearing loss, especially in children and patients with a palatal defect, may occur. The distinct disease-causing collagen genes are associated with a different prevalence of hearing impairment, but still large phenotypic variation exists. Regular auditory follow-up is strongly advised, particularly because many Stickler patients are visually impaired.