title: Inactivation of the WNT5A Alternative Promoter B Is Associated with DNA Methylation and Histone Modification in Osteosarcoma Cell Lines U2OS and SaOS-2.
abstract: WNT5A is a secreted ligand involved in Wnt pathway signaling and has a role in cell movement and differentiation. Altered WNT5A expression is associated with various cancers, although in most studies the focus has been on only one of the known WNT5A isoforms. In this study, we analyzed expression from two of the major WNT5A promoters, termed promoter A and promoter B, in normal human osteoblasts, SaOS-2 and U2OS osteosarcoma cell lines, and osteosarcoma tumor tissue. We found that both promoters A and B are active in normal osteoblasts with nearly 11-fold more promoter B than A transcripts. Promoter B but not promoter A transcripts are decreased or nearly undetectable in the SaOS-2 and U2OS cell lines and osteosarcoma tumor tissues. Transient transfection of promoter A and promoter B reporter constructs confirmed that SaOS-2 cells have the necessary factors to transcribe both promoters. Bisulfite sequencing analysis revealed that three CpG enriched regions upstream of the promoter B exon 1βare highly methylated in both SaOS-2 and U2OS cells. The CpG island sub-region R6 located in promoter B exon 1β was approximately 51% methylated in SaOS-2 and 25% methylated in U2OS. Region 3 was approximately 28% methylated in normal osteoblasts, whereas the others were unmethylated. Promoter B was re-activated by treatment of SaOS-2 cells with 1 μM 5-azacytidine, which was associated with only a small insignificant change in methylation of sub-region R6. ChIP analysis of U2OS and SaOS-2 cells indicated that the promoter B region is less enriched in the active histone mark H3K4me3, in comparison to promoter A and that there is increased enrichment of the repressive mark H3K27me3 in association with the promoter B genomic region in the cell line SaOS-2. These findings show that epigenetic inactivation of the WNT5A promoter B involves both DNA methylation and histone modifications and suggest that differential expression of the WNT5A alternative promoters A and B is a characteristic of osteosarcomas.
authors.affiliation: Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania, United States of America.
authors.forename: Himani
authors.initials: H
authors.lastname: Vaidya
authors.affiliation: Department of Biology, The University of North Carolina Greensboro, Greensboro, North Carolina, United States of America.
authors.forename: Candie
authors.initials: C
authors.lastname: Rumph
authors.affiliation: Department of Biology, The University of North Carolina Greensboro, Greensboro, North Carolina, United States of America.
authors.forename: Karen S
authors.initials: KS
authors.lastname: Katula
mesh_terms: D001374:Azacitidine / Q000494:pharmacology; D001859:Bone Neoplasms / Q000235:genetics / Q000473:pathology*; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000187:drug effects; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D005091:Exons / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D017930:Genes, Reporter; D042421:Histone Code; D006801:Humans; D049410:Luciferases, Renilla / Q000096:biosynthesis / Q000235:genetics; D008745:Methylation; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D010006:Osteoblasts / Q000378:metabolism / Q000473:pathology; D012516:Osteosarcoma / Q000235:genetics / Q000473:pathology*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014162:Transfection; D051153:Wnt Proteins / Q000096:biosynthesis / Q000235:genetics*; D060449:Wnt Signaling Pathway / Q000235:genetics; D000071818:Wnt-5a Protein
chemical_list: D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C085389:WNT5A protein, human; D051153:Wnt Proteins; D000071818:Wnt-5a Protein; D049410:Luciferases, Renilla; D001374:Azacitidine