Vaidya Himani, Rumph Candie, Katula Karen S
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania, United States of America.
Department of Biology, The University of North Carolina Greensboro, Greensboro, North Carolina, United States of America.
PLoS One. 2016 Mar 15;11(3):e0151392. doi: 10.1371/journal.pone.0151392. eCollection 2016.
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.
WNT5A是一种参与Wnt信号通路的分泌配体，在细胞运动和分化中发挥作用。WNT5A表达的改变与多种癌症相关，尽管在大多数研究中，重点仅放在已知的一种WNT5A异构体上。在本研究中，我们分析了正常人成骨细胞、SaOS-2和U2OS骨肉瘤细胞系以及骨肉瘤肿瘤组织中两个主要的WNT5A启动子（称为启动子A和启动子B）的表达情况。我们发现启动子A和B在正常成骨细胞中均有活性，启动子B的转录本比启动子A的多近11倍。在SaOS-2和U2OS细胞系以及骨肉瘤肿瘤组织中，启动子B而非启动子A的转录本减少或几乎检测不到。启动子A和启动子B报告基因构建体的瞬时转染证实，SaOS-2细胞具有转录这两个启动子所需的因子。亚硫酸氢盐测序分析显示，启动子B外显子1β上游的三个富含CpG的区域在SaOS-2和U2OS细胞中均高度甲基化。位于启动子B外显子1β的CpG岛亚区域R6在SaOS-2中甲基化程度约为51%，在U2OS中甲基化程度约为25%。区域3在正常成骨细胞中甲基化程度约为28%，而其他区域未甲基化。用1μM 5-氮杂胞苷处理SaOS-2细胞可使启动子B重新激活，这与亚区域R6甲基化的微小无显著变化相关。对U2OS和SaOS-2细胞的染色质免疫沉淀分析表明，与启动子A相比，启动子B区域的活性组蛋白标记H3K4me3富集程度较低，并且在细胞系SaOS-2中，与启动子B基因组区域相关的抑制性标记H3K27me3富集增加。这些发现表明，WNT5A启动子B的表观遗传失活涉及DNA甲基化和组蛋白修饰，并提示WNT5A替代启动子A和B的差异表达是骨肉瘤的一个特征。
