Unknown,Transcriptomics,Genomics,Proteomics

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Wnt inhibitory factor 1 (WIF1) is a marker of osteoblastic differentiation stage and is not silenced by DNA methylation in osteosarcoma (methylation)

ABSTRACT: Therapeutic targeting of the Wnt pathway is of high clinical interest for treating bone loss disorders such as osteoporosis. These therapies inhibit the action of negative regulators of osteoblastic Wnt signaling. The observation that Wnt inhibitory factor 1 (WNT1) was epigenetic silencing in osteosarcoma (OS) raised concerns for such a treatment approach. In this study, genome-wide methylation profiling of OS derived from mouse models demonstrated Wif1 silencing in OS is not driven by DNA methylation. Treatment of mouse and human OS cells with methylation and HDAC inhibitors showed Wif1 was unresponsive to methylation inhibition but responded robustly to HDAC inhibition. Consistent with an HDAC dependent mechanism of silencing, the Wif1 locus in OS was characterized by low levels of acetylation and a bivalent H3K4/H3K27 trimethylation state. Wif1 expression marked late stages of normal osteoblast development and stratified OS tumours based on differentiation stage across species. Culture of human and mouse OS cells under differentiation inductive conditions increased expression of Wif1 in parallel with known osteoblast differentiation markers. Together these results demonstrate that Wif1 is not targeted for silencing by DNA methylation in OS. The reduced expression of Wif1 in OS cells is in context with their stage in differentiation. 3 cell lines derived from primary tumors from p53 Rb Osterix-Cre:lox OS model, 3 Osteoblasts (differentiated Kusa4b10 cells (21 days under osteoblastic differentiation conditions)

ORGANISM(S): Mus musculus

SUBMITTER: Alistair Chalk

PROVIDER: E-GEOD-47571 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Embryogenesis is tightly regulated by multiple levels of epigenetic systems such as DNA methylation, histone modification, and chromatin remodeling. DNA methylation patterns are erased in primordial germ cells and in the interval immediately following fertilization. Subsequent reprogramming occurs by de novo methylation and demethylation. Variance of DNA methylation patterns between different cell types is not well understood. Here, using methylated DNA immunoprecipitation and tiling array technology, we have comprehensively analysed DNA methylation patterns at proximal promoter regions in mouse embryonic stem (ES) cells, ES cell-derived early germ layers (ectoderm, endoderm and mesoderm) and four adult tissues (brain, liver, skeletal muscle and sperm). Most of the methylated regions in the three germ layers and in the three adult somatic tissues are shared in common. This commonly methylated gene set is enriched in germ cell associated genes that are generally transcriptionally inactive in somatic cells. We also compared DNA methylation patterns with global mapping of histone H3 lysine 4/27 trimethylation, and found that gain of DNA methylation correlates with loss of histone H3 lysine 4 trimethylation. Taken together, our findings indicate that differentiation from ES cells to the three germ layers is accompanied by an increase in the number of commonly methylated DNA regions and that these tissue-specific alterations are present for only a small number of genes. Our findings indicate that DNA methylation at the proximal promoter regions of commonly methylated genes act as an irreversible mark which fixes somatic lineage by repressing transcription of germ cell specific genes. Using the MeDIP on chip protocol, we immunoprecipitated methylated DNA from R1 ES cell, SK7 cell, as well as SK7 derived-Ectoderm, - Endoderm, -Paraxial mesoderm, brain, liver, skeletal muscle, and sperm, and hybridized to a genome tiling array. The three germ layer lineages from ES cells were confirmed by the expression of specific marker genes in each germ layer (see related expression analysis).

Project description:To further our understanding of the role of DNA methylation in development, Methylated DNA Immunoprecipitation (MeDIP) was used in conjunction with a NimbleGen promoter plus CpG island array to identify Tissue and Developmental Stage specific Differentially Methylated DNA Regions (T-DMRs and DS-DMRs) on a genome-wide basis. Four tissues (brain, heart, liver, and testis) from C57BL/6J mice were analyzed at three developmental stages (15 day embryo, E15; new born, NB; 12 week adult, AD). Almost 5,000 adult T-DMRs and 10,000 DS-DMRs were identified. Surprisingly, almost all DS-DMRs were tissue specific (i.e., methylated and ummenthylated in one or more non-overlapping tissues), indicating that the vast majority of unique sequence DNA methylation has tissue specificity. Also, many DS-DMRs were methylated at early development stages (E15 and NB) but unmethylated in adult, indicating “demethylation” has a prominent role in tissue differentiation. The pattern of DNA methylation in adult testis was dramatically different from somatic tissues in many aspects, mostly notably with a very strong bias of methylation in non-CpGi (CpG island) promoter regions (94%). Although the majority of T-DMRs and DS-DMRs tended to be in non-CpGi promoter regions, a relatively large number were also located in CpGi in promoter, intra-genic and inter-genic regions (>15% of all CpGi). Gene Ontology analysis of genes with methylation in non-CpGi promoters indicates enrichment of genes related to membrane proteins and G-protein coupled receptors. Our data also suggest regulatory roles of DNA methylation outside of promoter regions and in alternative promoter selection. Overall, our studies indicate that change in DNA methylation during development is a dynamic, widespread and tissue-specific process involving both DNA methylation and demethylation. Comparison of DNA methylation across 3 developmental stages (15 day embryo, newborn, and adult) for four tissues (brain, heart, liver and testis)

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Wnt inhibitory factor 1 (WIF1) is a marker of osteoblastic differentiation stage and is not silenced by DNA methylation in osteosarcoma (methylation)

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