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)
Project description:The aim was to identify pathways and genes that are transcriptionally deregulated in osteosarcoma due to changes in CpG island DNA methylation. In order to identify candidates, we compared low passage cell cultures derived from a mouse model of osteosarcoma to mature osteoblasts derived by in vitro differentiation of the mouse bone marrow stromal cell line, Kusa4b10. Under cell culture osteoblastic differentiating conditions, Kusa4b10 cells acquire a mature osteoblastic phenotype (21 days). A potential role for DNA methylation in directing gene expression changes was established by integrating gene expression data with genome wide DNA methylation maps generated by methyl-DNA binding domain capture and NimbleGen promoter arrays (MBDCap-Chip). 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)
Project description:Bone morphogenetic protein 4 (BMP4) is essential for lung development. To define its intracellular signaling mechanisms by which BMP4 regulates lung development, BMP-specific Smad1 or Smad5 was selectively knocked out in fetal mouse lung epithelial cells. Abrogation of lung epithelial-specific Smad1, but not Smad5, resulted in retardation of lung branching morphogenesis and reduced sacculation, accompanied by altered distal lung epithelial cell proliferation and differentiation, and consequently severe neonatal respiratory failure. By combining cDNA microarray with ChIP-chip analyses, Wnt inhibitory factor-1 (Wif1) was identified as a novel target gene of Smad1 in the developing mouse lung epithelial cells. Loss of Smad1 transcriptional activation of Wif1 expression was associated with reduced Wif1 expression and increased Wnt/beta-catenin signaling activity in lung epithelia, resulting in specific fetal lung abnormalities. Therefore, a novel regulatory loop of BMP4-Smad1-Wif1-Wnt/beta-catenin in coordinating BMP and Wnt pathways to control fetal lung development is suggested. Overall design: mRNA profiling: Total RNA was isolated from left lobe lungs of three pair of E18.5 wild type and Smad1 lung epithelium-specific conditional knockout mice
Project description:Bone morphogenetic protein 4 (BMP4) is essential for lung development. To define its intracellular signaling mechanisms by which BMP4 regulates lung development, BMP-specific Smad1 or Smad5 was selectively knocked out in fetal mouse lung epithelial cells. Abrogation of lung epithelial-specific Smad1, but not Smad5, resulted in retardation of lung branching morphogenesis and reduced sacculation, accompanied by altered distal lung epithelial cell proliferation and differentiation, and consequently severe neonatal respiratory failure. By combining cDNA microarray with ChIP-chip analyses, Wnt inhibitory factor-1 (Wif1) was identified as a novel target gene of Smad1 in the developing mouse lung epithelial cells. Loss of Smad1 transcriptional activation of Wif1 expression was associated with reduced Wif1 expression and increased Wnt/beta-catenin signaling activity in lung epithelia, resulting in specific fetal lung abnormalities. Therefore, a novel regulatory loop of BMP4-Smad1-Wif1-Wnt/beta-catenin in coordinating BMP and Wnt pathways to control fetal lung development is suggested. mRNA profiling: Total RNA was isolated from left lobe lungs of three pair of E18.5 wild type and Smad1 lung epithelium-specific conditional knockout mice
Project description:BRMS1L (breast cancer metastasis suppressor 1 like，BRMS1-like) is a component of the SIN3A-HDAC corepressor complex that suppresses target gene transcription. Here, we show that reduced BRMS1L in breast cancer tissues is associated with tumor metastasis and poor patient survival. Functionally, BRMS1L inhibits migration and invasion of breast cancer cells by inhibiting epithelial-mesenchymal transition (EMT). These effects are mediated by epigenetic silencing of FZD10, a receptor for Wnt signaling, by facilitating the recruitment of HDAC1 to its promoter and enhancing histone H3K9 deacetylation. Consequently, BRMS1L-induced FZD10 silencing inhibits aberrant activation of WNT3-FZD10--catenin signaling. Furthermore, BRMS1L is a target of miR-106b and miR-106b upregulation leads to BRMS1L reduction in breast cancer cells. RNAi-mediated silencing of BRMS1L expression promotes metastasis of breast cancer xenografts in immunocompromised mice, while ectopic BRMS1L expression inhibits metastasis. Therefore, BRMS1L provides an epigenetic regulation of Wnt signaling in breast cancer cells and acts as a breast cancer metastasis suppressor. Ther transfection analysis used here were further desxribed in Chang Gong, eta al.2013. miR-106b expression determines the proliferation paradox of TGF-β in breast cancer cells. Oncogene. 2013 A two chip study using total RNA recovered from MDA-MB-231 breast cancer cells transfected with negative control vector or vector overexpressing BRMS1L for 24 hours. Each chip measures the expression 45033 genes were collected from the authoritative data source including NCBI.
Project description:Cellular differentiation entails loss of pluripotency and parallel gain of lineage-specific and ultimately cell-type specific characteristics. Using a murine system that progresses from stem cells to lineage-committed progenitors and further to terminally differentiated neurons we analyzed two repressive epigenetic pathways: DNA methylation and Polycomb-mediated methylation of histone H3 (H3K27me3). We show that several hundred promoters become DNA methylated in lineage-committed progenitor cells. Targets are selected for pluripotency and germline-specific genes, suggesting a role for DNA methylation in stabilizing loss of pluripotency already at the progenitor state. Conversely, we detect loss and acquisition of H3K27me3 at novel targets at both progenitor and terminal state. Surprisingly, many neuron-specific genes that are poised to be activated upon terminal differentiation become Polycomb targets only in progenitor cells. Moreover, promoters marked by H3K27me3 in stem cells frequently become DNA methylated during differentiation, suggesting context-dependent crosstalk between Polycomb and DNA methylation. This data suggest a new model how de novo DNA methylation and dynamic switches in Polycomb targets restrict pluripotency and define the developmental potential of progenitor cells. Keywords: MeDIP-chip, ChIP-chip, neuronal differentiation time-course MeDIP-chip and ChIP-chip experiments were performed with at least two independnet biological replicates. For each condition hybridizations include a dye-swap experiment.
Project description:Osteosarcoma (OS) is the most frequent malignant bone tumor with a high propensity for metastases. Through the tissue-specific alteration of p53 status, we have developed a genetically engineered mouse model of localized and metastatic OS. Gene expression analysis revealed naked cuticle homolog 2 (NKD2), a negative regulator of the Wnt signaling pathway, to be significantly downregulated in metastatic OS. An assessment of human OS tumors revealed downregulation of NKD2 in metastatic and recurrent OS. We determined that downregulation was secondary to methylation of the NKD2 promoter region for both mouse and human tumors. Furthermore, in vivo investigations indicate that NKD2 overexpression significantly diminishes OS tumor growth and metastasis and small molecule Wnt inhibitors can decrease OS growth and metastatic potential. Overall design: Development of novel osteoblast-specific alteration of a p53 mouse model that forms de novo localized and gross, macroscopic osteosarcoma that mimics the human disease allowing determination of novel genetic alterations involved in OS progression. Gene expression profiling of primary osteosarcomas (OS) and OS metastases to lung and liver. In order to identify genes and pathways that regulate OS metastasis, we performed an initial screen comparing the gene expression profiles of six localized, non-metastatic OS with those of 12 primary OS that produced metastatic lesions. In addition, seven lung metastatic lesion gene expression profiles were compared to those of the primary tumor from which they originated. multiple group comparison
Project description:Glioblastoma is the most aggressive primary brain tumor in adults and due to the invasive nature it cannot be completely removed. We have recently shown that the WNT inhibitory factor 1 (WIF1), a secreted inhibitor of WNTs, is downregulated in glioblastoma and acts as strong tumor suppressor. In search of a mediator for this function differential gene expression profiles of WIF1-expressing cells were performed. MALAT1, a long non-coding RNA and key positive regulator of invasion, emerged as the top downregulated gene. Indeed, knock-down of MALAT1 reduced migration in glioblastoma cells, without effect on proliferation. LN-229 cells induced with Doxocyclin to express WIF1 were compared to the non-induced control (two biological replicates each)
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)