TGF-β-dependent active demethylation and expression of the p15ink4b tumor suppressor are impaired by the ZNF217/CoREST complex

In this study we examine the mechanisms of dynamic DNA methylation of the p15(ink4b) tumor suppressor gene. Using conventional ChIP and ChiPseq, we identify the p15(ink4b) promoter as a target for the ZNF217 oncogene, the CoREST complex, and DNMT3A. Treatment of cells with TGF-β triggers active demethylation involving loss of ZNF217/CoREST/DNMT3A and the corecruitment of SMAD2/3, CBP, and the DNA glycosylase TDG. Knockdown of TDG, or its functional homolog MBD4, prevents TGF-β-dependent demethylation of p15(ink4b). DNA immunoprecipitation of 5mC and 5hmC indicates that 5mC undergoes conversion to 5hmC prior to activation of p15(ink4b). Remarkably, overexpression of ZNF217 inhibits active demethylation and expression of the p15(ink4b) gene by preventing recruitment of SMAD2/3 and TDG. These findings suggest that active demethylation is essential for regulating a subset of TGF-β-dependent genes. Importantly, disruption of active demethylation by the ZNF217 oncogene may be a paradigm for other oncogenic signals on DNA methylation dynamics.     

Hsa_circ_0069094 positively regulates the expression of oncogenic ZNF217 by competitively targeting miR-758–3p to promote the development of breast cancer

To investigate the functions and potential mechanisms of hsa_circ_0069094 in this cancer. The expression of hsa_circ_0069094, zinc finger protein 217 (ZNF217) and microRNA-758–3p (miR-758–3p) was detected by quantitative polymerase chain reaction (qPCR), and the protein level of ZNF217 was detected by western blot. Cell proliferation was assessed using cell counting kit-8 (CCK-8) assay and colony formation assay. Cell cycle progression and cell apoptosis were determined using flow cytometry assay. Cell invasion and cell migration were monitored using transwell assay and wound healing assay. The protein levels of apoptosis-related proteins were quantified by western blot. The putative relationship between miR-758–3p and hsa_circ_0069094 and ZNF217 was confirmed using dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Xenograft model was constructed in mice to explore the role of hsa_circ_0069094 on solid tumor growth.Hsa_circ_0069094 and ZNF217 were highly expressed, while miR-758–3p was poorly expressed in tissues and cells of breast cancer. Hsa_circ_0069094 knockdown or ZNF217 knockdown inhibited cell proliferation, invasion and migration and induced cell apoptosis and cell cycle arrest in breast cancer cells. The inhibitory effects of hsa_circ_0069094 knockdown on cell malignant behaviors were abolished by ZNF217 overexpression. Hsa_circ_0069094 competed with ZNF217 for the binding site of miR-758–3p, and hsa_circ_0069094 positively regulated ZNF217 expression by competitively binding to miR-758–3p. Hsa_circ_0069094 knockdown also blocked solid tumor growth in mice. Collectively, Hsa_circ_0069094 played oncogenic effects in breast cancer by activating the expression of ZNF217 via competitively binding to miR-758–3p, which might be a novel strategy for breast cancer suppression.     

SnoN co-repressor binds and represses smad7 gene promoter

SnoN and Ski oncoproteins are co-repressors for Smad proteins and repress TGF-beta-responsive gene expression. The smad7 gene is a TGF-beta target induced by Smad signaling, and its promoter contains the Smad-binding element (SBE) required for a positive regulation by the TGF-beta/Smad pathway. SnoN and Ski co-repressors also bind SBE but regulate negatively smad7 gene. Ski along with Smad4 binds and represses the smad7 promoter, whereas the repression mechanism by SnoN is not clear. Ski and SnoN overexpression inhibits smad7 reporter expression induced through TGF-beta signaling. Using chromatin immunoprecipitation assays, we found that SnoN binds smad7 promoter at the basal condition, whereas after a short TGF-beta treatment for 15-30 min SnoN is downregulated and no longer bound smad7 promoter. Interestingly, after a prolonged TGF-beta treatment SnoN is upregulated and returns to its position on the smad7 promoter, functioning probably as a negative feedback control. Thus, SnoN also seems to regulate negatively the TGF-beta-responsive smad7 gene by binding and repressing its promoter in a similar way to Ski.