Analysis of reprogramming of epigenome in TET1 knockdown cancer cell lines
ABSTRACT: Epigenetic changes associated with knockdown of TET1 in 2 cancer cell lines was assessed Overall design: Bisulphite converted DNA from the 4 samples were hybridized to the Illumina HumanMethylation450k Beadchip. Delta change in Beta value in TET1 kd compared to parent cell lines was calculated
Project description:Epigenetic modification of the mammalian genome by DNA methylation (5-methylcytosine) has a profound impact on chromatin structure, gene expression and maintenance of cellular identity. Recent demonstration that members of the Ten-eleven translocation (Tet) family proteins can convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) raised the possibility that Tet proteins are capable of establishing a distinct epigenetic state. We have recently demonstrated that Tet1 is specifically expressed in murine embryonic stem (ES) cells and is required for ES cell self-renewal and maintenance. Using chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq), here we show that Tet1 is preferentially bound to CpG-rich sequences at promoters of both transcriptionally active and Polycomb-repressed genes. Despite a general increase in levels of DNA methylation at Tet1 binding-sites, Tet1 depletion does not lead to down-regulation of all the Tet1 targets. Interestingly, while Tet1-mediated promoter hypomethylation is required for maintaining the expression of a group of transcriptionally active genes, it is also required for repression of Polycomb-targeted developmental regulators. Tet1 contributes to silencing of this group of genes by facilitating recruitment of PRC2 to CpG-rich gene promoters. Thus, our study not only establishes a role for Tet1 in modulating DNA methylation levels at CpG-rich promoters, but also reveals a dual function of Tet1 in promoting transcription of pluripotency factors as well as participating in the repression of Polycomb-targeted developmental regulators. To determine the genome-wide distribution of Tet1 in mouse ES cells, we have performed ChIP-seq experiments using Tet1 antibodies in control knockdown (KD) and Tet1 KD ES cells.
Project description:Glioblastomas are the most aggressive of malignant brain cancers with a median patient survival of approximately 18?months. We recently demonstrated that Tet methylcytosine dioxygenase 1(TET1) is involved in cellular responses to ionizing radiation (IR) in glial-, glioblastoma-, and non-tumor-derived cells. This study used a lentiviral-mediated knockdown of TET1 to further dissect the contribution of TET1 to the DNA damage response in glial cell lines by evaluating its role in DNA repair. TET1-deficient glial cell lines displayed attenuated cytotoxicity compared to non-targeted knockdown after treatment with IR but these differences were not observed between control and TET1 deficient in response to inhibitors of Na+/K+-ATPase. Additionally, the percentage of glial cells displaying ?H2A.x foci was greatly reduced in TET1-deficient glial cells compared to non-targeted knockdown conditions in response to IR and topoisomerase inhibitors. We also observed a lower percentage and a delay in 53BP1 foci formation, a marker of non-homologous end-joining, in response to IR and topoisomerase inhibitors in TET1-deficient glial cells. DNA-PK, another marker of non-homologous end-joining, was also lower in TET1-deficient glial cell lines. Interestingly, TET1-deficient glial cells displayed higher numbers of DNA strand breaks compared to control cells and repaired DNA breaks less efficiently in Comet assays. We suggest that attenuated DNA repair in TET1 deficient gliomas leads to genomic instability, which underlies poor patient survival.
Project description:Tet methylcytosine dioxygenases (TETs) catalyze the oxidative reactions of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). However, TET1 roles in ovarian cancer cell growth are unknown. Here, we show that ectopic expression of TET1 increased 5hmC levels, and inhibited proliferation and colony formation in ovarian cancer cell lines. Furthermore, in vitro and in vivo functional studies demonstrated that TET1 overexpression is necessary for the suppression of ovarian cancer growth, whereas depletion of TET1 expression had the opposite effect. Furthermore, the results of RNA-seq and qRT-PCR analyses identified a tumor suppressor, Ras association domain family member 5 (RASSF5), as the key downstream target of TET1. TET1 promotes RASSF5 expression by demethylating a CpG site within RASSF5 promoter. Up-regulated RASSF5 expression leads to the suppression of ovarian cancer cells growth. Additionally, we demonstrated that inhibition of CUL4-DDB1 ubiquitin ligase complex decrease 5hmC levels in ovarian cancer cells. These results provide new insights into the understanding of how ovarian cancers develop and grow, and identify TET1 as a key player in this process.
Project description:The role of transcriptional regulator ten-eleven translocation methylcytosine dioxygenease 1 (TET1) has not been well characterized in lung cancer. Here we show that TET1 is overexpressed in adenocarcinoma and squamous cell carcinomas. TET1 knockdown reduced cell growth in vitro and in vivo and induced transcriptome reprogramming independent of its demethylating activity to affect key cancer signaling pathways. Wild-type p53 bound the TET1 promoter to suppress transcription, while p53 transversion mutations were most strongly associated with high TET1 expression. Knockdown of TET1 in p53-mutant cell lines induced senescence through a program involving generalized genomic instability manifested by DNA single- and double-strand breaks and induction of p21 that was synergistic with cisplatin and doxorubicin. These data identify TET1 as an oncogene in lung cancer whose gain of function via loss of p53 may be exploited through targeted therapy-induced senescence. SIGNIFICANCE: These studies identify TET1 as an oncogene in lung cancer whose gain of function following loss of p53 may be exploited by targeted therapy-induced senescence.See related commentary by Kondo, p. 1751.
Project description:Aberrant DNA methylation is commonly observed in colorectal cancer (CRC), but the underlying mechanism is not fully understood. 5-hydroxymethylcytosine levels and TET1 expression are both reduced in CRC, while epigenetic silencing of TET1 is reportedly associated with the CpG island methylator phenotype. In the present study, we aimed to clarify the relationship between loss of TET1 and aberrant DNA methylation in CRC. Stable TET1 knockdown clones were established using Colo320DM cells, which express high levels of TET1, and HCT116 cells, which express TET1 at a level similar to that in normal colonic tissue. Infinium HumanMethylation450 BeadChip assays revealed increased levels of 5-methylcytosine at more than 10,000 CpG sites in TET1-depleted Colo320DM cells. Changes in DNA methylation were observed at various positions within the genome, including promoters, gene bodies and intergenic regions, and the altered methylation affected expression of a subset of genes. By contrast, TET1 knockdown did not significantly affect DNA methylation in HCT116 cells. However, TET1 depletion was associated with attenuated effects of 5-aza-2'-deoxycytidine on gene expression profiles in both cell lines. These results suggest that loss of TET1 may induce aberrant DNA methylation and may attenuate the effect of 5-aza-2'-deoxycytidine in CRC cells.
Project description:Hypermethylation-mediated tumor suppressor gene (TSG) silencing is a central epigenetic alteration in RAS-dependent tumorigenesis. Ten-eleven translocation (TET) enzymes can depress DNA methylation by hydroxylation of 5-methylcytosine (5mC) bases to 5-hydroxymethylcytosine (5hmC). Here, we report that suppression of TET1 is required for KRAS-induced DNA hypermethylation and cellular transformation. In distinct nonmalignant cell lines, oncogenic KRAS promotes transformation by inhibiting TET1 expression via the ERK-signaling pathway. This reduces chromatin occupancy of TET1 at TSG promoters, lowers levels of 5hmC, and increases levels of 5mC and 5mC-dependent transcriptional silencing. Restoration of TET1 expression by ERK pathway inhibition or ectopic TET1 reintroduction in KRAS-transformed cells reactivates TSGs and inhibits colony formation. KRAS knockdown increases TET1 expression and diminishes colony-forming ability, whereas KRAS/TET1 double knockdown bypasses the KRAS dependence of KRAS-addicted cancer cells. Thus, suppression of TET1-dependent DNA demethylation is critical for KRAS-mediated transformation.
Project description:Recent studies have shown that loss of TET1 may play a significant role in the formation of tumors. Because genomic instability is a hallmark of cancer, we examined the potential involvement of 10-11 translocation 1 (TET1) in the DNA damage response (DDR). Here we demonstrate that, in response to clinically relevant doses of ionizing radiation (IR), human glial cells made TET1-deficient with lentiviral vectors displayed greater numbers of colony forming units and lower levels of apoptotic markers compared with glial cells transduced with control vectors; yet, they harbored greater DNA strand breaks. The G<sub>2</sub>/M check point and expression of cyclin B1 were greatly diminished in TET1-deficient cells, and TET1-deficient cells displayed lower levels of ?H2A.x following exposure to IR. Levels of DNA-PKcs, which are DNA-PK complex members, were lower in TET1-deficient cells compared with control cell lines. However, levels of ATM were similar in both cell lines. Cyclin B1, DNA-PKcs, and ?H2A.x levels were each rescued by reintroduction of the TET1 catalytic domain. Finally, cytosine methylation within intron 1 of PRKDC, the gene encoding DNA-PKcs, was significantly higher upon depletion of TET1. Taken together, this study illustrates the involvement of TET1 in the different arms of the DDR and suggests its loss results in the continued survival of cells with genomic instability.
Project description:Background:Ten-eleven translocation (TET) enzymes catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) promoting demethylation in cells. However, the expression pattern and biologic significance of TET in papillary thyroid carcinoma (PTC) remain unclear. This study aimed to elucidate the biological functions of TET1 and the miRNA and mRNA expression levels in PTC cells with downregulated TET1. Methods:The expression of the TET family in 49 PTC tissues and corresponding tumor-adjacent tissues, as well as PTC cell lines (BCPAP, K1, and TPC-1) and the normal thyroid epithelial cell line (Nthy-ori 3-1), were detected using quantitative real-time polymerase chain reaction. The 5hmC level was detected in PTC tissues and cell lines using immunohistochemistry and dot blot assay, respectively. After silencing the TET1 gene with siRNAs in BCPAP and TPC-1?cells, cell proliferation was detected using EdU assay. Transwell assay was used to investigate cell migration and invasion. miRNA and mRNA expression arrays were conducted in TET1-depleted BCPAP cells. Results:The expression level of TET1 decreased in PTC tissues and cell lines and was consistent with the reduction in the 5hmC level. The knockdown of the TET1 gene with siRNAs in BCPAP and TPC-1?cells, cell proliferation was detected using EdU assay. Transwell assay was used to investigate cell migration and invasion. miRNA and mRNA expression arrays were conducted in TET1-depleted BCPAP cells. WNT4, FZD4, CDK6, MCF2L, and EDN1 was upregulated as potential target genes of dysregulated miRNAs. Conclusion:The study showed that TET1 dysfunction inhibited the migration and invasion of BCPAP cells and might have a potential role in the pathogenesis of PTC.