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Genomic DNA from MEF cells

ABSTRACT: Genome wide DNA methylation profiling of WT and Pml KO mEF cells treated W/O Doxorubicin for 24 hours. TET2 plays critical roles in the regulation of cell identity and inhibition of tumorigenesis through modulating DNA methylation. However, little is known about how TET2 is targeted to specific DNA-binding sites. It was found by us that TET2 is significantly recruited to specific sites by PML. Moreover, TET2 and PML synergistically inhibited cell proliferation. Further study indicated that multi chemotherapies, such as doxorubicin, were able to promote PML-NB, increase 5hmC and result in specific DNA demethylation. Due to the important role of inactivation of tumor suppressors by DNA methylation in cancer, the activity of most therapies depends to an extent on activation of tumor suppressors by demethylation. Consequently, it is not surprising that methylation of tumor suppressors has been reported to diminish in responder patients after end of chemotherapy. Little is known about mechanism of therapy-promoted DNA demethylation, although this therapy-promoted DNA demethylation potentially plays general roles in therapy response, resistance and recurrence. It was also found that knock out of Pml significantly diminished chemotherapy-promoted DNA demethylation. To identify PML-TET2 pathway-regulated genes, WT and Pml knock out MEF cells were treated W/O Doxorubicin. The genomic DNA was extracted and purified from samples using Qiagen DNeasy Kit according to manufacturer’s instruction. The resultant Genome DNA was cut by Restriction Enzyme. After DNA-end repair and 3’-dA overhang, the 40-220bp fragments were selected and treated with ZYMO EZ DNA Methylation-Gold kit according to manufacturer’s instruction. Then the bisulphite converted DNA from the 4 samples (WT, WT Dox, PML KO and Pml KO Dox) were sequenced using standard Illumina protocol. Standard recommended Illumina scanner setting was carried out in study.

ORGANISM(S): Mus musculus

PROVIDER: GSE75408 | GEO | 2015/11/26

SECONDARY ACCESSION(S): PRJNA304124

REPOSITORIES: GEO

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Project description:Genome wide DNA methylation profiling of WT and Pml KO mEF cells treated W/O Doxorubicin for 24 hours. TET2 plays critical roles in the regulation of cell identity and inhibition of tumorigenesis through modulating DNA methylation. However, little is known about how TET2 is targeted to specific DNA-binding sites. It was found by us that TET2 is significantly recruited to specific sites by PML. Moreover, TET2 and PML synergistically inhibited cell proliferation. Further study indicated that multi chemotherapies, such as doxorubicin, were able to promote PML-NB, increase 5hmC and result in specific DNA demethylation. Due to the important role of inactivation of tumor suppressors by DNA methylation in cancer, the activity of most therapies depends to an extent on activation of tumor suppressors by demethylation. Consequently, it is not surprising that methylation of tumor suppressors has been reported to diminish in responder patients after end of chemotherapy. Little is known about mechanism of therapy-promoted DNA demethylation, although this therapy-promoted DNA demethylation potentially plays general roles in therapy response, resistance and recurrence. It was also found that knock out of Pml significantly diminished chemotherapy-promoted DNA demethylation. To identify PML-TET2 pathway-regulated genes, WT and Pml knock out MEF cells were treated W/O Doxorubicin. The genomic DNA was extracted and purified from samples using Qiagen DNeasy Kit according to manufacturerâ??s instruction. The resultant Genome DNA was cut by Restriction Enzyme. After DNA-end repair and 3â??-dA overhang, the 40-220bp fragments were selected and treated with ZYMO EZ DNA Methylation-Gold kit according to manufacturerâ??s instruction. Then the bisulphite converted DNA from the 4 samples (WT, WT Dox, PML KO and Pml KO Dox) were sequenced using standard Illumina protocol. Standard recommended Illumina scanner setting was carried out in study. WT and Pml knock out MEF cells were treated with Doxorubicin. The genomic DNA was extracted and purified from samples using Qiagen DNeasy Kit according to manufacturerâ??s instruction. The resultant Genome DNA was cut by Restriction Enzyme. After DNA-end repair and 3â??-dA overhang, the 40-220bp fragments were selected and treated with ZYMO EZ DNA Methylation-Gold kit according to manufacturerâ??s instruction. Then the bisulphite converted DNA from the 4 samples (WT, WT Dox, PML KO and Pml KO Dox) were sequenced using standard Illumina protocol. Standard recommended Illumina scanner setting was carried out in study.

Project description:Ten-eleven translocation (TET) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytsosine (5fC), and 5-carboxylcytosine (5caC). 5fC/5caC can be excised and repaired by the base excision repair (BER) pathway, implicating 5mC oxidation in active DNA demethylation. Genome-wide DNA methylation is erased in the transition from metastable states to ground state of embryonic stem cells (ESCs) and in migrating primordial germ cells (PGCs), while some resistant regions become demethylated only in gonadal PGCs. Understanding the mechanisms underlying global hypomethylation in naïve ESCs and developing PGCs will be useful for realizing cellular pluripotency and totipotency. In this study, we found that PRDM14, the PR-domain-containing transcriptional regulator, accelerates the TET-BER cycle, resulting in the promotion of active DNA demethylation in ESCs. Induction of PRDM14 expression rapidly removed the 5mC associated with transient elevation of 5hmC at pluripotency-associated genes, germline-specific genes, and imprinted loci but not across the entire genome, which resemble second wave of DNA demethylation in gonadal PGCs. PRDM14 physically interacts with TET1/TET2 and enhances the recruitment of TET1/TET2 at target loci. Knockdown of Tet1/Tet2 impaired transcriptional regulation and DNA demethylation by PRDM14. The repression of the BER pathway by administration of pharmacological inhibitors against APE1 and PARP1 and the knockdown of thymine DNA glycosylase (TDG) also impaired DNA demethylation by PRDM14. Furthermore, DNA demethylation induced by PRDM14 normally takes place in the presence of aphidicolin, which is an inhibitor of G1/S progression. Together, our analysis provides mechanistic insight into DNA demethylation in naive pluripotent stem cells and developing PGCs. To investigate the function of TET1/TET2 in transcriptional regulation by PRDM14 in ESCs, we exploited microarray analysis using total mRNA derived from Scramble, Scramble + PRDM14, Tet1/Tet2 KD, Tet1/Tet2 KD + PRDM14 mouse ESC.

Project description:Silencing of genes that suppress the malignant phenotype by DNA methylation spurred an interest in the clinical use of epigenetic reprogramming agents. Single therapy is unlikely to be curative in the context of a heterogeneous disease such as Diffuse Large B cell Lymphomas (DLBCL). The combination of DNA demethylating drugs could increase the chance to respond to classical and new treatments. We found that DLBCL cell lines respond heterogeneously to DNA demethylating agents. In sensitive cell lines, 5-aza-2’-deoxycytidine induced a genomic signature similar to that of doxorubicin, the most important drug of the combinatorial chemotherapy regimen for DLBCL treatment. Accordingly, the combination of 5-aza-2’-deoxycytidine and doxorubicin proved to be synergistic in cell killing in vitro and in vivo for DLBCL cell lines individually responsive to these drugs. In doxorubicin resistant cell lines, long-term exposure to low-dose of 5-aza-2’-deoxycytidine induces DNA demethylation and subsequent doxorubicin sensitization in vitro and in vivo. This later effect correlates with SMAD1 demethylation. SMAD1 is epigenetically silenced in doxorubicin-resistant DLBCL cells and DLBCL patients with poor prognostic. In addition, we found that DNA demethylating agents can sensitize primary DLBCL cells to doxorubicin. Primary cells obtained from a DLBCL patient treated with 5-azacytidine shows SMAD1 demethylation and ex vivo sensitization to multiple drugs. Therefore, DNA demethylating drugs can reprogram otherwise resistant DLBCL cells to respond to chemotherapy agents without increasing the toxicity to normal tissues. Our data also indicate that DNA methylation and consequent suppression of SMAD1 expression represent a previously undescribed molecular mechanism of chemoresistance in DLBCL that can be further exploit for therapy. A microarray study using genomic DNA from different DLBCL cell lines before any treatment. Two to four biological replicates by cell line. The HELP data wil be used to find genes hypermethylated in resistant cell lines compared to sensitive cell lines to doxorubicin and other drugs.

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Genomic DNA from MEF cells

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