Unknown,Transcriptomics,Genomics,Proteomics

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Genome-wide maps of RNAPII Ser5(P) in HEK 293T cells after treatment with 5-aza-CdR

ABSTRACT: DNA methylation can be abnormally regulated in human disease and associated with effects on gene transcription that appear to be causally related to pathogenesis. The potential to use pharmacological agents that reverse this dysregulation is therefore an attractive possibility. To test how 5-aza-2M-bM-^@M-^Y-deoxycytidine (5-aza-CdR) influences the genome therapeutically, we exposed non-malignant cells in culture to the agent and used genome-wide assays to assess the cellular response. We found that cells allowed to recover from 5-aza-CdR treatment only partially recover DNA methylation levels, retaining an epigenetic M-bM-^@M-^\imprintM-bM-^@M-^] of drug exposure. We show very limited transcriptional responses to demethylation of not only protein-coding genes but also loci encoding non-coding RNAs, with a limited proportion of the induced genes acquiring new promoter activation within gene bodies. The data revealed an uncoupling of DNA methylation effects at promoters, with demethylation mostly unaccompanied by transcriptional changes. The limited panel of genes induced by 5-aza-CdR resembles those activated in other human cell types exposed to the drug, and represents loci targeted for Polycomb-mediated silencing in stem cells, suggesting a model for the therapeutic effects of the drug. Our results do not support the hypothesis of DNA methylation having a predominant role to regulate transcriptional noise in the genome, and indicate that DNA methylation acts only as part of a larger complex system of transcriptional regulation. The targeting of 5-aza-CdR effects with its clastogenic consequences to euchromatin raises concerns that the use of 5-aza-CdR has innate tumorigenic consequences, requiring its cautious use in diseases involving epigenetic dysregulation. Examination of RNAPII Ser5(P) localization by ChIP-seq in HEK 293T cell after treatment with 5-aza-CdR.

ORGANISM(S): Homo sapiens

SUBMITTER: Maria-Paz Ramos

PROVIDER: E-GEOD-62695 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background:Aberrant DNA methylation that silences tumor suppressor genes occurs frequently in patients with acute myeloid leukemia (AML). Treatment of AML patients with the inhibitor of DNA methylation, 5-aza-2'-deoxycytidine (5-AZA-CdR) can induce complete remissions, but most patients will relapse. The clinical efficacy of 5-AZA-CdR may be influenced by its limited capacity to activate tumor suppressor genes silenced by methylation of lysine 27 histone H3 (H3K27) by EZH2. In order to overcome this limitation, we investigated previously the antileukemic action of 5-AZA-CdR in combination with the EZH2 inhibitor, 3-deazaneplanocin A (DZNep) on HL-60 AML cells. We observed a remarkable synergistic interaction against these AML cells for this combination. In this study, we investigated in more depth the action of 5-AZA-CdR plus DZNep on gene expression in AML cells using RNA sequence analysis Result:In a colony assay, 5-AZA-CdR in combination with DZNep exhibited also a potent synergy against another human AML cell line: AML-3. The induction of apoptosis in HL-60 and AML3 leukemic cells by 5-AZA-CdR plus DZNep was also synergistic. RNA sequence analysis on HL-60 leukemic cells showed that the combination of 5-AZA-CdR plus DZNep increased the expression of thousands of genes. The genes upregulated by this combination included genes related to differentiation, development, senescence, apoptosis, and tumor suppressor function. Many of the genes activated by 5-AZA-CdR plus DZNep have the potential to suppress leukemogenesis. Conclusion: The activation of many genes by the combination of 5-AZA-CdR plus DZNep correlates with its synergistic antileukemic action. The block in differentiation is one of the hallmarks of AML.The activation of many genes that program differentiation and development by this combination of epigenetic agents has the potential to reverse this block. The reversal of these two epigenetic genesilencing mechanisms by 5-AZA-CdR plus DZNep merits clinical investigation in patients with AML

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Genome-wide maps of RNAPII Ser5(P) in HEK 293T cells after treatment with 5-aza-CdR

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