Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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PML regulates NF-ÎºB phosphorylation and transcriptional activity

ABSTRACT: The transcription factor NF-ÎºB is considered the master regulator of the immune response but also acts broadly to regulate gene expression that influences cell survival, proliferation and differentiation. Post-translational modification of NF-ÎºB, phosphorylation in particular, is essential for the transactivation activity of NF-ÎºB. Emerging evidence suggests that the regulation of NF-ÎºB in the nucleus is critical in controlling gene expression. Promyelocytic Leukemia (PML) is a nuclear protein that forms nuclear bodies (PML NBs), sub-nuclear structures that are associated with transcriptionally active genomic regions that have been implicated in multiple processes such as apoptosis, senescence and anti-viral responses. Chromosomal translocations leading to the expression of a PML-retinoic acid receptor-Î± (PML-RARÎ±) fusion protein are causative for acute promyelocytic leukemia (APL) characterised by a differentiation block at the promyelocytic state of myeloid development. Here we demonstrate that PML is required for phosphorylation of NF-ÎºB p65 and that PML is essential for NF-ÎºB- induced transcriptional responses. Our analysis of available transcriptional profiles of all-trans retinoic acid treated acute promyelocytic leukemia (APL) cells identifies a NF-ÎºB transcriptional programme suppressed by PML-RARÎ±. We further demonstrate that PML-RARÎ± inhibits NF-ÎºB phosphorylation and transcriptional activity. Our findings demonstrate a critical role for PML in promoting NF-ÎºB transcriptional activity which may contribute to APL initiation and maintenance. WT and PML-/- MEFs were analysed for gene expression analysis. Total of 12 samples, inlcluding triplicates were utilized. WT MEFs and PML-/- were stimulated with TNFÎ± for three hours and analysed for gene expresison using unstimulated WT MEFs as control.

ORGANISM(S): Mus musculus

SUBMITTER: Ruaidhri Carmody

PROVIDER: E-GEOD-47828 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The origin of aberrant DNA methylation in cancer remains largely unknown. In this study, we elucidated the DNA methylome in primary Acute Promyelocytic Leukemia (APL) and the role of PML-RARa in establishing these patterns. APL patients showed increased genome-wide DNA methylation with higher variability than healthy CD34+ cells, promyelocytes and remission bone marrow. A core set of differentially methylated regions in APL was identified. Age at diagnosis, Sanz score and Flt3-mutation status characterized methylation subtypes. Transcription factor binding sites, e.g. c-myc binding sites were associated with low methylation. SUZ12 and REST binding sites identified in embryonic stem cells were, however, preferentially DNA hypermethylated in APL. Unexpectedly, PML-RARa binding sites were also protected from aberrant DNA methylation in APL. In line, myeloid cells from pre-leukemic PML-RARa knock-in mice did not show altered DNA methylation and expression of PML-RARa in hematopoietic progenitor cells prevented differentiation without affecting DNA methylation. ATRA treatment of APL blasts did also not result in DNA methylation changes. These results suggest that aberrant DNA methylation is associated with leukemia phenotype but not required for PML-RARa-mediated initiation of leukemogenesis. We used Reduced Representation Bisulfite Sequencing (RRBS) to determine the genome-wide methylation signature of 18 primary APL patient samples. We then compared the APL methylation signature with methylation patterns found in CD34+ progenitor cells (n=4), promyelocytes (n=4) and remission bone marrow samples (n=8). Differentially methylated regions found in all three comparisons (APL vs. all three control specimens) were then further analyzed for genomic localization, variability and association with clinical parameters. Finally, the relationship between differentially methylated regions in APL and specific transcription factor binding sites was analyzed. For this purpose, ChiP-Sequencing of SUZ12 and REST was performed in primary APL patient blasts. To further determine the contribution of the leukemogenic transcription factor PML-RARa to methylation in APL, we also performed RRBS in pre-leukemic PML-RARa knock-in mice and hematopoetic progenitor cells retrovirally transduced with PML-RARa.

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PML regulates NF-ÎºB phosphorylation and transcriptional activity

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