Unknown,Transcriptomics,Genomics,Proteomics

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Deterministic direct reprogramming of somatic cells to pluripotency [RRBS]

ABSTRACT: Somatic cells can be directly reprogrammed to pluripotency by exogenous expression of transcription factors, classically Oct4, Sox2, Klf4 and c-Myc. While distinct types of somatic cells can be reprogramed with varying efficiencies and by different modified reprogramming protocols, induced pluripotent stem cell (iPSC) induction remains inefficient and stochastic where a fraction of the cells converts into iPSCs. The nature of rate limiting barrier(s) preventing majority of cells to convert into iPSCs remains elusive. Here we show that neutralizing Mbd3, a core member of the Mbd3/NURD co-repressor and chromatin-remodeling complex, results in deterministic and synchronized reprogramming of multiple differentiated cell types to pluripotency. 100% of Mbd3 depleted mouse and human somatic cells convert into iPSCs after seven days of reprogramming factor induction. Our findings delineate a critical pathway blocking the reestablishment of pluripotency, and offer a novel platform for future dissection of epigenetic dynamics leading to iPSC formation at high resolution. Reduced representation bisulfite sequencing (RRBS) was applied to mouse iPS cells and mouse embryonic fibroblast (MEF) before and after DOX induction (initiating reprogramming by OSKM factors) from randomly selected Mbd3+/+ and Mbd3flox/- clonal cell line series. Polyclonal donor cell cultures were harvested at days 0,4 and 8 after DOX reprogramming without selection or sorting for any marker or passaging, and mapped for similarity to subcloned iPSC lines.

ORGANISM(S): Mus musculus

SUBMITTER: Asaf Zviran

PROVIDER: E-GEOD-64115 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: to characterize epigenetic changes following Twist1 mediated Epithelial-Mesenchymal Transition in human Methods: we characterized the epigenetic and transcriptome landscapes using whole genome transcriptome analysis by RNA-seq, DNA methylation by digital restriction enzyme analysis of methylation (DREAM) and histone modifications by CHIP-seq of H3K4me3 and H3K27me3 in immortalized human mammary epithelial cells relative to cells induced to undergo EMT by Twist1. Results: EMT is accompanied by focal hypermethylation and widespread global DNA hypomethylation, predominantly within transcriptionally repressed gene bodies. At the chromatin level, the number of gene promoters marked by H3K4me3 increases by more than one fifth; H3K27me3 undergoes dynamic genomic redistribution characterized by loss at half of gene promoters and overall reduction of peak size by almost one-half. This is paralleled by increased phosphorylation of EZH2 at serine 21. Among genes with highly altered mRNA expression, 23.1% switch between H3K4me3 and H3K27me3 marks, and those point to the master EMT targets and regulators CDH1, PDGFRA and ESRP1. Strikingly, Twist1 increases the number of bivalent genes by more than two fold. Inhibition of the H3K27 methyltransferases EZH2 and EZH1, which form part of the PRC2 complex, results in blocking EMT and stemness properties. Conclusion: Our findings demonstrate that the EMT program requires epigenetic remodeling by the Polycomb/Trithorax complexes leading to increased cellular plasticity which suggests that its inhibition will prevent EMT, and the associated breast cancer metastasis. DREAM profiles of human mammary epithelial cells before (HMLE_parental) and after Twist1 transfection (HMLE_Twist) were generated in monolayer (HMLE_Twist2D) and sphere culture by deep sequencing using Illumina GAIIx or Illumina hiseq2000. Furthermore, DREAM profile was also obtained in parental human mammary epithelial cells transfected with GFP

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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Deterministic direct reprogramming of somatic cells to pluripotency [RRBS]

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