Somatic cell reprogramming to a pluripotent state continues to challenge many of our assumptions about cellular specification, and despite major efforts, we lack a complete molecular characterization of the reprograming process. To address this gap in knowledge, we generated extensive transcriptomic, epigenomic and proteomic data sets describing the reprogramming routes leading from mouse embryonic fibroblasts to induced pluripotency. Through integrative analysis, we reveal that cells transition through distinct gene expression and epigenetic signatures and bifurcate towards reprogramming transgene-dependent and -independent stable pluripotent states. Early transcriptional events, driven by high levels of reprogramming transcription factor expression, are associated with widespread loss of histone H3 lysine 27 (H3K27me3) trimethylation, representing a general opening of the chromatin state. Maintenance of high transgene levels leads to re-acquisition of H3K27me3 and a stable pluripotent state that is alternative to the embryonic stem cell (ESC)-like fate. Lowering transgene levels at an intermediate phase, however, guides the process to the acquisition of ESC-like chromatin and DNA methylation signature. Our data provide a comprehensive molecular description of the reprogramming routes and is accessible through the Project Grandiose portal at http://www.stemformatics.org.
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European Nucleotide Archive
Sequence Read Archive
Sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) under accession number SRP046744 for all RNA-seq and ChIP-seq experiments, and in the European Bioinformatics Institute under the European Nucleotide Archive (ENA) accession number ERP004116 for MethylC-sequencing. The global and cell surface mass spectrometry proteomics raw data have been deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository under data set identifiers PXD000413 and PXD001456, respectively.
We thank M. Gertsenstein and M. Pereira for chimaera production, C. Monetti for cell culture, R. Cowling for DNA purification, and K. Harpal for chimaera embryo sectioning and staining. We acknowledge the intellectual contributions of P. P. L. Tam and R. P. Harvey. A.N. is Tier 1 Canada Research Chair in Stem Cells and Regeneration. This work was supported by grants awarded to A.N., I.M.R. and P.W.Z. from the Ontario Research Fund Global Leadership Round in Genomics and Life Sciences grants (GL2-01-028), to A.N. from the Canadian stem cell network (9/5254 (TR3)) and from the Canadian Institutes of Health Research (CIHR MOP102575). This work received support from the Korean Ministry of Knowledge Economy (grant 10037410 to J.-S.S.), from the SNUCM Research Fund (grant 0411-20100074 to J.-S.S.), and from Macrogen Inc. (grant MGR03-11 and MGR03-12). The Stemformatics resource is supported by an Australian Research Council special research grant to Stem Cells Australia (C.A.W. and S.M.G.). The analysis of the miRNA was supported by grants from the National Health and Medical Research Council of Australia (1024852 to J.L.C. and T.P.) and the Australian Research Council (DP1300101928 to T.P.). W.R. is a Cancer Institute of NSW Fellow and with J.E.J.R. receives support from the Cancer Council of NSW and National Health & Medical Research Council (571156 and 1061906). J.E.J.R. receives funding from Cure the Future & Tour de Cure. K.-A.L.C. is supported, in part, by the Wound Management Innovation CRC (established and supported under the Australian Government’s Cooperative Research Centres Program). S.M.G. received support from the Australian Research Council (SR110001002). C.A.W. is a QLD Smart Futures Fellow. M.B., J.M. and A.J.R.H. are supported by the Netherlands Proteomics Centre, and by the European Community’s Seventh Framework Programme (FP7/2007-2013) by the PRIME-XS project grant agreement number 262067. P.W.Z. is the Canada Research Chair in Stem Cell Bioengineering. S.M.I.H. received a fellowship from the McEwen Centre of Regenerative Medicine.
Extended data figures
This zipped file contains Supplementary Tables 1-6.