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      Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells

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          Abstract

          Human induced pluripotent stem cells (hiPSCs) are capable of unlimited proliferation and can differentiate in vitro to generate derivatives of the three primary germ layers. Genetic and epigenetic abnormalities have been reported by Wissing and colleagues to occur during hiPSC derivation, including mobilization of engineered LINE-1 (L1) retrotransposons. However, incidence and functional impact of endogenous retrotransposition in hiPSCs are yet to be established. Here we apply retrotransposon capture sequencing to eight hiPSC lines and three human embryonic stem cell (hESC) lines, revealing endogenous L1, Alu and SINE-VNTR-Alu (SVA) mobilization during reprogramming and pluripotent stem cell cultivation. Surprisingly, 4/7 de novo L1 insertions are full length and 6/11 retrotransposition events occurred in protein-coding genes expressed in pluripotent stem cells. We further demonstrate that an intronic L1 insertion in the CADPS2 gene is acquired during hiPSC cultivation and disrupts CADPS2 expression. These experiments elucidate endogenous retrotransposition, and its potential consequences, in hiPSCs and hESCs.

          Abstract

          Genetic and epigenetic abnormalities have been found to result from reprogramming of differentiated cells into human induced pluripotent stem cells (hiPSCs). Here, Klawitter et al. identify endogenous L1, Alu and SVA mobilization during reprogramming, highlighting the risk of insertional mutagens in hiPSCs.

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          Most cited references52

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          RNA maps reveal new RNA classes and a possible function for pervasive transcription.

          Significant fractions of eukaryotic genomes give rise to RNA, much of which is unannotated and has reduced protein-coding potential. The genomic origins and the associations of human nuclear and cytosolic polyadenylated RNAs longer than 200 nucleotides (nt) and whole-cell RNAs less than 200 nt were investigated in this genome-wide study. Subcellular addresses for nucleotides present in detected RNAs were assigned, and their potential processing into short RNAs was investigated. Taken together, these observations suggest a novel role for some unannotated RNAs as primary transcripts for the production of short RNAs. Three potentially functional classes of RNAs have been identified, two of which are syntenically conserved and correlate with the expression state of protein-coding genes. These data support a highly interleaved organization of the human transcriptome.
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            Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution.

            Ectopic expression of the four transcription factors Oct4, Sox2, c-Myc, and Klf4 is sufficient to confer a pluripotent state upon the fibroblast genome, generating induced pluripotent stem (iPS) cells. It remains unknown if nuclear reprogramming induced by these four factors globally resets epigenetic differences between differentiated and pluripotent cells. Here, using novel selection approaches, we have generated iPS cells from fibroblasts to characterize their epigenetic state. Female iPS cells showed reactivation of a somatically silenced X chromosome and underwent random X inactivation upon differentiation. Genome-wide analysis of two key histone modifications indicated that iPS cells are highly similar to ES cells. Consistent with these observations, iPS cells gave rise to viable high-degree chimeras with contribution to the germline. These data show that transcription factor-induced reprogramming leads to the global reversion of the somatic epigenome into an ES-like state. Our results provide a paradigm for studying the epigenetic modifications that accompany nuclear reprogramming and suggest that abnormal epigenetic reprogramming does not pose a problem for the potential therapeutic applications of iPS cells.
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              Meiotic catastrophe and retrotransposon reactivation in male germ cells lacking Dnmt3L.

              Mammalian genomes employ heritable cytosine methylation in the long-term silencing of retrotransposons and genes subject to genomic imprinting and X chromosome inactivation. Little is known of the mechanisms that direct cytosine methylation to specific sequences. Here we show that DNA methyltransferase 3-like (Dnmt3L (ref. 1)) is expressed in testes during a brief perinatal period in the non-dividing precursors of spermatogonial stem cells at a stage where retrotransposons undergo de novo methylation. Deletion of the Dnmt3L gene prevented the de novo methylation of both long-terminal-repeat (LTR) and non-LTR retrotransposons, which were transcribed at high levels in spermatogonia and spermatocytes. Loss of Dnmt3L from early germ cells also caused meiotic failure in spermatocytes, which do not express Dnmt3L. Whereas dispersed repeated sequences were demethylated in mutant germ cells, tandem repeats in pericentric regions were methylated normally. This result indicates that the Dnmt3L protein might have a function in the de novo methylation of dispersed repeated sequences in a premeiotic genome scanning process that occurs in male germ cells at about the time of birth.
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                Author and article information

                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Publishing Group
                2041-1723
                08 January 2016
                2016
                : 7
                : 10286
                Affiliations
                [1 ]Division of Medical Biotechnology, Paul-Ehrlich-Institute , D-63225 Langen, Germany
                [2 ]Max-Delbrück-Center for Molecular Medicine , D-13125 Berlin, Germany
                [3 ]Mater Research Institute, University of Queensland , TRI Building, Woolloongabba, Brisbane, Queensland 4102, Australia
                [4 ]Department of Human DNA Variability, Pfizer/University of Granada and Andalusian Regional Government Center for Genomics and Oncology (GENYO), PTS Granada , 18016 Granada, Spain
                [5 ]Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiac, Thoracic, Transplantation, and Vascular Surgery; REBIRTH, Cluster of Excellence, Hannover Medical School , D-30625 Hannover, Germany
                [6 ]Department of Biophysics and Radiation Biology, Semmelweis University , H-1094 Budapest, Hungary
                [7 ]Australian Institute for Bioengineering and Nanotechnology, The University of Queensland , St Lucia, Queensland 4072, Australia
                [8 ]Queensland Brain Institute, University of Queensland , Brisbane, Queensland 4072, Australia
                Author notes
                [*]

                These authors contributed equally to this work.

                [†]

                Present address: Division of Inborn Metabolic Diseases, University Children's Hospital, D-69120 Heidelberg, Germany.

                Article
                ncomms10286
                10.1038/ncomms10286
                4729875
                26743714
                6b970826-9597-45cf-bddd-98374e195544
                Copyright © 2016, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 29 June 2015
                : 26 November 2015
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