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High-Resolution Dissection of Conducive Reprogramming Trajectory to Ground State Pluripotency

Asaf Zviran, Nofar Mor, Yoach Rais, Hila Gingold, Shani Peles, Elad Chomsky, Sergey Viukov, Jason D. Buenrostro, Leehee Weinberger, Yair S. Manor, Vladislav Krupalnik, Mirie Zerbib, Hadas Hezroni, Diego Adhemar Jaitin, David Larastiaso, Shlomit Gilad, Sima Benjamin, Awni Mousa, Muneef Ayyash, Daoud Sheban, Jonathan Bayerl, Alejandro Aguilera Castrejon, Rada Massarwa, Itay Maza, Suhair Hanna, Ido Amit, Yonatan Stelzer, Igor Ulitsky, William J. Greenleaf, Yitzhak Pilpel, Noa Novershtern, Jacob H. Hanna
doi: https://doi.org/10.1101/184135
Asaf Zviran
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
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Nofar Mor
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
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Yoach Rais
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
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  • For correspondence: jacob.hanna@weizmann.ac.il noa.novershtern@weizmann.ac.il yoach.rais@weizmann.ac.il
Hila Gingold
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
2Department of Neurobiology, Wise Faculty of Life Sciences and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 69978, Israel.
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Shani Peles
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
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Elad Chomsky
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
3Department of Biological Regulation, Weizmann Institute of Science, Rehovot 7610001, Israel.
4Department of Computer Science, Weizmann Institute of Science, Rehovot 7610001, Israel.
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Sergey Viukov
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
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Jason D. Buenrostro
5Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
6Harvard Society of Fellows, Harvard University, Cambridge, MA 02138, USA
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Leehee Weinberger
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
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Yair S. Manor
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
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Vladislav Krupalnik
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
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Mirie Zerbib
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
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Hadas Hezroni
3Department of Biological Regulation, Weizmann Institute of Science, Rehovot 7610001, Israel.
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Diego Adhemar Jaitin
7Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel.
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David Larastiaso
7Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel.
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Shlomit Gilad
8The Israel National Center for Personalized Medicine (INCPM), Weizmann Institute of Science, Rehovot 76100, Israel.
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Sima Benjamin
8The Israel National Center for Personalized Medicine (INCPM), Weizmann Institute of Science, Rehovot 76100, Israel.
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Awni Mousa
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
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Muneef Ayyash
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
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Daoud Sheban
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
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Jonathan Bayerl
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
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Alejandro Aguilera Castrejon
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
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Rada Massarwa
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
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Itay Maza
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
9Department of Gastroenterology, Rambam Health Care Campus & Bruce Rappaport School of Medicine, Technion Institute of Technology, Haifa, Israel
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Suhair Hanna
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
10Department of Pediatrics and the Pediatric Immunology Unit, Rambam Health Care Campus & Bruce Rappaport School of Medicine, Technion Institute of Technology, Haifa, Israel
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Ido Amit
7Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel.
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Yonatan Stelzer
11Department of Molecular and Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel.
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Igor Ulitsky
3Department of Biological Regulation, Weizmann Institute of Science, Rehovot 7610001, Israel.
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William J. Greenleaf
12Department of Applied Physics, Stanford University, Palo Alto, CA, USA
13Chan Zuckerberg Biohub, San Francisco, CA 94158, USA
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Yitzhak Pilpel
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
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Noa Novershtern
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
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  • For correspondence: jacob.hanna@weizmann.ac.il noa.novershtern@weizmann.ac.il yoach.rais@weizmann.ac.il
Jacob H. Hanna
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
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  • For correspondence: jacob.hanna@weizmann.ac.il noa.novershtern@weizmann.ac.il yoach.rais@weizmann.ac.il
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Abstract

The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) with four transcription factors Oct4, Sox2, Klf4 and cMyc (abbreviated as OSKM)1 has provoked interest to define the molecular characteristics of this process2-7. Despite important progress, the dynamics of epigenetic reprogramming at high resolution in correctly reprogrammed iPSCs and throughout the entire process remain largely undefined. This gap in understanding results from the inefficiency of conventional reprogramming methods coupled with the difficulty of prospectively isolating the rare cells that eventually correctly reprogram into iPSCs. Here we characterize cell fate conversion from fibroblast to iPSC using a highly efficient deterministic murine reprogramming system engineered through optimized inhibition of Gatad2a-Mbd3/NuRD repressive sub-complex. This comprehensive characterization provides single-day resolution of dynamic changes in levels of gene expression, chromatin modifications, TF binding, DNA accessibility and DNA methylation. The integrative analysis identified two transcriptional modules that dominate successful reprogramming. One consists of genes whose transcription is regulated by on/off epigenetic switching of modifications in their promoters (abbreviated as ESPGs), and the second consists of genes with promoters in a constitutively active chromatin state, but a dynamic expression pattern (abbreviated as CAPGs). ESPGs are mainly regulated by OSK, rather than Myc, and are enriched for cell fate determinants and pluripotency factors. CAPGs are predominantly regulated by Myc, and are enriched for cell biosynthetic regulatory functions. We used the ESPG module to study the identity and temporal occurrence of activating and repressing epigenetic switching during reprogramming. Removal of repressive chromatin modifications precedes chromatin opening and binding of RNA polymerase II at enhancers and promoters, and the opposite dynamics occur during repression of enhancers and promoters. Genome wide DNA methylation analysis demonstrated that de novo DNA methylation is not required for highly efficient conducive iPSC reprogramming, and identified a group of super-enhancers targeted by OSK, whose early demethylation marks commitment to a successful reprogramming trajectory also in inefficient conventional reprogramming systems. CAPGs are distinctively regulated by multiple synergystic ways: 1) Myc activity, delivered either endogenously or exogenously, dominates CAPG expression changes and is indispensable for induction of pluripotency in somatic cells; 2) A change in tRNA codon usage which is specific to CAPGs, but not ESPGs, and favors their translation. In summary, our unbiased high-resolution mapping of epigenetic changes on somatic cells that are committed to undergo successful reprogramming reveals interleaved epigenetic and biosynthetic reconfigurations that rapidly commission and propel conducive reprogramming toward naïve pluripotency.

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Posted September 07, 2017.
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High-Resolution Dissection of Conducive Reprogramming Trajectory to Ground State Pluripotency
Asaf Zviran, Nofar Mor, Yoach Rais, Hila Gingold, Shani Peles, Elad Chomsky, Sergey Viukov, Jason D. Buenrostro, Leehee Weinberger, Yair S. Manor, Vladislav Krupalnik, Mirie Zerbib, Hadas Hezroni, Diego Adhemar Jaitin, David Larastiaso, Shlomit Gilad, Sima Benjamin, Awni Mousa, Muneef Ayyash, Daoud Sheban, Jonathan Bayerl, Alejandro Aguilera Castrejon, Rada Massarwa, Itay Maza, Suhair Hanna, Ido Amit, Yonatan Stelzer, Igor Ulitsky, William J. Greenleaf, Yitzhak Pilpel, Noa Novershtern, Jacob H. Hanna
bioRxiv 184135; doi: https://doi.org/10.1101/184135
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High-Resolution Dissection of Conducive Reprogramming Trajectory to Ground State Pluripotency
Asaf Zviran, Nofar Mor, Yoach Rais, Hila Gingold, Shani Peles, Elad Chomsky, Sergey Viukov, Jason D. Buenrostro, Leehee Weinberger, Yair S. Manor, Vladislav Krupalnik, Mirie Zerbib, Hadas Hezroni, Diego Adhemar Jaitin, David Larastiaso, Shlomit Gilad, Sima Benjamin, Awni Mousa, Muneef Ayyash, Daoud Sheban, Jonathan Bayerl, Alejandro Aguilera Castrejon, Rada Massarwa, Itay Maza, Suhair Hanna, Ido Amit, Yonatan Stelzer, Igor Ulitsky, William J. Greenleaf, Yitzhak Pilpel, Noa Novershtern, Jacob H. Hanna
bioRxiv 184135; doi: https://doi.org/10.1101/184135

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