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

The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) with four transcription factors Oct4, Sox2, Klf4 and cMyc (abbreviated as OSKM)1has 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/offepigeneticswitching of modifications in theirpromoters (abbreviated as ESPGs), and the second consists of genes withpromoters in aconstitutivelyactive 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.