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

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 process 2-7 . 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. correlation above 0.5 between consecutive samples ( Extended Data...

Dynamic changes in tRNA modifications and abundance during T-cell activation

“…For tRNA Lys (UUU) we found RT-abortions in a similar pattern to tRNA Phe( GAA), without the appearance of mismatches in the activated cells (Fig 3D, Fig S3 -3B). The intermediate modification t 6 A, that might occur in the activated cells, cannot be detected using our tRNA-sequencing protocol, since it does not result in a detectable mismatch pattern (Fig S3 -1). Our data support the presence of the bulky modification 2-methylthio-6threonylcarbamoyl-A (ms 2 t 6 A) as shown before in mouse 18 and human 9,44 , as this modification was shown to cause RT abortion in human 13 .…”

“…However, the regulation of translation elongation, and especially tRNA availability was not explored. We have previously shown that genes that are upregulated in proliferating cancerous cells or upon induced pluripotency have a distinct translation program from that of arrested cells 5,6 . In particular, mRNAs corresponding to cell-autonomous functions, related to proliferating cells, are enriched with a specific set of codons, while mRNA of multicellular functions, related to non-dividing cells, are enriched with a different set of preferred codons.…”