Temporal resolution of global gene expression and DNA methylation changes in the final phases of reprogramming towards induced pluripotency

Abstract

The generation of induced pluripotent stem cells (iPSCs) involves activation of the endogenous 1 pluripotency circuitry and global DNA demethylation late in reprogramming, but temporal 2 resolution of these events using existing markers is insufficient. Here, we generated murine 3 transgenic lines harboring dual fluorescent reporters reflecting cell-state specific expression of 4 the master pluripotency factor Oct4 and the 5-methylcytosine dioxygenase Tet1. By assessing 5 reprogramming intermediates based on dual reporter patterns, we identified a sequential order of 6 Tet1 and Oct4 gene activation at proximal and distal regulatory elements following pluripotency 7 entry. Full induction of Tet1 marks a pivotal late intermediate stage occurring after a phase of 8 global gene repression, and preceding full activation of Oct4 along with late naive pluripotency 9 2 and germline-specific genes. Sequential activation of Tet1 further distinguishes two waves of 10 global DNA demethylation, targeting distinct genomic features and largely uncoupled from 11 transcriptional changes, with dynamics unique to iPSC reprogramming. Moreover, we 12 demonstrate that loss of Tet1 is compatible with reprogramming towards full Oct4 gene 13 activation, but generates iPSCs with aberrant DNA methylation, chromosomal instability during 14 lineage priming and defective differentiation potential. Therefore, the transcriptional logic of Tet1 15 expression signals a deterministic epigenetic roadmap towards generation of high quality iPSCs. 16 65 by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidation 66 products at CpG dinucleotides 21-23 . Of the three TET genes, Tet1 expression is barely detectable 67 in mouse embryonic fibroblasts (MEFs), but robustly up-regulated in iPSCs concomitantly with 68 5 endogenous Oct4 late in reprogramming 8,24 . Because Tet1 is a target gene regulated by OCT4 69 24,25 , a positive feedback logic involving Tet1 and Oct4 may function to jump-start the 70 pluripotency circuitry during reprogramming 26 . However, the sequential patterns of endogenous 71 Oct4 and Tet1 gene activation during reprogramming remain unknown. 72 During early embryogenesis, Tet1 expression is driven by two state-specific promoter-73 enhancer regions, using a similar cis-regulatory logic as for Oct4, where a distal promoter region 74 spanning 6 kb drives an alternative transcription start site (TSS) at exon 1b which is activated 75 exclusively in pre-implantation embryos and naive ESCs 25 . A proximal promoter-enhancer 76 coupling at exon 1a sustains Tet1 expression through primed pluripotency. Here, we generated 77 MEFs harboring distinct dual-fluorescent transgenic reporters for state-specific expression of 78 Oct4 and Tet1. By using live cell imaging and flow cytometry coupled with genome-scale 79 analyses, our studies revealed a distinct trajectory of epigenetic events marking cell state 80 transitions from early acquisition of pluripotency to terminal establishment of clonal iPSCs. We 81 further clar...