Dynamics of Tet1 and Oct4 gene activation identify temporally distinct late stages in reprogramming of induced pluripotency.

Pluripotency comprise two distinct developmental states: pre-implantation or "naive" state, displaying full developmental potency, including germ-line contribution in blastocyst-chimera and "primed" state, reflective of post- implantation stages. Although the master pluripotency regulator Oct4 is expressed in both states, it is regulated by cell-state distinct cis regulatory elements. A distal element drives naive-state Oct4 expression in pre-implantation embryos, while a proximal element regulates Oct4 expression in primed state. The Ten-Eleven-Translocation (TET) family of dioxygenases (TET1, TET2 and TET3) remove DNA methylation through active or passive mechanisms. Similarly to Oct4, Tet1 is highly expressed in pluripotent stem cells and it is downregulated in somatic cells Transcription factor-mediated reprogramming of somatic cells to induced pluripotent stem cells (iPSC) progresses via sequential events to gain full features of pluripotency. The use of markers to isolate and characterize reprogramming intermediates is essential to explore the underlying mechanisms, but existing markers do not provide sufficient temporal resolution of events late in reprogramming towards acquisition of naive pluripotency. Endogenous Oct4 and Tet1 are reactivated late in reprogramming, in association with global loss of DNA methylation. Thus, they are potential candidate marker genes to explore late reprogramming, when the formative iPSCs have entered a deterministic path towards pluripotency acquisition. First, we have defined two alternative transcription start sites (TSSs) for Tet1: the distal TSS at exon 1b is naive specific and distinct from a weaker but constitutively active proximal TSS at exon 1a. The distal embryonic stem cell (ESC) specific TSS is regulated by methylation and undergoes rapid silencing as cells exit from pluripotency, whereas an Oct4/Sox2-binding enhancer sustains the expression from the proximal TSS in both naive and primed pluripotent cells. Then, we have generated reporter ESC lines in which a fluorescent marker is driven by Tet1 naive-specific promoter region B (Tet1/B-mCherry) which is sufficient to drive expression of the mCherry reporter in ESC, loses expression during differentiation and is re-activated upon further conversion to primordial germ cells in vitro. During these cell state transitions, the transgene signals correlate tightly with endogenous transcript levels, thus demonstrating that the Tet1/B reporter line faithfully mimics endogenous promoter activity. In the second part of my project, we have generated murine transgenic lines harboring mCherry reporters driven either by the previously characterized naive-specific Tet1 distal promoter Tg(Tet1-mCherry)B or by total Tet1 gene activity. Then, by distinct coupling of these mCherry reporter lines with commercially available GFP reporters driven by naive-specific or total Oct4 gene expression, we have generated lines harboring dual fluorescent reporters reflecting cell-state specific endogenous gene expression of Tet1 and Oct4. By assessing reprogramming intermediates based on dual reporter patterns, we identified a sequential order of Tet1 and Oct4 gene activation at proximal and distal regulatory elements, beginning shortly after activation of NANOG, which signaled entry into pluripotency, and ending with activation of DPPA4, a late naive pluripotency marker. Transcriptome profiling of these sorted intermediates revealed five distinct cellular states reflecting transitions through first a gain of pattern specification processes, then a temporary stalling of cell migration and motility, followed by activation of meiotic genes and finally a restoration of cell motility and proliferation. Moreover, we identified global waves of DNA demethylation within those stage transitions: the first major wave of DNA demethylation is followed by a locus-specific loss, to coincide with activation of meiotic and germline-specific genes, in a manner reminiscent of germline reprogramming. Loss of Tet1 is compatible with reprogramming, but generates iPSCs with epigenetic defects. Our results offer insights into the basis for epigenetic differences between embryo-derived and in vitro induced pluripotent stem cells. Overall, we demonstrate that the transcriptional regulation of Tet1 expression can signal an epigenetic roadmap towards efficient reprogramming.

Jaar van publicatie:2019

Toegankelijkheid:Open