Tsix RNA and the Germline Factor, PRDM14, Link X Reactivation and Stem Cell Reprogramming

Abstract

SUMMARY Transitions between pluripotent and differentiated states are marked by dramatic epigenetic changes. Cellular differentiation is tightly linked to X-chromosome inactivation (XCI), whereas reprogramming to induced pluripotent stem cells (iPSCs) is associated with X-chromosome reactivation (XCR). XCR reverses the silent state of the inactive X, occurring in vivo in mouse blastocysts and the germline. In spite of its importance, little is known about underlying mechanisms. Here, we examine the role of the long noncoding Tsix RNA and the germline factor, PRDM14. In blastocysts, XCR is perturbed by mutation of either Tsix or Prdm14. In iPSCs, XCR is disrupted only by PRDM14-deficiency, which also affects iPSC derivation and maintenance. We show that Tsix and PRDM14 directly link XCR to pluripotency: First, PRDM14 represses Rnf12 by recruiting Polycomb repressive complex 2. Second, Tsix is required for PRDM14 to bind Xist. Thus, our study provides functional and mechanistic links between cellular and X-chromosomal reprogramming.

Coupling of X-Chromosome reactivation with the pluripotent stem cell state

“…Indeed, ChIP-experiments confirmed binding of OCT4, SOX2, NANOG and PRDM14 to the upstream regulatory region of Rnf12 in ESCs. 53,75 As a consequence, depletion of these pluripotency factors leads to an upregulation of Rnf12 in ESCs, confirming that the regulation of Rnf12 is directly linked to the pluripotent state.…”

“…83 Studying XCR in vivo in the epiblasts of Prdm14-and Tsix-mutant blastocysts, we observed a decrease in efficiency based on the failure to erase the X-chromosome-wide H3K27me3 mark. 53 In both Prdm14−/− and Tsix−/− E4.5 blastocysts, XCR occurred in only about half of the epiblast cells, in contrast to wildtype embryos in which XCR reached over 90% efficiency. In Tsix−/− mutants, there was a measurable delay in development, as the E4.5 blastocysts had fewer cells.…”

“…Xist regulation mechanisms in pluripotent (left) and differentiated (right) cell states. the mechanisms described in (A) 67 and (B) 64 have been described to occur both during the onset of XCi (red block arrows, left to right), while the mechanism given in (C) 53 during XCR (green block arrow, right to left). (A) in undifferentiated cells, Xist expression is blocked by binding of CtCf to the Xist promotor (left).…”

“…One of the most prominent regions is Xist intron 1, where abundant binding of OCT4, SOX2, NANOG and PRDM14 has been described in undifferentiated ESCs. [50][51][52][53] Indeed, depletion of OCT4, NANOG and PRDM14 using induced repression, conditional deletion or RNAi knockdown, leads to an upregulation of Xist expression, albeit not immediately, but rather concurrent with differentiation caused by depletion of the pluripotency factors. 50,52,54 57,58 In the absence of intron1, imprinted XCI is erased normally during development, as is random XCI during reprogramming to iPS-cells.…”

“…For this reason, we recently examined the XCR mechanism directly in mice and during iPSC repogramming, specifically investigating the pluripotency factor PRDM14 and the noncoding RNA Tsix as candidate regulators. 53 We hypothesized an involvement of Tsix and PRDM14 in XCR based on key observations made previously. For example, forced inducible Tsix expression from the paternal X-chromosome is sufficient to repress Xist in cis both in preimplantation embryos and in the placenta, resulting in a reversion of imprinted XCI.…”