|Roles for Prdm14 and Klf2 for Pluripotency and Totipotency? - Epiblast Stem Cell-Based System Reveals Reprogramming Synergy of Germline Factors|
Studies of the human germ line are, for obvious reasons, difficult to establish and therefore effective in vitro model systems to study the human germ line formation and subsequent specifications are attractive prospects. Primordial germ cells (PGCs) arise from the post-implantation epiblast of the embryo, and stem cells derived from these cells (Epiblast stem cells or EpiSCs) have the potential to revert to embryonic stem cells (ESCs) (Bao et al) or become specified to PGCs (Hayashi and Surani (2009b)) which, while distinct, share common features; inactive X chromosome (Xi) reactivation, distinct changes to epigenetic modifications and the expression of specific pluripotency genes (Hayashi and Surani (2009a)). Using this knowledge, researchers from the laboratory of M. Azim Surani at the Gurdon Institute, University of Cambridge, UK have designed a culture-based system for the reprogramming of EpiSCs to ESCs as a model to delineate important factors and mechanisms behind EpiSC reversion/PGC specification. This has allowed the discovery of a key role for the synergistic relationship between Prdm14-Klf2 in reprogramming which includes X-reactivation and key epigenetic changes (Gillich et al), providing a key insight into how germ cells can transit to the epigenetic ground state or the PGC fate.
Reporter lines utilised to study reprogramming of epiSCs utilised their greatest differences from ESCs and PGCs; the Xi in epiSCs, unlike ESCs and PGCs (Guo et al and Hayashi and Surani (2009b)) and their use of the proximal OCT4 enhancer rather than the distal enhancer as in ESCs and PGCs (Bao et al and Yeom et al). The epiSC reporter line was generated containing a GFP transgene on the Xi and was cultured on fibronectin in serum-free medium containing activin and bFGF to ensure a repressed Xi-GFP reporter. Analysis of these cells found them to demonstrate stable X inactivation in these conditions; high expression levels of epiblast genes (Fgf5 and Xist) and low levels of ESC- and PGC- associated genes (Klf2, Prdm14, Stella, Rex1, Nr0B1 and Tsix) and high levels of the repressive histone 3 lysine 27 trimethylation (H3K27me3) chromatin mark, which colocalized with monoubiquitinated H2A (ubH2A) and nuclear foci of Enhancer of Zeste (Ezh2).
Germ cell factors were then analysed for their ability to reprogram epiSCs to ESCs (reverted ESCs or rESCs) and reactivate the Xi, through stable expression of combinations of factors through piggyBac (PB) transposition. Despite increased mRNA and protein of these factors and some of their targets, no GFP expression was observed, Xist and Tsix remained expressed and repressed respectively and repressive chromatin domains remained, all suggesting that the Xi had not been reactivated. However, transfer of cells to LIF and serum (Lif-Stat3 conditions) on feeder cells allowed for GFP expression when Klf2 and Nanog or Prdm14 and Nanog were expressed for 7-8 days and after only 3-4 days when Prdm14 and Klf2 were expressed and allowed for the appearance of 500 GFP+ colonies from 50,000 plated cells by day 6. Importantly, Prdm14, which may possess histone methyltransferase activity, has previously been shown to be involved in the repression of the somatic program and the initiation of epigenetic reprogramming in early germ cells (Yamaji et al) while Klf2 has been previously linked to EpiSC reprogramming (Hall et al). Detailed analysis of Prdm14 and Klf2 reprogramming showed an initial mosaic GFP expression, which then spread out through colonies of cells. GFP+ colonies which were picked could be expanded and showed stable GFP expression suggesting that Prdm14 and Klf2 trigger rapid X reactivation in epiSCs upon transfer to LIF-Stat3. Prdm14 expression on its own in LIF-Stat3 conditions was unable to allow for Xi reactivation, however Klf2 did allow for GFP expression but this was slower (6-7 days) as compared to Prdm14 and Klf2 expression together suggesting a synergistic role for these two factors. Indeed expression of both factors in LIF-Stat3 conditions allowed for faster ESC-like colony formation, induction of Stella expression through enhanced DNA demethylation of its promoter, and earlier induction of Rex1, Nr0B1, and Nr5a2. Klf4 (expressed in ESCs and PGCs) and Klf5 (expressed only in PGCs) could substitute for Klf2 for the reprogramming of epiSCs, but the process was always higher when they were used in conjunction with Prdm14 and, further, Klf3 was the most potent of the Klf factors analysed. This suggests that re-entry into naive pluripotency, that is the formation or rESCs, could require different factors or factor combinations than maintenance of a naive pluripotent state, as Klf2, 3 and 5 had been previously shown to be redundant in mESCs (Jiang et al).
Prdm14 and Klf2 function in reprogramming was further examined in relation to enhancer switching at the Oct4 locus, another measure of reprogramming to rESCs. A previously described Oct4 distal enhancer-GFP reporter (Bao et al) was used which will activate GFP upon reversion of epiSCs to rESCs. Prdm14 and Klf2 under LIF-Stat3 conditions allowed for an extremely rapid (2 days) and efficient activation (5% of cells by day 6) of the reporter, suggesting fast and efficient epigenetic reprogramming. To exclude factors present in serum or secreted by feeder cells, reprogramming by Prdm14 and Klf2 was studied in serum- and feeder-free 2i/ LIF conditions (Ying et al), which demonstrated similar findings to before, suggesting a key role for Prdm14 and Klf2 in reprogramming.
Confirmation of complete reprogramming of epiSC to rESCs was established through analysis of their differentiation potential. rESCs expression profile was similar to control ESCs, and after integration into blastocysts, contributed to coat colour chimeras and contributed to cells of E13.5 genital ridges. Loss of nuclear foci of Xist, Ezh2, and H3K27me3 in rESCs indicating a fully reactivated X chromosome was also confirmed as was the silencing of any transgenes present.
Gene expression profiles of epiSCs found that cells grown in Activin A and bFGF were distinctly different to those grown in LIF/Stat3 conditions while, surprisingly, epiSCs maintained in Activin A, bFGF and Prdm14 alone were similar to cells with both Prdm14 and Klf2. Expression of Prdm14 led to the induction of 1,433 genes and also the repression of 1,310 genes compared to control with 1,135/1,433 upregulated genes and 1,088/1,310 downregulated genes being direct targets of Prdm14 (ChIP data from (Ma et al)). Many genes involved in early lineage specification were repressed (Nodal, Foxa2, Gata6, Hhex, Eomes, Foxh1, and Otx2) while genes associated with early epiblast or ESCs (Sox2, Gbx2, Esrrb, Fbxo15, Gdf3, Dppa2, and Dppa4) were induced. This suggests that Prdm14 may aid reprogramming to rESCs through its repression of differentiation and induction of pluripotency associated genes. ChIP-on-Chip analysis of Klf2 (Jiang et al) found that 95% of Klf2 targets were also Prdm14 targets while 39% were bound by both Prdm14 (Ma et al) and Stat3 (Chen et al). Two identified targets of Prdm14 and Klf2 were Nr5a2 and Oct4 DE, and subsequent ChIP analysis found that Klf2 bound in higher amounts at these sites in the presence of Prdm14 in LIF-Stat3 culture again indicating an important level of synergism for these two factors. Amongst other interesting genes induced by Prdm14 and Klf2 were Blimp1 (Prdm1), Stella (Dppa3), Fragilis (Ifitm1/3/5), and Nanos3 indicating that epiSC reprogramming may progress through a germ cell stage. However, knockout of Blimp1, a key determinant of PGC specification (Ohinata et al and Vincent et al), in epiSC did not affect any facet of reprogramming
In conclusion, this study has demonstrated the usefulness of a model system which has the ability to allow the complex mechanisms behind reprogramming and specification to be delineated; be they epigenetic changes, gene expression alterations or complex protein interactions. Prdm14 and Klf2 will no doubt be the first of many important findings utilising this model, allowing us to better understand the epigenetic and gene expression changes which occur during normal development and reprogramming to both pluripotency and totipotency. In the future, this may allow the effective differentiation of ESCs and EpiSCs to desirable cell types in humans, which has obvious potential in cell replacement and fertility problems.
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