In this study we verify the hypothesis if

In this study, we verify the hypothesis if TRIM28 plays a role in reprogramming of mouse embryonic fibroblasts into iPS cells. We stably silenced the expression of Trim28 in MEFs and submitted them to the reprogramming process by infection with lentiviral vector carrying the expression of Yamanaka factors. Then, we checked embryonic stem cell markers in several timepoints during reprogramming in Trim28 KD mpges-1 inhibitors and compared it to control cells. Additionally, we performed RPPA analysis to determine the deregulated proteins that occur in Trim28 KD cells and to evaluate proteomic changes during the course of somatic cell reprogramming. Here, we demonstrate surprising role of TRIM28 in induction and maintenance of pluripotency. Our results suggest that, Trim28 KD cells are defective in capability of reprogramming into stable iPS cells.
Results
Discussion Here, we demonstrate that Trim28 knockdown MEFs are not capable of reprogramming into stable pluripotent state. These results were obtained using RNA interference that guaranteed stable and efficient knockdown of epigenetic corepressor TRIM28 during further analyses. Trim28 KD MEFs and control MEFs were induced to dedifferentiate into iPS cells using efficient method of iPS cell generation with STEMCCA-tetO vector that provided fully pluripotent and functional iPS cells. For comprehensive observation of any changes during reprogramming process, we performed a time course analysis of endogenous pluripotency markers. To capture the first changes in cells induced to dedifferentiate, we focused on evaluation of alkaline phosphatase (AP) activity as it is reactivated early after induction of four reprogramming factors (Brambrink et al., 2008). However, even if it is detected first, AP does not necessarily mark fully reprogrammed cells (Brambrink et al., 2008; Si-Tayeb et al., 2010). Therefore, we also analyzed the expression of other ES cell marker, SSEA-1 that marks an intermediate state of reprogramming (Brambrink et al., 2008). Analysis of markers characteristic to the late state of pluripotency, such as OCT-3/4 and NANOG was limited to the OCT4-GFP reporter analysis because our preliminary results indicated that the most abundant changes in ES cell marker expression accompany the early reprogramming events. Several studies have elucidated the role of TRIM28 in self-renewal and regulation of pluripotency markers in mouse ES cells and iPS cells, but our research provides, for the first time, the results of a large scale analysis of proteomic changes occurring in Trim28 KD cells during reprogramming. TRIM28 was shown to be necessary for early postimplantation development (Cammas et al., 2000; Messerschmidt et al., 2012). Depletion of TRIM28 caused the differentiation into the primitive ectoderm lineage, stressing its essential function in self-renewal (Hu et al., 2009). The TRIM28-dependent chromatin relaxation was shown to be critical for the formation of a complex with OCT-3/4 and chromatin-modifying enzymes (Seki et al., 2010). Surprisingly, our results indicate that Trim28 KD-mediated euchromatization, obstructed the generation of iPS cells that could be stably maintained in cell culture and these results are consistent with previous reports (Friedli et al., 2014). Interestingly, recent studies indicated that depletion of Trim28 enhanced reprogramming by stimulation of the gene expression from H3K9me3 enriched regions and induction of expression of endogenous retroviruses (Miles et al., 2016). Importantly, the increase in reprogramming was more pronounced in immortalized MEFs when compared to wild- type MEFs (17-fold vs 2-fold) suggesting that MEFs with Trim28 knockdown cannot survive dedifferentiation (Miles et al., 2016). In our study, we also observed that Trim28 knockdown increased the population of AP-positive cells in the early stage of reprogramming (d5) but these cells have not expressed specific stem cell markers and disappeared very quickly. Additionally, utilizing RPPA analysis, we observed that Trim28 KD cells at the final stage of reprogramming (d14) clustered together with cells at the earliest steps of reprogramming (d5) and had not resembled intermediate state of Trim28 KD cell reprogramming. This observation suggests that Trim28 KD cells are not capable of reprogramming into fully pluripotent iPS cells because they differentiate spontaneously. These observations imply that even if depletion of Trim28 increases the relaxation of chromatin and in consequence facilitates the reset of differentiated state, Trim28 knockdown causes also that emerging iPS cells quickly lose their self-renewal ability and differentiate immediately, as suggested by previous reports (Seki et al., 2010).