prolyl hydroxylase Here we set out to evaluate the roles of
Here we set out to evaluate the roles of transcriptional regulators associated with EMT in iPSC reprogramming. We were intrigued by data from published reports that EMT factors (including SNAI1) increased during the early stages of reprogramming (Mikkelsen et al., 2008; Samavarchi-Tehrani et al., 2010). We were likewise perplexed by the continued prolyl hydroxylase of this and other EMT factors at time points when genes downregulated by them were strongly expressed. Thus, we examined the roles of SNAI1 during reprogramming.
Discussion Fibroblasts are the typical starting population for somatic cell reprogramming, and prior studies have indicated that reprogramming involves an MET. Paradoxically, however, transcription factors associated with EMT are expressed early in the reprogramming process and are not downregulated until the later stages (Samavarchi-Tehrani et al., 2010). While previously it was reported that keratinocytes could be reprogrammed with higher efficiency because of their pre-existing epithelial status (Maherali et al., 2008), a side-by-side comparison between cell types has not been done. In a secondary reprogramming system that enables direct comparison, we found mouse keratinocytes were reprogrammed less efficiently (0.02%) than fibroblasts (0.3%) (Figure S3E), and we have discovered ectopic expression of the EMT factor SNAI1 during early stages of reprogramming enhances efficiency in keratinocytes, an epithelial cell type. Thus, the effect of expression of EMT factors in the initial phase of reprogramming is not limited to mesenchymal target cell populations, but also occurs in epithelial cells, suggesting mesenchymal factor expression is an important aspect of reprogramming independent of starting cell type. Manipulating SNAI1 has led us to a multistep model of reprogramming whereby mesenchymal factors are expressed early and contribute to the reprogramming-amenable state (Koche et al., 2011), and only thereafter are pluripotency factors expressed en route to the pluripotent state seen in iPSCs (Figure 4H). Our data are in agreement with a recent study (Liu et al., 2013), which reported early enhancement of the mesenchymal state increased reprogramming efficiency. A role for let-7 in reprogramming has been established since its inhibition increases reprogramming efficiency (Melton et al., 2010). As shown here, SNAI1 binds several let-7 promoters, and SNAI1 expression is associated temporally with downregulation of let-7 miRs early in reprogramming, consistent with prior evidence that EMT factors suppress let-7 expression in cancer (Yang et al., 2012). Moreover, overexpressing SNAI1 in a poorly reprogramming strain augments both reprogramming efficiency and SNAI1 binding to the let-7 promoter, suggesting SNAI1 regulation of let-7 may be the basis for enhanced reprogramming efficiency. The downregulation of let-7 transcription by SNAI1 may be associated with upregulation of LIN28 by pluripotency factors, thereby potently reversing the differentiated state. While let-7 is downregulated in the first week of reprogramming, its expression appears to recover thereafter before again diminishing to near zero in the iPSC state (Figures 4E, 4F, and S4D). We have not studied this biphasic expression pattern, but we hypothesize the second wave is extinguished by LIN28. Prior studies have demonstrated that Twist promotes a stem cell phenotype in cancer, including self-renewal (Mani et al., 2008). We hypothesize that expression of SNAI1 might similarly promote a stem cell-like phenotype in fibroblasts and keratinocytes, moving them one step closer to dedifferentiation and making them more amenable to reprogramming. We propose that suppression of let-7 miRs is a mechanism whereby SNAI1 might be acting to confer these stem cell properties. Building on the model proposed by Samavarchi-Tehrani et al. and Li et al. demonstrating the role of MET in reprogramming, we show during the early phases of reprogramming, mesenchymal factors are expressed, and further ectopic expression of EMT factors enhances reprogramming efficiency. Our results provide a more nuanced view of the role of EMT factors in the reprogramming of both mesenchymal and epithelial cell types. Our data are corroborated by an independent study that identified SNAI1 in an unbiased shRNA screen as a factor that enhances conversion of pre-iPS cells to a fully reprogrammed state, thereby reinforcing the conclusion that SNAI1 acts to enhance reprogramming (Gingold et al., 2014). This improved understanding of the mechanism of reprogramming will provide strategies to improve its utility for modeling and treating disease and advance our insight into the regulation of gene expression and pluripotency.