For ESCs on the other hand we

For ESCs, on the other hand, we and others recently established that single factors, such as neurogenic differentiation factor 1 (NEUROD1) or neurogenin 2 (NGN2), alone are sufficient to rapidly induce the neuronal fate (Thoma et al., 2012; Zhang et al., 2013). In fibroblasts, however, we had originally observed that ASCL1 can induce neuronal XAV-939 cost only with very immature features, suggesting that single factors may initiate, but cannot complete, the reprogramming process (Vierbuchen et al., 2010). This raised interesting questions about the capacity and relative contribution of reprogramming factors toward neurogenesis from different cellular lineages. Our recent studies suggested a clear hierarchical role of the reprogramming factors, as ASCL1 alone, of the three BAM factors, immediately and directly accessed the majority of its cognate target sites in the fibroblast chromatin as a pioneer factor (Wapinski et al., 2013). BRN2 and MYT1L, on the other hand, bind to ectopic sites in a tight cell-context-specific manner and appear to be mainly required at later reprogramming stages. This suggests that ASCL1 might be the central driver of iN cell reprogramming, but it remained unclear whether ASCL1 is sufficient to induce generation of mature iN cells without further assistance from BRN2 and MYT1L.
Results
Discussion We previously found that the combined expression of three transcription factors (BAM) is required to induce fully functional iN cells from fibroblasts (Vierbuchen et al., 2010). Under the same conditions, single factors could only generate cells that had some neuronal characteristics but lacked critical others such as morphological and functional properties. Based on this observation, we had assumed that single factors can only initiate a partial reprogramming toward iN cells and additional factors are required to complete the reprogramming process. In this study, we challenged this hypothesis and demonstrate instead that cells reprogrammed with the single factor ASCL1 are in fact fully reprogrammed to a neuronal lineage but are simply less mature compared to cells reprogrammed with all three factors at early time points. Later in the reprogramming process, the single-factor iN cells can reach maturation levels almost equivalent to three-factor cells. This conclusion has important implications on how we view the molecular mechanism of iN cell reprogramming. Obviously, ASCL1 is the single most important driver of reprogramming, and success or failure of reprogramming of a given cell type will critically depend on the efficient engagement of ASCL1 with the proper chromatin targets. We recently identified an intriguing trivalent chromatin state (consisting of high H3K4 monomethylation, high H3K27 acetylation, and low H3K9 trimethylation levels) associated with ASCL1 targets in MEFs and potentially important for the correct targeting of ASCL1 to its proper sites (Wapinski et al., 2013). Now, with the knowledge that ASCL1 alone is sufficient to generate mature iN cells, these ASCL1-specific chromatin findings are even more relevant than originally assumed. In particular, for future attempts to generate iN cells from thus-far reprogramming-resistant cells such as keratinocytes or blood cells, the efforts should focus on targeting ASCL1 to its proper chromatin sites. The other two transcription factors, BRN2 and MYT1L, are not less important, but their predominant role appears to be to enhance neuronal maturation and less to contribute to the cell lineage conversion mechanism. These studies would predict that Pou-domain-containing and MYT-domain-containing transcription factors also act as maturation factors during normal neural development. Furthermore, ASCL1 can activate endogenous Myt1l and Brn2 expression, which supports the notion that these two transcription factors are responsible for neuronal maturation also in ASCL1-induced iN cells.