This study aimed to uncover the contribution of the reprogramming factors to the induction of pluripotency. During the reprogramming process, fibroblast markers are repressed and early embryonic markers like SSEA1 activated before expression of pluripotency regulators and the self-sustaining pluripotent state are attained (Brambrink et al., 2008
; Stadtfeld et al., 2008
). In agreement with these observations, fibroblast-specific genes are efficiently silenced in partially reprogrammed cells, while the embryonic program is not fully induced. Clones obtained in fibroblast reprogramming experiments with ectopic expression of only Oct4, Klf4 and c-Myc are also characterized by repression of fibroblast-specific genes although not to the same extent as in our four factor induced partially reprogrammed cell lines (Supp Fig 10
). Thus, silencing of the somatic cell expression program appears to be an important initial step required for the induction of the ES-like expression program. Our data indicate a major contribution of c-Myc to this first step. We found that c-Myc promotes the most ES-like expression changes, including the repression of fibroblast-specific genes, of the four factors (). Mechanisms by which c-Myc induces transcriptional repression are much less understood than its function as a transcriptional activator (Wanzel et al., 2003
). Global repressive effects of c-Myc could be mediated through its interaction with the transcription factor Miz (Wu et al., 2003
) or direct binding and activation of a transcriptional repressor. Interestingly, when murine fibroblasts were treated for seven days with valporic acid (VPA), a histone deacetylase inhibitor that can replace c-Myc function during reprogramming, their expression started to resemble an ES-like state, including the repression of highly transcribed fibroblast-specific genes (Huangfu et al., 2008
). Thus, c-Myc expression or VPA treatment may lay the framework for the efficient repression of the somatic expression and induction of the ES cell expression program.
Role of the four factors during reprogramming
The comparison of binding patterns of the factors in iPS/ES cells and partially reprogrammed cell lines further strengthens the conclusion that the contribution of c-Myc to the reprogramming process is separable from that of the other three factors. The metabolism-related embryonic expression program is induced by binding of combinations of all four factors (), while the activation of many regulators of the pluripotent state is dependent on co-binding of only Oct4, Klf4, and Sox2 (), which does not occur in partially reprogrammed cells (). Indeed, ectopic expression of c-Myc is only required for the first few days of the reprogramming process. We propose that lack of Oct4, Sox2 and Klf4 co-binding in partially reprogrammed cells contributes to the failure to establish the pluripotent state. During reprogramming, Oct4, Sox2 and Klf4 can likely only stochastically overcome this binding block contributing to the low efficiency of this conversion.
An important question is why Oct4, Sox2 and Klf4 cannot associate with their ES cell target genes in partially reprogrammed cells. One explanation could be the absence of other factors, such as Nanog, that allow targeting of the reprogramming factors. These factors could form large complexes necessary for cooperative binding to target genes or could induce a conformational change of DNA allowing subsequent binding by Oct4, Sox2 and Klf4. Alternatively, the target promoters may have repressive chromatin structures that prevent appropriate binding of the factors. Indeed, our data demonstrate that a gain in histone H3K4 methylation is specifically associated with the activation of many pluripotency regulators (). In partially reprogrammed cells, the methylation status is not in an ES-like pattern and the reprogramming factors do not bind appropriately suggesting that the establishment of the ES-like histone methylation pattern is either a requirement for or coincides with the recruitment of the factors. Elucidating how the methylation pattern transitions to an ES-like state and how Oct4, Sox2 and Klf4 are recruited to genes encoding pluripotency regulators should allow for the development of more efficient reprogramming strategies.
Interestingly, c-Myc is dispensable for fibroblast reprogramming as both human and murine iPS cells can be obtained in the absence of c-Myc, albeit with dramatically reduced efficiency and kinetics (Nakagawa et al., 2008
; Wernig et al., 2008
). The fact that activation of transcriptional regulators of pluripotency is largely independent of c-Myc binding provides an explanation for why c-Myc is not absolutely required for reprogramming. Our data indicate that reprogramming is delayed and inefficient in the absence of ectopic c-Myc expression because c-Myc greatly enhances the initial steps of reprogramming. Similarly, the fact that Oct4, Sox2 and Klf4 are targeted in a more ES-like pattern in partially reprogrammed cells to genes that are co-bound by c-Myc suggests that its presence facilitates the binding of the other factors. In addition, c-Myc normally fulfills many other functions, for example in regulating DNA replication (Dominguez-Sola et al., 2007
) and global histone acetylation (Knoepfler et al., 2006
) which may facilitate the reprogramming process more indirectly.
Once reprogramming is complete the binding pattern of the four factors is similar to that of ES cells suggesting that a common binding signature has to be achieved to attain pluripotent gene expression. Differences in binding between these ES and iPS cells occur generally in genes that are bound more weakly. In the future, it will be important to determine whether any of these binding differences reflect an epigenetic memory of the starting cell population that may influence differentiation behavior.