The transcription factor code in iPSC reprogramming

Abstract

Transcription factor (TF)-induced reprogramming of somatic cells across lineages and to induced pluripotent stem cells (iPSCs) has revealed a remarkable plasticity of differentiated cells and presents great opportunities for generating clinically relevant cell types for disease modeling and regenerative medicine. The understanding of iPSC reprogramming provides insights into the mechanisms that safeguard somatic cell identity, drive epigenetic reprogramming, and underlie cell fate specification in vivo. The combinatorial action of TFs has emerged as the key mechanism for the direct and indirect effects of reprogramming factors that induce the remodelling of the enhancer landscape. The interplay of TFs in iPSC reprogramming also yields trophectoderm- and extraembryonic endoderm-like cell populations, uncovering an intriguing plasticity of cell states and opening new avenues for exploring cell fate decisions during early embryogenesis.

Figure 1:. Cell state transitions and global gene expression changes in iPSC reprogramming

A) Roadmap of iPSC induction from somatic cells. Upon expression of OSKM, a diminishing pool of cells transitions through sequential stages towards the iPSC state. In addition to cells stalling along the productive reprogramming path, the formation of alternative cell states explains the low efficiency of iPSC generation. The proportion of cell states at each stage is strongly influenced by the experimental reprogramming system and culture medium [10-12,15]. B) Key gene expression dynamics during OSKM-induced reprogramming. Regardless of the starting somatic cell type, three broad gene expression changes occur on the productive path to iPSCs: somatic program silencing, transient program expression, and pluripotency program activation. Pluripotency program activation occurs gradually with the upregulation of cell cycle, biosynthesis, chromatin remodeling genes, and culminates in the activation of endogenous pluripotency-related TFs. Somatic gene repression and pluripotency gene activation, previously thought to be separated temporally, can overlap in individual cells [12]. It is still largely unclear how the expression of the transient program relates to the silencing of the somatic program and the activation of the pluripotency program.

Figure 2:. Enhancer reorganization during reprogramming is linked to distinct TF binding and motif patterns

A) Key enhancer and associated TF binding changes during reprogramming. Very early in reprogramming, OSK bind a fraction of somatic enhancers as well as transient enhancers and a subset of pluripotency enhancers. At transient and early-engaged pluripotency enhancers, OSK co-bind with somatic TFs. Over time, early engaged pluripotency enhancers gain the binding of additional TFs throughout reprogramming (such as NANOG), which replaces the binding of somatic TFs. The majority of pluripotency enhancers is engaged later in the process (late-engaged) by O and S (without K) and requires additional TFs (for instance ESRRB) that are activated during the reprogramming process. In starting fibroblasts, both early and late-engaged pluripotency enhancers lack hypersensitivity (based on ATAC-seq) and reprogramming factor binding coincides with substantial nucleosome removal. Based on the presence and absence of DNA sequence motifs (as shown on the right), it is thought that OSK engage transient and pluripotency enhancers through direct DNA binding and interact with somatic enhancers largely indirectly. B) Somatic TF redistribution model. Early in reprogramming, OSK recruit broadly expressed somatic TFs such as AP-1, CEBP, TEAD (orange symbols) to new sites in transient and pluripotency enhancers, depleting them from somatic enhancers. Since somatic cell-specific TF occupancy at somatic enhancers depends on the presence of broadly expressed somatic TFs, the binding of somatic cell-specific TFs (yellow symbols) is also decreased in this process. The redistribution of broad somatic TFs and the loss of somatic cell-specific TFs lead to the destabilization of fibroblast enhancers and the repression of the somatic gene program. In this model, OSK inactivate somatic enhancers indirectly, without the need for direct binding to somatic enhancers. C) Putative roles for somatic TF binding at early-engaged pluripotency enhancers. From left to right: broadly expressed somatic TFs may collaborate with OSK to remove nucleosomes if their binding sites are within one nucleosome length, and therefore be required for enhancer opening early in reprogramming; somatic TFs passively bind to DNA in regions opened by OSK; and somatic TFs indirectly bind through protein-protein interaction with OSK or co-factors. In the latter two cases, somatic TFs may not have a specific function or, alternatively, may block the activation of these enhancers.

Figure 3:. Strategies for producing iPSCs, iTSCs and iXENs

A) The existence of cells expressing endodermal TFs such as GATA4 and GATA6 in OSKM-induced iPSC reprogramming cultures can be exploited to, in addition to iPSCs, derive iXENs by exposing the reprogramming culture to a culture medium that supports iXENs [20]. Gata6 expression is required for iXEN formation. Similarly, iTSCs and iPSCs can be derived from human OSKM reprogramming cultures [10] (not shown). B) iTSCs, iXENs and iPSCs can also be derived from a reprogramming culture upon expression of an alternative TF cocktail (GETMS), when appropriate media are supplied [53]. Whether XEN-like cells arise in parallel to or on the path to iPSCs remains unclear. The balance of EOMES and ESRRB influences which cell states are formed during reprogramming. High EOMES levels favor iTSC induction, whilst high ESRRB favors iXEN and iPSC induction.