Direct lineage reprogramming via pioneer factors; a detour through developmental gene regulatory networks

Abstract

Although many approaches have been employed to generate defined fate in vitro, the resultant cells often appear developmentally immature or incompletely specified, limiting their utility. Growing evidence suggests that current methods of direct lineage conversion may rely on the transition through a developmental intermediate. Here, I hypothesize that complete conversion between cell fates is more probable and feasible via reversion to a developmentally immature state. I posit that this is due to the role of pioneer transcription factors in engaging silent, unmarked chromatin and activating hierarchical gene regulatory networks responsible for embryonic patterning. Understanding these developmental contexts will be essential for the precise engineering of cell identity.

Keywords: Cell fate engineering; Direct lineage reprogramming; Gene regulatory networks; Pioneer transcription factors.

Fig. 1.

Pioneer factors used in direct lineage conversions from fibroblasts. Pioneer transcription factors have been used in many instances of direct conversion of cell fate between differentiated states, both in mouse and human. These include the conversion of fibroblasts to cardiomyocytes (Ieda et al., 2010), hepatocytes (Huang et al., 2011; Sekiya and Suzuki, 2011; Zhu et al., 2014), glutamatergic neurons (Ambasudhan et al., 2011; Pang et al., 2011; Vierbuchen et al., 2010; Yoo et al., 2011), dopaminergic neurons (Caiazzo et al., 2011; Pfisterer et al., 2011), motor neurons (Son et al., 2011), hematopoietic progenitors (Pereira et al., 2013; Szabo et al., 2010), neural progenitors (Lujan et al., 2012; Thier et al., 2012), bipotent hepatic progenitors (Yu et al., 2013), angioblast-like progenitor cells (Kurian et al., 2013) and osteoblasts (Yamamoto et al., 2015). Other cell type conversions (not shown) include hepatocyte to neuron conversion (Marro et al., 2011), and cortical astrocytes to glutamatergic (Heinrich et al., 2010) and GABAergic (Berninger et al., 2007; Heinrich et al., 2010) neurons. This list is not exhaustive and continues to expand. Conversion from fibroblasts to progenitor states are circled in red. Pioneer factors involved in each conversion are shown in red text, cooperative factors are shown in black text.

Fig. 2.

Mechanism of pioneer factor activity. Pioneer factor binding results in the local opening of silent, unmarked chromatin, which confers lineage competence, although in the absence of any other transcription factors this in itself is insufficient to induce changes in gene expression. Subsequent pioneer factor-mediated recruitment of activators or repressors, which by themselves are unable to engage with silent chromatin, initiates transcriptional programs leading to cell fate change.

Fig. 3.

Gene regulatory network (GRN) hierarchy and pioneer factor activity. The embryonic body plan is initially laid down by upper-level GRNs (see Peter and Davidson, 2011). This creates a primary spatial organization within the embryo, enabling the initiation of regional fate decisions via the implementation of successive upper-level GRNs. Intermediate GRNs specify cell identity in the appropriate regions in order to define tissue pattern, laying the foundations for cell fate specification GRNs. Finally, the hierarchy concludes with GRN ‘batteries’ that control the final stages of cell differentiation. In direct lineage conversion, FoxA factors engage with upper-level GRNs to generate induced hepatocytes (iHeps). Ascl1 engages with downstream cell fate specification GRNs to produce induced neurons (iNs). Installation of GRN batteries is achieved via expression of maturation factors (Brn2 and Myt1l) or a period of engraftment within the in vivo niche. Pioneer factors are shown in red text, cooperative factors in green text.

Fig. 4.

Possible lineage reprogramming strategies. (A) Pioneer factor-mediated conversion of cell type A (orange) to cell type B (purple) depends on transition via a progenitor state (red circle), from which point cells must be matured. This approach is more feasible given the function of pioneer factors (red) to engage silent chromatin and recruit additional cooperative factors (green) to activate developmental GRNs. (B) Non-pioneer factor-mediated conversion aims to directly convert cells between fully differentiated states without reversion to a progenitor state. This could theoretically be achieved via the expression of terminal selector genes, which activate GRN batteries, although this approach is relatively infeasible given the low likelihood of these factors being able to engage silent chromatin in the host cell.