Exploring the reciprocity between pioneer factors and development Free

During development, the single-celled zygote divides and differentiates, eventually giving rise to all the cells of an adult organism. The cells of the adult are specialized and phenotypically distinct, but all share a common genome formed at fertilization. Therefore, it is the differential interpretation of the shared genome that determines cell fate. Transcription factors orchestrate this process by recognizing and binding to specific DNA motifs to drive gene expression. However, the packaging of DNA into chromatin is a barrier to transcription-factor binding. The nucleosome is the basic repeating unit of chromatin and is composed of approximately 147 base pairs of DNA wrapped twice around an octamer of histones, comprising two each of H2A, H2B, H3 and H4. H1 linker histones stabilize nucleosomes and help establish higher-order chromatin structure, including arrays of nucleosomes (Fyodorov et al., 2018; Kornberg and Thomas, 1974; Li et al., 2007). Nucleosome compaction enables the approximately two meters of DNA to fit in the eukaryotic nucleus. This compact chromatin structure prevents many transcription factors from accessing their binding motifs. Instead, transcription factors often bind their motifs within open and accessible chromatin. By contrast, a specialized class of transcription factors, termed pioneer factors, bind closed, nucleosomal DNA to promote regions of open chromatin that can be subsequently bound by other transcription factors. Because of the ability to define open chromatin, pioneer factors act at the top of gene-regulatory networks to define cell fate.

Despite their ability to target closed, nucleosome-occupied binding sites, pioneer factors do not bind all genomic instances of their target motif. Instead, they have cell type-specific patterns of binding and activity in promoting chromatin accessibility. Emerging evidence suggests that pioneer-factor occupancy and activity is regulated by the cell type in which it is expressed. Below, we explore the reciprocal interplay between pioneer factors and development. We initially provide a perspective on the role of pioneer factors in driving developmental transitions. We then discuss the mechanism by which pioneer factors bind chromatin and generate accessible cis-regulatory regions. Finally, we focus on how developmental context can influence the ability of pioneer factors to access the genome and promote gene expression.

Because of their ability to promote widespread changes in gene expression, pioneer factors drive developmental transitions, and their dysregulation results in disease.

Pioneer factors promote developmental transitions by defining the cis-regulatory regions of the gene-regulatory networks that drive specific cell fates. The first pioneer factor to be identified was Foxa1, discovered in mice based on its ability to bind and open the albumin enhancer, necessary for liver specification, prior to transcriptional activation of this gene (Gualdi et al., 1996). During endoderm differentiation, this initial opening of chromatin by Foxa1 facilitates the subsequent binding of tissue-specific transcription factors needed for lineage specification (Cirillo et al., 2002; Iwafuchi-Doi et al., 2016; Wang et al., 2015). Foregut endoderm-specific knockout of Foxa1 and Foxa2 results in the failure to initiate hepatic differentiation, highlighting the essential role of this family of pioneer factors in promoting the gene expression program required for normal development of the liver (Lee et al., 2005). In humans, the related factor FOXA2 is similarly required for pancreatic differentiation from pluripotent stem cells (Lee et al., 2019). Since the initial characterization of the pioneering functions of Foxa1, the vital roles of many different pioneer factors in promoting developmental transitions have been identified across a range of species (Balsalobre and Drouin, 2022; Barral and Zaret, 2024; Horisawa and Suzuki, 2023; Larson et al., 2021; Mayran and Drouin, 2018).

The importance of pioneer factors in development is evident from the conservation of their pioneering function across species. For example, in Caenorhabditis elegans, the FoxA ortholog PHA-4 is a pioneer factor required for specifying foregut fate. Like the mammalian FoxA family members, it binds nucleosomes in vitro and promotes chromatin accessibility and gene expression in vivo (Hsu et al., 2015). The Grainy head (GRH) family of transcription factors are a set of conserved pioneer factors defined by their similarity to the Grainy head protein that was originally discovered in the fruit fly (Drosophila melanogaster) (Bray et al., 1989; Dynlacht et al., 1989; Nüsslein-Volhard et al., 1984; Venkatesan et al., 2003). In Drosophila, Grh promotes chromatin accessibility and is required for expression of genes necessary for defining epithelial cell fate (Jacobs et al., 2018; Nevil et al., 2020; Wang and Samakovlis, 2012). The role of this pioneer factor in epithelial cell-fate specification is conserved in worms, frogs and mammals (Gasperoni et al., 2022). Mammals have three Grainy head-like (GRHL) proteins (Reese et al., 2019; Wilanowski et al., 2002). Expression of GRHL2 in naïve embryonic stem cells (ESCs) is required to maintain expression of epithelial genes as ESCs transition to formative epiblast-like cells (EpiLCs) (Chen et al., 2018). In the absence of GRHL2, EpiLCs fail to adopt an epithelial fate and instead transition to a mesenchymal state (Chen et al., 2018). These few examples highlight the conserved role of pioneer factors in promoting cell-fate specification in organisms ranging from nematodes to humans.

To enhance cell-fate transitions, pioneer factors not only activate specific gene-expression programs, but also shut off gene-expression networks for alternative cell fates. During mammalian pituitary development, Pax7 defines melanotrope identity by stimulating expression of melanotrope-specific genes and repressing genes that drive corticotrope fate (Budry et al., 2012). PU.1 (also known as SPI1) is a pioneer factor that plays a role in immune cell differentiation (Li et al., 2020) and functions through both activating and repressing gene expression (Li et al., 2020; Ungerbäck et al., 2018). This repression is thought to be indirect through the sequestration of factors away from non-PU.1 targets (Hosokawa et al., 2018). As hair follicle stem cells differentiate from multipotent embryonic progenitors, SOX9 opens enhancers of genes defining hair follicle stem cells. This results in the simultaneous silencing of epidermal enhancers through recruitment of co-factors away from these alternative-lineage enhancers (Yang et al., 2023). Pioneer factors can also directly facilitate repression. For example, all three FOXA proteins are important for silencing expression of alternative lineages during foregut differentiation. This is likely mediated by their recruitment of chromatin factors, including the enzyme PRDM1, which promotes a chromatin state that represses gene expression (Matsui et al., 2024). The pioneer factor OCT4 (also known as POU5F1) also interacts with a related chromatin modifying enzyme, PRDM14, suggesting this role in transcriptional repression may be shared amongst a distinct class of pioneer factors (Matsui et al., 2024). Thus, pioneer factors promote robust cell-fate changes by directly initiating chromatin accessibility at enhancers to drive gene expression and by repressing expression of genes that specify alternative cell fates.

The capacity of pioneer factors to prompt large-scale changes in gene expression is required for cellular reprogramming both in the early embryo and in cell culture. After fertilization, the genome of the unified gametes is reprogrammed to generate the totipotent state of the early embryo. This conserved developmental transition relies on maternally deposited mRNAs and proteins, as the zygotic genome is transcriptionally quiescent. Gradual activation of zygotic transcription is coordinated with the degradation of these maternal products during the maternal-to-zygotic transition (MZT) (Schulz and Harrison, 2019; Vastenhouw et al., 2019). In all organisms studied to date, pioneer factors drive the efficient reprogramming of the zygotic genome during these early developmental stages. The Drosophila protein Zelda was the first major activator of the zygotic genome to be discovered (Liang et al., 2008). Zelda is required broadly for restructuring chromatin accessibility and influences the 3D structure of the genome (Hug et al., 2017; Schulz and Harrison, 2019; Schulz et al., 2015; Sun et al., 2015; Brennan et al., 2023). Zelda is not the only pioneer factor essential for the progressive activation of zygotic transcription in Drosophila. Additional pioneer factors, including GAGA factor (GAF; also known as Trl) and CLAMP, function with Zelda to restructure and activate the early embryonic genome (Duan et al., 2021; Gaskill et al., 2021). Whereas Zelda is preferentially required for the earliest wave of zygotic gene expression, GAF is required slightly later in development (Gaskill et al., 2021). Following the MZT, embryonic patterning is established by another pioneer-like factor, Odd paired (Opa) (Koromila et al., 2020; Soluri et al., 2020). These studies highlight the importance of sequential pioneer-factor activity in promoting the highly conserved reprogramming that occurs following fertilization.

Since the discovery of Zelda in flies, the essential role of pioneer factors in early embryonic reprogramming has been established in numerous other organisms. In zebrafish (Danio rerio), the pioneer factors Pou5f3, Sox19b and Nanog cooperatively regulate zygotic genome activation (Lee et al., 2013; Leichsenring et al., 2013; Miao et al., 2022; Veil et al., 2019). Similarly, in frogs (Xenopus laevis), Pou5f3 (also known as Oct4), Sox3 and Foxh1 remodel early embryonic chromatin to regulate activation of the zygotic genome (Blitz and Cho, 2021; Charney et al., 2017; Gentsch et al., 2019; Paraiso et al., 2019). The conserved pioneer factors DUX4 and Dux, found in humans and mice, respectively, promote chromatin accessibility and gene expression during the earliest stages of embryo development (De Iaco et al., 2017; Hendrickson et al., 2017; Whiddon et al., 2017). However, some Dux mutant embryos are viable, indicating that Dux is important but not essential for mouse embryogenesis (Chen and Zhang, 2019; De Iaco et al., 2017). Like flies, zebrafish and frogs, mammalian embryonic reprogramming involves multiple pioneer factors. The PRD-like homeobox domain transcription factor family (Obox1-Obox8) and orphan nuclear receptor Nr5a2 are key activators of zygotic transcription in mice (Gassler et al., 2022; Ji et al., 2023). Although neither Dux nor Obox4 is individually essential for early mouse development, they are redundantly required for viability and zygotic genome activation (Guo et al., 2024 preprint). The parallel requirement for the cooperative action of pioneer factors in early embryonic reprogramming in many species highlights the importance of understanding how these proteins function together to robustly drive this conserved developmental transition.

In vitro reprogramming of differentiated cells into induced pluripotent stem cells (iPSCs) through the expression of a cocktail of transcription factors shares many features with the much more efficient in vivo reprogramming that occurs in the early embryo. The initial cocktail of transcription factors used to generate iPSCs from mammalian fibroblasts was Oct4, Sox2, Klf4 and Myc (OSKM) (Takahashi and Yamanaka, 2006). Although at the time the underlying mechanisms by which these factors drove reprogramming was unclear, it has since become evident that the unique ability of these factors to reprogram relies on their pioneering abilities. Oct4, Sox2 and Klf4 (OSK) are pioneer factors and are sufficient to drive reprogramming independently of Myc (Nakagawa et al., 2008; Soufi et al., 2012, 2015). Nonetheless, Myc enhances OSK binding (Soufi et al., 2012). As in early embryonic reprogramming, OSK function cooperatively in the generation of iPSCs by binding and opening enhancers that drive the pluripotency gene network as well as silencing the somatic program by promoting the decommissioning of somatic enhancers (Chronis et al., 2017; Li et al., 2017). Nucleosome binding is correlated with the reprogramming ability of transcription factors (Fernandez Garcia et al., 2019), suggesting that pioneering activity is a driver of efficient reprogramming. Together, these examples highlight the importance of collaborative interactions among multiple pioneer factors in activating a pluripotency network and erasing the features of a prior cellular state.

Pioneering activity is required for transdifferentiation, the process by which one mature somatic cell is transformed into another somatic cell type without transitioning through an intermediate, less-differentiated cell type. This is exemplified by the conversion of mouse or human fibroblasts into functional dopaminergic neurons through a cocktail of transcription factors that primarily depends on the pioneer factor Ascl1/ASCL1 (Marro and Yang, 2014; Vierbuchen et al., 2010; Wapinski et al., 2013). ASCL1 is a proneural basic helix-loop-helix (bHLH) transcription factor that induces neural differentiation in vivo (Bertrand et al., 2002). When exogenously expressed in fibroblasts, ASCL1 occupies most of the same sites it normally occupies in neurons and promotes chromatin accessibility. Binding to these sites persists as cells undergo transdifferentiation into neurons (Wapinski et al., 2013). FoxA proteins are also important in transdifferentiation. When expressed in mouse fibroblasts along with either Hnf1α and Gata4 or Hnf4α, FoxA proteins direct reprogramming into hepatocyte-like cells. In this context, all three members of the FoxA family function as pioneer factors (Huang et al., 2011; Sekiya and Suzuki, 2011). With the expression of other co-factors, Foxa2 can direct transdifferentiation of fibroblasts into dopaminergic neurons (Pfisterer et al., 2011).

During mitosis, the nuclear envelope breaks down, the genome becomes highly condensed, transcription ceases, the 3D genome organization is largely disrupted, and many transcription factors dissociate from the chromosomes (Antonin and Neumann, 2016; Gottesfeld, 1997; Naumova et al., 2013). Nonetheless, the gene-regulatory networks that maintain cell-fate specification must be re-established following division. How this process is achieved is not well understood. Even though most transcription factors do not remain associated with the mitotic chromosomes, a small subset of factors is retained. Some of these factors are required for proper reactivation of gene expression upon mitotic exit, a mechanism termed ‘mitotic bookmarking’. Pioneer factors comprise a large portion of those factors that are retained on mitotic chromosomes (Bellec et al., 2022; Caravaca et al., 2013; Chervova et al., 2024; Deluz et al., 2016; Festuccia et al., 2016, 2019; Liu et al., 2017; Nevil et al., 2020; Price et al., 2023; Silvério-Alves et al., 2023; Teves et al., 2016), suggesting that the capacity to bind closed chromatin may support retention on the compacted mitotic chromatin. However, not all pioneer factors are mitotically retained, for example both Zelda (in flies) and Ascl1 (in mice) are evicted from chromatin during mitosis (Dufourt et al., 2018; Soares et al., 2021). Additionally, some non-pioneer factors are retained on mitotic chromatin and retention is not correlated with pioneering activity (Raccaud et al., 2019). Thus, the features that define a pioneer factor are separable from those that enable mitotic retention.

Mitotic bookmarking by pioneer factors may promote rapid gene activation and the preservation of cellular identity following division. This is a specific challenge during early embryogenesis owing to the rapid division cycles. During the MZT in Drosophila, GAF is mitotically retained and required for the rapid activation of a reporter gene and transcriptional memory across mitosis (Bellec et al., 2022). By contrast, Zelda, which is not mitotically retained, is not necessary for maintaining this transcriptional memory through mitosis (Dufourt et al., 2018). In mouse ESCs, the pioneer factors Esrrb and Nr5a2 both coat mitotic chromosomes and, together, facilitate reactivation of gene expression following mitosis (Chervova et al., 2024). A challenge to these studies is the difficulty in specifically depleting proteins only during mitosis. Most studies have relied on generating proteins tagged with a domain of cyclin B1 that drives mitotic degradation during the metaphase-anaphase transition (Kadauke et al., 2012). This system has been used to demonstrate that mitotic expression of the reprogramming factors Oct4 and Sox2 is required for maintaining pluripotency and mouse ESC differentiation (Deluz et al., 2016; Liu et al., 2017). Although most of these studies have been performed in culture, mice homozygous for Gata2 fused with a mitotic degron phenocopied the Gata2 knockout, suggesting that mitotic expression of Gata2 is necessary for function (Silvério-Alves et al., 2023). However, it is not clear how rapidly protein expression is re-established following mitotic degradation, making it challenging to disentangle a role during mitosis from that during early interphase. Future, more acute methods of disrupting the mitotic localization of pioneer factors will be essential for determining the functional significance of their retention on mitotic chromatin.

Disturbances to the finely tuned processes governing cell-fate decisions culminate in developmental disorders and oncogenic transformations. As a result of the power of pioneer factors to reshape cell fate, their misexpression can promote disease states. Pioneer factor expression levels are indicators of poor prognosis for a growing list of cancers (Chiou et al., 2010; Jozwik and Carroll, 2012; Lemma et al., 2022; Matsuoka et al., 2012; Saigusa et al., 2009; Sholl et al., 2010; Sunkel and Stanton, 2021).

Cancer stem cells (CSCs) are drivers of new tumors in many cancers (Reya et al., 2001). Like ESCs, CSCs are capable of self-renewal, and this capacity is thought to promote tumor formation (Yu et al., 2012; Reya et al., 2001). The shared ability to self-renew suggests that common regulators may exist between CSCs and ESCs (Reya et al., 2001). Indeed, the core reprogramming factors OCT4 and NANOG have been identified as CSC markers (Chiou et al., 2008), and their overexpression leads to tumor formation in mice and is associated with aggressive nasopharyngeal carcinoma and metastasis in humans (Liu et al., 2024; Luo et al., 2013; Ohnishi et al., 2014; Wei et al., 2014). The pioneer factor DUX4 is commonly expressed in metastatic tumors, and its expression is correlated with poor survival rates (Pineda and Bradley, 2024). This suggests a causal relationship between pioneer factor-mediated reprogramming and tumor pathogenicity. These and numerous additional examples demonstrate how overexpression of a pioneer factor can drive a stem-cell-like fate, which, in the wrong context, leads to unwanted proliferation and cancer.

Pioneer factors also facilitate cancer progression through their ability to promote distinct cellular fates. The GRHL family of transcription factors suppress the epithelial-to-mesenchymal transition, a cell-fate transition that promotes metastasis (Cieply et al., 2012, 2013). Based on this, GRHL factors were thought to function as tumor suppressors. However, GRHL family members function as both tumor suppressors and oncogenes depending on the cell type, highlighting the complex nature of cancer progression (Reese et al., 2019). This may be due to the diversity of co-factors expressed in the different cell types from which these cancers arise. For example, in estrogen receptor (ER)-positive breast cancer cell lines, GRHL2 influences chromatin binding of phosphorylated ER, likely through the promotion of chromatin accessibility (Reese et al., 2022). In androgen receptor-expressing prostate cell lines, GRHL2 instead promotes binding and expression of this hormone receptor (Paltoglou et al., 2017). FOXA1 pioneer factors also influence hormone-receptor binding and may even function together with GRHL2 in some cell types (Carroll et al., 2005; Cocce et al., 2019; Jozwik et al., 2016; Seachrist et al., 2021; Wang and Yin, 2015). Through their capacity to promote chromatin accessibility, pioneer factors allow unique cell type-specific factors to bind DNA and activate gene expression. The role of pioneer factors in disease is further exemplified by the misexpression of DUX4 in myoblasts, which results in the re-activation of genes and retrotransposons typically expressed during early embryonic development (Mocciaro et al., 2021; Young et al., 2013). Thus, the dysregulation of pioneer factors can lead to disease by promoting improper cell fates.

Translocations can generate chimeric pioneer factors with neomorphic functions that promote the disease state. For example, a translocation resulting in the expression of a chimera between PAX3 and FOXO1 is associated with rhabdomyosarcoma. The PAX3-FOXO1 chimera has features of pioneer factors, such as recognition of motifs within nucleosomes and activation of gene-regulatory networks involved in tumor development (Galili et al., 1993; Sunkel and Stanton, 2021). Another example of a chimeric oncoprotein is EWS-FLI1, which acts as an oncogenic pioneer factor to initiate the Ewing sarcoma gene-regulatory program in mesenchymal stem cells and is required for the growth and survival of these cancer cells (Riggi et al., 2008, 2014).

The unique ability of pioneer factors to access the genome and promote widespread changes in gene expression allows them to drive broad developmental transitions, but dysregulation of this activity results in disease. Thus, determining the mechanisms by which these proteins function will have important implications for understanding normal developmental processes and advancing targeted therapeutics.

Pioneer factors elicit changes in gene expression and cell identity by defining cell type-specific cis-regulatory regions. Promoters typically maintain accessibility across cell types so, although some pioneer factors in flies and mammals are associated with broadly accessible promoters, this binding is unlikely to drive developmental transitions because accessibility is shared across tissue types (Biggin and Tjian, 1988; Fuda et al., 2015; Lee et al., 2007; Li et al., 1994). However, a set of promoters have been identified in Drosophila that gain paused RNA polymerase II and chromatin accessibility over embryonic development, and this is driven by the pioneer factor Lola-I (Gaertner et al., 2012; Ramalingam et al., 2023). In contrast to promoters, accessibility at enhancer regions is regulated. Open enhancers are indicative of active gene expression, and pioneer factors largely elicit changes in gene expression by promoting accessibility at distally located enhancer elements (Spitz and Furlong, 2012).

Although pioneer factors share the ability to bind and open closed chromatin, the mechanisms by which such factors engage nucleosomal DNA and drive accessibility are varied (Zaret, 2020; Stoeber et al., 2024). Beyond creating accessible regions, pioneer factors can also influence chromatin by recruiting enzymes that chemically modify the histone tails or DNA and by regulating the 3D structure of the genome (Fig. 1).

To promote changes in chromatin accessibility, pioneer factors must first access and bind to nucleosomal DNA. Although this is a defining feature, pioneer factors have a wide diversity of DNA-binding domains (DBDs) and access the DNA through distinct mechanisms (Barral and Zaret, 2024). Structural studies have suggested multiple mechanisms, including binding partial motifs, that enable pioneer factors to engage DNA even when it is wrapped around histones (Fernandez Garcia et al., 2019; Ozden et al., 2023; Soufi et al., 2015). The location of a pioneer factor binding motif on the nucleosome might also regulate pioneer-factor binding. High-throughput in vitro studies of hundreds of human transcription factors (413 DBD and 46 full-length proteins) identified a diversity of ways that pioneer factors bind nucleosomes (Zhu et al., 2018). Many factors prefer to bind to the entry/exit sites of nucleosomes, but others bind periodically to the solvent-exposed side of DNA or to the dyad axis (center of the nucleosomal DNA) (Zhu et al., 2018). For example, both mammalian and Drosophila pioneer factors, Nr5a2 and Lola-I, respectively, bind preferentially to motifs located at the edge of the nucleosome (Gassler et al., 2022; Ramalingam et al., 2023). Binding at the entry/exit site may be facilitated by transient exposure of DNA as it relaxes from the nucleosome (Isaac et al., 2016; Poirier et al., 2008). Although extensive work has characterized binding in vitro, how nucleosome positioning affects pioneer-factor binding in vivo remains less clear. Analysis of the binding preferences of Grh and Zelda in Drosophila cell culture using MNase (micrococcal nuclease digestion with deep sequencing) and ChIP-seq (chromatin immunoprecipitation followed by sequencing) data failed to identify a preferred position for their binding motif within the nucleosome (Gibson et al., 2024). Indeed, nucleosomes at silent regions of chromatin are not well-positioned, suggesting that the exact positioning of a binding motif on a nucleosome may not strongly regulate pioneer-factor binding within a cell (Stergachis et al., 2020; Abdulhay et al., 2020).

Some pioneer factors rely on engagement with histones for nucleosome binding. For example, Foxa1 interacts with the core histones through a C-terminal domain, and this domain is required for chromatin accessibility in the mouse embryo (Cirillo et al., 2002; Iwafuchi et al., 2020). Based on its crystal structure, the yeast pioneer factor Cbf1 is speculated to interact with the H2A tail (Donovan et al., 2023), and the pluripotency factor OCT4 interacts directly with the histone H3 tail through its DBD (Echigoya et al., 2020; MacCarthy et al., 2022). These examples highlight how the structure and composition of nucleosomes can affect pioneer factor–nucleosome interactions.

Co-factor interactions may also contribute to stable nucleosome binding, and nucleosome engagement can be regulated by the cooperative activities of multiple factors. In mouse ESCs, the nucleosome binding activity of Parp1 stabilizes occupancy of the pioneer factor Sox2, possibly by specifically promoting Sox2 binding to nucleosomes in which the DNA motif is positioned sub-optimally for Sox2 engagement (Liu and Kraus, 2017). In addition to proteins that generally stabilize interactions with chromatin, sequence-specific factors can also regulate pioneer-factor occupancy. OCT4 binding to the nucleosome edge is enhanced by SOX2 co-binding (Michael et al., 2020), and binding of ASCL1 to nucleosomes is promoted by heterodimerization with E12α (Fernandez Garcia et al., 2019). Similarly, the transcription factor AP-1 may facilitate binding of different pioneer factors, including Foxa1 and GATA3, in distinct cell types (Bi et al., 2020; Xu et al., 2023 preprint). The expression of multiple pioneer factors can also regulate occupancy. The pioneer factors Nanog, Pou5f3 and Sox19b are required for reprogramming of the zygotic genome in the zebrafish embryo and can bind either independently or cooperatively depending on the genomic locus (Miao et al., 2022). Thus, co-factors can direct pioneer-factor binding and are important for stable occupancy of pioneer factors at some loci.

Like all transcription factors, pioneer factors rely on their DBD for interactions with the genome. Indeed, the DBDs of many factors, including Foxa1, Nr5a2, Cbf1 and Zelda, are sufficient to bind mono-nucleosomes in vitro, demonstrating that these diverse binding domains can engage DNA when wrapped around histones (Cirillo, 1998; Donovan et al., 2023; Fernandez Garcia et al., 2019; Gassler et al., 2022; McDaniel et al., 2019). Nonetheless, these domains alone are often insufficient to stably occupy chromatin and additional protein regions are required for pioneering activity. For example, regions outside of the zinc-finger DBD of Zelda are required to facilitate robust binding to mono-nucleosomes in vitro (Fernandez Garcia et al., 2019; McDaniel et al., 2019). The in vivo chromatin environment is more complex than the nucleosomes used in vitro, and regions outside the DBD of pioneer factors are required for stable occupancy. For example, the DBD of Drosophila pioneer factors Grh and Zelda are not able to bind regions of closed chromatin that are pioneered by the full-length proteins (Gibson et al., 2024). Similarly, single-molecule assays of pioneer factors FOXA1 and SOX2 in human fibroblasts demonstrated that their DBDs alone are less efficient than the full-length proteins at scanning silent chromatin (Lerner et al., 2023). Overall, although pioneer factors share the ability to bind closed chromatin, they vary in the mechanisms by which they engage the genome.

Depending on how pioneer factors bind chromatin, they can promote accessibility either directly or indirectly. Pioneer factors can directly influence local chromatin opening by binding to the nucleosome (Cirillo et al., 2002; Donovan et al., 2019; Guan et al., 2023; Michael et al., 2020; Mivelaz et al., 2020; Sinha et al., 2023; Zhu et al., 2018). The DBD of FoxA family members are winged helix domains that are structurally similar to linker histones (Dai et al., 2021), allowing them to displace linker histone H1 and promote accessibility independently of additional factors both in vitro and in the mouse liver (Cirillo et al., 2002; Iwafuchi-Doi et al., 2016; Taube et al., 2010). In vitro binding of OCT4, SOX2 and SOX11 can destabilize the interactions of DNA with the core histones and more broadly shape inter-nucleosome interactions by repositioning histone tails (Dodonova et al., 2020; Guan et al., 2023; Michael et al., 2020). Regions outside the DBD may be important for this intrinsic ability to open chromatin, as is the case for PU.1 and OCT4 in vitro (Frederick et al., 2023; Sinha et al., 2023). Although the mechanisms of chromatin opening, like chromatin binding, are diverse, many pioneer factors share the intrinsic ability to directly open chromatin.

Pioneer factors also indirectly mediate chromatin accessibility via interactions with ATP-dependent chromatin remodelers, enzymes that can restructure chromatin by altering the interaction between histones and DNA (Fig. 1A) (Ahmad et al., 2024; Clapier et al., 2017). Even pioneer factors that can initially open chromatin independently of other factors may require chromatin remodelers to fully remodel the genome. For example, in vitro, PU.1 alone opens chromatin within an array of multiple nucleosomes, but recruitment of the chromatin remodeler SWI/SNF promotes further chromatin opening, which may lead to more stable, widespread opening of chromatin (Frederick et al., 2023; Minderjahn et al., 2020). Oct4 recruits the central catalytic subunit of SWI/SNF chromatin remodeling complex, Brg1 (Smarca4), to shape the chromatin accessibility at target sites in mouse ESCs (Huang et al., 2021; King and Klose, 2017). GATA3 similarly depends on BRG1 to create accessible chromatin regions during the mesenchymal-to-epithelial transition in human breast cancer cells (Takaku et al., 2016). Numerous other pioneer factors have been shown to interact with members of the SWI/SNF complex (Gouhier et al., 2024; Judd et al., 2021; Mivelaz et al., 2020; Păun et al., 2023; Wolf et al., 2023), suggesting that many pioneer factors may function through SWI/SNF to stably open chromatin and promote expression of gene-regulatory networks. A single pioneer factor can also synergize with multiple ATP-dependent chromatin remodelers to open chromatin and drive gene expression. For example, GAF interacts with remodelers from both the SWI/SNF family and ISWI families (Lomaev et al., 2017; Nakayama et al., 2012); its interactions with PBAP (SWI/SNF) promote chromatin accessibility and RNA polymerase II recruitment, whereas its interactions with NURF (ISWI) ensure the efficient release of elongating RNA polymerase II (Judd et al., 2021).

Pioneer factors recruit histone-modifying enzymes to elicit changes in the chromatin structure (Fig. 1A). Chromatin structure can be modified by multiple post-translational modifications of histone tails. These histone modifications act as molecular marks associated with the active and inactive cis-regulatory regions that are crucial for proper development (Dong and Weng, 2013; Kouzarides, 2007). The effects of histone modifications are widespread, including functioning as binding platforms for specific proteins, promoting formation of an open chromatin structure, and establishing enhancer–promoter communication.

Active enhancers are accessible regions marked by acetylation on lysine 27 of histone 3 (H3K27ac) (Wang et al., 2008). Recruitment of histone acetyltransferases, such as p300 (Ep300) and/or CBP (CREBBP), which are general transcription co-activators, is responsible for H3K27ac at nucleosomes flanking active enhancers (Calo and Wysocka, 2013). Deposition of histone acetylation marks, including H3K27ac, correlates with zygotic genome activation (Li et al., 2014; Vastenhouw et al., 2019). Indeed, the pioneer factors Nanog, Pou5f3 and Sox19b are necessary for occupancy of p300 at target genes in zebrafish embryos. p300 is recruited to the genome through these pioneer factors and is required for zygotic genome activation (Chan et al., 2019; Miao et al., 2022). NANOG interacts with p300 in mammalian ESCs (Fang et al., 2014), and recruitment of p300/CBP is required for DUX4-mediated transcriptional activation in human myoblasts (Choi et al., 2016), suggesting that there may be a general requirement for interactions with this histone acetyltransferase. In Drosophila, the dependency of enhancer-associated histone 3 lysine 18 acetylation (H3K18ac) on the pioneer factor Zelda suggests that Zelda may also recruit a histone acetyltransferase (Li et al., 2014). Despite these conserved interactions, in zebrafish, most Nanog, Pou5f3 and Sox19b target genes remain open upon inhibition of p300, suggesting that p300-mediated acetylation may not be required to promote chromatin accessibility during zygotic genome activation (Miao et al., 2022). Thus, pioneer factors that drive zygotic genome activation promote acetylation but the relationship between this chromatin mark, pioneer activity, and gene expression remains incompletely understood.

Pioneer factors also recruit chromatin-modifying enzymes to elicit changes in the histone methylation state. FOXA1 recruits the histone methyltransferase MLL3 (KMT2C) to promote methylation of lysine 4 on histone 3 (H3K4me1), which in turn increases activation of ER-responsive genes in breast cancer cells (Jozwik et al., 2016). In mouse pituitary cells, Pax7 first recruits a histone demethylase, KDM1A, to remove the repressive H3K9me2 mark followed by recruitment of the MLL complex to deposit the activating H3K4me1/3 mark (Gouhier et al., 2024; Kawabe et al., 2012; McKinnell et al., 2008).

Chromatin structure is also modified through methylation of DNA. Pioneer factors regulate DNA methylation actively by recruiting demethylase enzymes, or passively promote the loss of methylation as the cell cycle progresses. In mouse muscle stem cells, DNA demethylation is mediated by Pax7 and is a crucial event for gene activation (Carrió et al., 2016). FOXA1 exhibits physical interactions with the demethylases TET1 and TET2 in vitro and in vivo, and its knockdown in breast cancer cells leads to heightened methylation within FOXA1-bound regions, suggesting that FOXA1 has a role in inducing site-specific demethylation that is TET1/2 dependent (Lemma et al., 2022). Klf4 also promotes DNA demethylation through interactions with Tet2 in mouse ESCs (Sardina et al., 2018). By contrast, FOXA2 and SOX2 binding leads to a loss of DNA methylation that is replication dependent, suggesting a passive loss of DNA methylation upon pioneer-factor binding, without the active recruitment of DNA demethylases (Donaghey et al., 2018; Vanzan et al., 2021). In summary, pioneer factors regulate different developmental gene networks by modulating histone and DNA modifications in unique ways.

To package chromatin in the nucleus, it is structurally organized in 3D space. This packaging is organized at multiple scales from compartments of active (A) and inactive (B) chromatin to topologically associated domains (TADs) to loops (Theis and Harrison, 2023). Chromatin structure can govern long- and short-range DNA interactions, allowing for the proper regulation of gene expression and cell identity. TAD boundaries predominantly function to prevent unwanted interactions, whereas loop boundaries, which are governed by tethering elements, reflect enhancer–promoter interactions (Batut et al., 2022). These boundaries form sequentially during embryonic development, first at the level of chromatin loops and then further into TADs (Theis and Harrison, 2023). Pioneer-factor occupancy has been implicated in shaping this 3D genome organization. During the establishment of 3D chromatin structure in the early Drosophila embryo, binding sites for the pioneer factors Zelda and GAF are located at loop and TAD boundaries (Fig. 1B) (Batut et al., 2022; Hug et al., 2017; Levo et al., 2022; Ogiyama et al., 2018). Zelda is required for the initial formation of loops (Espinola et al., 2021) and later for defining individual TAD boundaries (Hug et al., 2017). GAF is similarly required for looping; depletion of the GAGA motif to which it binds results in loss of chromatin loops but does not seem to affect TAD formation (Gaskill et al., 2023; Li et al., 2023b; Ogiyama et al., 2018). The role of GAF in promoting 3D chromatin structure depends, in part, on the ability of GAF to interact with itself (Li et al., 2023b). The capacity of pioneer factors to multimerize, either through stable interactions or weak multivalent interactions, may promote the formation of sub-nuclear microenvironments that can shape 3D structure (Hayward-Lara et al., 2024). During somatic cell reprogramming of mouse embryonic fibroblasts, Oct4 facilitates TAD reorganization, and this depends on the ability of Oct4 to phase separate (Wang et al., 2021). In addition to their roles in forming TADs and loops, pioneer factors may influence whether regions of the genome are localized to the A or B compartments. For example, Pax7-mediated chromatin accessibility in mouse pituitary cells is correlated with a switch at pioneered loci from the inactive B compartment to the active A compartment through a process that requires cell division (Gouhier et al., 2024). Additional pioneer factors, such as Foxa2, AP-1 and KLF4, have been suggested to mediate changes to 3D chromatin structure (Di Giammartino et al., 2019; Hao et al., 2024; Phanstiel et al., 2017; Wolf et al., 2023).

Pioneer factors both directly, through binding nucleosomal DNA, and indirectly, through the recruitment of co-factors and chromatin-modifying enzymes, reshape the chromatin landscape at multiple levels. These modifications to the chromatin landscape result in robust changes to the gene-expression program and cellular state, driving normal developmental transitions and, when co-opted, leading to disease states. In the following section, we discuss how developmental state, in turn, feeds back on pioneer-factor activity.

Pioneer factors bind silenced regions of the genome, yet only a subset of their defined sequence-recognition motifs is bound in vivo, and these occupied binding sites vary between different cell types (Buecker et al., 2014; Cernilogar et al., 2019; Chronis et al., 2017; Donaghey et al., 2018; Gibson et al., 2024; Larson et al., 2021; Lupien et al., 2008; Mayran et al., 2018; Soufi et al., 2012). Both the binding and activity of pioneer factors can be influenced by developmental context (Gaskill et al., 2021; Gouhier et al., 2024; Maresca et al., 2023; Nevil et al., 2020; Schulz et al., 2015; Li et al., 2023a). The following examples highlight the role of cell fate in shaping pioneer factor function.

Distinct cell types possess unique chromatin architecture that often reflects the gene-expression profile of the cell. Promoters are generally accessible, whereas enhancers have cell type-specific accessibility that is correlated with gene expression (Andersson and Sandelin, 2020; Heinz et al., 2015; Rada-Iglesias et al., 2011). As discussed above, active enhancers are marked with H3K27ac. Silent enhancers are marked by post-translational modifications that are associated with repressed chromatin, such as H3K27me3. Although pioneer factors influence both chromatin accessibility and chromatin modifications, these same chromatin features also regulate pioneer-factor binding and activity.

Many pioneer factors prefer to bind naïve chromatin that is largely devoid of histone marks and are excluded from silent, condensed heterochromatic regions (Gibson et al., 2024; Soufi et al., 2012). H3K9me3 is enriched at constitutive heterochromatin and limits binding of OSK in human fibroblasts (McCarthy et al., 2023; Soufi et al., 2012); OSK binding can be promoted by knocking down a histone methyltransferase that deposits H3K9me3, and this, in turn, increases reprogramming efficiency (Kim et al., 2021; Onder et al., 2012). H3K9me3 also impedes mouse embryonic reprogramming following nuclear transfer, suggesting that it is a barrier to the pioneer factor-mediated events of early embryonic development (Matoba et al., 2014). H3K9 methylation may be a general barrier preventing pioneer factors from accessing the genome. High levels of H3K9me3 limit binding of Pax7 during mouse pituitary specification (Mayran et al., 2018). Similarly, human FOXA1 binding primarily occurs in regions depleted of H3K9me2, and FOXA2 binding is enriched at regions depleted of H3K9me3 (Donaghey et al., 2018; Lupien et al., 2008). Together, these data suggest that repressive chromatin modifications restrict pioneer-factor occupancy.

Whereas constitutive heterochromatin is shared across cell types, facultative heterochromatin, characterized by H3K27me3, is cell type specific. Unlike H3K9 methylation, H3K27me3-marked facultative heterochromatin does not appear to limit binding of either Zelda or Grainy head in Drosophila cell lines. Removal of H3K27me3, through inhibition of the PRC2 enzyme that deposits it, does not result in increased occupancy of either pioneer factor at regions previously marked by H3K27me3 (Gibson et al., 2024). However, there are cell type-specific features that may influence pioneer binding. During asymmetric neural stem cell divisions in Drosophila, Zelda-mediated reprogramming is progressively limited (Larson et al., 2021). Knockdown of proteins that recruit histone deacetylases enhance Zelda-mediated reprogramming, suggesting that histone acetylation state regulates Zelda pioneering activity (Larson et al., 2021). These examples indicate that chromatin context can restrict pioneer-factor occupancy in a cell type-specific manner.

In addition to histone modifications, other features of chromatin may impact pioneer factor binding. H1 linker histones function to compact the genome and may, like H3K9me2/3, limit pioneer factor binding. In vitro, OCT4 binding to nucleosomes is impeded by the addition of H1, and in cell culture H1-dependent chromatin compaction restricts Pax7 recruitment (Echigoya et al., 2020; Gouhier et al., 2024). The nuclear lamina lines the inner nuclear membrane, provides structure to the nucleus and is associated with condensed heterochromatin (Shevelyov, 2023). Association of loci with the nuclear lamina may similarly limit pioneer-factor binding. During foregut development in C. elegans, PHA-4 binding to a reporter gene is increased when the nuclear lamina protein EMR-1 is knocked down (Fakhouri et al., 2010). Similarly, Foxa2/FOXA2 binding is enhanced in multiple mammalian models upon disruption of the nuclear lamina (Whitton et al., 2018; Wei et al., 2022).

The ability of chromatin context to promote pioneer-factor binding is less well established than the restrictive contexts discussed above. FOXA1 binding to chromatin is correlated with marks of active modifications (Lupien et al., 2008; Wang et al., 2015), but there is little data to support an instrumental role for these marks in directly shaping the binding. Conflicting structural studies about the role of H3K27ac in regulating OCT4 nucleosome binding also further limit our understanding of whether active marks promote chromatin occupancy of pioneer factors (Lian et al., 2023 preprint; Sinha et al., 2023). By contrast to the unclear role of histone modifications in promoting pioneer-factor binding, nucleosomes promote binding. Both in the early embryo and upon exogenous expression, pioneer factors in multiple species preferentially bind and open chromatin regions enriched for nucleosomes (Ballaré et al., 2013; Gibson et al., 2024; Miao et al., 2022; Veil et al., 2019). These data suggest that pioneer factors may preferentially bind motifs wrapped around histones or that interactions with the histone proteins themselves promote pioneer-factor binding. Together, it is evident that, although pioneer factors can access their binding sites within closed chromatin, the chromatin structure itself can influence the ability of these factors to stably bind and promote accessibility (Fig. 2A).

Because co-factors influence pioneer-factor occupancy, developmentally regulated expression of these proteins impacts the ability of pioneer factors to stably bind and open the genome (Fig. 2B). Collaborative interactions between OSK and stage-specific transcription factors govern pluripotency-enhancer selection to reprogram somatic cells to pluripotency (Chronis et al., 2017). Consistent with this, Oct4 occupies different regions in the genome when it is expressed alone or with other reprogramming factors, suggesting that Oct4 cell type-specific binding is strongly influenced or redirected by additional co-factors (Chronis et al., 2017; Donaghey et al., 2018). This is similar to what is observed for the orthologs of these factors in zebrafish embryos (Miao et al., 2022). As stem cells transition between naïve and primed pluripotency, there is widespread relocalization of Oct4, which is regulated, in part, by expression of the transcription factor Otx2 in the primed pluripotent cells (Buecker et al., 2014). OCT4 and SOX2 cooperate in human neural crest specification but bind to regions distinct from those occupied by these factors in ESCs. This distinct binding is driven in part by binding with the neural crest transcription factor TFAP2 and highlights how the same pioneer factors can be co-opted by tissue-specific co-factors to drive distinct gene-expression programs (Hovland et al., 2022). Similarly, the capacity of SOX2 to promote the pluripotent fate versus the neuronal fate is regulated by the ability of neuronally expressed co-factors PAX6 and ATRX to promote localization of SOX2 to neuronal-specific regulatory regions (Bunina et al., 2020; Zhang et al., 2019). Thus, developmentally regulated expression of additional factors influences pioneer-factor occupancy. This contrasts with the relatively limited evidence that histone marks actively promote pioneer-factor binding. Indeed, upon exogenous expression of Zelda and Grh there is no clear chromatin profile that correlates with cell type-specific binding events, but motif analysis shows an enrichment for motifs bound by factors that are expressed in a tissue-specific manner. This suggests that expression of these transcription factors may modulate the binding of Zelda and Grh in a tissue-specific manner (Gibson et al., 2024). Similar data for FOXA2 suggest that coordinated involvement of multiple factors may be a general feature to help specify pioneer-factor binding (Meers et al., 2019).

Co-factors can also affect pioneer-factor activity. Despite the ability of pioneer factors to bind nucleosomal DNA and promote local opening, most pioneer factors only promote chromatin accessibility at a subset of bound regions (Gibson et al., 2024; Soufi et al., 2012; Veil et al., 2019). Thus, in many cases binding is separable from pioneer activity, and developmental state can regulate the capacity of pioneer factors to promote chromatin accessibility. For example, the co-factor GATA4 is co-expressed with FOXA2 in human endoderm and co-expression of this co-factor in a human fibroblast cell line promotes FOXA2 occupancy at endoderm-specific binding sites. However, this binding does not lead to accessibility, suggesting that additional factors must be recruited to promote accessibility (Donaghey et al., 2018). In Drosophila, Grh binding remains stable across developmental stages, but the gene expression pattern and chromatin accessibility driven by Grh varies through development (Nevil et al., 2017, 2020). This suggests that pioneering activity of Grh, rather than its binding, is regulated by tissue-specific features, such as co-factors. In pituitary stem cells, Pax7 can bind chromatin but requires the co-factor Tpit to promote accessibility (Mayran et al., 2018, 2019). Thus, during certain developmental stages, chromatin accessibility may be established through cooperation of pioneer factors with other non-pioneer factors.

In addition to extrinsic features, pioneer-factor activity is also regulated by protein-intrinsic properties of the pioneer factors themselves, including expression level, isoform expression and post-translational modifications (Fig. 2C-E). These properties are, in turn, regulated by developmental context.

Concentration is a key factor influencing pioneering activity (Fig. 2C), and when overexpressed pioneer factors can be oncogenic. Upon ectopic induction, chromatin binding and accessibility is correlated with the expression levels of Grh and Zelda (Gibson et al., 2024). Zelda overexpression in neural stem cells similarly results in novel occupancy of closed regions, suggesting that higher expression levels promote binding to closed chromatin (Gibson et al., 2024). Levels of Nanog, Pou5f3 and Sox19b expression have a concentration-dependent effect, with protein levels correlating with chromatin accessibility (Miao et al., 2022). In models of Pax7-induced melanotrope production, chromatin opening is similarly correlated with levels of Pax7 binding (Gouhier et al., 2024). Although overall expression levels clearly influence pioneer activity, the local concentration of pioneer factors may also be regulated at specific loci. Imaging of numerous pioneer factors, including FOXA1, KLF4, SOX2, GAF, Nanog and Zelda, demonstrate that they are not uniformly distributed in the nucleus but instead are concentrated in localized microenvironments (Dufourt et al., 2018; Gaskill et al., 2021, 2023; Hayward-Lara et al., 2024; Ji et al., 2024; Kuznetsova et al., 2023; Mir et al., 2018; Morin et al., 2022; Nguyen et al., 2022; Raff et al., 1994; Sharma et al., 2021). These pioneer factor microenvironments are formed by various mechanisms, including the multivalent interactions mediated by intrinsically disordered regions that are found in many transcription factors (Boeynaems et al., 2018; Ferrie et al., 2022; Peng et al., 2020; Rippe, 2022). These localized regions of relatively concentrated factors may promote chromatin opening and gene expression (Boija et al., 2018; Chong et al., 2018; Tang et al., 2022). Nonetheless, the relationship between concentration and ability to activate gene expression is not simple. Although hub formation can promote transcription, when concentrated above a certain threshold, proteins may promote transcriptional repression (Chong et al., 2022; Gaskill et al., 2023). Many mechanistic studies of pioneer factors rely on overexpression in culture. These conditions are similar to those applied for iPSC induction, which requires the exogenous expression of multiple pioneer factors to initiate the necessary reprogramming events. It is increasingly clear that the levels of expression can influence pioneer factor activity, and thus it is essential for studies to be performed in the context of normal development.

Because domains outside of the DBD influence pioneer-factor binding and activity, isoform expression can regulate function (Fig. 2D). Protein structure can vary with developmental context owing to splicing or alternative transcription start sites. For example, the Drosophila pioneer factor Grh has multiple isoforms that are regulated by both processes. There are two characterized splice isoforms that both contain the conserved DBD but differ in the N-terminal region and are expressed in different Drosophila tissues (Uv et al., 1997; Almeida and Bray, 2005; Bray and Kafatos, 1991). In intestinal stem cells, one isoform is thought to prevent differentiation whereas ectopic expression of the other promotes differentiation (Dominado et al., 2022 preprint). Similarly, the mammalian GRHL genes are differentially spliced, suggesting a conserved regulatory mechanism (Lambourne et al., 2024 preprint; Wilanowski et al., 2002). In addition to splicing, there are multiple transcription start sites that result in proteins with N-terminal domains of varying lengths, and these isoforms have tissue-specific expression patterns. Given that regions outside of the DBD strongly influence pioneer-factor activity, it is evident that these developmentally regulated isoforms could significantly impact pioneering function.

Protein activity can be influenced by post-translational modifications, and these modifications can differ depending on cellular context. Many pioneer factors are post-translationally modified (Brumbaugh et al., 2012; Cho et al., 2018; Park et al., 2021; Spelat et al., 2012; Sutinen et al., 2014; Wei et al., 2007; Williams et al., 2020; Wolfrum et al., 2003). Cellular signaling often results in downstream changes in post-translational modifications. For example, phosphorylation regulates conserved functions of Grh in a context-dependent manner. Grh is essential in both epidermal barrier formation and wound healing (Sundararajan et al., 2020; Wang and Samakovlis, 2012). Although phosphorylation is dispensable for maintenance of the epidermal barrier, it is required for the activation of wound enhancers upon wounding (Kim and McGinnis, 2011).

Post-translational modifications can influence pioneer activity by regulating total and local concentrations. For example, Sox2 levels are controlled through competing modifications; methylation of Sox2 resulting in ubiquitylation and subsequent degradation, whereas phosphorylation prevents methylation and therefore stabilizes the protein (Fang et al., 2014). Acetylation of Sox2 affects local concentration by disrupting the nuclear localization signal and promoting nuclear export (Baltus et al., 2009; Williams et al., 2020). Nuclear import of SOX11 is controlled by phosphorylation (Balta et al., 2018; Williams et al., 2020). Thus, the local and total concentrations of pioneer factors are regulated by post-translational modifications, which are subject to developmental regulation.

Post-translational modifications also regulate the interaction of pioneer factors with both the DNA and co-factors (Fig. 2E). The affinity of GAF, Pax7 and HMG-14 (HMGN1) for chromatin are reduced by acetylation and phosphorylation, which can influence interaction with both the negatively charged DNA as well as the nucleosomes (Aran-Guiu et al., 2010; Bergel et al., 2000; Bonet et al., 2005; Sincennes et al., 2021). During asymmetric division of satellite stem cells in mice, Pax7 is differentially regulated in the two resulting daughter cell lineages by methylation on the N terminus. This methylation allows Pax7 to interact with the MLL complex to deposit H3K4me3 and induce expression of a gene marking the committed satellite fate (Kawabe et al., 2012). Protein activity is broadly regulated through development by a myriad of different mechanisms, and pioneer factors are no exception.

The above examples highlight the reciprocal relationship between pioneer factors and development. Ultimately, understanding how this special class of transcription factors engages with the genome to drive cell-state transitions will have fundamental implications for our understanding of both normal development and disease progression.

The capacity of pioneer factors to promote chromatin accessibility enables them to drive major developmental transitions. However, this same function promotes disease states when dysregulated. Thus, it is imperative to understand the mechanisms governing pioneer-factor activity. Although extensive work has been done to characterize pioneer-factor function, many of these studies rely on in vitro and cell-culture systems. These systems have been essential for understanding the mechanisms by which pioneer factors bind and open chromatin, but provide limited insight into the relationship between pioneer factors and development. The emerging evidence that pioneering function is regulated by developmental context makes it clear that in vivo studies provide a necessary complement to studies in culture and in a test tube. To understand how these powerful factors drive development and disease, it will be essential to uncover the reciprocal relationship between pioneer factor-driven developmental transitions and how cellular state influences pioneering activity.

We thank Audrey Marsh, Annemarie Branks and members of the Harrison lab for helpful discussions. We apologize to those whose research we did not discuss or cite owing to space limitations.