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. Author manuscript; available in PMC: 2018 May 1.
Published in final edited form as: J Cell Biochem. 2017 Jan 11;118(5):953–958. doi: 10.1002/jcb.25723

Precocious Phenotypic Transcription-Factor Expression during Early Development

Jennifer J VanOudenhove 1,2, Ricardo Medina 2,‡, Prachi N Ghule 1, Jane B Lian 1, Janet L Stein 1, Sayyed K Zaidi 1, Gary Stein 1,*
PMCID: PMC5336526  NIHMSID: NIHMS816627  PMID: 27591551

Abstract

A novel role for phenotypic transcription factors in very early differentiation was recently observed and merits further study to elucidate what role this precocious expression may have in development. The RUNX1 transcription factor exhibits selective and transient upregulation during early mesenchymal differentiation. In contrast to phenotype-associated transcriptional control of gene expression to establish and sustain hematopoietic/myeloid lineage identity, precocious expression of RUNX1 is functionally linked to control of an epithelial to mesenchymal transition that is obligatory for development. This early RUNX1 expression spike provides a paradigm for precocious expression of a phenotypic transcription factor that invites detailed mechanistic study to fully understand its biological importance.

Keywords: Phenotypic transcription factor, Early development

Introduction

A fundamental, yet poorly understood dimension of developmental control is engagement of the phenotypic transcription factors in regulatory cascades that mediate gene expression to establish cell and tissue identity. When are phenotypic genes initially expressed during development? Is their control and are their regulatory activities modified to accommodate requirements for the initiation and progression of differentiation? Here we review recent evidence that selected phenotypic transcription factors are expressed during early differentiation—in advance of previously understood requirements for biological activity. Implications for how this precocious expression of phenotypic transcription factors contributes to mechanisms that mediate developmental processes associated with establishing cell and tissue specificity are explored. We focus on precocious, tissue-specific, expression of the RUNX1 transcription factor as a paradigm for this novel parameter of cellular control that is operative at the onset of differentiation.

The RUNX Transcription Factor Family

While the maintenance of pluripotency has been well studied [Boward et al., 2016; Boyer et al., 2005; Chambers et al., 2003; Huang et al., 2015; Kapinas et al., 2013], it is not well understood how differentiation signals regulate the transition from pluripotency to phenotypic establishment. Studies in human embryonic stem cells (hESCs) have shown that many genes responsible for early developmental events carry bivalent epigenetic modifications that poise them for rapid response to either activating or repressive transcription cues [Bernstein et al., 2006; Grandy et al., 2015; Szutorisz and Dillon, 2005]. Once a differentiation signal has been introduced, early factors are expressed that prime cells’ gene expression program for lineage determination [Zaret and Carroll, 2011]. Transcription factors are primary regulators of gene expression, with clear potential to influence cell fate determination and differentiation of hESCs; the RUNX (Runt-related transcription factor) transcription factors are of particular interest because they have well-documented, but distinct, roles in development [Chuang et al., 2013].

The RUNX gene family is also known as the acute myeloid leukemia (AML), core-binding factor α (CBFα) or polyoma enhancer-binding protein-2α (PEBP2α) family. It includes three members: RUNX1, RUNX2, and RUNX3 [Ito, 2004]. Each of the RUNX-family transcription factors have unique developmental roles: RUNX1 is necessary for definitive hematopoiesis [Okuda et al., 1996], RUNX2 for bone formation [Otto et al., 1997], and RUNX3 for gastrointestinal and nervous system development [Inoue et al., 2002; Levanon et al., 2002; Li et al., 2002]. All three RUNX-family members are implicated in malignancies as either tumor suppressors or oncogenes in a context-specific manner [Blyth et al., 2005; Ito, 2004].

RUNX genes are transcribed from two promoters, the distal P1 and the proximal P2 [Bangsow et al., 2001; Drissi et al., 2000; Fujiwara et al., 1999; Ghozi et al., 1996], and contain similar structural domains. There is a highly conserved 128 amino acid region in the N-terminal portion of the proteins known as the runt homology domain (RHD) due to its homology with the Drosophila Runt protein [Kagoshima et al., 1993; Ogawa et al., 1993b]. This RHD mediates both heterodimerization with the core binding factor β (CBFβ) protein, which stabilizes the protein complex, and binding target DNA at the RUNX family consensus sequence PyGPyGGTPy (Py represents either pyrimidine base, cytosine or thymine) [Melnikova et al., 1993; Ogawa et al., 1993a]. CBFβ alone does not bind DNA [Ogawa et al., 1993a]. A nuclear targeting signal (NLS) is on the C-terminal end of the RHD; it is required for the nuclear localization of RUNX proteins and consequently allows access to their DNA targets [Kanno et al., 1998].

The RUNX family members also have conserved regions on their C-termini. Each RUNX transcription factor has a nuclear matrix targeting signal (NMTS) in addition to the NLS. The NMTS is an ~31–38 amino acid sequence that is responsible for the subnuclear localization of RUNX proteins to distinct nuclear sites to facilitate gene regulation [Stein et al., 2003; Zeng et al., 1998; Zeng et al., 1997]. The C-terminus of RUNX proteins also include additional domains that mediate gene regulation, such as an activation and inhibitory domain, the PPxY (or PY) motif, and the VWRPY motif. The PPxY motif is a proline-rich peptide sequence that interacts with proteins, such as Yes-associated protein (YAP), that contain a WW domain, whereas the VWRPY motif interacts with proteins, such as Groucho/TLE transcription corepressors, that contain tryptophan-aspartic acid repeats [Aronson et al., 1997; Chuang et al., 2013; Ito, 2004; Javed et al., 2000].

RUNX1 in Development

Developmental hematopoiesis begins with primitive hematopoiesis, in which a limited number of blood lineages (mostly large erythroblasts) that sustain early embryonic development are produced primarily from the yolk sac [Chen et al., 2014]. A second wave of blood development, termed definitive hematopoiesis, occurs intra-embryonically in the aorta-gonad-mesonephros. During this stage of development, hematopoietic stem cells (HSCs) are formed. HSCs have the ability to produce any of the hematopoietic lineages and, importantly, have long-term repopulation capacity [Chen et al., 2014]. RUNX1 is required for definitive hematopoiesis, as no HSCs are formed in the absence of RUNX1. When RUNX1 was genetically deleted in mice, embryonic lethality resulted due to major defects in the formation of the fetal liver and hemorrhaging in the central nervous system [Okuda et al., 1996; Wang et al., 1996]. RUNX1 appears to be mostly dispensable once HSCs are formed, however, loss of RUNX1 has some effects on differentiation toward specific hematopoietic lineages [Growney et al., 2005; Ichikawa et al., 2004; Ichikawa et al., 2008]. It is hypothesized that RUNX1 alters chromatin, through unfolding and modification of the epigenetic landscape, to allow early hematopoiesis [Hoogenkamp et al., 2009; Lichtinger et al., 2012].

The RUNX1 transcript is expressed as three major isoforms, one from the distal P1 promoter (isoform c) and two from a proximal P2 promoter (isoforms a and b) [Ghozi et al., 1996; Ran et al., 2013; Sroczynska et al., 2009]. A study of Runx1 isoform expression in early mouse hematopoietic development found that primitive erythrocytes produced prior to definitive hematopoiesis express mainly the proximal P2 promoter–derived isoforms [Bee et al., 2009]. However, in HSCs formed during definitive hematopoiesis, transcription occurs from both the P1 and P2 promoters, with isoforms from each promoter exhibiting non-redundant functions [Bee et al., 2010]. Once cells have migrated to the fetal liver to establish adult hematopoiesis, RUNX1c, transcribed from the P1 promoter, gradually becomes the main RUNX1 isoform in hematopoietic cells [Bee et al., 2009].

In human ESCs, studies of hematopoietic differentiation reveal an increase in RUNX1c mRNA levels at 8–12 days of differentiation from embryoid bodies that correlates with the emergence of the definitive hematopoietic lineage marker, CD34. Low RUNX1b mRNA levels have been observed throughout hematopoietic differentiation [Challen and Goodell, 2010; Zambidis et al., 2005]. Of note, in undifferentiated hESCs, the RUNX1 P2 promoter, but not the P1 promoter, is bivalently marked with repressive H3K27me3 and activating H3K4me3 epigenetic modifications, indicating that it is poised for rapid activation [Grandy et al., 2015; Mikkelsen et al., 2007; Pope et al., 2014; Thurman et al., 2012]. This could indicate that RUNX1 transcription from the P2 promoter is more permissive early in development and may have a role that is not linked to emergence of HSCs. The role of RUNX1 in differentiation at very early time points in either directed or undirected differentiation of hESCs has been only minimally explored.

Emerging evidence indicates that RUNX1 has roles in non-hematopoietic cell lineages as well [Osorio et al., 2008; Scheitz and Tumbar, 2013; Stifani et al., 2008]. Multiple reports have detailed a role for RUNX1 in epithelial biology [Scheitz and Tumbar, 2013]. RUNX1 has been shown to modulate developmental activation and proliferation of hair follicle stem cells and inner olfactory nerve layer olfactory ensheathing cells [Hoi et al., 2010; Lee et al., 2014; Murthy et al., 2014; Osorio et al., 2008; Osorio et al., 2011]. RUNX1 is a key regulator of the differentiation of mammary epithelium stem cells from a state of ductal and lobular bipotency [Sokol et al., 2015]. Additional roles have been shown in other mesendodermal derivatives such as mesenchymal stem cells, myofibroblasts and skeletal progenitors [Kim et al., 2014; Lian et al., 2003].

Transient, Preemptive Expression of a Phenotypic Transcription Factor during Early Mesendodermal Differentiation

The recent report that, prior to their established functions in specifying lineage and cell type-specific identity, phenotype-associated transcription factors play a role in initial stages of differentiation is counterintuitive but significant [VanOudenhove et al., In Press, 2016]. This “preemptive” expression of RUNX1 as early as eight hours after initiation of differentiation, during mesenchymal differentiation, illustrates the contribution of a phenotypic transcription factor in initial parameters of developmental control (see overview in Fig. 1). The consequences are functional as well as potentially unique contributions of transcription factors to developmental stage specific gene expression.

Fig. 1.

Fig. 1

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Overview of the role of transient RUNX1 upregulation in early mesendodermal development.

The mechanistic role of RUNX1 in hematopoietic/myeloid differentiation has been extensively studied. Its role in genetic and epigenetic control has been established and validated through multiple lines of molecular, cellular, biochemical and in vivo genetic evidence. In contrast, a mechanistic understanding for the regulatory consequences of RUNX1 expression at the onset of mesendodermal differentiation is just beginning to emerge. The precocious burst of RUNX1 expression at the onset of mesendodermal differentiation is both selective and specific; other phenotypic transcription factors are not expressed during this developmental window. While RUNX1 expression from the distal P1 promoter is linked with the emergence of definitive hematopoietic stem cells [Chen et al., 2009; Lacaud et al., 2002; Okuda et al., 1996], there is limited understanding for the role of transcripts from the more ubiquitous proximal P2 promoter [Challen and Goodell, 2010; Fujita et al., 2001; Sroczynska et al., 2009]. The RUNX1 transcript that is synthesized during early mesendodermal differentiation is the RUNX1b isoform, transcribed from the P2 promoter. Interestingly, this P2 promoter is poised for expression, by virtue of bivalent H3K27me3 and H3K4me3 epigenetic modifications in undifferentiated hESCs, while the P1 promoter is not [Grandy et al., 2015; Mikkelsen et al., 2007; Pope et al., 2014; Thurman et al., 2012]. Additionally, the transcripts from the two promoters are translated through different primary mechanisms, with the P1-derived transcript translated through a cap-mediated mechanism and the P2 transcripts translated though an internal ribosome entry site (IRES)-mediated mechanism [Pozner et al., 2000], which is often invoked to accommodate cellular stress, facilitate mitotic progression, and support competency for differentiation [Komar and Hatzoglou, 2011]. This further supports the idea that the RUNX1 expression from the P2 promoter has evolved specifically to be expressed early under differentiating conditions, indicating it could have an important role in early development.

A rapid, substantial increase in RUNX1 expression early in differentiation to mesendoderm suggests a potential role for RUNX1 that is unrelated to hematopoiesis. A role for RUNX1 in early mesendodermal differentiation of hESCs through regulation of cell migration and adhesion is indicated by impairment of these processes upon RUNX1 depletion. These findings are consistent with the emerging role of RUNX1 in controlling cell motility and migration in other biological systems. RUNX1 depletion in breast cancer cells results in a decreased migration and invasion phenotype [Browne et al., 2015]; similar results were found in ovarian cancer cells [Keita et al., 2013]. Prior to release of mouse hematopoietic stem cells from hemogenic endothelium, RUNX1b is responsible for inducing a cell adhesion and migration program [Lie-A-Ling et al., 2014]. RUNX1 depletion results in decreased cell motility and de-repression of epithelial markers, which suggests that there is a requirement for RUNX1 for fidelity of epithelial-mesenchymal transition (EMT).

RUNX1 regulates transforming growth factor β (TGFβ) signaling during early mesendodermal differentiation in addition to its known roles in maintenance of hESC pluripotency [James et al., 2005; Wei et al., 2005], differentiation [Itoh et al., 2014; Watabe and Miyazono, 2009] and EMT [Xu et al., 2009]. The inhibition of motility and de-repression of epithelial genes observed upon RUNX1 depletion indicates that RUNX1 is upstream of the TGFβ pathway [VanOudenhove et al., In Press, 2016]. Moreover, RUNX1 specifically occupies the TGFβ2 promoter and regulates the expression of the TGFβ2 gene, which encodes one of the three TGFβ ligands. Though the TGFβ ligands share >70% homology [Kingsley, 1994], TGFβ-knockout mice show non-overlapping phenotypes, indicating that each ligand has specific functions in development [Sanford et al., 1997]. TGFβ2 knockout mice have cardiac, lung, craniofacial, limb, spinal column, eye, inner ear, and urogenital defects [Sanford et al., 1997], whereas the TGFβ1 knockout mice exhibit an autoimmune-like inflammatory disease or embryonic lethality due to defective yolk sac hematopoiesis and vasculogenesis, depending on the genetic background [Dickson et al., 1995; Kulkarni et al., 1993; Shull et al., 1992]. Consistent with the results from these mouse models, there is a specific requirement for TGFβ2, but not TGFβ1, to rescue the phenotype of RUNX1 depletion in hESCs [VanOudenhove et al., In Press, 2016].

In addition to a decrease in TGFβ2 ligand expression upon RUNX1 depletion, there is a decrease in phosphorylation of the downstream effector SMAD Family Member 2 (SMAD2) [VanOudenhove et al., In Press, 2016]. SMAD2 is required for competency to initiate gastrulation, as well as primitive streak/mesendoderm and mesoderm formation, with SMAD2 knockout being embryonic lethal before E8.5 [Nomura and Li, 1998; Weinstein et al., 1998]. This is consistent with a role for RUNX1 in mesendodermal regulation upstream of TGFβ2-SMAD2 signaling.

Conclusion and Perspectives

Transient expression of the phenotypic transcription factor RUNX1 during early mesendodermal differentiation of hESCs suggests that RUNX1 contributes to differentiation in addition to its established role in hematopoietic lineage identity. There is compelling evidence that RUNX1 has a defined role in the epithelial to mesenchymal transition, and the associated competency for cell mobility and motility required for development of the mesendodermal germ layer. The current knowledgebase indicates that RUNX1 regulates cell motility and gene expression during mesendodermal differentiation specifically through TGFβ2 signaling, supporting a novel, evidence-based role for RUNX1 in early development. While these findings provide confidence in the importance of RUNX1 expression and bioavailability of the RUNX1 protein during a developmental window in which mesendodermal differentiation is initiated, they also present several important unanswered questions that include: what upstream regulatory mechanisms activate the transient, precocious expression of RUNX1?; Are epigenetic regulatory mechanisms associated with precocious RUNX1 expression?; How does RUNX1 contribute to epigenetic regulation of target genes for which the RUNX protein provides a scaffold on which histone-modifying and signaling proteins at target gene promoters during early development are strategically localized; and, How is this precocious RUNX1 expression downregulated during mesenchymal progression? The exquisitely selective expression of the RUNX1 transcription factor during mesenchymal differentiation is impressive. While genome-wide expression analysis reinforces the uniqueness of RUNX1 expression at the onset of mesendodermal differentiation it will be important to investigate the extent to which transcription factors precociously exhibit transient expression throughout early development, within the context of establishing the three germ layers.

From a broad biological perspective, RUNX1-mediated regulation of TGFβ2 signaling provides mechanistic insights into early mesendodermal differentiation. Precociously expressed RUNX1 is a selective and specific regulator of cell motility and EMT-associated gene expression.

Acknowledgments

The authors declare no conflicts of interest.

Contract grant sponsor: National Cancer Institute; Contract grant number: P01 CA082834 (GSS, JLS)

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