Proc Natl Acad Sci U S A. 2017 Jun 6;114(23):5838-5845.

Dynamic regulation of Nanog and stem cell-signaling pathways by Hoxa1 during early neuro-ectodermal differentiation of ES cells

Bony De Kumara, Hugo J. Parkera, Mark E. Parrisha, Jeffrey J. Langea, Brian D. Slaughtera, Jay R. Unruha, Ariel Paulsona and Robb Krumlaufa,b

a   Stowers Institute for Medical Research, Kansas City, MO 64110;

b   Department of Anatomy and Cell Biology, Kansas University Medical Center, Kansas City,      KS 66160



Homeobox a1 (Hoxa1) is one of the most rapidly induced genes in ES cell differentiation and it is the earliest expressed Hox gene in the mouse embryo. In this study, we used genomic approaches to identify Hoxa1-bound regions during early stages of ES cell differentiation into the neuro-ectoderm. Within 2 h of retinoic acid treatment, Hoxa1 is rapidly recruited to target sites that are associated with genes involved in regulation of pluripotency, and these genes display early changes in expression. The pattern of occupancy of Hoxa1 is dynamic and changes over time. At 12 h of differentiation, many sites bound at 2 h are lost and a new cohort of bound regions appears. At both time points the genome-wide mapping reveals that there is significant co-occupancy of Nanog (Nanog homeobox) and Hoxa1 on many common target sites, and these are linked to genes in the pluripotential regulatory network. In addition to shared target genes, Hoxa1 binds to regulatory regions of Nanog, and conversely Nanog binds to a 3′ enhancer of Hoxa1. This finding provides evidence for direct cross-regulatory feedback between Hoxa1 and Nanog through a mechanism of mutual repression. Hoxa1 also binds to regulatory regions of Sox2 (sex-determining region Y box 2), Esrrb (estrogen-related receptor beta), and Myc, which underscores its key input into core components of the pluripotential regulatory network. We propose a model whereby direct inputs of Nanog and Hoxa1 on shared targets and mutual repression between Hoxa1 and the core pluripotency network provides a molecular mechanism that modulates the fine balance between the alternate states of pluripotency and differentiation.



Pluripotency and differentiation are two opposing cell states that must be balanced in controlling cellular homeostasis. Coordinating the proper regulation and cross-talk of these opposing states plays a fundamental role in the generation, homeostasis and repair of tissues in developing embryos and adult animals.  Studies on pluripotency in a wide variety of animal and cell culture model systems have described the presence of a highly conserved core pluripotency gene regulatory network (GRN) consisting of the transcription factors: Nanog, Oct4 and Sox2, while Klf4, c-Myc, Sal4, Esrrb, Utf1, tet2 and Glis1 comprise a second layer of regulatory components that function in elaboration of this network [1-4]. Major signaling pathways (e.g. Wnt, BMP4 and TGF-β also feed into this GRN to maintain cells in a pluripotent state by modulating the expression of the core of pluripotency network [5-8]. Part of the stability or robustness of the core pluripotency network arises from extensive feedback circuits where components in the pathway are modulated by complex auto- and cross-regulatory interactions. For example, Nanog is known to activate Oct4 and Sox2, while they in turn positively feedback to cross-regulate Nanog. Levels of Oct4 and Sox2 are also under positive auto-regulation, while Nanog maintains its level by negative auto-regulation [9-12]. This illustrates that the GRN for pluripotency has an ability to reinforce and self-perpetuate its regulatory state. Hence, mechanisms must exist to alter this state and initiate programs to promote differentiation.

Patterning and differentiation processes in developing embryos, which can be mimicked in embryonic stem (ES) cells, are initiated in response to gradients of signaling molecules, such as retinoic acid (RA), Fgfs and Wnt [13-16]. These pathways often oppose each other to induce the expression of master regulators of cellular fates, such as Hox genes. This leads to the progressive elaboration and patterning of the basic body plan.  The highly conserved family of transcription factors encoded by Hox genes are expressed during gastrulation and are involved in assigning regional identities to cells in a temporal and spatially-restricted manner. During RA induced differentiation of murine ES cells, Hox proteins and their cofactors Pbx and Meis, are amongst the most rapidly induced proteins, in accord with their roles as early determinants of differentiation process [17, 18]. In addition, the downstream targets of Hox proteins are enriched for signaling pathways that in turn can promote and expand differentiation [19]. This makes Hox proteins good candidates for participating in regulatory processes that counter the pluripotent state to promote programs of differentiation.

In the current study we used genomics approaches to characterize the genome-wide binding properties of murine Hoxa1 in ES cells programmed to differentiate into neural-like states. In characterizing the early targets of Hoxa1 there was a surprising enrichment for consensus Nanog binding motifs near Hoxa1 bound regions (Fig. 1A) [20]. Furthermore, we were able to experimentally demonstrate co-occupancy of Nanog near these Hoxa1 bound regions. These findings suggested the possibility that the close proximity of Hoxa1 and Nanog binding motifs in many genomic loci might reflect shared regulatory inputs that contribute to modulating the expression of genes containing these putative control elements. This type of regulatory logic is interesting because Nanog not only promotes the expression of genes required to maintain pluripotency, but it also represses genes that promote differentiation (Fig. 1B) [21]. We wondered whether Hoxa1 might do the reverse, promote differentiation and block pluripotency. The co-associated Nanog and Hoxa1 sites provide a means for integrating, at the level of cis-elements, the different outputs of these factors on shared target genes.

In support of this hypothesis, genomic analyses and single molecule FISH experiments revealed direct cross-regulation between Hoxa1, Nanog and core pluripotency genes providing insight into how they may regulate the balance between pluripotency and differentiation. Hoxa1 was rapidly recruited in and around genes involved in pluripotency network (e.g. Nanog and Sox2). A well characterized auto-regulatory region of Nanog shows rapid occupancy of Hoxa1 upon RA treatment which is correlated with a rapid decrease in nascent transcription . Conversely, Nanog occupies regions flanking the Hoxa1 locus. This lead to an idea that Hoxa1 and Nanog directly regulate each other’s expression through mutual repression which would alter the balance in GRNs controlling pluripotency and differentiation (Fig. 1B). Since many genes are found to be shared targets of Nanog and Hoxa1 these proteins may exert reciprocal actions on their shared targets further altering the programs that govern the balance between the pluripotency and differentiation state of cells.

Signaling pathways play a central role in modulating both pluripotency and differentiation states of cells.  In ES cell models and embryos, RA plays an important role in tipping cell fate towards differentiation through activating Hoxa1 and then Hoxa1 in turn regulates multiple pathways as downstream targets that drive differentiation [19, 22]. Many functional roles in patterning of Hox proteins are related to their ability to influence key signaling pathways in differentiating cells [23]. This sets up complex regulatory loops whereby signaling pathways induce Hox genes and them in turn modulate the outputs of other signaling cascades.

The importance of these interactions between Nanog and Hoxa1 in ES cells is not just limited to ES cells and has wider biological implications. Components of the pluripotency GRN are also utilized in other tissues and developmental contexts where progenitor cell populations possess the potential for generating multiple lineages. One example is the neural crest, which displays many of the features of stem cells, with a capacity for limited self-renewal and differentiation into a wide variety of cell fates (Fig. 1C) [24]. GRNs for neural crest biology have been characterized and core components of the pluripotency network are essential for their induction in the neural plate [24-26]. Furthermore, Hoxa1 has been shown to play an important role in the induction and patterning of neural crest in mouse and humans even though it is not expressed in neural crest cells [27-29]. Hence, Hoxa1 expression in the neural plate may play a role in downregulating the core pluripotency network and helping to promote in induction and differentiation of neural crest.

In case of human cancers, there is growing evidence to suggest roles of Hox genes in the etiology of various cancer types, through modulation of cell proliferation, invasion and metastasis [23, 30-32]. This maybe a result of a change in balance of pluripotency/multipotency towards a differentiation fate mediated by aberrant changes in Hox gene expression level.  In summary, the potential roles for Hoxa1 in altering the regulatory networks regulating pluripotency through mutual repression, which we uncovered in an ES cell model, offers insight into how similar cross-regulatory interactions between Hox genes and GRNs maintaining self-renewal and multipotency many operate in other tissues.



Figure 1. Hoxa1 and Nanog are involved in negative cross-regulatory interactions of their gene expression and co-occupy many common targets in ES cell models. A. An example of the close proximity of Nanog and Hoxa1 binding sites on putative downstream target genes. On a genome wide basis many Nanog sites are positioned from 2-100 base pairs adjacent to Hoxa1 binding sites. B. A model for direct cross-regulatory interactions between Hoxa1 and Nanog. Nanog binds to the Hoxa1 locus and conversely Hoxa1 to the Nanog locus in ES cells programmed to differentiate into neural fates. This provides the opportunity for mutual repression and hence modulation of balance between the gene regulatory programs directing pluripotency or differentiation. C. Pluripotent Neural Crest stem cells in the neural tube use core pluripotency genes to maintain their potential. These cells begin to differentiate and migrate from the neural tube into the branchial arches to make bone and connective tissues in the head. Hoxa1 is required in the neural tube for regulating this program in the second branchial arch.



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