The circuit of pluripotency 25

It is widely accepted that the most important players in pluripotency are Oct4, Sox2 and Nanog. These transcription factors are not only used to induce pluripotency [130] but their endogenous expression is key criterion in order to maintain pluripotency in cultured ESCs. Their unique expression pattern during early development suggests a major role of these transcription factors for the specification of embryonic stem cell identity [131–133]. The precise control of the master regulators’ expression is, for example, demonstrated by the restriction of Oct4 expression to ESCs, cells of the inner cell mass and to cells of the germ line [134]. Repression of Oct4 upon differentiation and lineage commitment and subsequent activation of Oct4-repressed developmental genes is crucial for normal development in mammalian organisms. Thus, massive expansion of poorly differentiated cells occurs upon systemic, ectopic Oct4-expression in adult mice underlining the importance of complete silencing of this pluripotency-associated factor during differentiation [135]. Regarding reprogramming, the importance of Nanog is demonstrated by proving that selecting for Nanog-expressing cells leads to superior reprogramming results [136] compared to initially established selection for Fbx15-positive cells [113]. Enhanced iPSC formation of Nanog- positive cells is observed and established Nanog-iPSCs mimic DNA methylation patterns of

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ESCs and can give rise to chimera formation indicating complete reprogramming into the ESC-state [136,137]. Nanog-selected iPSCs are also indistinguishable from ESCs regarding their global gene expression and their chromatin configuration in contrast to Fbx15-selected iPSCs [136]. In addition, reactivation of the inactivated X-chromosome in female cells is confirmed in Oct4- or Nanog-positive iPSCs [137]. These in vitro analyses of pluripotency are verified using the most stringent assays in order to define the stem-like state of these reprogrammed cells: Oct4- or Nanog-positive iPSCs generate postnatal chimeras upon blastocyst injection, contribute to the germ line [136–138] and generate “all iPSC-embryos” through tetraploid complementation [138].

Other studies showed that Sox2 is also required, not only for normal embryonic development and lineage specification but also for successful reprogramming. Embryos that carry a homozygous deletion of the Sox2 gene die soon after implantation of the embryo revealing the essential role of the regulator [139]. Interestingly, Sox2 requirement is phenotypically not manifested until after implantation due to sufficient maternal Sox2 protein in these early days of embryonic development [139]. Deeper insight was given by a study that used small interfering (si) RNA-mediated depletion of both, maternal and embryonic, Sox2 mRNA at the two-cell stage [140]. In vitro observation of embryonic development revealed that these embryos arrest at the morula stage and fail to form trophectoderm revealing a major role for Sox2 in establishment of the trophectoderm lineage. Interestingly, expression of the pluripotency-associated markers Oct4 and Nanog remained unaffected when Sox2 was depleted indicating that these three transcription factors exhibit independent functions despite of their high connectivity in regards to pluripotency [140]. The role of Sox2 in the process of reprogramming has also been investigated and studies showed that endogenous expression of Sox2 facilitates the conversion of melanocytes into iPSCs indicating that particular cell type are more susceptible to reprogramming than others, dependent on their endogenous gene expression signature [141].

Knowing that Oct4, Sox2 and Nanog are of individual importance in regards to pluripotency, also their direct or indirect interaction is of great interest. Transcription factors generally work in a complex interaction system including several different proteins rather than playing an isolated role during development and homeostasis. This holds true for the pluripotency- associated factors Oct4, Sox2 and Nanog, which demonstrate a high degree of complex interactions with promoter regions and other proteins. For instance, Sox2 supports maintenance of pluripotency in part by regulating Oct4 levels whereas Oct4 can heterodimerize with Sox2 showing the interconnected regulatory network [142]. On transcriptional level it is known that Oct4, Sox2 and Nanog bind together on their own promoters to form an auto-regulatory loop, which stabilizes the pluripotent state (summarized in Figure 2). This auto-regulatory circuitry in combination with para-regulatory effects on

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each of the factors suggests that the three factors function collaboratively to maintain their own and each other’s expression. Aside from direct binding to each other’s promoters, these three factors often co-occupy their target genes indicating a synergistic role in regulation of gene expression. One study using chromatin immunoprecipitation and a hybridization-based screening of promoter sequences established a model that includes a subset of active and a subset of repressed target genes, which promoters are co-occupied by Oct4, Sox2, and Nanog [143]. The active set includes genes encoding factors of chromatin-remodeling and histone-modifying complexes as well as genes that directly encode for transcription factors such as signal transducer and activator of transcription 3 (STAT3), which themselves are known to regulate specific genes [143]. These data indicate that the three key players of pluripotency guide a complex gene expression signature and work as a regulatory unit aside of their independent functions.

On protein level, direct interactions are shown for Oct4, Sox2 and Nanog. In ESCs a multi- protein complex containing Oct4 and Nanog has been identified by iterative immunoprecipitation [144]. In addition, Sox2, Oct4 and Nanog do not only associate with one another in pluripotent cells, they also cluster together with common transcription factors

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providing further evidence that multiple interacting proteins synergistically control pluripotency. For instance, using embryonic stem cells engineered for inducible expression of the four reprogramming factors (OKSM), more than 70 proteins that associate with Sox2 in ESC were identified [145]. Strikingly, 25 % of these proteins have been shown to be part of the Oct4-interactome studied previously [145]. In an Oct4-centered analysis a larger dense interaction network was detected, which contains 166 proteins and includes Sall4, Tcfcp2l1, Dax1, and Esrrb. The network is not only comprised of transcription factors and chromatin- modifying complexes that are known to possess roles in self-renewal, but many factors have not been previously associated with the pluripotency network [146]. In addition, a comprehensive study in murine embryonic stem cells revealed a complex DNA-protein interactome of Oct4, Nanog and Sox2. The three transcription factors co-occupy promoters of genes, whose translated proteins in turn are present in protein-complexes containing Oct4 ([147], simplified in Figure 2). Sall4, for instance, is a downstream target of all three master

regulators and was found in the Oct4-centered protein-network purified from mouse embryonic stem cells [147]. This further underlines the interconnectivity of regulatory loops used by Oct4, Sox2 and Nanog in order to maintain pluripotency.

Considering the tight interplay between these three master regulators of pluripotency, it is plausible that overexpression of one or two transcription factors, for instance in OSKM- directed reprogramming, re-activates expression of endogenous Oct4, Sox2 and Nanog, the products of which in turn contribute to maintenance of their own gene expression guiding the cellular state towards pluripotency (reviewed in [148]).