OCT4/SOX2-independent<i>Nanog</i>autorepression modulates heterogeneous<i>Nanog</i>gene expression in mouse ES cells

Navarro, Pablo; Festuccia, Nicola; Colby, Douglas; Gagliardi, Alessia; Mullin, Nicholas P.; Zhang, Wensheng; Karwacki-Neisius, Violetta; Osorno, Rodrigo; Kelly, David A.; Robertson, Marcus; Chambers, Ian

doi:10.1038/emboj.2012.321

Cited by 126 publications

(164 citation statements)

References 43 publications

(90 reference statements)

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“…This reporter line was also used to assess the effect of transcription factor overexpression. This confirmed that NANOG overexpression increased Esrrb‐GFP expression, and reduced Nanog‐mCherry levels, consistent with autorepression (Navarro et al , 2012). In contrast, ESRRB overexpression showed minimal effects (Fig 1G).…”

Section: Resultssupporting

confidence: 73%

Esrrb extinction triggers dismantling of naïve pluripotency and marks commitment to differentiation

et al. 2018

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Self‐renewal of embryonic stem cells (ESCs) cultured in LIF/fetal calf serum (FCS) is incomplete with some cells initiating differentiation. While this is reflected in heterogeneous expression of naive pluripotency transcription factors (TFs), the link between TF heterogeneity and differentiation is not fully understood. Here, we purify ESCs with distinct TF expression levels from LIF/FCS cultures to uncover early events during commitment from naïve pluripotency. ESCs carrying fluorescent Nanog and Esrrb reporters show Esrrb downregulation only in Nanoglow cells. Independent Esrrb reporter lines demonstrate that Esrrbnegative ESCs cannot effectively self‐renew. Upon Esrrb loss, pre‐implantation pluripotency gene expression collapses. ChIP‐Seq identifies different regulatory element classes that bind both OCT4 and NANOG in Esrrbpositive cells. Class I elements lose NANOG and OCT4 binding in Esrrbnegative ESCs and associate with genes expressed preferentially in naïve ESCs. In contrast, Class II elements retain OCT4 but not NANOG binding in ESRRB‐negative cells and associate with more broadly expressed genes. Therefore, mechanistic differences in TF function act cumulatively to restrict potency during exit from naïve pluripotency.

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Section: Resultssupporting

confidence: 73%

Esrrb extinction triggers dismantling of naïve pluripotency and marks commitment to differentiation

et al. 2018

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“…It is interesting that Hoxa1 shows occupancy on both the Nanog transcription start site (TSS) and a 4-kb region upstream ( Fig. 2A), which corresponds to a previously identified autoregulatory enhancer (ARE) of Nanog (12). Binding of Nanog and Zfp281 to adjacent sites recruits the NuRD complex and mediates autorepression through this ARE in stem cells to maintain the appropriate levels of Nanog (16,17).…”

Section: Resultsmentioning

confidence: 99%

“…For example, Nanog is known to directly activate Oct4 and Sox2, whereas they in turn positively cross-regulate Nanog. Oct4 and Sox2 positively feedback to maintain their own expression via direct autoregulation, whereas Nanog modulates its level of gene expression by negative autoregulation mediated by interactions with Zfp281 (zinc finger protein 281), which recruits the NuRD repressor complex (12)(13)(14)(15)(16)(17).…”

mentioning

confidence: 99%

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

Kumar

Parker

Parrish

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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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.

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“…Other mechanisms to justify the observed NANOG heterogeneity in mESCs have been proposed, coinciding in the view that fluctuations are controlled at the transcriptional level and emerge from particular configurations of the pluripotency gene regulatory network, involving various types of feedback loops (Niwa et al, 2009;Lanner and Rossant, 2010;MacArthur et al, 2012). For instance, transcriptional autorepression has been proposed to generate fluctuations in Nanog expression (Navarro et al, 2012), whereas other authors propose that these fluctuations result from complex network interactions involving feedback and crosstalk between various signalling pathways and pluripotency TFs (Adachi and Niwa, 2013;Posfai et al, 2014). An intriguing mechanism based on dynamic alleleswitching of Nanog in mESCs has also been proposed to underlie its fluctuations (Miyanari and Torres-Padilla, 2012), although recent evidence does not support a contribution of this mechanism to the observed heterogeneity of NANOG expression in mESCs (Faddah et al, 2013;Filipczyk et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

Stochastic NANOG fluctuations allow mouse embryonic stem cells to explore pluripotency

et al. 2014

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Heterogeneous expression of the transcription factor NANOG has been linked to the existence of various functional states in pluripotent stem cells. This heterogeneity seems to arise from fluctuations of Nanog expression in individual cells, but a thorough characterization of these fluctuations and their impact on the pluripotent state is still lacking. Here, we have used a novel fluorescent reporter to investigate the temporal dynamics of NANOG expression in mouse embryonic stem cells (mESCs), and to dissect the lineage potential of mESCs at different NANOG states. Our results show that stochastic NANOG fluctuations are widespread in mESCs, with essentially all expressing cells showing fluctuations in NANOG levels, even when cultured in ground-state conditions (2i media). We further show that fluctuations have similar kinetics when mESCs are cultured in standard conditions (serum plus leukemia inhibitory factor) or ground-state conditions, implying that NANOG fluctuations are inherent to the pluripotent state. We have then compared the developmental potential of low-NANOG and high-NANOG mESCs, grown in different conditions, and confirm that mESCs are more susceptible to enter differentiation at the low-NANOG state. Further analysis by gene expression profiling reveals that low-NANOG cells have marked expression of lineage-affiliated genes, with variable profiles according to the signalling environment. By contrast, high-NANOG cells show a more stable expression profile in different environments, with minimal expression of lineage markers. Altogether, our data support a model in which stochastic NANOG fluctuations provide opportunities for mESCs to explore multiple lineage options, modulating their probability to change functional state.

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OCT4/SOX2-independentNanogautorepression modulates heterogeneousNanoggene expression in mouse ES cells

Cited by 126 publications

References 43 publications

Esrrb extinction triggers dismantling of naïve pluripotency and marks commitment to differentiation

Esrrb extinction triggers dismantling of naïve pluripotency and marks commitment to differentiation

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

Stochastic NANOG fluctuations allow mouse embryonic stem cells to explore pluripotency

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