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Embryonic stem (ES) cell self‐renewal efficiency is determined by the Nanog protein level. However, the protein partners of Nanog that function to direct self‐renewal are unclear. Here, we identify a Nanog interactome of over 130 proteins including transcription factors, chromatin modifying complexes, phosphorylation and ubiquitination enzymes, basal transcriptional machinery members, and RNA processing factors. Sox2 was identified as a robust interacting partner of Nanog. The purified Nanog–Sox2 complex identified a DNA recognition sequence present in multiple overlapping Nanog/Sox2 ChIP‐Seq data sets. The Nanog tryptophan repeat region is necessary and sufficient for interaction with Sox2, with tryptophan residues required. In Sox2, tyrosine to alanine mutations within a triple‐repeat motif (S X T/S Y) abrogates the Nanog–Sox2 interaction, alters expression of genes associated with the Nanog‐Sox2 cognate sequence, and reduces the ability of Sox2 to rescue ES cell differentiation induced by endogenous Sox2 deletion. Substitution of the tyrosines with phenylalanine rescues both the Sox2–Nanog interaction and efficient self‐renewal. These results suggest that aromatic stacking of Nanog tryptophans and Sox2 tyrosines mediates an interaction central to ES cell self‐renewal. This paper features a comprehensive proteomic view on the Nanog interactome. Further, it molecularly and functionally defines the intimate interplay of Nanog with another pluripotency determinant Sox2.

Tet1 , Tet2 , and Tet3 encode DNA demethylases that play critical roles during stem cell differentiation and reprogramming to pluripotency. Although all three genes are transcribed in pluripotent cells, little is known about the expression of the corresponding proteins. Here, we tagged all the endogenous Tet family alleles using CRISPR/Cas9, and characterised TET protein expression in distinct pluripotent cell culture conditions. Whereas TET1 is abundantly expressed in both naïve and primed pluripotent cells, TET2 expression is restricted to the naïve state. Moreover, TET2 is expressed heterogeneously in embryonic stem cells (ESCs) cultured in serum/leukemia inhibitory factor, with expression correlating with naïve pluripotency markers. FACS-sorting of ESCs carrying a Tet2 Flag-IRES-EGFP reporter demonstrated that TET2-negative cells have lost the ability to form undifferentiated ESC colonies. We further show that TET2 binds to the transcription factor NANOG. We hypothesize that TET2 and NANOG co-localise on chromatin to regulate enhancers associated with naïve pluripotency genes.

Deletion of Sox2 from mouse embryonic stem cells (ESCs) causes trophectodermal differentiation. While this can be prevented by enforced expression of the related SOXB1 proteins, SOX1 or SOX3, the roles of SOXB1 proteins in epiblast stem cell (EpiSC) pluripotency are unknown. Here, we show that Sox2 can be deleted from EpiSCs with impunity. This is due to a shift in the balance of SoxB1 expression in EpiSCs, which have decreased Sox2 and increased Sox3 compared to ESCs. Consistent with functional redundancy, Sox3 can also be deleted from EpiSCs without eliminating self-renewal. However, deletion of both Sox2 and Sox3 prevents self-renewal. The overall SOXB1 levels in ESCs affect differentiation choices: neural differentiation of Sox2 heterozygous ESCs is compromised, while increased SOXB1 levels divert the ESC to EpiSC transition towards neural differentiation. Therefore, optimal SOXB1 levels are critical for each pluripotent state and for cell fate decisions during exit from naïve pluripotency.

An analysis whether death qualification--the process of removing potential jurors who are unwilling to impose the death penalty--survives an originalist assessment is presented. It begins with the background of death qualification and then analyzes whether the process survives; a number of potential originalist objections. Ultimately, it concludes that although there was no direct analogue for death qualification at common law or in criminal procedure at the time of the ratification of the Constitution and Bill of Rights, death qualification does not violate an originalist understanding of the Sixth Amendment right to an impartial jury or of a constitutional criminal trial.

Nanog changes tack In 2003 the transcription factor Nanog was identified as a key contributor to the property that makes embryonic stem cells unique: pluripotency. Nanog, named after Tir nan Og, the 'land of the forever-young' of Celtic myth, was thought to be required for stem cells to multiply while retaining the potential to differentiate. New work in mouse embryonic stem cells suggests a rather different picture. In fact Nanog is not essential for maintaining pluripotency; its levels fluctuate, but Nanog appears to stabilize the pluripotent state by resisting or reversing alternative states of gene expression. The transcription factor Nanog is considered a hallmark of pluripotent cells in vivo and in vitro , and loss of Nanog an early marker of differentiation. This is now revised by the demonstration that Nanog is not essential for maintaining pluripotency, but acts in stabilizing the pluripotent state. Nanog is a divergent homeodomain protein found in mammalian pluripotent cells and developing germ cells 1 , 2 . Deletion of Nanog causes early embryonic lethality 2 , whereas constitutive expression enables autonomous self-renewal of embryonic stem cells 1 . Nanog is accordingly considered a core element of the pluripotent transcriptional network 3 , 4 , 5 , 6 , 7 . However, here we report that Nanog fluctuates in mouse embryonic stem cells. Transient downregulation of Nanog appears to predispose cells towards differentiation but does not mark commitment. By genetic deletion we show that, although they are prone to differentiate, embryonic stem cells can self-renew indefinitely in the permanent absence of Nanog. Expanded Nanog null cells colonize embryonic germ layers and exhibit multilineage differentiation both in fetal and adult chimaeras. Although they are also recruited to the germ line, primordial germ cells lacking Nanog fail to mature on reaching the genital ridge. This defect is rescued by repair of the mutant allele. Thus Nanog is dispensible for expression of somatic pluripotency but is specifically required for formation of germ cells. Nanog therefore acts primarily in construction of inner cell mass and germ cell states rather than in the housekeeping machinery of pluripotency. We surmise that Nanog stabilizes embryonic stem cells in culture by resisting or reversing alternative gene expression states.

NANOG, OCT4 and SOX2 form the core network of transcription factors supporting embryonic stem (ES) cell self‐renewal. While OCT4 and SOX2 expression is relatively uniform, ES cells fluctuate between states of high NANOG expression possessing high self‐renewal efficiency, and low NANOG expression exhibiting increased differentiation propensity. NANOG, OCT4 and SOX2 are currently considered to activate transcription of each of the three genes, an architecture that cannot readily account for NANOG heterogeneity. Here, we examine the architecture of the Nanog ‐centred network using inducible NANOG gain‐ and loss‐of‐function approaches. Rather than activating itself, Nanog activity is autorepressive and OCT4/SOX2‐independent. Moreover, the influence of Nanog on Oct4 and Sox2 expression is minimal. Using Nanog :GFP reporters, we show that Nanog autorepression is a major regulator of Nanog transcription switching. We conclude that the architecture of the pluripotency gene regulatory network encodes the capacity to generate reversible states of Nanog transcription via a Nanog ‐centred autorepressive loop. Therefore, cellular variability in self‐renewal efficiency is an emergent property of the pluripotency gene regulatory network. The discovery of Nanog autorepression offers a new perspective on the transcriptional networks that govern Nanog's heterogeneous expression in ES cells.

Cell identity relies upon transcription factors (TFs). The concentration of the TF NANOG determines the efficiency of maintenance of mouse embryonic stem cell (ESC) identity. However, the mechanisms by which NANOG acts are not fully understood. Models of mammalian transcription generally propose that TFs bind DNA and connect to the transcriptional machinery indirectly via intermediary proteins. Accordingly, examples of direct contact between cell-type specific TFs and RNA synthesis enzymes in mammalian cells remain elusive. Here we show that NANOG directly contacts RNA Polymerase II (RNAPII) via aromatic residues within the low complexity domains of both proteins. NANOG can localize RNAPII to a specific DNA site, with RNAPII enhancing NANOG DNA affinity. The NANOG-RNAPII complex is dissociated by the transcriptional pause-release enzyme, CDK9. Inhibition of CDK9 enhances NANOG chromatin binding, while induction of NANOG localizes RNAPII to specific NANOG chromatin sites. Induced RNAPII localization is selective for targets activated by NANOG. A NANOG mutant that retains DNA, but not RNAPII binding, cannot stimulate RNAPII localization to chromatin, shows an impaired transcriptional response and does not drive LIF-independent ESC self-renewal. These results identify a novel, regulatable interaction between a cell-type specific TF and RNAPII that can change transcription and alter cell fate.

The Nanog gene plays a key role in the pluripotency of early embryonic cells in vitro and in vivo. In this article retrotransposed copies of Nanog, termed NanogPc and NanogPd, are identified on mouse Chromosomes 4 and 7, respectively. In contrast to the two previously characterized mouse Nanog retrogenes that contain multiple frameshifts and point mutations, NanogPc and NanogPd are 98% identical to NANOG within the open reading frame and encode proteins with activity in an embryonic stem cell self-renewal assay. Mutations common to all four retrotransposed genes but distinct from Nanog suggest divergence from a common progenitor that appears likely to be Nanog because transcripts derived from Nanog but not from the retrogenes are detected in germ-line cells. The possibility that expression of Nanog could be erroneously attributed to novel cellular sources is suggested by the high homology among Nanog, NanogPc, and NanogPd. Analysis of distinct Mus species suggests that NanogPc and NanogPd arose between divergence of M. caroli and M. spretus and indicates that Nanog retrotransposition events continue to occur at a high frequency, a property likely to extend to other germ-line transcripts.