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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.

X-inactivation, the molecular mechanism enabling dosage compensation in mammals, is tightly controlled during mouse early embryogenesis. In the morula, X-inactivation is imprinted with exclusive silencing of the paternally inherited X-chromosome. In contrast, in the post-implantation epiblast, X-inactivation affects randomly either the paternal or the maternal X-chromosome. The transition from imprinted to random X-inactivation takes place in the inner cell mass (ICM) of the blastocyst from which embryonic stem (ES) cells are derived. The trigger of X-inactivation, Xist , is specifically downregulated in the pluripotent cells of the ICM, thereby ensuring the reactivation of the inactive paternal X-chromosome and the transient presence of two active X-chromosomes. Moreover, Tsix , a critical cis -repressor of Xist , is upregulated in the ICM and in ES cells where it imposes a particular chromatin state at the Xist promoter that ensures the establishment of random X-inactivation upon differentiation. Recently, we have shown that key transcription factors supporting pluripotency directly repress Xist and activate Tsix and thus couple Xist / Tsix control to pluripotency. In this manuscript, we report that Rnf12 , a third X-linked gene critical for the regulation of X-inactivation, is under the control of Nanog , Oct4 and Sox2 , the three factors lying at the heart of the pluripotency network. We conclude that in mouse ES cells the pluripotency-associated machinery exerts an exhaustive control of X-inactivation by taking over the regulation of all three major regulators of X-inactivation: Xist , Tsix , and Rnf12 .

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.

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.

SOX2 is of central importance for the acquisition and maintenance of pluripotency. Investigation of the ability of SOX2 to regulate transcription in pluripotent cells has centered mainly on the cooperative binding of SOX2 and OCT4 to deoxyribonucleic acid in embryonic stem (ES) cells. However, SOX2 interacts with a large number of protein partners and is more than simply an OCT4 cofactor. Here we discuss the interaction of SOX2 with partners other than OCT4, particularly NANOG. We also discuss the consequences of deletion and overexpression of SOX2 for pluripotency of ES cells and discuss the role that SOX2 has in the induction of pluripotency and the pluripotency of epiblast stem cells.

Hyperemesis gravidarum (HG), severe nausea and vomiting of pregnancy, occurs in 0.3–2% of pregnancies and is associated with maternal and fetal morbidity. The cause of HG remains unknown, but familial aggregation and results of twin studies suggest that understanding the genetic contribution is essential for comprehending the disease etiology. Here, we conduct a genome-wide association study (GWAS) for binary (HG) and ordinal (severity of nausea and vomiting) phenotypes of pregnancy complications. Two loci, chr19p13.11 and chr4q12, are genome-wide significant ( p  < 5 × 10 −8 ) in both association scans and are replicated in an independent cohort. The genes implicated at these two loci are GDF15 and IGFBP7 respectively, both known to be involved in placentation, appetite, and cachexia. While proving the casual roles of GDF15 and IGFBP7 in nausea and vomiting of pregnancy requires further study, this GWAS provides insights into the genetic risk factors contributing to the disease. Hyperemesis gravidarum (HG) is a severe form of nausea and vomiting associated with unfavourable outcomes during pregnancy. Here, Fejzo et al. perform genome-wide scans for HG and pregnancy nausea and vomiting and identify genetic associations at two loci implicating the genes GDF15 and IGFBP7 .

Background Inherited retinal degeneration is a leading cause of incurable vision loss in the developed world. While autologous iPSC mediated photoreceptor cell replacement is theoretically possible, the lack of commercially available technologies designed to enable high throughput parallel production of patient specific therapeutics has hindered clinical translation. Methods In this study, we describe the use of the Cell X precision robotic cell culture platform to enable parallel production of clinical grade patient specific iPSCs. The Cell X is housed within an ISO Class 5 cGMP compliant closed aseptic isolator (Biospherix XVivo X2), where all procedures from fibroblast culture to iPSC generation, clonal expansion and retinal differentiation were performed. Results Patient iPSCs generated using the Cell X platform were determined to be pluripotent via score card analysis and genetically stable via karyotyping. As determined via immunostaining and confocal microscopy, iPSCs generated using the Cell X platform gave rise to retinal organoids that were indistinguishable from organoids derived from manually generated iPSCs. In addition, at 120 days post-differentiation, single-cell RNA sequencing analysis revealed that cells generated using the Cell X platform were comparable to those generated under manual conditions in a separate laboratory. Conclusion We have successfully developed a robotic iPSC generation platform and standard operating procedures for production of high-quality photoreceptor precursor cells that are compatible with current good manufacturing practices. This system will enable clinical grade production of iPSCs for autologous retinal cell replacement.

Abstract Context Circulating concentrations of free triiodothyronine (fT3), free thyroxine (fT4), and thyrotropin (TSH) are partly heritable traits. Recent studies have advanced knowledge of their genetic architecture. Epigenetic modifications, such as DNA methylation (DNAm), may be important in pituitary-thyroid axis regulation and action, but data are limited. Objective To identify novel associations between fT3, fT4, and TSH and differentially methylated positions (DMPs) in the genome in subjects from 2 Australian cohorts. Method We performed an epigenome-wide association study (EWAS) of thyroid function parameters and DNAm using participants from: Brisbane Systems Genetics Study (median age 14.2 years, n = 563) and the Raine Study (median age 17.0 years, n = 863). Plasma fT3, fT4, and TSH were measured by immunoassay. DNAm levels in blood were assessed using Illumina HumanMethylation450 BeadChip arrays. Analyses employed generalized linear mixed models to test association between DNAm and thyroid function parameters. Data from the 2 cohorts were meta-analyzed. Results We identified 2 DMPs with epigenome-wide significant (P < 2.4E−7) associations with TSH and 6 with fT3, including cg00049440 in KLF9 (P = 2.88E−10) and cg04173586 in DOT1L (P = 2.09E−16), both genes known to be induced by fT3. All DMPs had a positive association between DNAm and TSH and a negative association between DNAm and fT3. There were no DMPs significantly associated with fT4. We identified 23 differentially methylated regions associated with fT3, fT4, or TSH. Conclusions This study has demonstrated associations between blood-based DNAm and both fT3 and TSH. This may provide insight into mechanisms underlying thyroid hormone action and/or pituitary-thyroid axis function.