Sex differences exist in a myriad of human diseases, including disorders of cardiac electrophysiology. These differences are poorly understood and have been traditionally attributed to the role of gonadal sex hormones. Recent work studying the evolution of sex chromosomes has brought to light the potential contribution of sex chromosome complement in sexual dimorphism. Natural sex chromosome complement variants in the form of Turner syndrome (45, XO) and Klinefelter syndrome (47, XXY) display an increased propensity for congenital heart disease and changes in electrophysiological parameters. An example of this includes QTc, which is prolonged in Turner syndrome and shortened in Klinefelter syndrome. We propose that studying phenotypes such as QTc in sex chromosome aneuploidy patients will not only be essential for appropriate clinical management of these patients but will also provide a model to help to elucidate the complex interplay between the contributions of genetics and hormones in euploid male–female differences.

Mouse and human embryonic stem cells (ESCs) are derived from the inner cell mass of blastocysts however are distinct in their molecular and biological properties. Historically these discrepancies have been attributed to speciesspecific differences. Recent work capturing human ESC-like cells from postimplantation mouse embryos instead suggests that they represent distinct developmental stages of the pluripotent epiblast. Mouse ESCs resemble the preimplantation epiblast where cells are “naive” and have no differentiation bias. In contrast human ESCs represent a developmental stage postimplantation “primed” for lineage specification. Here a systematic approach was taken to identify conditions to induce and maintain naive human ESCs by screening for small molecules that support self-renewal based on the maintenance of endogenous OCT4 distal enhancer activity, a molecular signature of naive pluripotency. Iterative chemical screening identified a combination of five kinase inhibitors, 5i (MEK, GSK3, BRAF, SRC, ROCK) that induce and maintain OCT4 distal enhancer activity when applied directly to conventional human ESCs. Furthermore, these conditions allowed the capture of naive pluripotent cells from human embryos and somatic cells through reprogramming. The inhibitors described herein capture a population of human pluripotent cells in which transcription factors specific to ground state pluripotency are highly upregulated and bivalent chromatin domains are depleted. Comparison to published reports of naive human ESCs suggest that previous attempts to isolate naive human pluripotent cells fall short of recapitulating the transcriptional foundations of the naive state. In contrast the conditions presented in this thesis capture a distinct state of human pluripotency that very closely resembles mouse ESCs. Given the stringency of the reporter used and the systematic approach taken, this study should provide a framework for future work in characterizing and consolidating naive human pluripotency.

Nature 550, 67–73 (2017); doi:10.1038/nature24033 In this Article, the received date appeared wrongly in the advance online publication (AOP) version as 12 June 2016 rather than 12 June 2017. This error was corrected online on 22 September 2017; the print version is also correct.

Having the correct number of chromosomes is vital for normal development and health. Sex chromosome trisomy affects 0.1% of the human population and is associated with infertility. We show that during reprogramming to induced pluripotent stem cells (iPSCs), fibroblasts from sterile trisomic XXYand XYYmice lose the extra sex chromosome through a phenomenon we term trisomy-biased chromosome loss (TCL). Resulting euploid XY iPSCs can be differentiated into the male germ cell lineage and functional sperm that can be used in intracytoplasmic sperm injection to produce chromosomally normal, fertile offspring. Sex chromosome loss is comparatively infrequent during mouse XX and XY iPSC generation. TCL also applies to other chromosomes, generating euploid iPSCs from cells of a Down syndrome mouse model. It can also create euploid iPSCs from human trisomic patient fibroblasts. The findings have relevance to overcoming infertility and other trisomic phenotypes.

Nature 525, E6–E9 (2015); doi:10.1038/nature15513 During extensive revisions of this BCA, we inadvertently omitted a citation by Takaho A. Endo that used variant calls from RNA-seq data to conclude that the purported Fgf4-induced stem cells (FI-SCs) described in Obokata et al.1 constituted a mixtureof trophoblastic and embryonic stem cells2.

In this study, we describe the 3D chromosome regulatory landscape of human naive and primed embryonic stem cells. To devise this map, we identified transcriptional enhancers and insulators in these cells and placed them within the context of cohesin-associated CTCF-CTCF loops using cohesin ChIA-PET data. The CTCF-CTCF loops we identified form a chromosomal framework of insulated neighborhoods, which in turn form topologically associating domains (TADs) that are largely preserved during the transition between the naive and primed states. Regulatory changes in enhancer-promoter interactions occur within insulated neighborhoods during cell state transition. The CTCF anchor regions we identified are conserved across species, influence gene expression, and are a frequent site of mutations in cancer cells, underscoring their functional importance in cellular regulation. These 3D regulatory maps of human pluripotent cells therefore provide a foundation for future interrogation of the relationships between chromosome structure and gene control in development and disease.

Nature 525, E6–E9 (2015); doi:10.1038/nature15513 During extensive revisions of this BCA, we inadvertently omitted a citation by Takaho A. Endo that used variant calls from RNA-seq data to conclude that the purported Fgf4-induced stem cells (FI-SCs) described in Obokata et al.1 constituted a mixtureof trophoblastic and embryonic stem cells2.

Tet enzymes (Tet1/2/3) convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and are dynamically expressed during development. Whereas loss of individual Tet enzymes or combined deficiency of Tet1/2 allows for embryogenesis, the effect of complete loss of Tet activity and 5hmC marks in development is not established. We have generated Tet1/2/3 triple-knockout (TKO) mouse embryonic stem cells (ESCs) and examined their developmental potential. Combined deficiency of all three Tets depleted 5hmC and impaired ESC differentiation, as seen in poorly differentiated TKO embryoid bodies (EBs) and teratomas. Consistent with impaired differentiation, TKO ESCs contributed poorly to chimeric embryos, a defect rescued by Tet1 reexpression, and could not support embryonic development. Global gene-expression and methylome analyses of TKO EBs revealed promoter hypermethylation and deregulation of genes implicated in embryonic development and differentiation. These findings suggest a requirement for Tet- and 5hmC-mediated DNA demethylation in proper regulation of gene expression during ESC differentiation and development.

Embryonic stem cells (ESCs) of mice and humans have distinct molecular and biological characteristics, raising the question of whether an earlier, "naive" state of pluripotency may exist in humans. Here we took a systematic approach to identify small molecules that support self-renewal of naive human ESCs based on maintenance of endogenous OCT4 distal enhancer activity, a molecular signature of ground state pluripotency. Iterative chemical screening identified a combination of five kinase inhibitors that induces and maintains OCT4 distal enhancer activity when applied directly to conventional human ESCs. These inhibitors generate human pluripotent cells in which transcription factors associated with the ground state of pluripotency are highly upregulated and bivalent chromatin domains are depleted. Comparison with previously reported naive human ESCs indicates that our conditions capture a distinct pluripotent state in humans that closely resembles that of mouse ESCs. This study presents a framework for defining the culture requirements of naive human pluripotent cells.

Tet enzymes (Tet1/2/3) convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in various embryonic and adult tissues. Mice mutant for either Tet1 or Tet2 are viable, raising the question of whether these enzymes have overlapping roles in development. Here we have generated Tet1 and Tet2 double-knockout (DKO) embryonic stem cells (ESCs) and mice. DKO ESCs remained pluripotent but were depleted of 5hmC and caused developmental defects in chimeric embryos. While a fraction of double-mutant embryos exhibited midgestation abnormalities with perinatal lethality, viable and overtly normal Tet1/Tet2-deficient mice were also obtained. DKO mice had reduced 5hmC and increased 5mC levels and abnormal methylation at various imprinted loci. Nevertheless, animals of both sexes were fertile, with females having smaller ovaries and reduced fertility. Our data show that loss of both enzymes is compatible with development but promotes hypermethylation and compromises imprinting. The data also suggest a significant contribution of Tet3 to hydroxylation of 5mC during development.

Recent advances have highlighted the central role of DNA methylation in leukemogenesis and have led to clinical trials of epigenetic therapy, notably hypomethylating agents, in myelodysplasia and acute myeloid leukemia. However, despite these advances, our understanding of the dynamic regulation of the methylome remains poor. We have attempted to address this shortcoming by producing a dynamic, six-compartmental model of DNA methylation levels based on the activity of the Dnmt methyltransferase proteins. In addition, the model incorporates the recently discovered Tet family proteins which enzymatically convert methylcytosine to hydroxymethylcytosine. A set of first order, partial differential equations comprise the model and were solved via numerical integration. The model is able to predict the relative abundances of unmethylated, hemimethylated, fully methylated, and hydroxymethylated CpG dyads in the DNA of cells with fully functional Dnmt and Tet proteins. In addition, the model accurately predicts the experimentally measured changes in these abundances with disruption of Dnmt function. Furthermore, the model reveals the mechanism whereby CpG islands are maintained in a hypomethylated state via local modulation of Dnmt and Tet activities without any requirement for active demethylation. We conclude that this model provides an accurate depiction of the major epigenetic processes involving modification of DNA.

The Tet family of enzymes (Tet1/2/3) converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Mouse embryonic stem cells (mESCs) highly express Tet1 and have an elevated level of 5hmC. Tet1 has been implicated in ESC maintenance and lineage specification in vitro but its precise function in development is not well defined. To establish the role of Tet1 in pluripotency and development, we have generated Tet1 mutant mESCs and mice. Tet1(-/-) ESCs have reduced levels of 5hmC and subtle changes in global gene expression, and are pluripotent and support development of live-born mice in tetraploid complementation assay, but display skewed differentiation toward trophectoderm in vitro. Tet1 mutant mice are viable, fertile, and grossly normal, though some mutant mice have a slightly smaller body size at birth. Our data suggest that Tet1 loss leading to a partial reduction in 5hmC levels does not affect pluripotency in ESCs and is compatible with embryonic and postnatal development.