Deletion of the Polycomb-Group Protein EZH2 Leads to Compromised Self-Renewal and Differentiation Defects in Human Embryonic Stem Cells

Through the histone methyltransferase EZH2, the Polycomb complex PRC2 mediates H3K27me3 and is associated with transcriptional repression. PRC2 regulates cell-fate decisions in model organisms; however, its role in regulating cell differentiation during human embryogenesis is unknown. Here, we report the characterization of $\small \textit{EZH2}$-deficient human embryonic stem cells (hESCs). H3K27me3 was lost upon $\small \textit{EZH2}$ deletion, identifying an essential requirement for EZH2 in methylating H3K27 in hESCs, in contrast to its non-essential role in mouse ESCs. Developmental regulators were derepressed in $\small \textit{EZH2}$-deficient hESCs, and single-cell analysis revealed an unexpected acquisition of lineage-restricted transcriptional programs. $\small \textit{EZH2}$-deficient hESCs show strongly reduced self-renewal and proliferation, thereby identifying a more severe phenotype compared to mouse ESCs. $\small \textit{EZH2}$-deficient hESCs can initiate differentiation toward developmental lineages; however, they cannot fully differentiate into mature specialized tissues. Thus, $\small \textit{EZH2}$ is required for stable ESC self-renewal, regulation of transcriptional programs, and for late-stage differentiation in this model of early human development.Wellcome Trust (Grant ID: WT093736), Biotechnology and Biological Sciences Research Council (Grant ID: BBS/E/B/000C0402), Medical Research Council (DTG Studentships, Grant ID: MR/J003808/1

The pluripotency factor Nanog regulates pericentromeric heterochromatin organization in mouse embryonic stem cells.

An open and decondensed chromatin organization is a defining property of pluripotency. Several epigenetic regulators have been implicated in maintaining an open chromatin organization, but how these processes are connected to the pluripotency network is unknown. Here, we identified a new role for the transcription factor NANOG as a key regulator connecting the pluripotency network with constitutive heterochromatin organization in mouse embryonic stem cells. Deletion of Nanog leads to chromatin compaction and the remodeling of heterochromatin domains. Forced expression of NANOG in epiblast stem cells is sufficient to decompact chromatin. NANOG associates with satellite repeats within heterochromatin domains, contributing to an architecture characterized by highly dispersed chromatin fibers, low levels of H3K9me3, and high major satellite transcription, and the strong transactivation domain of NANOG is required for this organization. The heterochromatin-associated protein SALL1 is a direct cofactor for NANOG, and loss of Sall1 recapitulates the Nanog-null phenotype, but the loss of Sall1 can be circumvented through direct recruitment of the NANOG transactivation domain to major satellites. These results establish a direct connection between the pluripotency network and chromatin organization and emphasize that maintaining an open heterochromatin architecture is a highly regulated process in embryonic stem cells.We thank Ludovic Vallier for constitutive Nanog-EpiSC, Gabrielle Brons for 129S2 EpiSC, Prim Singh for H3K9me3 antibody, Maria Elena Torres Padilla for TALE-mClover and luciferase plasmids, Wellcome Trust Sanger Institute for pCyL43 plasmid and Andras Nagy for PB-TET and rtTA plasmids. We are grateful to David Oxley and Judith Webster Novo et al. for mass spectrometry support, Simon Walker for imaging support and Anne Segonds- Pichon for statistical advice. We thank Wolf Reik and Jon Houseley for comments on the manuscript and members of Wolf Reik’s group for helpful discussions. P.J.R.-G. is supported by the Wellcome Trust [WT093736], BBSRC [M022285] and the European Commission Network of Excellence EpiGeneSys [HEALTH-F4-2010-257082]. The work was also supported with funds from the Canadian Institutes of Health Research to J.E. [Team Grant EPS-129129] and D.P.B.-J. D.P.B-J. holds the Canada Research Chair in Molecular and Cellular Imaging. I.C. is supported by the MRC

Comprehensive Cell Surface Protein Profiling Identifies Specific Markers of Human Naive and Primed Pluripotent States

Human pluripotent stem cells (PSCs) exist in naive and primed states and provide important models to investigate the earliest stages of human development. Naive cells can be obtained through primed-to-naive resetting, but there are no reliable methods to prospectively isolate unmodified naive cells during this process. Here we report comprehensive profiling of cell surface proteins by flow cytometry in naive and primed human PSCs. Several naive-specific, but not primed-specific, proteins were also expressed by pluripotent cells in the human preimplantation embryo. The upregulation of naive-specific cell surface proteins during primed-to-naive resetting enabled the isolation and characterization of live naive cells and intermediate cell populations. This analysis revealed distinct transcriptional and X chromosome inactivation changes associated with the early and late stages of naive cell formation. Thus, identification of state-specific proteins provides a robust set of molecular markers to define the human PSC state and allows new insights into the molecular events leading to naive cell resetting.Imaging was performed at the Live Cell Imaging Facility/Nikon Center of Excellence, Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden, supported by grants from the Knut and Alice Wallenberg Foundation, the Swedish Research Council, the Centre for Innovative Medicine, and the Jonasson donation to the School of Technology and Health, Royal Institute of Technology, Sweden. We would like to acknowledge the MedH Flow Cytometry facility at Karolinska Institutet, supported by grants from Karolinska Institutet and the Stockholm County Council. We thank Céline Vallot and Claire Rougeulle at the Université Paris Diderot for providing X chromosome SNP coordinates. We are grateful to Rudolph Jaenisch at the Whitehead Institute for Biomedical Research for providing WIBR3 cells and Austin Smith at the WT–MRC Cambridge Stem Cell Institute for providing H9 NK2 and FiPS cells. We thank all couples who donated embryos to this study. S.P., A.P.R., J.P.S., and F.L. are supported by grants from the Swedish Research Council (2013-2570), Ragnar Söderberg Foundation (M67/13), Swedish Foundation for Strategic Research (ICA-5), Knut and Alice Wallenberg Foundation (4-1205/2016 and 4-148/2017), and Centre for Innovative Medicine and by a Lau fellowship. R.W. is an ISAC Shared Resource Laboratory Emerging Leader. A.J.C. is supported by an MRC DTG Studentship (MR/J003808/1). P.J.R.G. is supported by the Wellcome Trust (WT093736) and BBSRC (BBS/ E/B/000C0402)

Satellite repeat transcripts modulate heterochromatin condensates and safeguard chromosome stability in mouse embryonic stem cells

Heterochromatin maintains genome integrity and function, and is organised into distinct nuclear domains. Some of these domains are proposed to form by phase separation through the accumulation of HP1ɑ. Mouse heterochromatin contains noncoding major satellite repeats (MSR), which are highly transcribed in mouse embryonic stem cells (ESCs). Here, we report that MSR transcripts can drive the formation of HP1ɑ droplets in vitro, and modulate heterochromatin into dynamic condensates in ESCs, contributing to the formation of large nuclear domains that are characteristic of pluripotent cells. Depleting MSR transcripts causes heterochromatin to transition into a more compact and static state. Unexpectedly, changing heterochromatin’s biophysical properties has severe consequences for ESCs, including chromosome instability and mitotic defects. These findings uncover an essential role for MSR transcripts in modulating the organisation and properties of heterochromatin to preserve genome stability. They also provide insights into the processes that could regulate phase separation and the functional consequences of disrupting the properties of heterochromatin condensates

Critique of the review of 'Water fluoridation for the prevention of dental caries' published by the Cochrane Collaboration in 2015

The Cochrane Review on water fluoridation for the prevention of dental caries was published in 2015 and attracted considerable interest and comment, especially in countries with extensive water fluoridation programmes. The Review had two objectives: (i) to evaluate the effects of water fluoridation (artificial or natural) on the prevention of dental caries, and (ii) to evaluate the effects of water fluoridation (artificial or natural) on dental fluorosis. The authors concluded, inter alia, that there was very little contemporary evidence, meeting the Review's inclusion criteria, that evaluated the effectiveness of water fluoridation for the prevention of dental caries. The purpose of this critique is to examine the conduct of the above Review, and to put it into context in the wider body of evidence regarding the effectiveness of water fluoridation. While the overall conclusion that water fluoridation is effective in caries prevention agrees with previous reviews, many important public health questions could not be answered by the Review because of the restrictive criteria used to judge adequacy of study design and risk of bias. The potential benefits of using wider criteria in order to achieve a fuller understanding of the effectiveness of water fluoridation are discussed

DNA Methylation Dynamics in Human Induced Pluripotent Stem Cells over Time

Epigenetic reprogramming is a critical event in the generation of induced pluripotent stem cells (iPSCs). Here, we determined the DNA methylation profiles of 22 human iPSC lines derived from five different cell types (human endometrium, placental artery endothelium, amnion, fetal lung fibroblast, and menstrual blood cell) and five human embryonic stem cell (ESC) lines, and we followed the aberrant methylation sites in iPSCs for up to 42 weeks. The iPSCs exhibited distinct epigenetic differences from ESCs, which were caused by aberrant methylation at early passages. Multiple appearances and then disappearances of random aberrant methylation were detected throughout iPSC reprogramming. Continuous passaging of the iPSCs diminished the differences between iPSCs and ESCs, implying that iPSCs lose the characteristics inherited from the parent cells and adapt to very closely resemble ESCs over time. Human iPSCs were gradually reprogrammed through the “convergence” of aberrant hyper-methylation events that continuously appeared in a de novo manner. This iPS reprogramming consisted of stochastic de novo methylation and selection/fixation of methylation in an environment suitable for ESCs. Taken together, random methylation and convergence are driving forces for long-term reprogramming of iPSCs to ESCs

Cancer Genes Hypermethylated in Human Embryonic Stem Cells

Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation

A regression analysis of gene expression in ES cells reveals two gene classes that are significantly different in epigenetic patterns

<p>Abstract</p> <p>Background</p> <p>To understand the gene regulatory system that governs the self-renewal and pluripotency of embryonic stem cells (ESCs) is an important step for promoting regenerative medicine. In it, the role of several core transcription factors (TFs), such as Oct4, Sox2 and Nanog, has been intensively investigated, details of their involvement in the genome-wide gene regulation are still not well clarified.</p> <p>Methods</p> <p>We constructed a predictive model of genome-wide gene expression in mouse ESCs from publicly available ChIP-seq data of 12 core TFs. The tag sequences were remapped on the genome by various alignment tools. Then, the binding density of each TF is calculated from the genome-wide bona fide TF binding sites. The TF-binding data was combined with the data of several epigenetic states (DNA methylation, several histone modifications, and CpG island) of promoter regions. These data as well as the ordinary peak intensity data were used as predictors of a simple linear regression model that predicts absolute gene expression. We also developed a pipeline for analyzing the effects of predictors and their interactions.</p> <p>Results</p> <p>Through our analysis, we identified two classes of genes that are either well explained or inefficiently explained by our model. The latter class seems to be genes that are not directly regulated by the core TFs. The regulatory regions of these gene classes show apparently distinct patterns of DNA methylation, histone modifications, existence of CpG islands, and gene ontology terms, suggesting the relative importance of epigenetic effects. Furthermore, we identified statistically significant TF interactions correlated with the epigenetic modification patterns.</p> <p>Conclusions</p> <p>Here, we proposed an improved prediction method in explaining the ESC-specific gene expression. Our study implies that the majority of genes are more or less directly regulated by the core TFs. In addition, our result is consistent with the general idea of relative importance of epigenetic effects in ESCs.</p

A direct physical interaction between Nanog and Sox2 regulates embryonic stem cell self-renewal

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

Cerebellum Abnormalities in Idiopathic Generalized Epilepsy with Generalized Tonic-Clonic Seizures Revealed by Diffusion Tensor Imaging

Although there is increasing evidence suggesting that there may be subtle abnormalities in idiopathic generalized epilepsy (IGE) patients using modern neuroimaging techniques, most of these previous studies focused on the brain grey matter, leaving the underlying white matter abnormalities in IGE largely unknown, which baffles the treatment as well as the understanding of IGE. In this work, we adopted multiple methods from different levels based on diffusion tensor imaging (DTI) to analyze the white matter abnormalities in 14 young male IGE patients with generalized tonic-clonic seizures (GTCS) only, comparing with 29 age-matched male healthy controls. First, we performed a voxel-based analysis (VBA) of the fractional anisotropy (FA) images derived from DTI. Second, we used a tract-based spatial statistics (TBSS) method to explore the alterations within the white matter skeleton of the patients. Third, we adopted region-of-interest (ROI) analyses based on the findings of VBA and TBSS to further confirm abnormal brain regions in the patients. At last, considering the convergent evidences we found by VBA, TBSS and ROI analyses, a subsequent probabilistic fiber tractography study was performed to investigate the abnormal white matter connectivity in the patients. Significantly decreased FA values were consistently observed in the cerebellum of patients, providing fresh evidence and new clues for the important role of cerebellum in IGE with GTCS