Yoach Rais

Derivation of novel human ground state naive pluripotent stem cells

Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3β signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo.

Deterministic direct reprogramming of somatic cells to pluripotency

Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.

m6A mRNA methylation facilitates resolution of naïve pluripotency toward differentiation

mRNA modification regulates pluripotency When stem cells progress from an embryonic pluripotent state toward a particular lineage, molecular switches dismantle the transcription factor network that keeps the cell pluripotent. Geula et al. now show that N6-methyladenosine (m6A), a messenger RNA (mRNA) modification present on transcripts of pluripotency factors, drives this transition. Methylation destabilized mRNA transcripts and limited their translation efficiency, which promoted the timely decay of naïve pluripotency. This m6A methylation was also critical for mammalian development. Science, this issue p. 1002 A messenger RNA epigenetic modification regulates stem cell progression from the pluripotent to the differentiated state. Naïve and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here, we identify Mettl3, an N6-methyladenosine (m6A) transferase, as a regulator for terminating murine naïve pluripotency. Mettl3 knockout preimplantation epiblasts and naïve embryonic stem cells are depleted for m6A in mRNAs, yet are viable. However, they fail to adequately terminate their naïve state and, subsequently, undergo aberrant and restricted lineage priming at the postimplantation stage, which leads to early embryonic lethality. m6A predominantly and directly reduces mRNA stability, including that of key naïve pluripotency-promoting transcripts. This study highlights a critical role for an mRNA epigenetic modification in vivo and identifies regulatory modules that functionally influence naïve and primed pluripotency in an opposing manner.

The H3K27 demethylase Utx regulates somatic and germ cell epigenetic reprogramming

Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by ectopic expression of different transcription factors, classically Oct4 (also known as Pou5f1), Sox2, Klf4 and Myc (abbreviated as OSKM). This process is accompanied by genome-wide epigenetic changes, but how these chromatin modifications are biochemically determined requires further investigation. Here we show in mice and humans that the histone H3 methylated Lys 27 (H3K27) demethylase Utx (also known as Kdm6a) regulates the efficient induction, rather than maintenance, of pluripotency. Murine embryonic stem cells lacking Utx can execute lineage commitment and contribute to adult chimaeric animals; however, somatic cells lacking Utx fail to robustly reprogram back to the ground state of pluripotency. Utx directly partners with OSK reprogramming factors and uses its histone demethylase catalytic activity to facilitate iPSC formation. Genomic analysis indicates that Utx depletion results in aberrant dynamics of H3K27me3 repressive chromatin demethylation in somatic cells undergoing reprogramming. The latter directly hampers the derepression of potent pluripotency promoting gene modules (including Sall1, Sall4 and Utf1), which can cooperatively substitute for exogenous OSK supplementation in iPSC formation. Remarkably, Utx safeguards the timely execution of H3K27me3 demethylation observed in embryonic day 10.5–11 primordial germ cells (PGCs), and Utx-deficient PGCs show cell-autonomous aberrant epigenetic reprogramming dynamics during their embryonic maturation in vivo. Subsequently, this disrupts PGC development by embryonic day 12.5, and leads to diminished germline transmission in mouse chimaeras generated from Utx-knockout pluripotent cells. Thus, we identify Utx as a novel mediator with distinct functions during the re-establishment of pluripotency and germ cell development. Furthermore, our findings highlight the principle that molecular regulators mediating loss of repressive chromatin during in vivo germ cell reprogramming can be co-opted during in vitro reprogramming towards ground state pluripotency.

p53 Regulates the Ras Circuit to Inhibit the Expression of a Cancer-Related Gene Signature by Various Molecular Pathways

In this study, we focus on the analysis of a previously identified cancer-related gene signature (CGS) that underlies the cross talk between the p53 tumor suppressor and Ras oncogene. CGS consists of a large number of known Ras downstream target genes that were synergistically upregulated by wild-type p53 loss and oncogenic H-Ras(G12V) expression. Here we show that CGS expression strongly correlates with malignancy. In an attempt to elucidate the molecular mechanisms underling the cooperation between p53 loss and oncogenic H-Ras(G12V), we identified distinguished pathways that may account for the regulation of the expression of the CGS. By knocking-down p53 or by expressing mutant p53, we revealed that p53 exerts its negative effect by at least two mechanisms mediated by its targets B-cell translocation gene 2 (BTG2) and activating transcription factor 3 (ATF3). Whereas BTG2 binds H-Ras(G12V) and represses its activity by reducing its GTP loading state, which in turn causes a reduction in CGS expression, ATF3 binds directly to the CGS promoters following p53 stabilization and represses their expression. This study further elucidates the molecular loop between p53 and Ras in the transformation process.

Clonal allelic predetermination of immunoglobulin-κ rearrangement.

Although most genes are expressed biallelically, a number of key genomic sites—including immune and olfactory receptor regions—are controlled monoallelically in a stochastic manner, with some cells expressing the maternal allele and others the paternal allele in the target tissue. Very little is known about how this phenomenon is regulated and programmed during development. Here, using mouse immunoglobulin-κ (Igκ) as a model system, we demonstrate that although individual haematopoietic stem cells are characterized by allelic plasticity, early lymphoid lineage cells become committed to the choice of a single allele, and this decision is then stably maintained in a clonal manner that predetermines monoallelic rearrangement in B cells. This is accompanied at the molecular level by underlying allelic changes in asynchronous replication timing patterns at the κ locus. These experiments may serve to define a new concept of stem cell plasticity.

Transcriptional activity of ATF3 in the stromal compartment of tumors promotes cancer progression.

Compelling evidences have rendered the tumor microenvironment a crucial determinant in cancer outcome. Activating transcription factor 3 (ATF3), a stress response transcription factor, is known to have a dichotomous role in tumor cells, acting either as a tumor suppressor or an oncogene in a context-dependent manner. However, its expression and possible role in the tumor microenvironment are hitherto unknown. Here we show that ATF3 is upregulated in the stromal compartment of several types of cancer. Accordingly, Cancer-associated fibroblasts (CAFs) ectopically expressing ATF3 proliferated faster as indicated by increased colony-forming capacity and promoted the growth of adjacent tumor cells when co-injected into nude mice. Utilizing a genome-wide profiling approach, we unraveled a robust gene expression program induced by ATF3 in CAFs. Focusing on a specific subset of genes, we found that the ability of stromal ATF3 to promote cancer progression is mediated by transcriptional repression of CLDN1 and induction of CXCL12 and RGS4. In addition, regulation of LIF, CLDN1, SERPINE2, HSD17B2, ITGA7 and PODXL by ATF3 mediated the increased proliferation capacity of CAFs. In sum, our findings implicate ATF3 as a novel stromal tumor promoter and suggest that targeting ATF3 pathway might be beneficial for anticancer therapy.

Trained Memory of Human Uterine NK Cells Enhances Their Function in Subsequent Pregnancies

SUMMARY Natural killer cells (NKs) are abundant in the human decidua, regulating trophoblast invasion and angiogenesis. Several diseases of poor placental development are associated with first pregnancies, so we thus looked to characterize differences in decidual NKs (dNKs) in first versus repeated pregnancies. We discovered a population found in repeated pregnancies, which has a unique transcriptome and epigenetic signature, and is characterized by high expression of the receptors NKG2C and LILRB1. We named these cells Pregnancy Trained decidual NK cells (PTdNKs). PTdNKs have open chromatin around the enhancers of IFNG and VEGFA. Activation of PTdNKs led to increased production and secretion of IFN‐&ggr; and VEGF&agr;, with the latter supporting vascular sprouting and tumor growth. The precursors of PTdNKs seem to be found in the endometrium. Because repeated pregnancies are associated with improved placentation, we propose that PTdNKs, which are present primarily in repeated pregnancies, might be involved in proper placentation. Graphical Abstract Figure. No Caption available. HighlightsA unique subset of human natural killer cells exists in repeated pregnanciesThese NK cells, termed PTdNKs, express increased amounts of NKG2C and LILRB1PTdNKs secrete increased levels of IFN‐&ggr; and VEGF&agr;; the latter supports vascularization &NA; Natural killer cells are present in the human decidua, regulating trophoblast invasion and angiogenesis. Here, Gamliel et al. report on a special subset of human decidual natural killer cells, which “remember” pregnancy and better support subsequent pregnancies. This might explain why first pregnancies are at increased risk of developing diseases of poor placentation.

Mbd3/NuRD is a Key Inhibitory Module During the Induction and Maintenance of Naïve Pluripotency

Our group has published a study on induced pluripotent stem cell (iPSC) reprogramming (Rais et al. Nature 20131) that reached the following conclusions: a) Mbd3/NuRD is a repressor of inducing naïve pluripotency from mouse Epiblast stem cells (EpiSCs), primordial germ cells (PGCs), murine somatic cells and human secondary fibroblasts; b) Up to 100% iPSC formation efficiency can be achieved via optimized Mbd3/NuRD depletion, in concert with optimized OKSM delivery and naïve pluripotency conditions (2i supplement applied only after 48 hours, human LIF, hypoxia and Vitamin C containing Knockout serum replacement)1. This represented the first proof for deterministic/near-deterministic iPSC reprogramming, and highlighted a previously unappreciated role for Mbd3/NuRD in hampering the re-establishment of pluripotency. Recent reports have seemingly provided contradictory results and attempted to dispute our iPSC efficiency quantifications and/or the role of Mbd3/NuRD in blocking reprogramming2,3. Here we provide a detailed response to these reports based on extended discussions and providing new data. The synthesis presented herein disagrees with claims made by Silva, Hendrich, Bertone and colleagues2,3, and reconfirms that Mbd3/NuRD is a major pathway that inhibits the maintenance and induction of pluripotency1. Further, we foresee that its controlled manipulation is likely to become an integral pathway for inducing and maintaining naïve pluripotency in a variety of species.

High-Resolution Dissection of Conducive Reprogramming Trajectory to Ground State Pluripotency

The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) with four transcription factors Oct4, Sox2, Klf4 and cMyc (abbreviated as OSKM)1 has provoked interest to define the molecular characteristics of this process2-7. Despite important progress, the dynamics of epigenetic reprogramming at high resolution in correctly reprogrammed iPSCs and throughout the entire process remain largely undefined. This gap in understanding results from the inefficiency of conventional reprogramming methods coupled with the difficulty of prospectively isolating the rare cells that eventually correctly reprogram into iPSCs. Here we characterize cell fate conversion from fibroblast to iPSC using a highly efficient deterministic murine reprogramming system engineered through optimized inhibition of Gatad2a-Mbd3/NuRD repressive sub-complex. This comprehensive characterization provides single-day resolution of dynamic changes in levels of gene expression, chromatin modifications, TF binding, DNA accessibility and DNA methylation. The integrative analysis identified two transcriptional modules that dominate successful reprogramming. One consists of genes whose transcription is regulated by on/off epigenetic switching of modifications in their promoters (abbreviated as ESPGs), and the second consists of genes with promoters in a constitutively active chromatin state, but a dynamic expression pattern (abbreviated as CAPGs). ESPGs are mainly regulated by OSK, rather than Myc, and are enriched for cell fate determinants and pluripotency factors. CAPGs are predominantly regulated by Myc, and are enriched for cell biosynthetic regulatory functions. We used the ESPG module to study the identity and temporal occurrence of activating and repressing epigenetic switching during reprogramming. Removal of repressive chromatin modifications precedes chromatin opening and binding of RNA polymerase II at enhancers and promoters, and the opposite dynamics occur during repression of enhancers and promoters. Genome wide DNA methylation analysis demonstrated that de novo DNA methylation is not required for highly efficient conducive iPSC reprogramming, and identified a group of super-enhancers targeted by OSK, whose early demethylation marks commitment to a successful reprogramming trajectory also in inefficient conventional reprogramming systems. CAPGs are distinctively regulated by multiple synergystic ways: 1) Myc activity, delivered either endogenously or exogenously, dominates CAPG expression changes and is indispensable for induction of pluripotency in somatic cells; 2) A change in tRNA codon usage which is specific to CAPGs, but not ESPGs, and favors their translation. In summary, our unbiased high-resolution mapping of epigenetic changes on somatic cells that are committed to undergo successful reprogramming reveals interleaved epigenetic and biosynthetic reconfigurations that rapidly commission and propel conducive reprogramming toward naïve pluripotency.