(2020) Genes and Development. 34, Abstract[All authors]
The N6-methyladenosine (m6A) modification is the most prevalent post-transcriptional mRNA modification, regulating mRNA decay and splicing. It plays a major role during normal development, differentiation, and disease progression. The modification is regulated by a set of writer, eraser, and reader proteins. The YTH domain family of proteins, consists of three homologous m6A-binding proteins, Ythdf1, Ythdf2, and Ythdf3, which were suggested to have different cellular functions. However, their sequence similarity and their tendency to bind the same targets suggest that they may have overlapping roles. We systematically knocked out (KO) the Mettl3 writer, each of the Ythdf readers, and the three readers together (triple-KO). We then estimated the effect in vivo in mouse gametogenesis, postnatal viability, and in vitro in mouse embryonic stem cells (mESCs). In gametogenesis, Mettl3-KO severity is increased as the deletion occurs earlier in the process, and Ythdf2 has a dominant role that cannot be compensated by Ythdf1 or Ythdf3, due to differences in readers' expression pattern across different cell types, both in quantity and in spatial location. Knocking out the three readers together and systematically testing viable offspring genotypes revealed a redundancy in the readers' role during early development that is Ythdf1/2/3 gene dosage-dependent. Finally, in mESCs there is compensation between the three Ythdf reader proteins, since the resistance to differentiate and the significant effect on mRNA decay occur only in the triple-KO cells and not in the single KOs. Thus, we suggest a new model for the Ythdf readers function, in which there is profound dosage-dependent redundancy when all three readers are equivalently coexpressed in the same cell types.
(2020) Epigenomes. 4, 1, 5. Abstract
The rising field of RNA modifications is stimulating massive research nowadays. m(6)A, the most abundant mRNA modification is highly conserved during evolution. Through the last decade, the essential components of this dynamic mRNA modification machinery were found and classified into writer, eraser and reader proteins. m(6)A modification is now known to take part in diverse biological processes such as embryonic development, cell circadian rhythms and cancer stem cell proliferation. In addition, there is already firm evidence for the importance of m(6)A modification in stem cell differentiation and gametogenesis, both in males and females. This review attempts to summarize the important results of recent years studying the mechanism underlying stem cell differentiation and gametogenesis processes.
Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules(2019) Cell Stem Cell. 24, 2, p. 328-341.e9 Abstract
The epigenetic dynamics of induced pluripotent stem cell (iPSC) reprogramming in correctly reprogrammed cells at high resolution and throughout the entire process remain largely undefined. Here, we characterize conversion of mouse fibroblasts into iPSCs using Gatad2a-Mbd3/NuRD-depleted and highly efficient reprogramming systems. Unbiased high-resolution profiling of dynamic changes in levels of gene expression, chromatin engagement, DNA accessibility, and DNA methylation were obtained. We identified two distinct and synergistic transcriptional modules that dominate successful reprogramming, which are associated with cell identity and biosynthetic genes. The pluripotency module is governed by dynamic alterations in epigenetic modifications to promoters and binding by Oct4, Sox2, and Klf4, but not Myc. Early DNA demethylation at certain enhancers prospectively marks cells fated to reprogram. Myc activity drives expression of the essential biosynthetic module and is associated with optimized changes in tRNA codon usage. Our functional validations highlight interweaved epigenetic- and Myc-governed essential reconfigurations that rapidly commission and propel deterministic reprogramming toward naive pluripotency.[All authors]
Neutralizing Gatad2a-Chd4-Mbd3/NuRD Complex Facilitates Deterministic Induction of Naive Pluripotency(2018) Cell Stem Cell. 23, 3, p. 412-425.e10 Abstract
Mbd3, a member of nucleosome remodeling and deacetylase (NuRD) co-repressor complex, was previously identified as an inhibitor for deterministic induced pluripotent stem cell (iPSC) reprogramming, where up to 100% of donor cells successfully complete the process. NuRD can assume multiple mutually exclusive conformations, and it remains unclear whether this deterministic phenotype can be attributed to a specific Mbd3/NuRD subcomplex. Moreover, since complete ablation of Mbd3 blocks somatic cell proliferation, we aimed to explore functionally relevant alternative ways to neutralize Mbd3-dependent NuRD activity. We identify Gatad2a, a NuRD-specific subunit, whose complete deletion specifically disrupts Mbd3/NuRD repressive activity on the pluripotency circuitry during iPSC differentiation and reprogramming without ablating somatic cell proliferation. Inhibition of Gatad2a facilitates deterministic murine iPSC reprogramming within 8 days. We validate a distinct molecular axis, Gatad2a-Chd4-Mbd3, within Mbd3/NuRD as being critical for blocking reestablishment of naive pluripotency and further highlight signaling-dependent and post-translational modifications of Mbd3/NuRD that influence its interactions and assembly. Optimized partial depletion of Mbd3 had been implicated in deterministic reprogramming. Hanna and colleagues now dissect the subcomplex within Mbd3/NuRD that underlies this outcome. Gatad2a is identified as a flexible component that can be entirely ablated without compromising somatic cell proliferation and yet still similarly yields deterministic mouse iPSC formation.[All authors]
(2017) BioRxiv. Abstract
The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) with four transcription factors Oct4, Sox2, Klf4 and cMyc (abbreviated as OSKM) has provoked interest to define the molecular characteristics of the process. 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 synergetic 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 naive pluripotency.[All authors]
(2016) Nature Reviews Molecular Cell Biology. 17, 3, p. 155-169 Abstract
The molecular mechanisms and signalling pathways that regulate the in vitro preservation of distinct pluripotent stem cell configurations, and their induction in somatic cells by direct reprogramming, constitute a highly exciting area of research. In this Review, we integrate recent discoveries related to isolating unique naive and primed pluripotent stem cell states with altered functional and molecular characteristics, and from different species. We provide an overview of the pathways underlying pluripotent state transitions and interconversion in vitro and in vivo. We conclude by highlighting unresolved key questions, future directions and potential novel applications of such dynamic pluripotent cell states.
Transient acquisition of pluripotency during somatic cell transdifferentiation with iPSC reprogramming factors(2015) Nature Biotechnology. 33, 7, p. 769-774 Abstract
Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors(1,2). Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation(3-6). Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced 'transdifferentiation' pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by various methods.[All authors]
(2015) Science. 347, 6225, p. 1002-1006 Abstract
Naive and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here, we identify Mettl3, an N-6-methyladenosine (m(6)A) transferase, as a regulator for terminating murine naive pluripotency. Mettl3 knockout preimplantation epiblasts and naive embryonic stem cells are depleted for m(6)A in mRNAs, yet are viable. However, they fail to adequately terminate their naive state and, subsequently, undergo aberrant and restricted lineage priming at the postimplantation stage, which leads to early embryonic lethality. m(6)A predominantly and directly reduces mRNA stability, including that of key naive pluripotency-promoting transcripts. This study highlights a critical role for an mRNA epigenetic modification in vivo and identifies regulatory modules that functionally influence naive and primed pluripotency in an opposing manner.[All authors]
(2013) Nature. 504, 7479, p. 282-286 Abstract
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 GSK3 beta signalling (termed 2i/LIF conditions)(1,2). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation Xchromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters(3). Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast(4). Although human ES cells share several molecular features with naive mouse ES cells(5), they also share a variety of epigenetic properties with primed murine epiblast stemcells (EpiSCs)(6,7). 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(7). 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(1). 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 com[All authors]
(2013) Nature. 502, 7469, p. 65-+ Abstract
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.[All authors]
(2012) Nature. 488, 7411, p. 409-+ Abstract
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)(1). This process is accompanied by genome-wide epigenetic changes(2-5), 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(6-9) (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 germcells (PGCs)(10), and Utx-deficientPGCs 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[All authors]
(2011) Nature Cell Biology. 13, 8, p. 886-888 Abstract
How the unique chromatin configuration of embryonic stem cells (ESCs) integrates inputs from exogenous stimuli to maintain pluripotency remains largely unknown. The ESC-specific ATP-dependent chromatin-remodelling (esBAF) complex maintains the accessibility of the target sites of Stat3, a leukaemia inhibitory factor (LIF) signalling effector, by preventing repressive localized polycomb-mediated trimethylation of Lys 27 of histone 3 (H3K27me3).