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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.

Author(s): Alejandro De Los Angeles [1, 2, 3]; Francesco Ferrari [4]; Yuko Fujiwara [1, 2, 3]; Ronald Mathieu [5]; Soohyun Lee [4]; Semin Lee [4]; Ho-Chou Tu [1, 2, 3]; [...]

Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive innercell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSKβ 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 X chromosome 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 stem cells (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 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 invitro and invivo., Naive 2i/LIF conditions are not sufficient to elicit a self-renewal response in human ES cells/human iPS cells and result in their differentiation (7). Nevertheless, the proof of concept for the [...]

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. This study shows that the combination of naive pluripotency growth conditions, Oct4, Sox2, Klf4 and Myc (OSKM) overexpression, and depleting the Mbd3/NuRD co-repressor results in deterministic and synchronized reprogramming to pluripotency. Efficient and near-complete conversion to iPS cells Somatic cells can be reprogrammed to pluripotency by expression of exogenous factors,classically Oct4, Sox2,Klf4 and c-Myc (OSKM). During reprogramming, only a fraction of the cells converts into induced pluripotent stem (iPS) cells. The nature of rate limiting barrier(s) that prevent the majority of cells to convert into iPS cells remains elusive and it is unknown whether iPS cell reprogramming can be rendered deterministic and very efficient. Jacob Hanna and colleagues now show that the combination of 2i/LIF growth conditions, OSKM overexpression and neutralizing the Mbd3/NURD co-repressor results in deterministic and synchronized reprogramming to pluripotency. Using this approach, they found that almost 100% of mouse and human somatic cells convert into naive iPS cells after only seven days of OSKM induction., Author(s): Yoach Rais [sup.1] , Asaf Zviran [sup.1] , Shay Geula [sup.1] , Ohad Gafni [sup.1] , Elad Chomsky [sup.1] , Sergey Viukov [sup.1] , Abed AlFatah Mansour [sup.1] , [...]

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 [...]

Author(s): Ohad Gafni; Leehee Weinberger; Abed AlFatah Mansour; Yair S. Manor; Elad Chomsky; Dalit Ben-Yosef; Yael Kalma; Sergey Viukov; Itay Maza; Asaf Zviran; Yoach Rais; Zohar Shipony; Zohar Mukamel; Vladislav [...]

Author(s): Yoach Rais; Asaf Zviran; Shay Geula; Ohad Gafni; Elad Chomsky; Sergey Viukov; Abed AlFatah Mansour; Inbal Caspi; Vladislav Krupalnik; Mirie Zerbib; Itay Maza; Nofar Mor; Dror Baran; Leehee Weinberger; [...]

The article informs that beta-Catenin safeguards the ground state of mouse pluripotency by strengthening the robustness of the transcriptional apparatus. Topics include transcriptional mechanisms governing this pluripotent ground state are unresolved; and release of promoter-proximal paused RNA polymerase II (Pol2) is a multistep process necessary for pluripotency and cell cycle gene transcription in serum/leukemia (SL).

The article focuses on the role of the N6methyladenosine (m6A) transferase Mettl3 in regulating the state transitions of naïve and primed pluripotency toward cell differentiation. It states a small interfering RNA screen was conducted against transcriptional regulators and tested if a primed EpiSC harboring a green fluorescent protein reporter knockin allele might rely on some of the factors. It comments that M6A messenger RNA methylation was a regulator acting at molecular switches.

The N6-methyladenosine (m 6 A) 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 m 6 A-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 Lasman et al.; Published by Cold Spring Harbor Laboratory Press.)