Keeping your options open: insights from Dppa2/4 into how epigenetic priming factors promote cell plasticity.

The concept of cellular plasticity is particularly apt in early embryonic development, where there is a tug-of-war between the stability and flexibility of cell identity. This balance is controlled in part through epigenetic mechanisms. Epigenetic plasticity dictates how malleable cells are to change by adjusting the potential to initiate new transcriptional programmes. The higher the plasticity of a cell, the more readily it can adapt and change its identity in response to external stimuli such as differentiation cues. Epigenetic plasticity is regulated in part through the action of epigenetic priming factors which establish this permissive epigenetic landscape at genomic regulatory elements to enable future transcriptional changes. Recent studies on the DNA binding proteins Developmental Pluripotency Associated 2 and 4 (Dppa2/4) support their roles as epigenetic priming factors in facilitating cell fate transitions. Here, using Dppa2/4 as a case study, the concept of epigenetic plasticity and molecular mechanism of epigenetic priming factors will be explored. Understanding how epigenetic priming factors function is key not only to improve our understanding of the tight control of development, but also to give insights into how this goes awry in diseases of cell identity, such as cancer

Regulation of the ESC transcriptome by nuclear long non-coding RNAs

Long noncoding (lnc)RNAs have recently emerged as key regulators of gene expression. Here, we performed high-depth poly(A)+ RNA sequencing across multiple clonal populations of mouse embryonic stem cells (ESCs) and neural progenitor cells (NPCs) to comprehensively identify differentially regulated lncRNAs. We establish a biologically robust profile of lncRNA expression in these two cell types and further confirm that the majority of these lncRNAs are enriched in the nucleus. Applying weighted gene co-expression network analysis, we define a group of lncRNAs that are tightly associated with the pluripotent state of ESCs. Among these, we show that acute depletion of PAT-14 using antisense oligonucleotides impacts the differentiation- and development-associated gene expression program of ESCs. Furthermore, we demonstrate that Firre, a lncRNA highly enriched in the nucleoplasm and previously reported to mediate chromosomal contacts in ESCs, controls a network of genes related to RNA processing. Together, we provide a comprehensive, up-to-date and high resolution compilation of lncRNA expression in ESCs and NPCs and show that nuclear lncRNAs are tightly integrated into the regulation of ESC gene expression

Random Monoallelic Gene Expression Increases upon Embryonic Stem Cell Differentiation

SummaryRandom autosomal monoallelic gene expression refers to the transcription of a gene from one of two homologous alleles. We assessed the dynamics of monoallelic expression during development through an allele-specific RNA-sequencing screen in clonal populations of hybrid mouse embryonic stem cells (ESCs) and neural progenitor cells (NPCs). We identified 67 and 376 inheritable autosomal random monoallelically expressed genes in ESCs and NPCs, respectively, a 5.6-fold increase upon differentiation. Although DNA methylation and nuclear positioning did not distinguish the active and inactive alleles, specific histone modifications were differentially enriched between the two alleles. Interestingly, expression levels of 8% of the monoallelically expressed genes remained similar between monoallelic and biallelic clones. These results support a model in which random monoallelic expression occurs stochastically during differentiation and, for some genes, is compensated for by the cell to maintain the required transcriptional output of these genes

IMPLICON: an ultra-deep sequencing method to uncover DNA methylation at imprinted regions

Babraham Institute Translational Advisory Group award (to M.E.-M. and F.v.M.); M.E.-M. is supported by a BBSRC Discovery Fellowship [BB/T009713/1]; EMBO Fellowship [ALTF938-2014]; Marie Sklodowska-Curie Individual Fellowship; Work in S.T.d.R.’s team at iMM JLA was supported by Fundac¸ao para a Ci ˜ encia e Tecnologia ˆ(FCT) Ministerio da Cincia, Tecnologia e Ensino Supe- ˆrior (MCTES), Portugal [PTDC/BEX-BCM/2612/2014, PTDC/BIA-MOL/29320/2017 IC&DT]; S.T.d.R. has a CEECUIND/01234/207 assistant research contract from FCT/MCTES; T.K.’s work was supported by Erasmus+and University Foundation of eng. Lenarciˇ c Milan at ˇthe University of Ljubljana. Funding for open access charge: accounts payable, Babraham Institute.publishersversionpublishe

Imprinting fidelity in mouse iPSCs depends on sex of donor cell and medium formulation

Funding Information: We would like to thank Sérgio de Almeida, Miguel Casanova and Inês Milagre for critical reading of the manuscript, and the members of the S.T.d.R.’s team for helpful discussions. We also thank Tânia Carvalho and Pedro Ruivo for their help in histological analysis; Judith Webster at Babraham Institute for LC-MS measurements; Bethan Hussey at Sanger Sequencing and Kristina Tabbada at Babraham Institute for assistance with high-throughput sequencing; and the Bioimaging unit as well as Andreia Santos, Rute Gonçalves and Mariana Fernandes of the Flow Cytometry Facility of Instituto de Medicina Molecular João Lobo Antunes for their services and assistance. Work in S.T.d.R.’s team was supported by Fundação para a Ciência e Tecnologia (FCT) Ministério da Ciência, Tecnologia e Ensino Superior (MCTES), Portugal [IC&DT projects PTDC/BEX-BCM/2612/2014 and PTDC/BIA-MOL/29320/2017 as well as projects UIDB/04565/2020 and UIDP/04565/2020 of the Research Unit Institute from Bioengineering and Biosciences – iBB and LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy – i4HB]; S.T.d.R. and A.-V.G. are supported by assistant research contracts from FCT/MCTES (CEECIND/01234/2017 and CEECIND/02085/2018, respectively); M.A and A.C.R. are supported, respectively, by SFRH/BD/151251/2021 and SFRH/BD/137099/2018 PhD fellowships from FCT/MCTES. J.V.G.L is supported by COVID/BD/152624/2022 from FCT/MCTES. MAE-M was supported by a BBSRC Discovery Fellowship (BB/T009713/1) and is now supported by a Snow Medical Fellowship. F.K. is supported by the Babraham Institute Strategic Core Funding and A.M. by BBSRC BBS/E/B/000C0421. B.B.J. work was funded by Fundação para a Ciência e Tecnologia (FCT), and FEDER, LISBOA-01-0145-FEDER-028534, project co-funded by FEDER, through POR Lisboa 2020—Programa Operacional Regional de Lisboa. T.K. is supported by Janko Jamnik Doctoral Scholarship from National Institute of Chemistry. Funding Information: We would like to thank Sérgio de Almeida, Miguel Casanova and Inês Milagre for critical reading of the manuscript, and the members of the S.T.d.R.’s team for helpful discussions. We also thank Tânia Carvalho and Pedro Ruivo for their help in histological analysis; Judith Webster at Babraham Institute for LC-MS measurements; Bethan Hussey at Sanger Sequencing and Kristina Tabbada at Babraham Institute for assistance with high-throughput sequencing; and the Bioimaging unit as well as Andreia Santos, Rute Gonçalves and Mariana Fernandes of the Flow Cytometry Facility of Instituto de Medicina Molecular João Lobo Antunes for their services and assistance. Work in S.T.d.R.’s team was supported by Fundação para a Ciência e Tecnologia (FCT) Ministério da Ciência, Tecnologia e Ensino Superior (MCTES), Portugal [IC&DT projects PTDC/BEX-BCM/2612/2014 and PTDC/BIA-MOL/29320/2017 as well as projects UIDB/04565/2020 and UIDP/04565/2020 of the Research Unit Institute from Bioengineering and Biosciences – iBB and LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy – i4HB]; S.T.d.R. and A.-V.G. are supported by assistant research contracts from FCT/MCTES (CEECIND/01234/2017 and CEECIND/02085/2018, respectively); M.A and A.C.R. are supported, respectively, by SFRH/BD/151251/2021 and SFRH/BD/137099/2018 PhD fellowships from FCT/MCTES. J.V.G.L is supported by COVID/BD/152624/2022 from FCT/MCTES. MAE-M was supported by a BBSRC Discovery Fellowship (BB/T009713/1) and is now supported by a Snow Medical Fellowship. F.K. is supported by the Babraham Institute Strategic Core Funding and A.M. by BBSRC BBS/E/B/000C0421. B.B.J. work was funded by Fundação para a Ciência e Tecnologia (FCT), and FEDER, LISBOA-01-0145-FEDER-028534, project co-funded by FEDER, through POR Lisboa 2020—Programa Operacional Regional de Lisboa. T.K. is supported by Janko Jamnik Doctoral Scholarship from National Institute of Chemistry. Publisher Copyright: © 2022, The Author(s).Reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs) is a major leap towards personalised approaches to disease modelling and cell-replacement therapies. However, we still lack the ability to fully control the epigenetic status of iPSCs, which is a major hurdle for their downstream applications. Epigenetic fidelity can be tracked by genomic imprinting, a phenomenon dependent on DNA methylation, which is frequently perturbed in iPSCs by yet unknown reasons. To try to understand the causes underlying these defects, we conducted a thorough imprinting analysis using IMPLICON, a high-throughput method measuring DNA methylation levels, in multiple female and male murine iPSC lines generated under different experimental conditions. Our results show that imprinting defects are remarkably common in iPSCs, but their nature depends on the sex of donor cells and their response to culture conditions. Imprints in female iPSCs resist the initial genome-wide DNA demethylation wave during reprogramming, but ultimately cells accumulate hypomethylation defects irrespective of culture medium formulations. In contrast, imprinting defects on male iPSCs depends on the experimental conditions and arise during reprogramming, being mitigated by the addition of vitamin C (VitC). Our findings are fundamental to further optimise reprogramming strategies and generate iPSCs with a stable epigenome.publishersversionpublishe

ATR expands embryonic stem cell fate potential in response to replication stress

Fondazione Italiana per la Ricerca sul Cancro FIRC 18112 Sina Atashpaz.Fondazione Umberto Veronesi Sina Atashpaz Associazione Italiana per la Ricerca sul Cancro AIRC 5xmille METAMECH program Vincenzo Costanzo Giovanni Armenise-Harvard Foundation Vincenzo Costanzo European Research Council Consolidator grant 614541 Vincenzo Costanzo Associazione Italiana per la Ricerca sul Cancro Fellowship 23961 Negar ArghavanifarDanish Cancer Society KBVU-2014 Andres Joaquin Lopez-Contreras Danish Council for Independent Research Sapere Aude, DFF Starting Grant 2014 Andres Joaquin Lopez-Contreras European Research Council ERC-2015-STG-679068 Andres Joaquin Lopez-Contreras Danish National Research Foundation DNRF115 Andres Joaquin Lopez-Contreras The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.Unrepaired DNA damage during embryonic development can be potentially inherited by a large population of cells. However, the quality control mechanisms that minimize the contribution of damaged cells to developing embryos remain poorly understood. Here, we uncovered an ATR- and CHK1-mediated transcriptional response to replication stress (RS) in mouse embryonic stem cells (ESCs) that induces genes expressed in totipotent two-cell (2C) stage embryos and 2C-like cells. This response is mediated by Dux, a multicopy retrogene defining the cleavage-specific transcriptional program in placental mammals. In response to RS, DUX triggers the transcription of 2C-like markers such as murine endogenous retrovirus-like elements (MERVL) and Zscan4. This response can also be elicited by ETAA1-mediated ATR activation in the absence of RS. ATR-mediated activation of DUX requires GRSF1-dependent post-transcriptional regulation of Dux mRNA. Strikingly, activation of ATR expands ESCs fate potential by extending their contribution to both embryonic and extra-embryonic tissues. These findings define a novel ATR dependent pathway involved in maintaining genome stability in developing embryos by controlling ESCs fate in response to RS.S

Quantitative analysis of chromatin interaction changes upon a 4.3 Mb deletion at mouse 4E2

BACKGROUND: Circular chromosome conformation capture (4C) has provided important insights into three dimensional (3D) genome organization and its critical impact on the regulation of gene expression. We developed a new quantitative framework based on polymer physics for the analysis of paired-end sequencing 4C (PE-4Cseq) data. We applied this strategy to the study of chromatin interaction changes upon a 4.3 Mb DNA deletion in mouse region 4E2. RESULTS: A significant number of differentially interacting regions (DIRs) and chromatin compaction changes were detected in the deletion chromosome compared to a wild-type (WT) control. Selected DIRs were validated by 3D DNA FISH experiments, demonstrating the robustness of our pipeline. Interestingly, significant overlaps of DIRs with CTCF/Smc1 binding sites and differentially expressed genes were observed. CONCLUSIONS: Altogether, our PE-4Cseq analysis pipeline provides a comprehensive characterization of DNA deletion effects on chromatin structure and function

MERVL/Zscan4 Network Activation Results in Transient Genome-wide DNA Demethylation of mESCs.

Mouse embryonic stem cells are dynamic and heterogeneous. For example, rare cells cycle through a state characterized by decondensed chromatin and expression of transcripts, including the Zscan4 cluster and MERVL endogenous retrovirus, which are usually restricted to preimplantation embryos. Here, we further characterize the dynamics and consequences of this transient cell state. Single-cell transcriptomics identified the earliest upregulated transcripts as cells enter the MERVL/Zscan4 state. The MERVL/Zscan4 transcriptional network was also upregulated during induced pluripotent stem cell reprogramming. Genome-wide DNA methylation and chromatin analyses revealed global DNA hypomethylation accompanying increased chromatin accessibility. This transient DNA demethylation was driven by a loss of DNA methyltransferase proteins in the cells and occurred genome-wide. While methylation levels were restored once cells exit this state, genomic imprints remained hypomethylated, demonstrating a potential global and enduring influence of endogenous retroviral activation on the epigenome