Recent evolution of a TET-controlled and DPPA3/STELLA-driven pathway of passive DNA demethylation in mammals

AbstractGenome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. Here, we describe a recently evolved pathway in which global hypomethylation is achieved by the coupling of active and passive demethylation. TET activity is required, albeit indirectly, for global demethylation, which mostly occurs at sites devoid of TET binding. Instead, TET-mediated active demethylation is locus-specific and necessary for activating a subset of genes, including the naïve pluripotency and germline marker Dppa3 (Stella, Pgc7). DPPA3 in turn drives large-scale passive demethylation by directly binding and displacing UHRF1 from chromatin, thereby inhibiting maintenance DNA methylation. Although unique to mammals, we show that DPPA3 alone is capable of inducing global DNA demethylation in non-mammalian species (Xenopus and medaka) despite their evolutionary divergence from mammals more than 300 million years ago. Our findings suggest that the evolution of Dppa3 facilitated the emergence of global DNA demethylation in mammals.

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TDG regulates cell cycle progression in human neural progenitors

F1000Research ◽

10.12688/f1000research.13801.1 ◽

2018 ◽

Vol 7 ◽

pp. 497

Author(s):

Igal Germanguz ◽

Jenny C. Park ◽

Jessica Cinkornpumin ◽

Aryeh Solomon ◽

Minori Ohashi ◽

...

Keyword(s):

Cell Cycle ◽

Dna Methylation ◽

Stem Cells ◽

Pluripotent Stem Cells ◽

Methylation Status ◽

Human Pluripotent Stem Cells ◽

Dna Demethylation ◽

Neural Progenitors ◽

Specific Cell ◽

Active Demethylation

Background: As cells divide, they must both replicate their DNA and generate a new set of histone proteins. The newly synthesized daughter strands and histones are unmodified, and must therefore be covalently modified to allow for transmission of important epigenetic marks to daughter cells. Human pluripotent stem cells (hPSCs) display a unique cell cycle profile, and control of the cell cycle is known to be critical for their proper differentiation and survival. A major unresolved question is how hPSCs regulate their DNA methylation status through the cell cycle, namely how passive and active demethylation work to maintain a stable genome. Thymine-DNA glycosylase (TDG), an embryonic essential gene, has been recently implicated as a major enzyme involved in demethylation. Methods: We use human pluripotent stem cells and their derivatives to investigate the role of TDG in differentiation and proliferation. To perform loss of function of TDG, RNA Interference was used. To study the cell cyle, we engineered human pluripotent stem cells to express the FUCCI tool which marks cells at various stages of the cell cycle with distinct patterns of fluorescent proteins. We also used cell cycle profiling by FACS, and DNA methylation analysis to probe a connection between DNA demethylation and cell cycle. Results: Here we present data showing that TDG regulates cell cycle dynamics in human neural progenitors (NPCs) derived from hPSCs, leading to changes in cell cycle related gene expression and neural differentiation capacity. These data show that loss of TDG function can block differentiation by driving proliferation of neural progenitors. We also identify specific cell cycle related genes whose expression changes upon loss of TDG expression. Conclusions: These observations suggest that TDG and active demethylation play an important role in hPSC cell cycle regulation and differentiation.

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Recent evolution of a TET-controlled and DPPA3/STELLA-driven pathway of passive demethylation in mammals

10.1101/321604 ◽

2018 ◽

Cited By ~ 2

Author(s):

Christopher B. Mulholland ◽

Atsuya Nishiyama ◽

Joel Ryan ◽

Ryohei Nakamura ◽

Merve Yiğit ◽

...

Keyword(s):

Dna Methyltransferase ◽

Ectopic Expression ◽

Dna Demethylation ◽

Mammalian Development ◽

Dna Hypomethylation ◽

Tet Proteins ◽

Genome Wide ◽

Recent Emergence ◽

High Conservation ◽

Early Mammalian Development

AbstractGenome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. So far, it was unclear how mammals specifically achieve global DNA hypomethylation, given the high conservation of the DNA (de-)methylation machinery among vertebrates. We found that DNA demethylation requires TET activity but mostly occurs at sites where TET proteins are not bound suggesting a rather indirect mechanism. Among the few specific genes bound and activated by TET proteins was the naïve pluripotency and germline marker Dppa3 (Pgc7, Stella), which undergoes TDG dependent demethylation. The requirement of TET proteins for genome-wide DNA demethylation could be bypassed by ectopic expression of Dppa3. We show that DPPA3 binds and displaces UHRF1 from chromatin and thereby prevents the recruitment and activation of the maintenance DNA methyltransferase DNMT1. We demonstrate that DPPA3 alone can drive global DNA demethylation when transferred to amphibians (Xenopus) and fish (medaka), both species that naturally do not have a Dppa3 gene and exhibit no post-fertilization DNA demethylation. Our results show that TET proteins are responsible for active and - indirectly also for - passive DNA demethylation; while TET proteins initiate local and gene-specific demethylation in vertebrates, the recent emergence of DPPA3 introduced a unique means of genome-wide passive demethylation in mammals and contributed to the evolution of epigenetic regulation during early mammalian development.

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A human tissue map of 5-hydroxymethylcytosines exhibits tissue specificity through gene and enhancer modulation

Nature Communications ◽

10.1038/s41467-020-20001-w ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Xiao-Long Cui ◽

Ji Nie ◽

Jeremy Ku ◽

Urszula Dougherty ◽

Diana C. West-Szymanski ◽

...

Keyword(s):

Human Tissue ◽

Tissue Specificity ◽

Dna Demethylation ◽

Tissue Type ◽

Specific Gene ◽

Mammalian Development ◽

Tissue Specific ◽

Genome Wide ◽

Organ Systems

AbstractDNA 5-hydroxymethylcytosine (5hmC) modification is known to be associated with gene transcription and frequently used as a mark to investigate dynamic DNA methylation conversion during mammalian development and in human diseases. However, the lack of genome-wide 5hmC profiles in different human tissue types impedes drawing generalized conclusions about how 5hmC is implicated in transcription activity and tissue specificity. To meet this need, we describe the development of a 5hmC tissue map by characterizing the genomic distributions of 5hmC in 19 human tissues derived from ten organ systems. Subsequent sequencing results enabled the identification of genome-wide 5hmC distributions that uniquely separates samples by tissue type. Further comparison of the 5hmC profiles with transcriptomes and histone modifications revealed that 5hmC is preferentially enriched on tissue-specific gene bodies and enhancers. Taken together, the results provide an extensive 5hmC map across diverse human tissue types that suggests a potential role of 5hmC in tissue-specific development; as well as a resource to facilitate future studies of DNA demethylation in pathogenesis and the development of 5hmC as biomarkers.

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Erasure of DNA methylation, genomic imprints, and epimutations in a primordial germ-cell model derived from mouse pluripotent stem cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1610259113 ◽

2016 ◽

Vol 113 (34) ◽

pp. 9545-9550 ◽

Cited By ~ 24

Author(s):

Norikatsu Miyoshi ◽

Jente M. Stel ◽

Keiko Shioda ◽

Na Qu ◽

Junko Odajima ◽

...

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Pluripotent Stem Cells ◽

Cell Model ◽

Methylation Status ◽

Primordial Germ Cell ◽

Dna Demethylation ◽

Imprinted Genes ◽

Sequencing Data ◽

Genome Wide

The genome-wide depletion of 5-methylcytosines (5meCs) caused by passive dilution through DNA synthesis without daughter strand methylation and active enzymatic processes resulting in replacement of 5meCs with unmethylated cytosines is a hallmark of primordial germ cells (PGCs). Although recent studies have shown that in vitro differentiation of pluripotent stem cells (PSCs) to PGC-like cells (PGCLCs) mimics the in vivo differentiation of epiblast cells to PGCs, how DNA methylation status of PGCLCs resembles the dynamics of 5meC erasure in embryonic PGCs remains controversial. Here, by differential detection of genome-wide 5meC and 5-hydroxymethylcytosine (5hmeC) distributions by deep sequencing, we show that PGCLCs derived from mouse PSCs recapitulated the process of genome-wide DNA demethylation in embryonic PGCs, including significant demethylation of imprint control regions (ICRs) associated with increased mRNA expression of the corresponding imprinted genes. Although 5hmeCs were also significantly diminished in PGCLCs, they retained greater amounts of 5hmeCs than intragonadal PGCs. The genomes of both PGCLCs and PGCs selectively retained both 5meCs and 5hmeCs at a small number of repeat sequences such as GSAT_MM, of which the significant retention of bisulfite-resistant cytosines was corroborated by reanalysis of previously published whole-genome bisulfite sequencing data for intragonadal PGCs. PSCs harboring abnormal hypermethylation at ICRs of the Dlk1-Gtl2-Dio3 imprinting cluster diminished these 5meCs upon differentiation to PGCLCs, resulting in transcriptional reactivation of the Gtl2 gene. These observations support the usefulness of PGCLCs in studying the germline epigenetic erasure including imprinted genes, epimutations, and erasure-resistant loci, which may be involved in transgenerational epigenetic inheritance.

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Generation of human oogonia from induced pluripotent stem cells in vitro

Science ◽

10.1126/science.aat1674 ◽

2018 ◽

Vol 362 (6412) ◽

pp. 356-360 ◽

Cited By ~ 59

Author(s):

Chika Yamashiro ◽

Kotaro Sasaki ◽

Yukihiro Yabuta ◽

Yoji Kojima ◽

Tomonori Nakamura ◽

...

Keyword(s):

Stem Cells ◽

Germ Cell ◽

Pluripotent Stem Cells ◽

Primordial Germ Cell ◽

Dna Demethylation ◽

Inactive X Chromosome ◽

Genome Wide ◽

Aberrant Dna Methylation ◽

Induced Pluripotent

Human in vitro gametogenesis may transform reproductive medicine. Human pluripotent stem cells (hPSCs) have been induced into primordial germ cell–like cells (hPGCLCs); however, further differentiation to a mature germ cell has not been achieved. Here, we show that hPGCLCs differentiate progressively into oogonia-like cells during a long-term in vitro culture (approximately 4 months) in xenogeneic reconstituted ovaries with mouse embryonic ovarian somatic cells. The hPGCLC-derived oogonia display hallmarks of epigenetic reprogramming—genome-wide DNA demethylation, imprint erasure, and extinguishment of aberrant DNA methylation in hPSCs—and acquire an immediate precursory state for meiotic recombination. Furthermore, the inactive X chromosome shows a progressive demethylation and reactivation, albeit partially. These findings establish the germline competence of hPSCs and provide a critical step toward human in vitro gametogenesis.

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Strand-specific single-cell methylomics reveals distinct modes of DNA demethylation dynamics during early mammalian development

10.1101/804526 ◽

2019 ◽

Cited By ~ 1

Author(s):

Maya Sen ◽

Dylan Mooijman ◽

Jean-Charles Boisset ◽

Alex Chialastri ◽

Mina Popovic ◽

...

Keyword(s):

Single Cell ◽

Single Cells ◽

Embryonic Stem ◽

Cost Effective ◽

Dna Demethylation ◽

Preimplantation Embryos ◽

Mammalian Development ◽

Cell Stage ◽

Stage Of Development ◽

Genome Wide

AbstractDNA methylation (5mC) is central to cellular identity and the global erasure of 5mC from the parental genomes during preimplantation mammalian development is critical to reset the methylome of terminally differentiated gametes to the pluripotent cells in the blastocyst. While active and passive modes of demethylation have both been suggested to play a role in this process, the relative contribution of these two mechanisms to genome-wide 5mC erasure remains unclear. Here, we report a new high-throughput single-cell method (scMspJI-seq) that enables strand-specific quantification of 5mC, thereby allowing us to systematically probe the dynamics of global demethylation. First, when applied to hybrid mouse embryonic stem cells, we identified substantial cell-to-cell strand-specific 5mC heterogeneity, with a small group of cells displaying asymmetric levels of 5mCpG between the two DNA strands of a chromosome suggesting loss of maintenance methylation. Next, using scMspJI-seq in preimplantation mouse embryos, we discovered that methylation maintenance is active till the 16-cell stage followed by passive demethylation in a fraction of cells within the early blastocyst at the 32-cell stage of development. Finally, we found that human preimplantation embryos qualitatively show temporally delayed yet similar demethylation dynamics as mouse preimplantation embryos. Collectively, these results demonstrate that scMspJI-seq is a sensitive and cost-effective method to map the strand-specific genome-wide patterns of 5mC in single cells, thereby enabling quantitative investigation of methylation dynamics in developmental systems.

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