Epigenetic regulation and transcriptional repression by Tet1 in the early post-implantation mouse embryo: WG Bisulfite sequencing
ABSTRACT: The mammalian TET dioxygenases contribute to global waves of DNA demethylation in the zygote and in primordial germ cells, but their involvement during de novo DNA methylation at peri/post-implantation development is unknown. Here, we show novel physiological functions of Tet1 in the pre-primitive streak stage mouse embryo, where it is expressed not only in the primed-state epiblast, but also in the extra-embryonic ectoderm. In the epiblast, Tet1 contributes to DNA methylation patterning, which indirectly results in dominant transcriptional repression involving a Jumonji-family gene Jmjd8. In the extra-embryonic ectoderm, Tet1 suppresses expression of metabolic genes involved in oxidative phosphorylation. These lineage-specific gene repressive functions, involving distinct modes of regulation by DNA methylation, counteract precocious differentiation of the embryo prior to the onset of gastrulation. Such dysregulation in the absence of Tet1 are surprisingly tolerated in an inbred strain but results in full embryonic lethality in non-inbred mice, thus implicating a complex but essential role of Tet1 in normal gestational development. This dataset includes the WGBS/oxWGBS: Methylation and hydroxymethylation profiling of epiblast-like cells (EpiLCs) in presence or absence of TET1. Whole genome bisulfite sequencing and oxidative whole genome bisulfite sequencing were performed on two wt EpiLCs (male) and two TET1 knock-out EpiLCs (one male and one female).
Project description:Primordial germ cells (PGCs) are specified from epiblast cells in mice. Genes associated with naïve pluripotency are transiently repressed in the transition from inner cell mass (ICM) to epiblast cells, followed by their upregulation soon after PGC specification. However, the molecular mechanisms underlying the reactivation of pluripotency genes are poorly characterized. Here, we exploited in vitro differentiation of epiblast-like cells (EpiLCs) from embryonic stem cells (ESCs) to elucidate the molecular and epigenetic functions of PR domain-containing 14 (PRDM14). We found that Prdm14 overexpression in EpiLCs induced their conversion to ESC-like cells even in the absence of leukemia inhibitory factor (LIF). This was impaired by the loss of Kruppel-like factor 2 (Klf2) and ten-eleven translocation (TET) proteins. Furthermore, PRDM14 recruited OCT3/4 to the enhancer regions of naïve pluripotency genes via TET-base excision-repair-mediated demethylation. Our results provide evidence that PRDM14 establishes a transcriptional network for naïve pluripotency via active DNA demethylation. Overall design: To investigate the function of TET1/TET2 in the transition form primed to naïve pluripotency, we exploited microarray analysis using total mRNA derived from Scramble, Scramble + PRDM14, Tet1/Tet2 KD, Tet1/Tet2 KD + PRDM14 mouse ESCs and EpiLCs.
Project description:Loss of Tet1 expression causes global 5mC and 5hmC changes in stem and progenitor cells in mice and causes enhanced Pro-B cell self-renewal, increased DNA damage and B-lymphomageneis. In this study we performed reduced representative bisulfite sequencing (RRBS) of DNA from WT and Tet1 KO LSK cells. DNA methylation sequencing was performed and analyzed using an enhanced reduced representation (ERRBS) methodology as previously described. DNA was extracted from purified LSK cells of 6-month old WT and Tet1 KO mice, Bisulphite treatment was performed using the EZ DNA Methylation Kit (Zymo Research). Libraries were amplified according to illumina protocols and sequenced on an Illumina HiSeq2500 machine DNA methylation profiling of LSK cells in WT and Tet1 KO mice.
Project description:Mammary gland development and luminal differentiation occur largely postnatally during puberty and pregnancy. We found that pregnancy had the most significant effects on stem cells, inducing a distinct epigenetic state that remained stable through life. Mammary glands were collected from mice at non-pregnant and pregnant stages for DNA extraction and DNA methylation analysis via mRRBS (multiplexed reduced representation bisulfite sequencing).
Project description:Direct lineage conversion is a promising approach to generate therapeutically important cell types for disease modeling and tissue repair. However, it is often unclear whether lineage-reprogrammed cells remain stable long-term and whether the properties of the reprogrammed cells evolve over time. Here, using an improved method of converting pancreatic acinar cells to beta-cells, we show that induced beta-cells persist in the adult pancreas for up to 14 months and form islet-like structures. Detailed analyses of induced cells over 7 months reveal that global DNA methylation changes occur rapidly whereas transcription network remodeling evolves over two months to resemble that of endogenous beta-cells and then stabilizes thereafter. Progressive gain of beta-cell function by converted cells during the 7 month period coincides with both transcriptional changes and the formation of islet-like structures. These studies demonstrate the ability of lineage-reprogrammed cells to achieve a stable state and identify key cellular and molecular milestones during their long-term evolution. Acinar cells and beta cells were collected as control, as well as induced beta cell samples at day 10, day 30, day 60, and 7 months
Project description:In this study, we show that by simple modulation of extrinsic signaling pathways, a new class of pluripotent stem cells, referred to as region selective epiblast stem cells (rsEpiSCs), could be efficiently derived from different stages of the early embryo. rsEpiSCs share features of primed pluripotency yet are distinct from EpiSCs in their molecular characteristics and ability to colonize post-implantation embryos. We performed whole-genome bisulfite sequencing (WGBS) experiments to compare the DNA methylation landscapes of conventional EpiSCs and rsEpiSCs. Compare the DNA methylation profiles in 2 pluripotent stem cell types (LP-EpiSCs and conventional EpiSCs) in mouse. Two replicates are examined for each cell type.
Project description:Purpose: To investigate the effect of Tet1 depletion on global DNA methylation, we performed whole-genome bisulfite sequencing (WGBS). Methods: Starting with as little as 1400-5251 manually micro-dissected PGCs, we used an ultra-low input method, Tn5mC-seq. Results: We generated 945 million reads for Tet1Gt/Gt PGCs and 302 million reads for wild-type PGCs. We obtained 14-16 million CpG sites per genotype at 1.76-2.66x genome coverage, which enables a comprehensive view of genome-wide DNA methylation patterns in E13.5 PGCs. PGCs are almost completely unmethylated genome-wide. Loss of Tet1 led to a subtle increase of methylation level in various genomic elements including promoters, exons, introns and repetitive elements in Tet1Gt/Gt PGCs (p<0.01). Local analysis identified 4,337 differentially methylated regions (DMRs) between Tet1-/- PGCs and wild-type cells. These DMRs are associated with 5,261 genes, among which 271 genes also exhibited differential gene expression and enriched for the cell cycle pathway (FDR=0.02). Conclusions: This result revealed that demethylation of certain set of cell cycle genes is largely abolished in the Tet1-/- PGCs. Genome-wide methylation profiles of primordial germ cells derived from the wild type (WT) and Tet1-null female embryos at E13.5 were generated by whole genome bisulfite sequencing using Illumina Hiseq.
Project description:In this study, we analyzed the DNA methylation levels of 4799 IAP LTRs in three murine cell types: AB2.2 ES cells, somatic cells and a neuroblastoma cell line Neuro2A. According to the results, half of the IAP LTR retrotransposons show constant methylation patterns between the three cell types whereas the remaining half display variable levels of methylation. About half of the variably methylated IAP LTRs tend to be hypomethylated in ES cells, and nearly all of this group are hypomethylated in Neuro2A cells. Interestingly, the observed hypomethylation in both cell types occur in a non-uniform, locus-specific manner and to various degrees of severity, with some of them being easily detectible by COBRA. Overall, this study demonstrates the feasibility of HT-TREBS to study alterations in DNA methylation at retrotransposons in a locus-specific manner in multiple cell types and further suggests the potential utility of this technique in developing epigenetic biomarkers for tracking disease progression. HT-TREBS has been used with the Ion Torrent PGM platform to analyze the DNA methylation of 4799 IAP LTRs in a locus-specific manner in 3 cell types: somatic cells (previously submitted under GEO Accession GSE49222), AB2.2 ES cells and Neuro2A cells
Project description:The epiblast (EPI) is the origin of all somatic and germ cells in mammals, and of the spectrum of pluripotent stem cells (PSCs) in vitro. To explore the ontogeny of human/primate pluripotency, we performed comprehensive single-cell RNA sequencing for pre- and post-implantation EPI development in cynomolgus monkeys. Here we show that after specification in the blastocysts [embryonic day (E)7], cyEPI undergoes major transcriptome changes upon implantation. Thereafter, cyEPI, while generating gastrulating cells (~E13), maintains its transcriptome relatively stably over a week, retaining a unique set of pluripotency genes while acquiring properties for “neuron differentiation.” h/cyPSCs show the highest similarity to post-implantation late cyEPI (~E17), which, despite co-existing with gastrulating cells, bears characteristics of pre-gastrulating mouse EPI (E5.5) and epiblast-like cells (EpiLCs) in vitro. These findings not only reveal divergence/coherence of EPI development, but also identify a developmental coordinate of the spectrum of pluripotency among key species, providing a basis for better regulation of human pluripotency in vitro. Single cell transcriptome analysis of cynomolgus monkey embryo E6-17 and mouse embryo E4.5 - E6.5 as well as of cynomogus monkey embryonic stem cells (ESCs) and mouse ESC, epiblast like cells (EpiLCs), using SC3-seq technology.
Project description:Nanog, a core pluripotency factor in the inner cell mass of blastocysts, is also expressed in unipotent primordial germ cells (PGC) in mice1, where its precise role is yet unclear2-4. We investigated this in an in vitro model, where naïve pluripotent embryonic stem cells (ESCs) cultured in bFGF/ActivinA develop as epiblast-like cells (EpiLCs), and gain competence for PGC-like fate5. Consequently, bone morphogenetic protein (BMP4), or ectopic expression of key germline transcription factors Prdm1/ Prdm14/ Tfap2c, directly induce PGC-like cells (PGCLCs) in EpiLCs, but not in ESCs6-8. Here we report an unexpected discovery that Nanog alone can induce PGCLCs in EpiLCs, independently of BMP4. We propose that following the dissolution of the naïve ESC pluripotency network during establishment of EpiLCs9,10, the epigenome is reset for cell fate determination. Indeed, we found genome-wide changes in NANOG binding pattern between ESCs and EpiLCs, indicating epigenetic resetting of regulatory elements. Accordingly, we show that NANOG can bind and activate enhancers of Prdm1 and Prdm14 in EpiLCs in vitro; BLIMP1 (encoded by Prdm1) then directly induces Tfap2c. Furthermore, while SOX2 and NANOG promote the pluripotent state in ESCs, they show contrasting roles in EpiLCs since Sox2 specifically represses PGCLC induction by Nanog. This study demonstrates a broadly applicable mechanistic principle for how cells acquire competence for cell fate determination, resulting in the context-dependent roles of key transcription factors during development. Nanog ChIP-seq
Project description:To determine whether differences between background strains or housing conditions altered the hepatic methylome, We report the generation and analysis of genome-wide DNA methylation profiles at nucleotide resolution in mouse liver from two male mice on a mixed background (mixed-1, mixed-2) and two males on a pure Black-6 (B6-1, B62) background. Using Enhanced high-throughput Reduced Representation Bisulfite Sequencing (ERRBS), we enriched CpG islands in mouse liver, and covered a representative sampling of conserved non-coding elements, transposons and other genomic features, for mouse liver. We found that the total CpG methylation of each methylome was strikingly similar among the 4 mouse liver samples from two different genetic backgrounds. Analysis of all CpG sites with at least 10x coverage showed a bimodal distribution of methylation, with all samples having 25% of hypermethylated CpG sites and 60% as hypomethylated CpG sites. Given the high percent of genome coverage and robust depth at single nucleotide level, these datasets provide a resource for investigation into changes in DNA methylation patterns in liver disease, tumorigenesis and regeneration. Ehanced reduced representation bisulfite sequencing (MspI 70~320bp size fraction) of liver tissue