Journal: eLife

Article Title: Dynamic enhancer partitioning instructs activation of a growth-related gene during exit from naïve pluripotency

doi: 10.7554/eLife.44057

Figure Lengend Snippet: ( A ) Imprinted regulation of Liz and Zdbf2 in vivo. The Liz promoter is DNA methylated and silent on the maternal allele. Conversely, the paternal allele is expressed, leading to de novo DNA methylation at the paternal sDMR, and Zdbf2 activation. The epigenetic setting is programmed around the time of implantation in embryogenesis, but is then stably maintained throughout life. ( B ) Expression dynamics of promoter switch during protracted EpiLC differentiation. The pLiz -to- pZdbf2 promoter switch occurs at roughly D4. pLiz is shut down and pZdbf2 is active by D7. ( C ) Hierarchical clustering using correlation-based distance of WT RNA-seq data during EpiLC differentiation. Samples generated in this study are highlighted in bold and italic type. In comparison with publicly available data , D4 and D7 EpiLCs cluster more closely with EpiSCs, indicating a primed pluripotency state. ( D ) Plots of reads per kilobase million (RPKM) from RNA-seq of WT cells during EpiLC differentiation for selected markers. Upregulation of markers of primed pluripotency indicate that D7 EpiLCs have entered a primed pluripotency state.

Article Snippet: WGBS data from ESCs were previously generated ( ) and EpiLCs were prepared from 50 ng of bisulfite-converted genomic DNA using the EpiGnome/Truseq DNA Methylation Kit (Illumina) following the manufacturer instructions.

Techniques: In Vivo, Methylation, DNA Methylation Assay, Activation Assay, Stable Transfection, Expressing, RNA Sequencing, Generated, Comparison

Journal: eLife

Article Title: Dynamic enhancer partitioning instructs activation of a growth-related gene during exit from naïve pluripotency

doi: 10.7554/eLife.44057

Figure Lengend Snippet: ( A ) Chromatin and expression landscape at the Zdbf2 locus in ESCs (top) and D7 EpiLCs (bottom). In hypomethylated naïve ESCs, Zdbf2 initiates from the pLiz promoter while a ~ 25 kb block of H3K27me3 extends through pZdbf2 . Upon EpiLC differentiation, the H3K27me3 signal depletes while DNA methylation is gained; the pLiz ATAC-seq peak decreases concomitantly with decreased expression, while pZdbf2 becomes the main promoter. Four prominent ATAC-seq peaks of accessible chromatin (E1 to E4) lie between pLiz and pZdbf2 promoters. WGBS: Whole genome bisulfite sequencing. H3K27me3 ChIP-seq data is from . All other genomics data were generated for this study. One representative biological replicate is displayed for each RNA-seq track. ( B ) H3K27ac ChIP-qPCR shows enrichment for this mark at E1 to E3 in ESCs and D7 EpiLCs, while E4 only becomes enriched in EpiLCs. Data shown as ±s.e.m. from three biological replicates. ( C ) 4C-seq tracks from the pLiz (top) and pZdbf2 (bottom) viewpoints (VPs) in ESCs and D7 EpiLCs. ESC/EpiLC ratio between 4C-seq signals is indicated. pLiz interactions with E1-3 are more frequent in ESCS, but remain high in EpiLCs, likely due to proximity. pLiz seems less restricted in EpiLCs, with interactions spreading on the ‘right’ side of the locus. pZdbf2 exhibits increased interactions at E1 to E4 in EpiLCs over ESCs. Data from one representative biological replicate (two total). Statistical analyses were performed by two-tailed unpaired t-test: n.s = not significant, **p≤0.01, ***p≤0.001. 10.7554/eLife.44057.006 Figure 1—source data 1. Source data for .

Article Snippet: WGBS data from ESCs were previously generated ( ) and EpiLCs were prepared from 50 ng of bisulfite-converted genomic DNA using the EpiGnome/Truseq DNA Methylation Kit (Illumina) following the manufacturer instructions.

Techniques: Expressing, Blocking Assay, DNA Methylation Assay, Methylation Sequencing, ChIP-sequencing, Generated, RNA Sequencing, ChIP-qPCR, Two Tailed Test

Journal: eLife

Article Title: Dynamic enhancer partitioning instructs activation of a growth-related gene during exit from naïve pluripotency

doi: 10.7554/eLife.44057

Figure Lengend Snippet: ( A ) Model for enhancer regulation based on 4C-seq data. ( B ) Model for deletion of E1-3.. ( C ) RT-qPCR of Liz (left) and Zdbf2 (right) during EpiLC differentiation in WT and the ∆E1-3 mutant. The ∆E1-3 mutation significantly reduces both transcripts. Data are shown as ±s.e.m. from five and three biological replicates for WT and mutant, respectively. ( D ) DNA methylation of pLiz (left) and the sDMR (right) during EpiLC differentiation as measured by bisulfite conversion followed by pyrosequencing (BS-pyro) in WT and the ∆E1-3 mutant. When Liz fails to activate, DNA methylation is acquired faster at pLiz , and fails to properly accumulate at the sDMR. Data shown as ±s.e.m. from five and three biological replicates for WT and mutant, respectively. ( E ) Model for deletion of E4. ( F ) RT-qPCR of Liz (left) and Zdbf2 (right) during EpiLC differentiation in WT and the ∆E4 mutant. There is no effect on Liz expression dynamics, but Zdbf2 does not properly activate. Data shown as ±s.e.m. from four biological replicates for each genotype. ( G ) DNA methylation of pLiz (left) and the sDMR (right) during EpiLC differentiation as measured by BS-pyro in WT and the ∆E4 mutant. DNA methylation is unperturbed in the ∆E4 mutant. Data are shown as ±s.e.m. from four biological replicates for each genotype. ( H ) H3K27me3 ChIP-qPCR in ESCs (left) and EpiLCs (right). There is no significant effect on polycomb dynamics in ∆E4 mutation, except mild ectopic spreading upstream of the sDMR region. pPax5 and pOct4 are positive and negative controls, respectively. Data shown as ±s.e.m. from three biological replicates for each genotype. Statistical analyses were performed by two-tailed unpaired t-test: n.s = not significant, *p≤0.05, **p≤0.01, ***p≤0.001. 10.7554/eLife.44057.009 Figure 2—source data 1. Source data for .

Article Snippet: WGBS data from ESCs were previously generated ( ) and EpiLCs were prepared from 50 ng of bisulfite-converted genomic DNA using the EpiGnome/Truseq DNA Methylation Kit (Illumina) following the manufacturer instructions.

Techniques: Quantitative RT-PCR, Mutagenesis, DNA Methylation Assay, Expressing, ChIP-qPCR, Two Tailed Test

Journal: eLife

Article Title: Dynamic enhancer partitioning instructs activation of a growth-related gene during exit from naïve pluripotency

doi: 10.7554/eLife.44057

Figure Lengend Snippet: ( A ) Alleles generated by CRISPR/Cas9 mediated deletions of enhancer elements. ( B ) RT-qPCR of Liz (left) and Zdbf2 (right) during EpiLC differentiation in WT and the ∆E3 mutant. There is no effect on Liz or Zdbf2 expression. Data shown as ±s.e.m. from at least four biological replicates for each genotype. ( C ) DNA methylation of pLiz (left) and the sDMR (right) during EpiLC differentiation as measured by BS-pyro in WT and the ∆E3 mutant. DNA methylation is unperturbed. Data shown as ±s.e.m. from four biological replicates for each genotype. ( D ) H3K27me2 ChIP-qPCR at E4 in WT ESCs and EpiLCs. E4 is depleted for the mark in both cell types. Data shown as ±s.e.m. from three biological replicates. Ctsh and Oct4 are positive and negative controls, respectively.

Article Snippet: WGBS data from ESCs were previously generated ( ) and EpiLCs were prepared from 50 ng of bisulfite-converted genomic DNA using the EpiGnome/Truseq DNA Methylation Kit (Illumina) following the manufacturer instructions.

Techniques: Generated, CRISPR, Quantitative RT-PCR, Mutagenesis, Expressing, DNA Methylation Assay, ChIP-qPCR

Journal: eLife

Article Title: Dynamic enhancer partitioning instructs activation of a growth-related gene during exit from naïve pluripotency

doi: 10.7554/eLife.44057

Figure Lengend Snippet: ( A ) 4C-seq tracks from the CTCF_PS VP in WT ESCs and EpiLCs. Ratios between 4C-seq signals is indicated between the samples, and gene and CTCF binding tracks are below. The CTCF_PS forms a loop with two CTCF sites downstream of the Gpr1 gene. The looping diminishes in EpiLCs. ( B ) CTCF ChIP-qPCR in WT ESCs and EpiLCs and Eed-/- ESCs. CTCF binding remains unchanged in all conditions on the two sites downstream of Gpr1 (termed CTCF Left_1 and Left_2). At the CTCF_PS, CTCF binding reduces from WT ESCs to EpiLCs, which is correlated with decreased interactions between CTCF_PS and the two upstream CTCF sites. CTCF binding remains enriched in Eed-/- ESCs, consistent with the maintained loop structure. Data shown as ±s.e.m. from three biological replicates. ( C ) CTCF ChIP-qPCR in WT, Dnmt tKO and ∆Liz ESCs and EpiLCs. CTCF binding is depleted at the CTCF_PS in EpiLCs in all three contexts. Data shown as ±s.e.m. from three biological replicates. ( D ) Model for CTCF-mediated partitioning of the locus. In ESCs, pLiz and E1-3 are active, and physically separated from the silent E4 and pZdbf2 . During differentiation, the partition is diminished, allowing E1-3 to bolster pZdbf2 activation, while pLiz has become silent. ( E ) RT-qPCR of Liz (left) and Zdbf2 (right) during EpiLC differentiation in WT and the ∆CTCF_PS mutant. Liz is less expressed in mutant ESCs, but reaches WT levels of expression during differentiation. Nevertheless, Zdbf2 does not properly activate in the mutant. Data are shown as ±s.e.m. from four biological replicates for each genotype. ( F ) DNA methylation of pLiz (left) and the sDMR (right) during EpiLC differentiation as measured by BS-pyro in WT and the ∆ mutant. DNA methylation is unperturbed in the mutant at pLiz , but is reduced at the sDMR. Data shown as ±s.e.m. from four biological replicates for each genotype. ( G ) RT-qPCR of Zdbf2 in ESCs in absence of CTCF partition and/or PRC2. While Zdbf2 is already upregulated in the Eed mutant, this effect is exacerbated in the absence of the partition, likely because pZdbf2 is less restrained from interacting with E1-3. Data shown as ±s.e.m. from three biological replicates for each genotype. Statistical analyses were performed by two-tailed unpaired t-test: n.s. = not significant, *p≤0.05, **p≤0.01, ***p≤0.001. 10.7554/eLife.44057.017 Figure 4—source data 1. Source data for .

Article Snippet: WGBS data from ESCs were previously generated ( ) and EpiLCs were prepared from 50 ng of bisulfite-converted genomic DNA using the EpiGnome/Truseq DNA Methylation Kit (Illumina) following the manufacturer instructions.

Techniques: Binding Assay, ChIP-qPCR, Activation Assay, Quantitative RT-PCR, Mutagenesis, Expressing, DNA Methylation Assay, Two Tailed Test

Journal: Acta Neuropathologica

Article Title: “De novo replication repair deficient glioblastoma, IDH-wildtype” is a distinct glioblastoma subtype in adults that may benefit from immune checkpoint blockade

doi: 10.1007/s00401-023-02654-1

Figure Lengend Snippet: “De novo replication repair deficient glioblastoma, IDH-wildtype” has a distinct global hypomethylation epigenetic signature compared to conventional glioblastoma, IDH-wildtype. a Violin plot of DNA methylation data showing the mean beta-value for each of approximately 850,000 CpG sites across 7 de novo RRD glioblastomas and 98 conventional glioblastomas. b Heatmap of DNA methylation profiles for 7 de novo RRD glioblastoma alongside 98 conventional glioblastoma. Shown are the 5000 most differentially methylated probes amongst the 105 glioblastomas revealing extensive hypomethylation of CpG sites in the de novo RRD glioblastoma compared to the cohort of conventional glioblastomas. See Supplementary Table S5 for detailed annotations of these 5000 most differentially methylated CpG sites

Article Snippet: In addition to the chromosomal copy number data obtained from the targeted next-generation DNA sequencing platform, chromosomal copy number profiles were derived from the Infinium EPIC DNA methylation array data using the conumee package (v.1.26.0) [ ].

Techniques: DNA Methylation Assay, Methylation

Journal: Acta Neuropathologica

Article Title: “De novo replication repair deficient glioblastoma, IDH-wildtype” is a distinct glioblastoma subtype in adults that may benefit from immune checkpoint blockade

doi: 10.1007/s00401-023-02654-1

Figure Lengend Snippet: “De novo replication repair deficient glioblastoma, IDH-wildtype” has a unique epigenetic signature distinct from all established reference methylation classes of IDH-wildtype glioblastomas in adults. a tSNE dimensionality reduction plot of genome-wide DNA methylation profiles for 7 de novo RRD glioblastomas alongside 1143 reference samples spanning 25 CNS tumor methylation groups and 3 control tissue methylation groups. See Supplementary Table S7 for sample manifest. b Results of DNA methylation-based classification for the 7 de novo RRD glioblastoma samples using version 12.7 of the DKFZ Molecular Neuropathology classifier. See Supplementary Table S8 for further details

Article Snippet: In addition to the chromosomal copy number data obtained from the targeted next-generation DNA sequencing platform, chromosomal copy number profiles were derived from the Infinium EPIC DNA methylation array data using the conumee package (v.1.26.0) [ ].

Techniques: Methylation, Genome Wide, DNA Methylation Assay, Control

Journal: Acta Neuropathologica

Article Title: “De novo replication repair deficient glioblastoma, IDH-wildtype” is a distinct glioblastoma subtype in adults that may benefit from immune checkpoint blockade

doi: 10.1007/s00401-023-02654-1

Figure Lengend Snippet: Deconvolution analysis reveals a unique cellular composition of “De novo replication repair deficient glioblastoma, IDH-wildtype” compared to conventional glioblastoma, IDH-wildtype. a Cellular composition of 7 de novo RRD glioblastoma and 98 conventional glioblastoma was estimated by methyCIBERSORT deconvolution of Infinium EPIC DNA methylation profiles at time of initial diagnostic surgery before any adjuvant therapy or immune checkpoint blockade. The de novo RRD glioblastomas demonstrated a greater proportion of microglia and CD8 + T-cells, and lower proportion of regulatory T-cells (Tregs) compared to conventional glioblastoma. See Supplementary Table S9 for source data. b Representative photomicrographs of immunohistochemistry for CD8 and CD163 on a de novo RRD glioblastoma and a conventional glioblastoma for comparison

Article Snippet: In addition to the chromosomal copy number data obtained from the targeted next-generation DNA sequencing platform, chromosomal copy number profiles were derived from the Infinium EPIC DNA methylation array data using the conumee package (v.1.26.0) [ ].

Techniques: DNA Methylation Assay, Diagnostic Assay, Adjuvant, Immunohistochemistry, Comparison

Journal: Advances in Laboratory Medicine

Article Title: Liquid biopsy for cancer management: a revolutionary but still limited new tool for precision medicine

doi: 10.1515/almed-2020-0009

Figure Lengend Snippet: Liquid biopsy FDA-approved tests.

Article Snippet: Epi proColon DNA-methylation blood test [ ] , Epigenomics AG , FDA-approved (April 2016) , Detection of methylated cytosine residues in the SEPTIN9 gene in ctDNA by real-time PCR , Screening for colon cancer , The company has developed a kit to assess SEPTIN9 methylation that will be completed in around 32 h. According to the literature, its sensitivity ranges from 69-72%..

Techniques: Marker, Methylation, Real-time Polymerase Chain Reaction, Mutagenesis, Selection, Multiplex Assay, Next-Generation Sequencing

Journal: Epigenomics

Article Title: Maternal lipodome across pregnancy is associated with the neonatal DNA methylome

doi: 10.2217/epi-2020-0234

Figure Lengend Snippet: Differentially methylated CpG sites were classified fitting linear models using empirical Bayes methodology. Models were adjusted for sex, EPIC bead array sample plate, nRBC and B cells. Represented by Manhattan plot, (A) 45 CpG sites were significantly related to maternal term (M3) lysophosphatidylcholine (LysoPE)-saturated (sat) (false discovery rate [FDR] <0.05) and (B) 18 CpG probes were significantly related to M3 LysoPE-sat (FDR <0.05). Line signifies the -log(p-value) corresponding to the FDR <0.05. Differentially methylated CpG sites were classified by genomic location. (C) Percentage of CpG sites within a CpG island, shore, shelf and open sea in the EPIC bead array. Percentage of statistically significant CpG sites within a CpG island, shore, shelf and open sea reported for association with (D) M3 LysoPC-sat and (E) M3 LysoPE-sat.

Article Snippet: Cord blood leukocyte DNA methylation was profiled using the Infinium EPIC bead array and empirical Bayes modeling identified differential DNA methylation related to maternal lipid groups.

Techniques: Methylation