Journal: Epigenetics & Chromatin

Article Title: Depletion of DNMT1 in differentiated human cells highlights key classes of sensitive genes and an interplay with polycomb repression

doi: 10.1186/s13072-018-0182-4

Figure Lengend Snippet: Loss of methylation at the protocadherin γ ( PCDHG ) cluster of neuroepithelial identity genes. a Structure of the PCDHG cluster showing the 5′ variable exons (A, B and C classes) which are spliced to the 3′ constant exons (right). The top track (amber) shows absolute β values in the WT fibroblast cells from the 450K array, which range from 1(fully methylated) to zero (unmethylated). Only the sites showing significant differences from WT (FDR < 0.05) in each cell line are shown in the three tracks below, with decreases in red representing loss of methylation, and gains in blue. The size of the bar is proportional to the magnitude of change: maxima and minima are indicated on the scales at left. The locations of CpG islands (CGI) are also shown. Pyroassay locations are boxed. b Median β values for all variable exons. Significant differences (Mann–Whitney U ) are indicated: * p < 0.05; ** p < 0.1; n.s., not significant. c Methylation at each exon in WT and d16 cells obtained by taking the median of the absolute β value for all probes at that exon. The variable class C exons are underlined. d Average methylation values in WT and KD cells obtained from a pyrosequencing assay (pyroassay) designed to cover CpGs in the A2 exon. Bars represent SEM; *** p < 0.001, t -test. e Methylation at the C4 variable exon by pyroassay, shown as a control

Article Snippet: Key to tracks as before; pyroassay locations ( UGT1A1 and UGT1A4 ) are boxed. b Median β values for all first exons: though medians are higher in KD lines these failed to reach statistical significance. c Median absolute β values at individual first exons in WT and d16 cells. d Average methylation values in WT and KD cells obtained from a pyrosequencing assay (pyroassay) designed to cover CpGs in the A4 exon. e Methylation by pyroassay at the A1 exon, shown as a control

Techniques: Methylation, MANN-WHITNEY, Pyrosequencing Assay, Control

Journal: Epigenetics & Chromatin

Article Title: Depletion of DNMT1 in differentiated human cells highlights key classes of sensitive genes and an interplay with polycomb repression

doi: 10.1186/s13072-018-0182-4

Figure Lengend Snippet: Gains in methylation on the X chromosome. a Sites reliably showing gain in methylation and located in promoters were analysed to identify those common to all three KD lines ( n = 201). Some of these sites showing the greatest change in methylation were located on the X chromosome including MAGEA12 and GABRQ . b Schematic showing the locations of the two genes adjacent to each other on X in a region showing gain in methylation. Tracks indicate the locations of all 450K probes and CGI; the positions of the pyroassays are also indicated; the scale bar pertains to the bottom part of the schematic; ∆ β , change in beta value. c Methylation as determined by pyroassay at the two genes indicated in a , b . d Clonal analysis of GABRQ in WT and d8. Filled circles represent methylated sites, open circles unmethylated. The CpG which were also analysed by the pyroassay (pyro) and the 450K array (asterisk) are indicated

Article Snippet: Key to tracks as before; pyroassay locations ( UGT1A1 and UGT1A4 ) are boxed. b Median β values for all first exons: though medians are higher in KD lines these failed to reach statistical significance. c Median absolute β values at individual first exons in WT and d16 cells. d Average methylation values in WT and KD cells obtained from a pyrosequencing assay (pyroassay) designed to cover CpGs in the A4 exon. e Methylation by pyroassay at the A1 exon, shown as a control

Techniques: Methylation

Journal: Epigenetics & Chromatin

Article Title: Depletion of DNMT1 in differentiated human cells highlights key classes of sensitive genes and an interplay with polycomb repression

doi: 10.1186/s13072-018-0182-4

Figure Lengend Snippet: Methylation loss is concentrated at regions normally repressed by polycomb. a Distribution of probes showing significant loss per chromatin state—numbers of probes are shown at left, chromatin states below: tcn, transcription; heterochrom/Lo, heterochromatin or low signal; repetitive, repeat DNA. b Region around the LEP gene: tracks as before, with the addition of data from cells treated with siRNA for 72 h (top). A track showing ChromHMM chromatin states from NHLF foetal lung fibroblasts is shown at bottom: grey, polycomb-repressed; green, transcriptionally active (full colour key at top right). c DNMT1 mRNA levels by qPCR following treatment with siRNA (+) for 72 h compared with scrambled control (Scr). ACTB is shown as a control; ladder as above. d Median β values for all regions (WT) compared to medians for polycomb-repressed regions (Polycomb), or all other regions (Other) in the cell lines indicated at top; remeth, remethylated. e Numbers of probes showing loss and gain in methylation in hTERT cells following treatment with siRNA for 72 h compared with the shRNA lines (averaged); #, number. f mRNA levels for the indicated genes in shRNA lines treated with the EzH2 inhibitor DZNeP; UNT, untreated; bars represent SEM, experiment carried out in duplicate. g Median β values for all variable exons at the PCDHG locus (left) and for fat/body mass genes (FBM, right): compare d16 shRNA lines with cells treated with siRNA. h DNMT1 mRNA levels in WT cells exposed to siRNA for 48 h, then allowed to recover in normal medium; comparisons were made to a scrambled siRNA negative control (Scr). i Methylation levels by pyroassay at the loci indicated during the transient KD and recovery shown in ( h ); timepoints are in days. All loci showed significant loss of methylation: LEP and SNRPN showed no significant gain versus lowest methylation level, while PCDHGA2 showed no significant gain between d22 and d36

Article Snippet: Key to tracks as before; pyroassay locations ( UGT1A1 and UGT1A4 ) are boxed. b Median β values for all first exons: though medians are higher in KD lines these failed to reach statistical significance. c Median absolute β values at individual first exons in WT and d16 cells. d Average methylation values in WT and KD cells obtained from a pyrosequencing assay (pyroassay) designed to cover CpGs in the A4 exon. e Methylation by pyroassay at the A1 exon, shown as a control

Techniques: Methylation, Control, shRNA, Negative Control

Journal: GeroScience

Article Title: DNA methylation and histone acetylation changes to cytochrome P450 2E1 regulation in normal aging and impact on rates of drug metabolism in the liver

doi: 10.1007/s11357-020-00181-5

Figure Lengend Snippet: Illustration showing the transcription start site (TSS) and upstream regulatory regions of the human CYP2E1 and the homologous mouse Cyp2e1 gene. Upstream regulatory element positions were obtained from GeneHancer (Fishilevich et al. 2017) and ORegAnno (Lesurf et al. 2016). DNA methylation assays were conducted in the current study at both the TSS and upstream regulatory region in mouse at positions marked by the arrows. Reference histone 3 lysine 9 acetylation (H3K9ac) and histone 3 lysine 27 acetylation (H3K27ac) in 8-week-old mouse livers are from the ENCODE/LICR track in UCSC Genome Browser. Two loci with high liver histone acetylation levels were chosen for analysis using ChIP-qPCR in the current study, at positions marked by the arrows. For exact assay coordinates, see Supplementary Tables S2 and S3

Article Snippet: The CpG at position 5 (chr7: 147,949,754, mm9) was the most significantly hypermethylated ( p = 0.007) with the largest beta value of 0.84% increase per month of age (Fig. ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 4 caption a7 Pyrosequencing data for Cyp2e1 . a Scatter plot of percent methylation of cytosine–phosphate–guanine (CpG) and all investigated CpG positions with superimposed line plot for each age group connecting the average methylation percentage at each CpG ( n = 20 per CpG, N = 80 total).

Techniques: DNA Methylation Assay, ChIP-qPCR

Journal: GeroScience

Article Title: DNA methylation and histone acetylation changes to cytochrome P450 2E1 regulation in normal aging and impact on rates of drug metabolism in the liver

doi: 10.1007/s11357-020-00181-5

Figure Lengend Snippet: Box plots with regression line (blue) of age-associated changes to Cyp2e1a 5′UTR percent methylation (n = 19) and b gene expression (n = 20). c Box plot of age-associated changes to Cyp2e1 protein expression with representative western blot (n = 20). Data represent median (middle hinge), 25% (lower hinge), and 75% (upper hinge) quantiles. Data points beyond upper or lower 1.5 × interquantile range are represented as individual black dots

Article Snippet: The CpG at position 5 (chr7: 147,949,754, mm9) was the most significantly hypermethylated ( p = 0.007) with the largest beta value of 0.84% increase per month of age (Fig. ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 4 caption a7 Pyrosequencing data for Cyp2e1 . a Scatter plot of percent methylation of cytosine–phosphate–guanine (CpG) and all investigated CpG positions with superimposed line plot for each age group connecting the average methylation percentage at each CpG ( n = 20 per CpG, N = 80 total).

Techniques: Methylation, Gene Expression, Expressing, Western Blot

Journal: GeroScience

Article Title: DNA methylation and histone acetylation changes to cytochrome P450 2E1 regulation in normal aging and impact on rates of drug metabolism in the liver

doi: 10.1007/s11357-020-00181-5

Figure Lengend Snippet: Pyrosequencing data for Cyp2e1. a Scatter plot of percent methylation of cytosine–phosphate–guanine (CpG) and all investigated CpG positions with superimposed line plot for each age group connecting the average methylation percentage at each CpG (n = 20 per CpG, N = 80 total). X-axis not drawn to scale. chr 7 chromosome 7, mm9 mouse genome assembly NCBI37/build 9, July 2007. b Scatter plot of CpG methylation and age of individual CpG positions with regression line and statistics under each location. A simple linear regression was performed on all 20 data points for each CpG (n = 20) against each age 4, 18, 24, and 32 months (N = 80). Positions 1–8: chr7: 147,942,492; 147,949,679; 147,949,684; 147,949,743; 147,949,754; 147,949,770; 147,949,791; 147,949,806, mm9

Article Snippet: The CpG at position 5 (chr7: 147,949,754, mm9) was the most significantly hypermethylated ( p = 0.007) with the largest beta value of 0.84% increase per month of age (Fig. ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 4 caption a7 Pyrosequencing data for Cyp2e1 . a Scatter plot of percent methylation of cytosine–phosphate–guanine (CpG) and all investigated CpG positions with superimposed line plot for each age group connecting the average methylation percentage at each CpG ( n = 20 per CpG, N = 80 total).

Techniques: Methylation, CpG Methylation Assay

Journal: GeroScience

Article Title: DNA methylation and histone acetylation changes to cytochrome P450 2E1 regulation in normal aging and impact on rates of drug metabolism in the liver

doi: 10.1007/s11357-020-00181-5

Figure Lengend Snippet: Chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) data. Box plots with regression line (blue) of age-associated changes to percentage of input occupancy of histone 3 lysine 9 acetylation (H3K9ac) (n = 20 per region), histone 3 lysine 27 acetylation (H3K27ac) (n = 20 per region) at Cyp2e1 intron 1 (region 1, chr7: 147,950,223–147,950,367, mm9) and promoter (region 2, chr7: 147,942,350–147,942,468, mm9). IgG percentage of input shows a low background noise signal for each of the sample’s age groups (n = 20 per region). Data represent median (middle hinge), 25% (lower hinge), and 75% (upper hinge) quantiles. Data points beyond upper or lower 1.5 × interquantile range are represented as individual black dots

Article Snippet: The CpG at position 5 (chr7: 147,949,754, mm9) was the most significantly hypermethylated ( p = 0.007) with the largest beta value of 0.84% increase per month of age (Fig. ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 4 caption a7 Pyrosequencing data for Cyp2e1 . a Scatter plot of percent methylation of cytosine–phosphate–guanine (CpG) and all investigated CpG positions with superimposed line plot for each age group connecting the average methylation percentage at each CpG ( n = 20 per CpG, N = 80 total).

Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR

Journal: GeroScience

Article Title: DNA methylation and histone acetylation changes to cytochrome P450 2E1 regulation in normal aging and impact on rates of drug metabolism in the liver

doi: 10.1007/s11357-020-00181-5

Figure Lengend Snippet: Correlation matrix reporting Pearson correlation statistical test result (r). White blank cells indicate non-significant association (p > 0.05). Refer to Supplementary Table S5 for individual p values of each Pearson correlation test of a given pair. Color gradient indicates the direction of effect of the association with dark pink representing the strongest positive association of 1 while dark gray representing the strongest negative association of − 1. Only lower half of the plot is shown to prevent redundancy in reporting the results. Age: chronological age, Pos 1–8: Positions 1–8 chr7: 147,942,492; 147,949,679; 147,949,684; 147,949,743; 147,949,754; 147,949,770; 147,949,791; 147,949,806, mm9. Vmax: maximal rate of hydroxylation reaction of chlorzoxazone by Cyp2e1, Km: chlorzoxazone concentration at half maximal rate, CL.Int: intrinsic clearance, K9: histone 3 lysine 9 acetylation in Cyp2e1 intron 1 (chr7: 147,950,223–147,950,367, mm9), R2K9 histone 3 lysine 9 acetylation in Cyp2e1 promoter (chr7: 147,942,350–147,942,468, mm9)

Article Snippet: The CpG at position 5 (chr7: 147,949,754, mm9) was the most significantly hypermethylated ( p = 0.007) with the largest beta value of 0.84% increase per month of age (Fig. ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 4 caption a7 Pyrosequencing data for Cyp2e1 . a Scatter plot of percent methylation of cytosine–phosphate–guanine (CpG) and all investigated CpG positions with superimposed line plot for each age group connecting the average methylation percentage at each CpG ( n = 20 per CpG, N = 80 total).

Techniques: Concentration Assay

Journal: Journal of Hematology & Oncology

Article Title: Downregulation of miR-130b~301b cluster is mediated by aberrant promoter methylation and impairs cellular senescence in prostate cancer

doi: 10.1186/s13045-017-0415-1

Figure Lengend Snippet: Differentially methylated microRNAs in prostate cancer. a Unsupervised hierarchical clustering of microRNAs’ promoters displaying significant alterations in DNA methylation as determined by Infinium HumanMethylation450 BeadChip in 25 prostate cancer (PCa) and 5 morphologically normal prostate tissue (MNPT) samples. Overall, 51 miRNA promoters were differentially methylated in PCa versus MNPT. b Validation of miR-130b~301b by pyrosequencing and ( c ) by RT-qPCR in 111 primary PCa and 14 MNPT cases, indicated that promoter hypermethylation was associated with miR-130b~301b downregulation. d LNCaP, DU145 and PC3 cell lines retain basal expression of miR-130b and miR-301b. e Reversal of DNA methylation in LNCaP cells using 5-aza-2-deoxycytidine (5-AZA-CdR) increased the expression of miR-130b and, in combination, with TSA augmented miR-301b expression. Mann-Whitney U test: * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: DNA methylation changes in microRNAs´ promoters in prostate cancer (PCa), determined by Infinium HumanMethylation450 BeadChip in 25 PCa tissues and 5 morphologically normal prostate tissue (MNPT) samples. (A) Schematic representation of the DNA methylation mapping approach used to identify new aberrantly methylated miRNAs. (B) β values from representative microRNAs. (C) Genomic location of the differentially methylated microRNAs. (D) DNA methylation levels determined by pyrosequencing in PCa cell lines, showing that all cell lines tested display aberrant DNA methylation in the promoter of miR-130b~301b cluster. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Techniques: Methylation, DNA Methylation Assay, Quantitative RT-PCR, Expressing, MANN-WHITNEY