Journal: Nature genetics

Article Title: TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells

doi: 10.1038/s41588-017-0002-y

Figure Lengend Snippet: a , Schematic for analysis of 5mC, 5hmC and TET1 binding at the PAX6 locus. Arrows represent the P0 and P1 promoter, the grey box represents the PAX6 mRNA transcript and the black box represents the PAX6 protein. The region analyzed for 5mC using MassArray and for 5hmC using hMe-Seal profiling is shown by a green box. b , Heat map of MassArray analysis of 5mC at the PAX6 P0 promoter. The location of each row of CpGs with respect to the P0 TSS is shown to the left of the heat map. For each cell line three independent experiments are shown as three columns. (NE D4) Neuroectoderm differentiation day 4; (NE D10) Neuroectoderm Differentiation day 10. Statistical analysis: Student’s t test (two sided), **** P <0.0001. c , Diagram of homology-directed repair (HDR) of the TET1 locus in TKO hESCs. Red letters indicate mutations of the WT sequence. The sequences of the two repaired lines (TKO-r1, TKO-r2) are shown below. d , Mass spectrometry analysis of 5hmC levels in WT, TET KO mutant lines, lines in which one allele of TET1 has been repaired (TKO-R) and lines which underwent HDR targeting but retained the TKO mutations in the TET1 locus (TKO-nr). For all mass spectrometry analysis, 2 mutant lines were used for all genotypes except for TKO. For TKO lines, 2 different passages of the same line were used for mass spectrometry measurements. Data presented are mean ± STD. Statistical analysis: black lines indicate comparisons to WT, one-way ANOVA, *** P <0.001. e , Analysis of percent 5hmC at the PAX6 P0 promoter by Epimark, n = 3 independent experiments. Data presented are mean ± STD. Statistical analysis: Student’s t test (two sided), **** P <0.0001. f , Top panel: Analysis of 5hmC peak at the PAX6 P0 promoter by hMe-Seal in WT hESCs. Bottom panel: Analysis of TET1 peak at the PAX6 P0 promoter by TET1 ChIP-Seq in WT hESCs. Shaded area represents the region of the PAX6 P0 promoter assayed for 5hmC by Epimark ( e ) and TET1 binding by ChIP-qPCR ( g ). g , ChIP-qPCR for TET1 in WT and TKO hESCs, n = 3 independent experiments. Data presented are mean ± STD. Statistical analysis: Student’s t test (two sided), * P <0.05, ** P <0.01, *** P <0.001. h , Analysis of 5hmC and TET1 peaks at bivalent promoters in WT hESCs. The height above the x-axis reflects the normalized tag count. i , Percent DNA methylation change (TKO – WT) in bivalent promoters that have 5hmC peaks compared to bivalent promoters that don’t have 5hmC peaks.

Article Snippet: MassArray Epityper analysis was performed using the WCMC Epigenomics core.

Techniques: Binding Assay, Sequencing, Mass Spectrometry, Mutagenesis, ChIP-sequencing, ChIP-qPCR, DNA Methylation Assay

Journal: Nature genetics

Article Title: TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells

doi: 10.1038/s41588-017-0002-y

Figure Lengend Snippet: a , Schema for rescue of the NE differentiation defect in TKO hESCs using PAX6 overexpression or targeted demethylation of the PAX6 P0 promoter. b , qPCR analysis of neuroectoderm ( SOX1 ) and pluripotency ( OCT4 ) markers in WT and TKO cells without doxycycline (TKO) and with doxycycline treatment (TKO +PAX6) at D10 of NE differentiation, n = 3 independent experiments. Data presented are mean ± STD. Statistical analysis: one-way ANOVA, * P <0.05, ** P <0.01, *** P <0.001. c , Immunofluorescence of PAX6, SOX1 and OCT4 at the endpoint of differentiation (D10) of TKO cells without doxycycline treatment (TKO) and with doxycycline treatment (TKO + PAX6). Scale bar indicates 100 µm. d , Heat map of MassArray analysis of 5mC at the PAX6 P0 promoter for TKO hESCs that express PAX6 targeting gRNAs with either a dCas9-TET1CD/Mut (left) or a dCas9-TET1CD (right) fusion protein. The location of each row of CpGs with respect to the P0 TSS is shown to the left of the heat map. Methylation analysis at the PAX6 P0 promoter was analyzed for these cell lines with and without doxycycline treatment, n = 3 independent experiments. Statistical analysis: Student’s t test (two sided), **** P <0.0001. e , qPCR of PAX6 expression on D10 of NE differentiation for TKO hESCs that express PAX6 targeting gRNAs with either a dCas9-TET1CD/Mut or a dCas9-TET1CD fusion protein. PAX6 expression was analyzed for these cell lines with and without doxycycline treatment prior to differentiation, n = 3 independent experiments. Data presented are mean ± STD. Statistical analysis: Student’s t test (two sided), *** P <0.001. f , Immunofluorescence of PAX6 on D10 of NE differentiation for TKO hESCs that express PAX6 targeting gRNAs with either a dCas9-TET1CD/Mut or a dCas9-TET1CD fusion protein. TKO hESCs expressing the dCas9-TET1CD fusion and a non-targeting gRNA were also used as a control. PAX6 immunofluorscence was analyzed for these cell lines with and without doxycycline treatment prior to differentiation. Scale bar indicates 100 µm.

Article Snippet: MassArray Epityper analysis was performed using the WCMC Epigenomics core.

Techniques: Over Expression, Immunofluorescence, Methylation, Expressing, Control

Journal: Nature genetics

Article Title: TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells

doi: 10.1038/s41588-017-0002-y

Figure Lengend Snippet: a , ChIP-qPCR for DNMT3B at the PAX6 locus in WT and TKO hESCs, n = 3 independent experiments. Data presented are mean ± STD. Statistical analysis: Student’s t test (two sided), * P <0.05, ** P <0.01. b , Heat map of MassArray analysis of 5mC at the PAX6 P0 promoter in WT, TKO and QKO hESCs. WT and TKO hESCs are passage-matched with the QKO hESCs. The location of each row of CpGs with respect to the TSS is shown to the left of the heat map. For each cell line three independent experiments are shown as three columns. Quantification of the percent methylation is shown on the right, n = 3 independent experiments. Data presented are mean ± STD. Statistical analysis: Student’s t test (two sided), **** P <0.0001. c , Immunofluorescence of PAX6, SOX1 and OCT4 at D10 of NE differentiation in WT, TKO and QKO cells. Scale bar indicates 100 µm. d , qPCR analysis of neuroectoderm ( PAX6 and SOX1 ) and pluripotency ( OCT4 and NANOG ) markers in WT, TKO and QKO cells at D10 of NE differentiation, n = 3 independent experiments. Data presented are mean ± STD. Statistical analysis: black lines indicate comparisons to WT, one-way ANOVA, * P <0.05,** P <0.01,*** P <0.001, **** P <0.0001.

Article Snippet: MassArray Epityper analysis was performed using the WCMC Epigenomics core.

Techniques: ChIP-qPCR, Methylation, Immunofluorescence

Journal: Oncotarget

Article Title: DNA methylation promotes paired box 2 expression via myeloid zinc finger 1 in endometrial cancer

doi: 10.18632/oncotarget.12626

Figure Lengend Snippet: A. Bisulfite sequencing results showed fragment B1 (-764 to -447 bp) of the PAX2 promoter was hypermethylated in endometrial cancer cell lines and tissues: rows represent clones (10 for each sample), columns represent CpG sites. Black squares represent methylated CpGs, and white squares represent unmethylated CpGs. EEC: endometrial epithelial cell. EnCa: endometrial cancer. N: normal. B. MassARRAY results indicate that PAX2 was hypermethylated in endometrial cancer tissues compared with normal endometrial tissues. M1 is an amplicon of a 280-bp fragment from -723 bp to -443 bp; M2 is an amplicon of a 379-bp fragment from -468 bp to -89 bp. Hypermethylated CpG sites were centralized in the 5’ of M1 (CpG 1 to 7).

Article Snippet: The PCR annealing Tm was 56°C, and sample preparation was performed according to “Training Instructions for EpiTYPER Quantitative Methylation Analysis Using MassCLEAVE for MassARRAY” (Sequenom).

Techniques: Methylation Sequencing, Clone Assay, Methylation, Amplification

Journal: Cancers

Article Title: BRCA1 Promoter Hypermethylation is Associated with Good Prognosis and Chemosensitivity in Triple-Negative Breast Cancer

doi: 10.3390/cancers12040828

Figure Lengend Snippet: Correlation between methylation-specific PCR and EpiTYPER® MassARRAY® methylation evaluation ( n = 153). Black columns indicate discordant cases ( n = 4, methylation percentage assessed by MassARRAY® = 9.5, 6, 4, and 4), using a positivity cut-off of 10%.

Article Snippet: The key secondary endpoint was the evaluation of the concordance between the determination of BRCA1 promoter methylation status by the quantitative EpiTyper ® MassARRAY ® (Agena Bioscience, Sans Diego, CA, USA) and by MS-PCR.

Techniques: Methylation

Journal: Cancers

Article Title: BRCA1 Promoter Hypermethylation is Associated with Good Prognosis and Chemosensitivity in Triple-Negative Breast Cancer

doi: 10.3390/cancers12040828

Figure Lengend Snippet: Univariate clinicopathological correlations with BRCA1 promoter methylation using MS-PCR.

Article Snippet: The key secondary endpoint was the evaluation of the concordance between the determination of BRCA1 promoter methylation status by the quantitative EpiTyper ® MassARRAY ® (Agena Bioscience, Sans Diego, CA, USA) and by MS-PCR.

Techniques: Methylation, Expressing

Journal: Cancers

Article Title: BRCA1 Promoter Hypermethylation is Associated with Good Prognosis and Chemosensitivity in Triple-Negative Breast Cancer

doi: 10.3390/cancers12040828

Figure Lengend Snippet: Distribution of CpG sites analyzed in the promoter region of the BRCA1 gene. ( A ) The BRCA1–NBR2 locus: the position of the first exons of the BRCA1 is indicated by shaded boxes. The first exon of NBR2 gene is indicated by a black box. ( B ) Position of CpG sites analyzed Cytosine on CpG sites tested by MS-PCR are underlined. Cytosine on CpG sites tested by MassARRAY® EpiTYPER® assay are in bold. The positive strand of the BRCA1 gene is shown based on GenBank accession number U37574. The transcription start site of the BRCA1 exon 1A is marked by an arrow. Exon 1A sequence is in capital, and both 5’upstream sequence and intron 1 sequence are in lower case characters.

Article Snippet: The key secondary endpoint was the evaluation of the concordance between the determination of BRCA1 promoter methylation status by the quantitative EpiTyper ® MassARRAY ® (Agena Bioscience, Sans Diego, CA, USA) and by MS-PCR.

Techniques: MassARRAY EpiTYPER assay, Sequencing