Journal: Scientific Reports

Article Title: Epigenetic silencing of the MUPCDH gene as a possible prognostic biomarker for cyst growth in ADPKD

doi: 10.1038/srep15238

Figure Lengend Snippet: ( a ) The altered DNA methylation pattern in the MUPCDH promoter region was confirmed using the EpiTYPER® assay. As shown in the EpiTYPER® heat map, methylated CpG sites (yellow) were changed to unmethylated CpG sites (red) after treatment of WT9-7 cystic epithelial cells with 5-aza-dC (5 μM, 72 h). Green box indicate CpG sites within −543 and +228, which was more sensitive to 5-aza-dC, compared to those in other regions. The translation start ATG is indicated by a black arrow. P < 0.05. ( b ) Altered mRNA and protein expression levels upon treatment of WT9-7 cells with 5-aza-dC were confirmed by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis, and ( c ) western blot analysis. The band density was measured using the MultiGauge software. The height of each bar represents the mean, and the error bars indicate ± SD. β-Actin was used as an internal control in real-time qRT-PCR and western blot analyses. Each experiment was performed in triplicate. * P < 0.05; ** P < 0.001.

Article Snippet: PCR amplification was carried out in a total volume of 20 μl, containing the following: 10× Buffer, 1.65 mM MgCl 2 , 200 μM each of the four dNTPs, 500 nM each primer, 0.04 U HotStarTaq DNA polymerase (QIAGEN, Hilden, Germany), ResoLight dye (Roche, Branchburg, NJ, USA), and 10 ng of bisulfite-modified template.

Techniques: DNA Methylation Assay, EpiTYPER Assay, Methylation, Expressing, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Software, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Epigenetic and post-transcriptional regulation of CD16 expression during human natural killer cell development 1

doi: 10.4049/jimmunol.1701128

Figure Lengend Snippet: DNA methylation and isoform-specific expression within the promoter regions of FCGR3A and FCGR3B. A) Representation of the FCGR3 locus spanning approximately 100 kb of chromosome 1q23.3 displaying transcripts annotated in the RefSeq database (adapted from the UCSC Genome Browser). Below, promoter regions of FCGR3A and FCGR3B are enlarged displaying the various transcript variants annotated in the RefSeq database that differ in the 5′ region. MassARRAY amplicons (green bars) used are shown along with the positions of CpG dinucleotides (green markers). TSS, transcriptional start site. B) The MassARRAY EpiTYPER assay was used to interrogate the DNA methylation levels of CpGs across the promoter region of FCGR3A and FCGR3B in a gene-specific manner in sorted NK CD16a+ and CD16a- fractions. Methylation plots are aligned to enlargements of gene promoters (in B) according to the hg19 genome assembly. C, D) DNA methylation levels in NK cell lines and neutrophils, respectively. E) The expression ratio of FCGR3A to FCGR3B in CD16a+ NK cells and neutrophils as determined by the MassARRAY iPLEX assay. F) Quantitative RT-PCR analysis of 5′ transcript variants using primers specific for variant 1 or variants 3 and 2 indicating promoter usage in NK cells, neutrophils, and NK cell lines YT and NKL (both primer pairs do not distinguish FCGR3A vs. FCGR3B). D1-4 represents donor numbers 1–4. Expression values expressed relative to the average of four housekeeping (HK) genes.

Article Snippet: Antibodies and flow cytometric analysis The following antibodies were used to stain human peripheral blood cells: CD3 (SK7, BD Biosciences), CD14 (TÜK4, Miltenyi), CD15 (VIMC6, Miltenyi), CD16 (VEP13, Miltenyi), CD16 (3G8, BD Biosciences), and CD56 (N901, Beckman Coulter).

Techniques: DNA Methylation Assay, Expressing, MassARRAY EpiTYPER assay, Methylation, Quantitative RT-PCR, Variant Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Epigenetic and post-transcriptional regulation of CD16 expression during human natural killer cell development 1

doi: 10.4049/jimmunol.1701128

Figure Lengend Snippet: Analysis of DNA methylation- and lineage-specific activity of FCGR3 promoter sequences. A) Illustration of FCGR3A and FCGR3B sequences cloned into luciferase constructs (black bars). B) Four separate promoter sequence fragments were cloned from either FCGR3A (left) or FCGR3B (right) and transfected in various cell lines. Luciferase assays showing sequence- and gene-specific activity (relative to empty-vector control). Pmed1-A and Pmed1-B were additionally methylated in vitro prior to transfection. Error bars represent SEM of n=3 individual experiments; ND, not done. C) Sequence alignment of Pmed1-A and Pmed1-B with numbered CpGs. All sequence variants are enlarged below; an 8 bp repeat occurring in the vicinity of CpG#6 is highlighted in blue, asterisks below the sequence indicate homology. D) Luciferase activity following site-directed mutagenesis of CpG#s 1&2 of Pmed1-A compared to unaltered FCGR3A and FCGR3B sequences in YT and K562 cells.

Article Snippet: Antibodies and flow cytometric analysis The following antibodies were used to stain human peripheral blood cells: CD3 (SK7, BD Biosciences), CD14 (TÜK4, Miltenyi), CD15 (VIMC6, Miltenyi), CD16 (VEP13, Miltenyi), CD16 (3G8, BD Biosciences), and CD56 (N901, Beckman Coulter).

Techniques: DNA Methylation Assay, Activity Assay, Clone Assay, Luciferase, Construct, Sequencing, Transfection, Plasmid Preparation, Control, Methylation, In Vitro, Mutagenesis

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Epigenetic and post-transcriptional regulation of CD16 expression during human natural killer cell development 1

doi: 10.4049/jimmunol.1701128

Figure Lengend Snippet: Identification of miR-218 as a potential regulator of FCGR3A. (A) Expression ratio of predicted miRNAs that were present in the miRNA expression array comparing CD16a+ and CD16a- NK cells freshly isolated from adult peripheral blood. A ratio <1 indicates low expression in CD16a+ NK cells while a ratio >1 indicates high expression in CD16a+ NK cells. (B) Predicted miRNA regulators of FCGR3A have putative binding sites in the FCGR3A 3′ UTR. (C) Validation of expression of predicted miRNA regulators of FCGR3A by qPCR (n=3). (D) Luciferase expression as a ratio of firefly/renilla for each expression vector (n=2). Data are presented as mean±SD, * indicates p<0.05.

Article Snippet: Antibodies and flow cytometric analysis The following antibodies were used to stain human peripheral blood cells: CD3 (SK7, BD Biosciences), CD14 (TÜK4, Miltenyi), CD15 (VIMC6, Miltenyi), CD16 (VEP13, Miltenyi), CD16 (3G8, BD Biosciences), and CD56 (N901, Beckman Coulter).

Techniques: Expressing, Isolation, Binding Assay, Biomarker Discovery, Luciferase, Plasmid Preparation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Epigenetic and post-transcriptional regulation of CD16 expression during human natural killer cell development 1

doi: 10.4049/jimmunol.1701128

Figure Lengend Snippet: MiR-218 negatively regulates CD16a in primary human NK cells. Primary human NK cells were enriched by magnetic selection to >70% purity and infected with lentivirus containing either miR-218 or empty vector. 48hr after infection, NK cells were sorted as live/CD3-/CD56+/GFP+ lymphocytes. (A) Representative histogram plot (of one of six donors) of CD16a expression in live/CD3-/CD56+/GFP+ primary human NK cells infected with miR-218 or empty vector virus. (B) CD16a expression in primary human NK cells infected with miR-218 or empty vector virus (* indicates p=0.05). (C) Validation of miR-218 over-expression by real-time PCR (** indicates p<0.01). (D) FCGR3A mRNA expression assessed by real-time RT-PCR in sorted NK cells infected with either miR-218 or empty vector (*** indicates p<0.005). (B–D) Data are presented as mean±SD, n=6.

Article Snippet: Antibodies and flow cytometric analysis The following antibodies were used to stain human peripheral blood cells: CD3 (SK7, BD Biosciences), CD14 (TÜK4, Miltenyi), CD15 (VIMC6, Miltenyi), CD16 (VEP13, Miltenyi), CD16 (3G8, BD Biosciences), and CD56 (N901, Beckman Coulter).

Techniques: Selection, Infection, Plasmid Preparation, Expressing, Virus, Biomarker Discovery, Over Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Epigenetic silencing of the MUPCDH gene as a possible prognostic biomarker for cyst growth in ADPKD

doi: 10.1038/srep15238

Figure Lengend Snippet: ( a ) CpG sites on the MUPCDH promoter region are shown as vertical bars. The position of the translation start ATG is indicated by a black arrow. The exons are presented as thick black horizontal bars. Black horizontal dotted lines indicate the primer sites for methylation-sensitive high resolution melting analysis. Blue solid lines indicate the primer sites for the EpiTYPER® assay. ( b ) The DNA methylation status of the MUPCDH promoter CpG islands, covering approximately 1 kb upstream from the ATG, was quantitated by EpiTYPER® analysis in renal tissue and ( c ) renal epithelial cells. As shown in the EpiTYPER® heat map, hypermethylated CpG sites (yellow) were observed in ADPKD renal tissue (n = 4) and WT9-7 renal cystic epithelial cells, while CpG sites in non-ADPKD kidney tissue (n = 3) and human renal cortical epithelial cells were hypomethylated (red). P < 0.001.

Article Snippet: The EZ DNA Methylation-Gold TM kit (Zymo Research, Orange, CA, USA) was used for bisulfite treatment of genomic DNA, according to the manufacturer’s instructions.

Techniques: Methylation, EpiTYPER Assay, DNA Methylation Assay

Journal: Scientific Reports

Article Title: Epigenetic silencing of the MUPCDH gene as a possible prognostic biomarker for cyst growth in ADPKD

doi: 10.1038/srep15238

Figure Lengend Snippet: ( a ) The altered DNA methylation pattern in the MUPCDH promoter region was confirmed using the EpiTYPER® assay. As shown in the EpiTYPER® heat map, methylated CpG sites (yellow) were changed to unmethylated CpG sites (red) after treatment of WT9-7 cystic epithelial cells with 5-aza-dC (5 μM, 72 h). Green box indicate CpG sites within −543 and +228, which was more sensitive to 5-aza-dC, compared to those in other regions. The translation start ATG is indicated by a black arrow. P < 0.05. ( b ) Altered mRNA and protein expression levels upon treatment of WT9-7 cells with 5-aza-dC were confirmed by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis, and ( c ) western blot analysis. The band density was measured using the MultiGauge software. The height of each bar represents the mean, and the error bars indicate ± SD. β-Actin was used as an internal control in real-time qRT-PCR and western blot analyses. Each experiment was performed in triplicate. * P < 0.05; ** P < 0.001.

Article Snippet: The EZ DNA Methylation-Gold TM kit (Zymo Research, Orange, CA, USA) was used for bisulfite treatment of genomic DNA, according to the manufacturer’s instructions.

Techniques: DNA Methylation Assay, EpiTYPER Assay, Methylation, Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Software

Journal: Scientific Reports

Article Title: Epigenetic silencing of the MUPCDH gene as a possible prognostic biomarker for cyst growth in ADPKD

doi: 10.1038/srep15238

Figure Lengend Snippet: ( a,b ) Methylation-sensitive high resolution melting (MS-HRM) analysis was carried out using urine sediment genomic DNA from patients with autosomal dominant polycystic kidney disease (ADPKD). Bisulfite converted human control DNA (0, 50, 100% methylated) was used as quantification standards for MS-HRM analysis (green lines). Based on control DNA, ADPKD patients were classified into five groups as a methylation level of MUPCDH promoter region. Dark blue and blue lines indicate unmethylated and hemi-methylated samples. Red line showed fully methylation pattern. ( c ) Baseline TKV and ( d ) rate of annual change of TKV according to MUPCDH promoter methylation level were shown. ( e ) When the subjects were grouped into 0–75% (n = 46) vs. 100% (n = 7) methylation level, percent change in TKV per year was significantly faster in 100% group than 0–75% (median 14.6% vs. 5.9% per year, * P = 0.019 by Mann-Whitney test).

Article Snippet: The EZ DNA Methylation-Gold TM kit (Zymo Research, Orange, CA, USA) was used for bisulfite treatment of genomic DNA, according to the manufacturer’s instructions.

Techniques: Methylation, MANN-WHITNEY

Journal: Clinical Epigenetics

Article Title: DNA methylation at an enhancer of the three prime repair exonuclease 2 gene ( TREX2 ) is linked to gene expression and survival in laryngeal cancer

doi: 10.1186/s13148-019-0666-5

Figure Lengend Snippet: Identification of differential methylation of the TREX2 gene in laryngeal cancer. a Quantitative DNA methylation analysis using EpiTYPER assay in a cohort of 161 laryngeal cancer tumor tissues (T), 58 adjacent non-cancerous normal tissues (A), and 24 normal mucosa samples from non-cancer patients who underwent tonsillectomy (N). Average methylation data for EpiTYPER TREX2_2 amplicon (see Fig. 1b) are shown; the p value refers to ANOVA test across the three sample subsets. b Upper panel: map of the TREX2 gene locus with EpiTYPER PCR amplicons, single CpG dinucleotides analyzed in EpiTYPER (black), Illumina Infinium BeadChIP CpG probe locations (cg18879010 and cg 09364317), TREX2 transcript (blue), and CpG islands (green) indicated. Informative CpG unit 3.4 of TREX2_2 is marked with a red box. Lower panel: heat map showing EpiTYPER results for amplicons TREX2_1 and TREX2_2 in laryngeal cancer tumors ( n = 58, red) and adjacent normal tissue controls ( n = 25, green), with DNA methylation at individual CpGs depicted as a color gradient ranging from white (non-methylated) to orange (fully methylated). Corresponding EpiTYPER amplicons are listed above, and DNA methylation standard values (std) are shown below. Grey: data unavailable/excluded. c Methylation analysis of matched pairs of laryngeal tumor and adjacent tissue samples at the informative CpG unit 3.4 of the TREX2_2 amplicon; p value for two-tailed Student’s unpaired t test. d Average TREX2 _2 methylation in colorectal cancer tumor tissues (T) and adjacent normal tissues ( a ). p value for two-tailed Student’s paired t test. Box-whisker plots show mean with 10 to 90 percentile

Article Snippet: Total cellular RNA was isolated using TRIzol (Invitrogen,) according to standard protocols. mRNA expression was measured using complementary DNA samples generated from 1.0 μg DNase I-treated RNA with SuperScript III Reverse Transcriptase (Invitrogen) and random hexamers (QIAGEN).

Techniques: Methylation, DNA Methylation Assay, EpiTYPER Assay, Amplification, Two Tailed Test, Whisker Assay

Journal: Clinical Epigenetics

Article Title: DNA methylation at an enhancer of the three prime repair exonuclease 2 gene ( TREX2 ) is linked to gene expression and survival in laryngeal cancer

doi: 10.1186/s13148-019-0666-5

Figure Lengend Snippet: TREX2 methylation loss as a frequent event in multiple cancer studies. a Differential DNA methylation and b mRNA expression of TREX2 in multiple cancer studies (for abbreviations of cancer types, see Additional file : Table S1A). Graph shows a DNA methylation average at Illumina Infinium CpG probes cg09364317 and cg18879010 located at the TREX2 DMR and b TCGA Illumina HiSeq2000 RNAseq cohorts separated by primary tumor and normal tissue type. mRNA expression data depict RNA expression of the TREX2 transcript as log2( x + 1) transformed RSEM-normalized count. * p < 0.05. In addition, laryngeal cancer patients, a subgroup of HNSC patients with the ICD10-code C32, were evaluated for overall and pairwise differences in TREX2 DNA methylation ( c ) and mRNA expression ( d )

Article Snippet: Total cellular RNA was isolated using TRIzol (Invitrogen,) according to standard protocols. mRNA expression was measured using complementary DNA samples generated from 1.0 μg DNase I-treated RNA with SuperScript III Reverse Transcriptase (Invitrogen) and random hexamers (QIAGEN).

Techniques: Methylation, DNA Methylation Assay, Expressing, RNA Expression, Transformation Assay

Journal: Clinical Epigenetics

Article Title: DNA methylation at an enhancer of the three prime repair exonuclease 2 gene ( TREX2 ) is linked to gene expression and survival in laryngeal cancer

doi: 10.1186/s13148-019-0666-5

Figure Lengend Snippet: TREX2 DNA methylation in tumor tissue and overall survival of cancer patients. Samples from the German laryngeal cancer study and TCGA cancer patients with the ICD10-code C32 for laryngeal cancer are shown. For TCGA laryngeal cancer patients, overall survival was also analyzed in relation to TREX2 mRNA expression (given as log 2 (normalized expression + 1)). HRs with p values < 0.05 are presented in italic

Article Snippet: Total cellular RNA was isolated using TRIzol (Invitrogen,) according to standard protocols. mRNA expression was measured using complementary DNA samples generated from 1.0 μg DNase I-treated RNA with SuperScript III Reverse Transcriptase (Invitrogen) and random hexamers (QIAGEN).

Techniques: DNA Methylation Assay, Expressing, Amplification

Journal: Clinical Epigenetics

Article Title: DNA methylation at an enhancer of the three prime repair exonuclease 2 gene ( TREX2 ) is linked to gene expression and survival in laryngeal cancer

doi: 10.1186/s13148-019-0666-5

Figure Lengend Snippet: The TREX2 DMR displays gene enhancer characteristics in multiple cell lines. a DNA methylation at individual CpG sites at the TREX2 locus across different cancer cell lines and normal human epidermal keratinocyte (NHEK) specimens, matched with respective mRNA expression values (black bars). EHEB was included as a control with low TREX2 expression. Dark gray: data unavailable. b Correlation of average DNA methylation at TREX2 _1 EpiTYPER and TREX2 mRNA expression determined by qRT-PCR in cell lines from a . LS174T was excluded due to reported X-chromosomal aberrations . c 5-Aza-dC treatment in three cell lines and effects on TREX2 DNA methylation and mRNA expression. Bar charts show cell lines after 6 days of treatment with 0.5 μM 5-aza-dC, depicting average TREX2 DNA methylation (upper panel) and TREX2 mRNA expression (lower panel). Average expression ratios of TREX2 versus the housekeeping gene HPRT1 are depicted below each data point for approximate estimation of TREX2 mRNA abundance. d Upper panel: map of the TREX2 gene with ChIP-qPCR amplicons and regions used for luciferase reporter assays. Lower panel: heat map depicting relative luciferase signals in cell lines transfected with TREX2 reporter constructs. Schematic view of reporters is shown to the left. Signals depict mean of quadruplicate measurements normalized to empty vector (pGl4.23). minP/luc2, minimal promoter/luc2 luciferase. e HEK293T cells transfected with CpG-free reporter vectors carrying TREX2 PROM1 and DMR (with minimal promoter) regions. Data show luciferase signal of in vitro CpG-methylated and non-methylated reporter plasmids. EV, empty vector (pCpGfree-promoter-lucia). f ChIP-qPCR assays at the TREX2 locus. Bars show H3K27ac signal at four regions (see d ) of the TREX2 gene with mean and standard deviation from four replicates. Dot plot shows correlation of H3K27ac signal at the TREX2 DMR (ChIP region 3) and relative TREX2 mRNA expression in cell lines ( n = 6). All data depict mean of three replicates with standard deviation, unless stated otherwise. p values refer to unpaired Student’s t test. For correlations, Pearson coefficient ( R ) is shown

Article Snippet: Total cellular RNA was isolated using TRIzol (Invitrogen,) according to standard protocols. mRNA expression was measured using complementary DNA samples generated from 1.0 μg DNase I-treated RNA with SuperScript III Reverse Transcriptase (Invitrogen) and random hexamers (QIAGEN).

Techniques: DNA Methylation Assay, Expressing, Control, Quantitative RT-PCR, ChIP-qPCR, Luciferase, Transfection, Construct, Plasmid Preparation, In Vitro, Methylation, Standard Deviation

Journal: Translational Psychiatry

Article Title: What do DNA methylation studies tell us about depression? A systematic review

doi: 10.1038/s41398-019-0412-y

Figure Lengend Snippet: PRISMA flow diagram: DNA methylation and depression. Some selected studies had more than one study topic (i.e., BDNF ); therefore, the total of these subgroups were bigger than the final number eligible for the review

Article Snippet: 41 , Numata et al. , 2015 , Japan , 63 , Hospital-based cases and matched controls , Case–control , MDD , Whole blood , Bisulfite conversion using EZ DNA methylation Kit (ZYMO research), Infinium Human Methylation 450 Beadchips , Genome-wide , 363 (313 CGIs) , 363 CpG sites demonstrated lower DNA methylation in MDD patients than in controls. 18 MDD-associated DNA methylation markers to discriminate cases from controls.

Techniques: DNA Methylation Assay

Journal: Translational Psychiatry

Article Title: What do DNA methylation studies tell us about depression? A systematic review

doi: 10.1038/s41398-019-0412-y

Figure Lengend Snippet: A summary of selected articles in this systematic review

Article Snippet: 41 , Numata et al. , 2015 , Japan , 63 , Hospital-based cases and matched controls , Case–control , MDD , Whole blood , Bisulfite conversion using EZ DNA methylation Kit (ZYMO research), Infinium Human Methylation 450 Beadchips , Genome-wide , 363 (313 CGIs) , 363 CpG sites demonstrated lower DNA methylation in MDD patients than in controls. 18 MDD-associated DNA methylation markers to discriminate cases from controls.

Techniques: DNA Methylation Assay, Genome Wide, Methylation, Variant Assay, Derivative Assay, Biomarker Assay, Expressing, Software, Binding Assay, Sequencing, EpiTYPER Assay, CpG Methylation Assay, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Methylated DNA Immunoprecipitation Sequencing, Real-time Polymerase Chain Reaction, SYBR Green Assay, Marker

Journal: Cancers

Article Title: Epigenetic Modulation of Radiation-Induced Diacylglycerol Kinase Alpha Expression Prevents Pro-Fibrotic Fibroblast Response

doi: 10.3390/cancers13102455

Figure Lengend Snippet: DNA demethylation of the DGKA enhancer region results in increased DGKA expression after irradiation. ( A ) Upper panel: Schematic presentation of the epigenetic regulation at the DGKA locus. Black dots represent methylated CpG sites and white circles unmethylated CpG sites. Lower panel: Map of the interrogated DGKA enhancer region indicating the two EGR1-binding sites (EGR1_1 and EGR1_2, gray), the differentially methylated region (DGKA_DMR) and the amplicon for the EpiTYPER assay (green), regions for EMSA probes (E1 and E2, blue), four sgRNA-targeting sites for CRISPR/dCas9-VPR-TET3 gene editing (red), and amplicons for antibody-guided chromatin tagmentation (ACT)-qPCR (Promo, EGR1-BD1 and EGR1-BD2, Ctrl, purple) and DGKA transcripts (navy). Amplicons for ACT-qPCR cover the promoter region (Promo), the two EGR1-binding regions (EGR1_BD1 and EGR1_BD2) and the 3‘ downstream control region (Ctrl). ( B ) DNA-binding activity of EGR1 at the DGKA enhancer measured by EMSA. E1/E1me and E2/E2me denote the biotin-labeled probes (BTN probes) that contain unmethylated/methylated oligonucleotides, which cover the first or second EGR1-binding site. Ratios indicated are based on the E1 or E2 band shifts highlighted with blue brackets. ( C ) Methylation average across DGKA DMR-associated CpGs in HEK293T WT cells as well as dCas9-VPR-TET3 expressing cells with or without cumate (300 µg/mL) treatment. Methylation was measured as β-values by EpiTYPER technology. ( D ) Heatmap of DNA methylation of all CpG sites measured as described in ( C ). For each cell type, three replicates (1,2,3) are shown. Arrows indicate the location of the EGR1-binding motifs. The first EGR1-binding site (EGR1_1) covers CpG_6, and the second binding site (EGR1_2) covers CpG_14, which is located between CpG_11 and CpG_16 and not detected by EpiTYPER. ( E ) Relative mRNA expression of DGKA in HEK293T and dCas9-VPR-TET3-expressing cells treated with cumate (300 µg/mL). Cells were harvested 48 h after irradiation (6 Gy). ( F – H ) ACT-qPCR signals for the histone modifications H3K4me1 ( F ), H3K27ac ( G ), and H3K27me3 ( H ) at the EGR1-binding sites of DGKA enhancer (EGR1_BD1 and EGR1_BD2), gene promoter (Promo) and a downstream control region (Ctrl) in HEK293T and cumate-treated dCas9-VPR-TET3-expressing cells. Results from ( C ) and ( E – H ) are shown as mean ± SEM from three biological replicates. Statistical significance (* p < 0.05 and ** p < 0.01) was determined by one-way ANOVA analysis followed by Tukey’s test ( C ) or one-tailed Student’s t -test ( E – H ).

Article Snippet: Human foreskin BJ fibroblasts (CRL-2522) and HEK293T cells (CRL-11268) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Irradiation, Methylation, Binding Assay, Amplification, EpiTYPER Assay, CRISPR, Control, Activity Assay, Labeling, DNA Methylation Assay, One-tailed Test