Journal: Cell Reports Methods

Article Title: Engineering CpG island DNA methylation in pluripotent cells through synthetic CpG-free ssDNA insertion

doi: 10.1016/j.crmeth.2023.100465

Figure Lengend Snippet: CIMR overview (A) Briefly, CG-free DNA is integrated in a target CGI by ssDNA/Cas RNP reaction, cells are clonally isolated after CG-free antibiotic selection, and de novo DNAMe assessed. (B) Schematic of CpG-free plasmid-integrated DNA (PI) and shorter CpG-free ssDNA (ssI), with insert-testing primer sites and expected PCR products displayed. (C) Both NeoR gels indicate successful integration of PI and ssI DNA by PCR, as well pilot Cre mRNA mediated removal 48 h post-transfection (right). (D) The first 6 candidate PI and ssI clones are shown. (E) Visualization of EPIC Illumina Infinium array data and target-region-induced DNAme using IgV Viewer (v.2.3). Individual CpG probes with peak intensity corresponding to DNAme level are displayed at the target CGI (DMR in black box). Blue arrows mark bisulfite (BSF)-PCR primers ( Figure S1 C). (F) Global target DNAme specificity among all probes analyzed. Scatterplots display Infinium probe values for each pairwise comparison (Pearson correlation displayed). Red dots highlight CGs falling within the CIMR DMR (N = 38). (G) The most significant DMR, target MLH1, was specifically and consistently induced across all insert-containing clones ( Table S2 B). DMRs (N = 28) observed between H1 controls and ssI-C1 are displayed by heatmap of mean individual DMR DNAme levels. (H) qRT-PCR analysis of MLH1 expression post CpG-free PI or ssI by the standard curve method. Mean normalized expression ± SE (error bars) to internal control gene TPT1 is displayed (∗p < 0.05, t test).

Article Snippet: To easily visualize strong candidate regions, already available bisulfite PCR primer design software is helpful (e.g., methprimer ), as candidate primers are designed specifically to bind more complex sequences of lower GC content and minimal CpGs.

Techniques: Isolation, Selection, Plasmid Preparation, Transfection, Clone Assay, Comparison, Quantitative RT-PCR, Expressing, Control

Journal: Cell Reports Methods

Article Title: Engineering CpG island DNA methylation in pluripotent cells through synthetic CpG-free ssDNA insertion

doi: 10.1016/j.crmeth.2023.100465

Figure Lengend Snippet: CIMR testing in other PSCs and CIMP cancer lines, and CIMR testing guidelines (A) Visualization of DMRs by heatmap of DNAme levels across samples. Known CIMP lines have higher CGI DNAme and cluster separately from hypomethylated PSCs. For inclusion, a CGI required a DNAme level of >0.5 in ≥2 samples and an SD of >0.25 (N = 6,062 CGIs). (B) CIMR DNAme occurs in multiple PSCs and embryonal carcinoma Nt2d1 cells but not CIMP cancer lines. See Figure 1 E for IgV. (C) CIMR DNAme testing guidelines. Briefly, after CGI selection, gRNAs can be ranked for cutting specificity, prevalidated by Surveyor or T7E1 assays, and then paired with a candidate ssDNA repair template. CIMR RNP/ssDNA reactions are followed by antibiotic selection, FACS isolation of single-cell clones as desired, inserts validated by PCR, and DNAme assessed by Infinium analysis or another suitable technique. (D) ONECUT1 CIMRs were tested at 2 different CGIs ( Figure S4 G). (E) Specificity of the edited ONECUT1 CGI is shown by x/y scatterplot of all tested CGs, with 8 altered CGs highlighted in red. (F) TP53 CGI CIMR testing. 4/12 TP53 clones harbored full-length CG-free inserts. (G) Global specificity is shown as in (E), with the 4 CGI spanning probes marked in red. (H and I) TP53 CGI DNAme clones have repressed TP53 (H) and antisense WRAP53 (I) expression (mean normalized expression ± SE (error bars) to internal control TPT1 , ∗p < 0.05, t test).

Article Snippet: To easily visualize strong candidate regions, already available bisulfite PCR primer design software is helpful (e.g., methprimer ), as candidate primers are designed specifically to bind more complex sequences of lower GC content and minimal CpGs.

Techniques: Selection, Isolation, Clone Assay, Expressing, Control

Journal: OncoTargets and therapy

Article Title: The clinicopathological significance of HES1 promoter hypomethylation in patients with colorectal cancer

doi: 10.2147/OTT.S151857

Figure Lengend Snippet: Promoter methylation status of HES1 in CRC tissues and paired normal samples. Notes: ( A ) A schematic illustration of the CpG islands in the HES1 promoter region. ( B ) Representative data of MSP analysis in CRC patients. Experiments were performed in triplicate. T, tumor tissue; N, normal tissue; H 2 O, water control; M, methylated HES1 ; U, unmethylated HES1 ; the numbers at the very top of part B represent the case numbers. Abbreviations: BSP, bisulfite-sequencing PCR; CpG, cytosine-phosphate-guanine; CRC, colorectal cancer; DM, DNA marker; GC, guanine and cytosine; HES1 , hairy/enhancer of split 1; MF1, methylated forward primer 1; MR1, methylated reverse primer 1; MSP, methylation-specific PCR; O/E, observed/expected CpG ratio; PCR, polymerase chain reaction; UF1, unmethylated forward primer 1; UR1, unmethylated reverse primer 1.

Article Snippet: The sequences of bisulfite-sequencing PCR (BSP) primers (GeneCopoeia) were as follows: (forward) 5′-GGAATATTGTATTAAAGGGTAGGTAGG-3′ and (reverse) 5′-TCAATAATTCCTAACTCTAAATA ACC-3′.

Techniques: Methylation, Methylation Sequencing, Marker, Polymerase Chain Reaction

Journal: Frontiers in Oncology

Article Title: DNA Demethylation Switches Oncogenic ΔNp63 to Tumor Suppressive TAp63 in Squamous Cell Carcinoma

doi: 10.3389/fonc.2022.924354

Figure Lengend Snippet: Decitabine reduces DNA methylation at the TAp63 promoter. The indicated cell lines were untreated (CTR, DMSO only) or treated with 0.01 µM or 10 µM decitabine for four days. DNA was extracted and bisulfite converted, and PCR products were sequenced to analyze CpG methylation at each of the three individual CpG sites (A–C) . Plots show percentage methylation (n = 2 to 3 biological replicates). Statistical comparisons compare each CpG site in control cells with the same site in decitabine treated cells. **p < 0.01; ***p < 0.001.

Article Snippet: Bisulfite converted PCR primers (Generi Biotech, Hradec Kralove, Czech Republic) were designed according to MethPrimer 2.0 (urogene.org/cgi-bin/methprimer2/MethPrimer.cgi) ( ) ( ) to amplify a 134 bp region beginning 111 bp upstream of the TAP63 transcription start site and containing three CpG sites.

Techniques: DNA Methylation Assay, CpG Methylation Assay, Methylation, Control

Journal: Frontiers in Oncology

Article Title: DNA Demethylation Switches Oncogenic ΔNp63 to Tumor Suppressive TAp63 in Squamous Cell Carcinoma

doi: 10.3389/fonc.2022.924354

Figure Lengend Snippet: DNMT1 reduces methylation at the TAp63 promoter but with a lesser effect than decitabine. Parental HaCaT and FaDu cells were treated with 10 µM decitabine for four days, or DNMT1 -shRNA cells were induced with 2 µg/ml doxycycline for six days. DNA was bisulfite converted and PCR products were sequenced to analyze the extent of CpG methylation at site A. The plot shows the percentage increase in non-methylated cytosine (average changes in HaCaT and FaDu combined) after treatment, compared to matched control cells without decitabine or doxycycline.

Article Snippet: Bisulfite converted PCR primers (Generi Biotech, Hradec Kralove, Czech Republic) were designed according to MethPrimer 2.0 (urogene.org/cgi-bin/methprimer2/MethPrimer.cgi) ( ) ( ) to amplify a 134 bp region beginning 111 bp upstream of the TAP63 transcription start site and containing three CpG sites.

Techniques: Methylation, shRNA, CpG Methylation Assay, Control

Journal: Oncology Letters

Article Title: DNMT3B regulates proliferation of A549 cells through the microRNA-152-3p/NCAM1 pathway

doi: 10.3892/ol.2021.13129

Figure Lengend Snippet: Primers for RT-quantitative PCR of miR-152-3p and NCAM1 and sequences of miR-152-3p mimics and inhibitor.

Article Snippet: Bisulfite sequencing PCR primers were synthesized by Invitrogen; Thermo Fisher Scientific, Inc. ( ).

Techniques: Sequencing

Journal: Oncology Letters

Article Title: DNMT3B regulates proliferation of A549 cells through the microRNA-152-3p/NCAM1 pathway

doi: 10.3892/ol.2021.13129

Figure Lengend Snippet: Primers for bisulfite sequencing PCR of the miR-152-3p promoter.

Article Snippet: Bisulfite sequencing PCR primers were synthesized by Invitrogen; Thermo Fisher Scientific, Inc. ( ).

Techniques: Methylation Sequencing, Sequencing

Journal: Oncology Letters

Article Title: DNMT3B regulates proliferation of A549 cells through the microRNA-152-3p/NCAM1 pathway

doi: 10.3892/ol.2021.13129

Figure Lengend Snippet: DNMT3B binds to and methylates the miR-152-3p core region. (A) Bisulfite treated DNA samples from A549, A549/DDP and A549/ADM cells were amplified with methylated and non-methylated primers at the same time, and the amplification level was detected by PCR. (B) Bisulfite sequencing was used to detect the methylation level of miR-152-3p in A549, A549/DDP and A549/ADM cells. Five respective clones from each group were sequenced. (C) ChIP assay detected the binding of DNMT3B, DNMT3A and DNMT1 proteins to the core region of miR-152-3p. (D) Bisulfite sequencing results of the methylation levels in the core region of miR-152-3p in A549 cells were detected after treatment with DNMT3B/DNMT1 methylase inhibitors in three independent experiments. (E) Relative mRNA expression levels of miR-152-3p and NCAM1 in A549 cells treated with DNMT3B methylase inhibitor. Two-tailed Student's t-test was used for statistical analysis. *P<0.05, ***P<0.001. DNMT, DNA methyltransferase; miR, microRNA; IP, immunoprecipitation, ChIP, chromatin IP; NCAM1, neural cell adhesion molecule 1; U, unmethylated; M, methylated.

Article Snippet: Bisulfite sequencing PCR primers were synthesized by Invitrogen; Thermo Fisher Scientific, Inc. ( ).

Techniques: Amplification, Methylation, Methylation Sequencing, Clone Assay, Binding Assay, Expressing, Two Tailed Test, Immunoprecipitation, Chromatin Immunoprecipitation

Journal: Orphanet Journal of Rare Diseases

Article Title: Distinct promoter methylation patterns of LKB1 in the hamartomatous polyps of Peutz-Jeghers syndrome and its potential in gastrointestinal malignancy prediction

doi: 10.1186/s13023-020-01502-9

Figure Lengend Snippet: Bisulfite PCR-Sanger sequencing revealed elevated methylation level in the hamartomatous polyps of PJS patients compared with normal mucosa. a , b Histology of PJS polyp samples used in this study , magnificatio n = 100x, n = 50 c , d Histology of normal colon mucosa used in this study, magnification = 100x, n = 50 e Bisulfite PCR Primer design from LKB1 promoter by MethPrimer. f Representative of gel image after bisufite PCR amplifications. The PCR product is 259 bp, n = 100 ( g ) Average methylation level for LKB1 promoter region, comparison between 50 PJS polyps and 50 normal mucosa samples revealed the gap between two groups. Means ± SEM, * P < 0.05. h The methylation analysis per each CpG site indicated that instead of randomly distributed, DNA methylation was evenly distributed across the whole region. Data presented as means. All bars = 100 μm

Article Snippet: Fig. 1 Bisulfite PCR-Sanger sequencing revealed elevated methylation level in the hamartomatous polyps of PJS patients compared with normal mucosa. a , b Histology of PJS polyp samples used in this study , magnificatio n = 100x, n = 50 c , d Histology of normal colon mucosa used in this study, magnification = 100x, n = 50 e Bisulfite PCR Primer design from LKB1 promoter by MethPrimer. f Representative of gel image after bisufite PCR amplifications.

Techniques: Sequencing, Methylation, Comparison, DNA Methylation Assay

Journal: Molecular Medicine

Article Title: Down-regulated miR-495 can target programmed cell death 10 in ankylosing spondylitis

doi: 10.1186/s10020-020-00157-3

Figure Lengend Snippet: Methylation detection of miR-495 promoter region. All data was presented as the mean + SD. a Sequence schema of the bisulfite-specific PCR sequenced region in the miR-495 promoter region. Numbers (1–10) indicate potential CpG islands. b Methylation status of CpGs in the miR-495 promoter region. White and black circles indicated un-methylated and methylated CpGs, respectively. AS1, AS2, AS3, AS4: different patients with AS. HC1, HC2, HC3, HC4: different healthy controls. c The percentage of miR-495 promoter methylation. The percentage of miR-495 promoter methylation in healthy controls and patients with AS was 0.79 + 3.30 and 0.18 + 2.63, respectively. d MeDIP-qPCR of GADPH , miR-495 , and PDCD10 promoters. The values of GADPH promoter methylation in healthy controls and patients with AS were 1.01 + 0.025 and 1.10 + 0.064, respectively. The values of miR-495 promoter methylation in healthy controls and patients with AS were 0.97 + 0.036 and 6.68 + 0.224, respectively. The values of PDCD10 promoter methylation in healthy controls and patients with AS were 0.93 + 0.11, and 0.94 + 0.028, respectively. n = 150 or 20: tested samples in each group. AS: patients with ankylosing spondylitis. HC: healthy controls. ***: P value < 0.01

Article Snippet: Bisulfite-specific PCR sequencing primers were synthesized by Shanghai Sangon Biotech.

Techniques: Methylation, Sequencing, Methylated DNA Immunoprecipitation