Journal: PLoS Genetics

Article Title: Two cis -regulatory SNPs upstream of ABCG2 synergistically cause the blue eggshell phenotype in the duck

doi: 10.1371/journal.pgen.1009119

Figure Lengend Snippet: (A) Supershift EMSA was performed using CTCF antibody. CTCF antibody (1 μg) was incubated with 0.5 μg recombinant human CTCF protein prior to the addition of the binding buffer and probe. G and A represent labelled probes containing the white eggshell and blue eggshell allele, respectively, and G cold and A cold represent cold probes containing the white eggshell and blue eggshell allele, respectively. (B) ChIP-qPCR analysis of CTCF binding on variation M5 in uteruses from blue-eggshelled and white-eggshelled ducks. Chromatin extracts from four individuals of each genotype were immunoprecipitated with CTCF antibody. ChIP-enriched DNA was quantified by qPCR with primers specific for variation M5. Rabbit IgG was used as a negative control. Values of immunoprecipitated samples were normalized to that of the input DNA. Data are presented as mean±SD from four independent individuals.

Article Snippet: CTCF antibody (1μg) was incubated with 0.5 μg recombinant human CTCF protein (Novus biologicals, Littleton, USA) prior to addition of the binding buffer and probe.

Techniques: Incubation, Recombinant, Binding Assay, ChIP-qPCR, Immunoprecipitation, Negative Control

Journal: PLoS Genetics

Article Title: Two cis -regulatory SNPs upstream of ABCG2 synergistically cause the blue eggshell phenotype in the duck

doi: 10.1371/journal.pgen.1009119

Figure Lengend Snippet: (A) Effect of knockdown of CTCF on the promoter activity of causative sites. The pGL3-Basic vectors containing the blue eggshell or white eggshell alleles inserted in their upstream region were transfected into DEF cells 6 hours after transfection of CTCF siRNA1 or negative control siRNA (scramble siRNA) or without siRNA. Cells were collected for luciferase activity analysis 30 hours after siRNA transfection. The group only transfected with the blue eggshell or white eggshell vector was used as control. Data represent the mean±SD from three biological repeats per vector. ** indicates P<0.01. (B) Schematic map of CpGs sites used in the DNA methylation analysis. The causative site M5 was referred as position +1. The black bars represented the CpG dinucleotides upstream or downstream of the predicted CTCF binding sites which were marked in grey background. (C) Heatmap of uterine DNA methylation levels of CpG sites in region A and B of blue-eggshelled and white-eggshelled ducks. The DNA methylation level was determined by sequencing 8 clones of bisulfite treated genomic DNA for each individual. The DNA methylation level of each CpG site was represented by the average methylation of 8 clones. The DNA methylation level from 0 to 1 is shown in the color from blue to red.

Article Snippet: CTCF antibody (1μg) was incubated with 0.5 μg recombinant human CTCF protein (Novus biologicals, Littleton, USA) prior to addition of the binding buffer and probe.

Techniques: Knockdown, Activity Assay, Transfection, Negative Control, Luciferase, Plasmid Preparation, Control, DNA Methylation Assay, Binding Assay, Sequencing, Clone Assay, Methylation

Journal: Clinical Epigenetics

Article Title: CTCF loss mediates unique DNA hypermethylation landscapes in human cancers

doi: 10.1186/s13148-020-00869-7

Figure Lengend Snippet: Knockdown of CTCF protein results in DNA hypermethylation preferentially at CTCF sites. a Workflow of methylated DNA immunoprecipitation followed by copy number array application (MeDIP-chip) for detecting methylation alterations. NspI restriction fragments were bound to anti-5-methylcytosine antibody, eluted, and hybridized to a Affymetrix Cytoscan HD probe. An unenriched total input fraction was processed for comparison. b Short hairpin mediated CTCF knockdown in two separate shRNA targeting CTCF verified by western blotting after 3 and 5 days of shRNA induction including shRNA non-silencing control (shNSC). Data shown are one representative of 3 independent experiments using immortalized HPECs. Percentage knockdown compared to shCTCF -Dox control, quantified by ImageJ. c Volcano plot of detected methylation changes in CTCF knockdown HPECE6/E7 after 5 days of dox exposure (cut-point, methylation Abs. Log2FC > 1.5, P < 0.01). d De novo motif analysis results using HOMER. Fold change enrichment of hypermethylated sequences was compared to array background. The top 3 transcription factor motifs included CTCF, BORIS (a CTCF paralogue), and NFκB-p65 (all P < 0.001)

Article Snippet: CTCF shRNA and controls were analyzed by western blotting using anti-CTCF rabbit monoclonal antibody (Cell Signaling #3418) to verify knockdown.

Techniques: Knockdown, Methylation, Immunoprecipitation, Methylated DNA Immunoprecipitation, Comparison, shRNA, Western Blot, Control

Journal: Clinical Epigenetics

Article Title: CTCF loss mediates unique DNA hypermethylation landscapes in human cancers

doi: 10.1186/s13148-020-00869-7

Figure Lengend Snippet: Transcriptional profiling of genes altered with CTCF knockdown. a Heat map of differentially expressed (DE) transcripts following 5 days of CTCF shRNA induction (Dox) versus uninduced vehicle control (vehicle). CTCF knockdown in HPECE6/E7 leads to 1308 significantly altered gene transcripts (FDR < 0.1). b Gene ontology (GO) analysis of DE genes, pathways with FDR q -value < 0.05. c Prostate cell CTCF binding sites (LNCaP ChIP-Seq) are enriched near transcription start sites (TSS) of DE genes identified after CTCF knockdown ( P = 0.0001, Chi-square test for + 2 kb from TSS). d Venn diagram displaying overlap between differentially methylated genes and differentially expressed genes identified by arrays. Detected DMRs found within a promoter or transcribed region represented 3650 genes. Compared with 865 genes (865 genes from 1308 transcripts with gene annotation data), 249 genes were differentially expressed and contained a DMR ( P = 1.1e−5; hypergeometric test)

Article Snippet: CTCF shRNA and controls were analyzed by western blotting using anti-CTCF rabbit monoclonal antibody (Cell Signaling #3418) to verify knockdown.

Techniques: Knockdown, shRNA, Control, Binding Assay, ChIP-sequencing, Methylation

Journal: Clinical Epigenetics

Article Title: CTCF loss mediates unique DNA hypermethylation landscapes in human cancers

doi: 10.1186/s13148-020-00869-7

Figure Lengend Snippet: DNA methylation alterations occur at CTCF binding sites after CTCF knockdown in vitro and methylation inhibition reintroduces gene expression. Stable E6/E7 cell lines expressing CTCF shRNAs were cultured up to 10 days. a Validation of LTBP2 transcriptional silencing after 10 days of shCTCF induction by qPCR. Data are shown mean ± SD of technical triplicates from one representative experiment of three. b ChIP-qPCR for CTCF at LTBP2 promoter associated CTCF binding site (pCBS) ~ 400 bp upstream of LTBP2 transcription start site. Showing a reduction in CTCF binding after 10 days of shCTCF induction. Data shown are mean + SD of technical triplicates from one representative experiment of three. * P < 0.05 and ** P < 0.01. c Pyrosequencing of bisulfite DNA demonstrating increased methylation at LTBP2 promoter CTCF binding site after 10 days of shCTCF1 induction. Data shown are mean ± SD of technical triplicates from one representative experiment of three. shCTCF2 induction and controls are shown in Supp Fig S4. d Decreased TNFAIP3, FGF5, EPHA3, and AMIGO2 transcriptional silencing after 10 days of CTCF knockdown. At day 5, Dox + cells were also exposed to 5-aza-2 deoxycytidine a methyltransferase inhibitor at a low 0.2 uM dose that does not result in significant growth inhibition. Data shown are mean ± SD of technical triplicates from one representative experiment of three. * P < 0.05 and ** P < 0.01. e ChIP-qPCR for CTCF demonstrating decreased binding activity at promoter associated CTCF binding sites of candidate genes after 10 days of CTCF knockdown (for controls and expanded results see Supp Fig S3). f MeDIP-qPCR of promoter associated CTCF binding sites exhibiting loss of CTCF binding. Methylation increases were detected accompanying reduced CTCF binding. g Methylation B -values and mRNA (log2 RSEM) expression levels compared for LTBP2 gene in TCGA prostate tumors (Cell 2015). Pearson correlation R -value shown. Data was downloaded from cBioPortal for PRAD TCGA samples (Cell 2015). Decreased mRNA expression correlates with greater LTBP2 methylation

Article Snippet: CTCF shRNA and controls were analyzed by western blotting using anti-CTCF rabbit monoclonal antibody (Cell Signaling #3418) to verify knockdown.

Techniques: DNA Methylation Assay, Binding Assay, Knockdown, In Vitro, Methylation, Inhibition, Gene Expression, Expressing, Cell Culture, Biomarker Discovery, ChIP-qPCR, Activity Assay, Methylated DNA Immunoprecipitation

Journal: Clinical Epigenetics

Article Title: CTCF loss mediates unique DNA hypermethylation landscapes in human cancers

doi: 10.1186/s13148-020-00869-7

Figure Lengend Snippet: Prostate and breast tumors of the TCGA harboring CTCF copy number loss demonstrate hypermethylation events. Alterations exhibit a distinct DNA methylation profile. a Primary prostate tumors from TCGA ( n = 333) segregated by CTCF CN status, boxplots of RNA-Seq for CTCF mRNA demonstrating significantly altered expression in diploid versus deletion cancers ( P < 0.03). b Volcano plot of Illumina Methylation 450k Array data for CTCF CN loss tumors versus CTCF diploid tumors reveals increased primarily hypermethylation events, prostate tumors. Dots represent individual probes; Black, above cut point (Absolute value log2-FC loss/diploid B -values > 0.5, Adj P < 0.01). c PCa cell line LNCaP CTCF ChIP-Seq (GSE33213) identified putative CTCF binding sites. The black bar is percentage of differentially methylated probes, and gray bar is percentage of total probes from HM450 array were calculated with respect to proximity to CTCF binding sites. d Primary breast tumors from the TCGA ( n = 816) segregated by CTCF CN status, boxplots of RNA-Seq for CTCF mRNA. e Volcano plot of 450k Array for BRCA tumors; black, above cut point (absolute value log2-FC loss/diploid B -values > 0.5, Adj P < 0.01). f BCa cell line MCF7 CTCF ChIP-Seq (GSE30263) identified putative CTCF binding sites for BRCA samples. The black bar is percentage of differentially methylated probes, and gray bar is percentage of total probes from HM450 array were calculated with respect to proximity to CTCF binding sites. g Overlap comparisons of differentially methylated (DM) CTCF binding sites in prostate and breast tumors demonstrate distinct methylation profiles at CTCF sites, related to Additional file : Table S2

Article Snippet: CTCF shRNA and controls were analyzed by western blotting using anti-CTCF rabbit monoclonal antibody (Cell Signaling #3418) to verify knockdown.

Techniques: DNA Methylation Assay, RNA Sequencing, Expressing, Methylation, ChIP-sequencing, Binding Assay

Journal: Genome Research

Article Title: Widespread plasticity in CTCF occupancy linked to DNA methylation

doi: 10.1101/gr.136101.111

Figure Lengend Snippet: CTCF in vivo binding exhibits widespread plasticity. ( A–C ) Constitutive and variable CTCF sites. ( A ) The H19/IGF2 imprinted locus in multiple human cell types. Note the total silencing in two cell lines of the seven CTCF sites in the differentially methylated region (DMR; yellow box at left ), and the complex pattern of cell-selective CTCF binding flanked by constitutive sites. Location (hg19), chr11:2,015,000–2,184,000. ( B , C ) Additional examples of variable sites. ( D ) Genome-wide analysis of CTCF binding in 19 cell types reveals 77,811 distinct binding sites; 27,662 sites are constitutively present in all cell types; 50,149 variable sites exhibiting a wide range of selectivity are present in a subset of one to 18 cell types ( below ). ( E ) Genomic distribution of variable sites is similar to constitutive sites (Supplemental Fig. S2A).

Article Snippet: The chromatin was incubated with Dynabeads (M-280, sheep anti-rabbit IgG, Invitrogen)-conjugated anti-CTCF polyclonal antibody (Cell Signaling no. 2899).

Techniques: In Vivo, Binding Assay, Methylation, Genome Wide

Journal: Genome Research

Article Title: Widespread plasticity in CTCF occupancy linked to DNA methylation

doi: 10.1101/gr.136101.111

Figure Lengend Snippet: CTCF occupancy distinguishes similar cell types. ( A ) Unsupervised hierarchical clustering of binding at all CTCF sites. ( B ) CTCF occupancy at 4146 variable binding sites that distinguish immortal cell lines, epithelia, fibroblasts and endothelia (Methods). x -axis, CTCF binding sites in chromosomal order, separated into sites that are up-regulated and down-regulated (arrows) in each of the three groups (immortal, epithelial, fibroblast, and endothelial). Color corresponds to Z -score of normalized ChIP-seq density.

Article Snippet: The chromatin was incubated with Dynabeads (M-280, sheep anti-rabbit IgG, Invitrogen)-conjugated anti-CTCF polyclonal antibody (Cell Signaling no. 2899).

Techniques: Binding Assay, ChIP-sequencing

Journal: Genome Research

Article Title: Widespread plasticity in CTCF occupancy linked to DNA methylation

doi: 10.1101/gr.136101.111

Figure Lengend Snippet: Impact of DNA methylation on cell-selective CTCF binding. ( A ) Example CTCF binding sites, where occupancy ( above ) quantitatively increases as local CpG methylation decreases ( below ). Green indicates CpG is 0% methylated; yellow, 50%; and red, 100%. ( B ) Quantitative analysis of methylation at the boxed CTCF binding site in A . ( C ) Global impact of methylation at variable CTCF sites monitored by RRBS. Sixty-five percent of sites with cell-type selective patterns of methylation also exhibited differences in occupancy. ( D ) At methylated binding sites, occupancy was reduced on average by 87% compared with cell lines without methylation at the same site. Shown are sites where increased methylation was associated with decreased occupancy (98% of all significant sites).

Article Snippet: The chromatin was incubated with Dynabeads (M-280, sheep anti-rabbit IgG, Invitrogen)-conjugated anti-CTCF polyclonal antibody (Cell Signaling no. 2899).

Techniques: DNA Methylation Assay, Binding Assay, CpG Methylation Assay, Methylation

Journal: Genome Research

Article Title: Widespread plasticity in CTCF occupancy linked to DNA methylation

doi: 10.1101/gr.136101.111

Figure Lengend Snippet: Sites significantly affected by methylation are enriched for CpGs at two positions. Frequency of a CpG ( y -axis) at positions relative to the CTCF motif ( x -axis) is shown for sites with variable methylation that is associated (red) and is not associated (gray) with occupancy changes. Note that at positions 1 and 11, there is a 2.2- and 1.8-fold enrichment, respectively, for the presence of a CpG at sites where the variable methylation was not associated with occupancy. Twenty-nine percent of CTCF motifs genome-wide contain a CpG at one or both of these positions.

Article Snippet: The chromatin was incubated with Dynabeads (M-280, sheep anti-rabbit IgG, Invitrogen)-conjugated anti-CTCF polyclonal antibody (Cell Signaling no. 2899).

Techniques: Methylation, Genome Wide

Journal: Genome Research

Article Title: Widespread plasticity in CTCF occupancy linked to DNA methylation

doi: 10.1101/gr.136101.111

Figure Lengend Snippet: Cell-selective patterns of methylation associated with occupancy differences. ( A ) Methylation status at 1969 CTCF sites where differential methylation is significantly associated with occupancy differences. Color corresponds to the percentage of bisulfite sequencing tags at each site overlapping methylated CpG positions. Dendrogram ( left ) highlights pattern of hypermethylation in immortal cell lines. ( Right ) Smoothed plot of number of immortal lines exhibiting hypermethylation at each site. ( B ) Immortal lines show no significant difference in number of occupied CTCF sites ( y -axis, mean). Error bars, SD. ( C ) immortal lines demonstrate increased CTCF transcript levels ( y -axis, mean). Error bars, SD. ( D ) Immortal lines exhibit increased methylation relative to the other cell types, though significant promoter methylation is rarely observed in normal lines. y -axis, genome-wide median of per-site methylation. P -values, Wilcoxon. Promoter, ±2.5 kb of RefSeq transcription start site.

Article Snippet: The chromatin was incubated with Dynabeads (M-280, sheep anti-rabbit IgG, Invitrogen)-conjugated anti-CTCF polyclonal antibody (Cell Signaling no. 2899).

Techniques: Methylation, Methylation Sequencing, Genome Wide

Journal: Molecular Cell

Article Title: ZNF143 is a transcriptional regulator of nuclear-encoded mitochondrial genes that acts independently of looping and CTCF

doi: 10.1016/j.molcel.2024.11.031

Figure Lengend Snippet: ZFP143 depletion has no detectable effect on 3D genome structure and CTCF binding (A) Average Hi-C loops in DMSO-treated and dTAG-V1-treated cells. Value in the upper-right corner indicates the interaction strength of the loop over the background. (B) Same as in (A), but for the average ZFP143-associated Hi-C loops. (C) 4C-seq data generated for the Cpox and Cldn1 (left) and Zfp111 and Zfp108 (right) loci. The matrix in the top panel represents interaction frequencies in a previously published high-resolution Micro-C dataset. The arrows point to detected Micro-C chromatin loops. The bottom panel shows 4C contact profiles in DMSO-treated (blue) and dTAG-V1-treated (orange) cells. Genomic tracks show ZFP143-HA ChIP-seq (red), calibrated CTCF ChIP-seq (blue), TT-seq nascent transcription (yellow for sense and purple for antisense transcription) in DMSO-treated and dTAG-V1-treated cells. (D) Tornado plots of calibrated CTCF ChIP-seq signal centered at CTCF peaks in DMSO-treated and dTAG-V1-treated cells. (E) Genomic tracks showing ZFP143-HA ChIP-seq (red) in DMSO-treated cells and calibrated CTCF ChIP-seq (blue) in DMSO-treated and dTAG-V1-treated cells. (F) Venn diagram showing the overlap between ZFP143-HA (red) and CTCF (blue) peaks.

Article Snippet: CTCF ChIP-seq and ZNF143 ChIP-seq generated with Proteintech (this study) and HA antibodies were mapped to the hg38 mouse reference genome assembly using bwa mem v0.7.17-r1188.

Techniques: Binding Assay, Hi-C, Generated, ChIP-sequencing

Journal: Molecular Cell

Article Title: ZNF143 is a transcriptional regulator of nuclear-encoded mitochondrial genes that acts independently of looping and CTCF

doi: 10.1016/j.molcel.2024.11.031

Figure Lengend Snippet: Re-analysis of publicly available ChIP-seq data reveals ZNF143 antibody cross-reactivity with CTCF (A) Overlap between ZNF143 peaks from re-analyzed publicly available data and CTCF peaks from CISTROME for human (left) and mouse (right) datasets. Each dot represents the overlap between the indicated ZNF143 peak set with an individual CTCF peak set. Colors represent the antibody used for chromatin immunoprecipitation. (B) Venn diagram showing the overlap between ZNF143 peaks detected by Proteintech (light pink) and FLAG (light green) antibodies in K562 cells. (C) Heatmap showing the enrichment of ZNF143 SBS and CTCF motifs in common, Proteintech-specific, and FLAG-specific peaks in K562 cells. (D) Tornado plots of ChIP-seq signals detected by Proteintech (light pink), FLAG (light green), and custom (orange) antibodies, and CTCF signal (blue) in K562 cells. The ChIP-seq signals are centered on common (top) and Proteintech-specific (bottom) peaks. (E) Genomic tracks showing ChIP-seq signals for CTCF (blue) and signals detected by Proteintech (pink), FLAG (light green), and custom (orange) antibodies in K562 cells. Rectangles indicate common (left) and Proteintech-specific (middle and right) peaks in the region. (F) Scatterplot of the percentage of loop anchors overlapping the peak (x axis) against the fold enrichment of peaks in loop anchors (y axis) for a number of DNA-binding proteins and for Proteintech-specific, FLAG-specific, and common peaks in K562 cells.

Article Snippet: CTCF ChIP-seq and ZNF143 ChIP-seq generated with Proteintech (this study) and HA antibodies were mapped to the hg38 mouse reference genome assembly using bwa mem v0.7.17-r1188.

Techniques: ChIP-sequencing, Chromatin Immunoprecipitation, DNA Binding Assay

Journal: Molecular Cell

Article Title: ZNF143 is a transcriptional regulator of nuclear-encoded mitochondrial genes that acts independently of looping and CTCF

doi: 10.1016/j.molcel.2024.11.031

Figure Lengend Snippet:

Article Snippet: CTCF ChIP-seq and ZNF143 ChIP-seq generated with Proteintech (this study) and HA antibodies were mapped to the hg38 mouse reference genome assembly using bwa mem v0.7.17-r1188.

Techniques: Virus, Bacteria, Recombinant, Western Blot, Flow Cytometry, Purification, Plasmid Preparation, Bradford Protein Assay, Multiplex Assay, Microscopy, Cell Counting, Software

Journal: Nature

Article Title: Altered chromosomal topology drives oncogenic programs in SDH-deficient GISTs

doi: 10.1038/s41586-019-1668-3

Figure Lengend Snippet: (a) Specimen collection and generation of PDX model. (b) Scatter plot compares expression of genes (points) between primary tumor S1 (x-axis) and PDX (y-axis) per RNA-seq. Pearson correlation indicated. (c) Venn diagram depicts overlap between strong H3K27ac ChIP-seq peaks in primary tumor (black) and PDX (green). (d) Venn Diagram depicts overlap between hypermethylated CpG islands in primary tumor (black) and PDX (red) per bisulfite-sequencing. (e) Scatter plot depicts principle component analysis (PCA) on top 1,000 differential CTCF sites for primary tumors (colored by subtype) and PDX (star). PDX and originating tumor (S1) both cluster with SDH-deficient GISTs (red). (f) Experimental design of pre-clinical trial. Following xenograft implantation and growth, mice were randomized to four treatment groups and treated with the indicated regimen daily for 28 days. Observation was continued until clinical endpoint (tumor volume of 2,000 mm 3 ). (g) Plot depicts tumor volume during treatment and observation periods. Points represent mean tumor volume, error bars represent S.E.M., and shading represents range of tumor volumes for n=8 biologically-independent xenografts per group. Relative tumor volume immediately following treatment cessation (day 29) is indicated at right. P-values reflect difference in tumor growth between group per two-way ANOVA (* P < 0.05, ** P < 0.001, *** P < 0.0001).

Article Snippet: Chromatin immunoprecipitation was performed with 30 ul of monoclonal rabbit CTCF antibody, clone D31H2, (Cell Signaling 3418).

Techniques: Expressing, RNA Sequencing, ChIP-sequencing, Methylation Sequencing

Journal: Nature

Article Title: Altered chromosomal topology drives oncogenic programs in SDH-deficient GISTs

doi: 10.1038/s41586-019-1668-3

Figure Lengend Snippet: (a) Proposed mechanism of epigenetic oncogene activation. Left: Oncogene shielded from super-enhancer by CTCF insulator, which creates a topological boundary. Right: CTCF insulator displaced by DNA methylation, allowing super-enhancer to contact and induce oncogene. (b) Violin plots depict DNA methylation levels over the 10,000 most variable CpG island promoters (top) and CTCF sites (bottom) in normal stomach muscle (NSM; n=2), and KIT mutant (n=9), PDGFRA mutant (P; n=2) and SDH-deficient GISTs (n=6). Yellow bars indicate mean. (c) Volcano plot depicts differential CTCF occupancy between SDH-deficient (n=6) and SDH-intact (n=8) GISTs. Sites that gain DNA methylation in SDH-deficient GISTs are indicated in red (>25% increase, two-sided t-test FDR < 5%). (d) Plot depicts H3K27ac peaks near lost CTCF insulators (y-axis) rank ordered by signal strength. (e) Scatter plot depicts genes (points) separated from a super-enhancer by a CTCF loop anchor that is lost in SDH-deficient GIST. Genes are positioned according to their relative (y-axis) and absolute median expression (x-axis) in SDH-deficient GISTs. Potentially deregulated gene targets (outliers) include oncogenes FGF3, FGF4 and KIT (red); see also . (f) Box plot depicts average expression of MAPK signature genes in RNA-seq data for normal stomach (n=262), and KIT mutant (n=10), PDGFRA mutant (n=3), and SDH-deficient GIST (n=8). Boxes depict 25 th , 50 th and 75 th percentiles, and whiskers depict extreme values. (g) Radial phylogenetic tree depicts tyrosine kinase gene expression in SDH-deficient GISTs. Each branch is one tyrosine kinase, arranged by similarity, and with major families depicted by color. The area of each red circle is proportional to the average expression of the kinase. (h) Scatter plot depicts average expression of FGF ligands in SDH-intact (x-axis) and SDH-deficient (y-axis) GISTs. For all panels, n values indicate number of biologically-independent specimens.

Article Snippet: Chromatin immunoprecipitation was performed with 30 ul of monoclonal rabbit CTCF antibody, clone D31H2, (Cell Signaling 3418).

Techniques: Activation Assay, DNA Methylation Assay, Mutagenesis, Expressing, RNA Sequencing, Gene Expression

Journal: Nature

Article Title: Altered chromosomal topology drives oncogenic programs in SDH-deficient GISTs

doi: 10.1038/s41586-019-1668-3

Figure Lengend Snippet: (a) ChIP-seq profiles for H3K27ac were compared for GIST specimens and other gastrointestinal tract tumor specimens (GAC: gastric adenocarcinoma, CRC: colorectal cancer, PDAC: pancreatic ductal adenocarcinoma). Heatmap depicts pairwise Pearson correlations between the top 10,000 most variable peaks (yellow indicates high correlation; blue indicates low correlation). Dendrogram (left) was derived by unweighted average distance linkage. Enhancer patterns are relatively consistent across GIST subtypes, compared to other tumor types. (b) DNA methylation levels in the vicinity of CTCF sites were profiled genomewide by hybrid-selection bisulfite sequencing. CTCF sites are binned according to the amount their methylation increased in SDH-deficient GISTs, relative to SDH-intact GISTs (methylation change computed over 250 bp window centered on the motif). For each bin, bar graphs depict the percentage of sites that lose CTCF binding in SDH-deficient GISTs, per ChIP-seq. Separate plots are shown for CTCF sites whose motifs do or do not contain a CpG. Increased methylation over CTCF sites is associated with more frequent loss of CTCF binding, even when the CTCF motif lacks a CpG. (c) Plot depicts correlation between CTCF occupancy and DNA methylation in SDH-deficient GISTs. Red points show Spearman correlations between CTCF ChIP-seq signal and methylation of CpGs at indicated positions relative to the center of the CTCF motif. Red line reflects correlation to average methylation over 10bp windows. Randomly permuted data (black) are shown for comparison. Anti-correlation between CTCF occupancy and methylation is evident over a ~250 bp binding footprint. (d) Genomic views of a representative 10 MB region on chromosome 21 depict chromosome topology (HiC, red), CTCF binding (ChIP-seq, orange) and CTCF-CTCF loop interactions (HiChIP, black) for the SDH-intact GIST model, GIST-T1. TADs are visible as triangles of enhanced interaction in HiC data, flanked by boundaries that correspond to loop interactions in HiChIP data. Genes (blue) are also indicated.

Article Snippet: Chromatin immunoprecipitation was performed with 30 ul of monoclonal rabbit CTCF antibody, clone D31H2, (Cell Signaling 3418).

Techniques: ChIP-sequencing, Derivative Assay, DNA Methylation Assay, Selection, Methylation Sequencing, Methylation, Binding Assay, Comparison, HiChIP

Journal: Nature

Article Title: Altered chromosomal topology drives oncogenic programs in SDH-deficient GISTs

doi: 10.1038/s41586-019-1668-3

Figure Lengend Snippet: (a) Genomic views of the FGF3/4 and ANO1 loci depict baseline chromosome topology (HiC, red), genes (blue), CTCF-CTCF loop interactions (HiChIP, arcs, with darkness indicating significance), CTCF binding (ChIP-seq, orange) and candidate enhancers (H3K27ac ChIP-seq, green). HiC/HiChIP data are for the SDH-intact model GIST-T1, while CTCF and H3K27ac data are for representative clinical specimens (see also ). ANO1 super-enhancer (green bar) and FGF insulator (orange shading) are indicated. (b) Traces depict 4C-seq interaction frequency (y-axis) between the ANO1 super-enhancer viewpoint (dashed white line) and genomic positions in the FGF3/4-ANO1 locus (x-axis). Data shown for SDH-intact GISTs (n=4; top), normal stomach muscle (n=1; gray line, top) and SDH-deficient GISTs (n=3; bottom). CTCF binding profiles for representative SDH-intact (top) and SDH-deficient (bottom) tumors also shown (orange). Genes (blue) and CTCF sites in the FGF insulator (orange) are highlighted. (c,d) Plots depict relative FGF4 (c) and FGF3 (d) expression in GIST-T1 cells expressing CRISPR/Cas9 and control sgRNA (black) or sgRNAs targeting the two CTCF sites in the FGF insulator (red). Bar indicates mean of 3 biologically-independent replicates (dots). P-values by two-sided T-test.

Article Snippet: Chromatin immunoprecipitation was performed with 30 ul of monoclonal rabbit CTCF antibody, clone D31H2, (Cell Signaling 3418).

Techniques: HiChIP, Binding Assay, ChIP-sequencing, Expressing, CRISPR, Control

Journal: Nature

Article Title: Altered chromosomal topology drives oncogenic programs in SDH-deficient GISTs

doi: 10.1038/s41586-019-1668-3

Figure Lengend Snippet: (a) Genomic views of PDGFRA and KIT loci depict baseline chromosome topology (HiC, red), genes (blue), CTCF-CTCF loop interactions (HiChIP, arcs), CTCF binding (ChIP-seq, orange) and candidate enhancers (H3K27ac ChIP-seq, green). HiC/HiChIP data are for the SDH-intact GIST model GIST-T1, while CTCF and H3K27ac data are for representative clinical specimens (see also ). KIT super-enhancer (green bar) and KIT insulator (orange shading) are indicated. (b) Traces depict 4C-seq interaction frequency (y-axis) between the KIT super-enhancer viewpoint (dashed white line) and genomic positions in the KIT / PDGFRA locus (x-axis). Data shown for SDH-intact GISTs (n=6, top), and SDH-deficient GISTs (n=3, bottom). CTCF profiles for representative SDH-intact (top) and SDH-deficient (bottom) tumors also shown. Genes (blue) and CTCF binding sites in the KIT insulator (orange) are highlighted. (c) Traces depict 4C-seq interaction signal between the KIT super-enhancer viewpoint and the KIT gene in GIST-T1 cells expressing Cas9 and control (black) or KIT insulator targeting sgRNAs (red). (d) Plot depicts relative KIT expression in GIST-T1 cells expressing Cas9 and control (black) or KIT insulator targeting sgRNAs (red). Bar indicates mean of three biologically-independent replicates (dots). P-values by two-sided T-test. (e,f) FGF and KIT insulator methylation evaluated in an expanded cohort of GIST tumors by locus-specific bisulfite sequencing. (e) Bar plot depicts average methylation levels across six CpGs within FGF insulator CTCF peak 2 in normal stomach muscle (n=2), SDH-intact GISTs (n=17) and SDH-deficient GISTs (n=11). (f) Bar plot depicts average methylation across four CpGs within KIT insulator CTCF peak 2 in normal stomach muscle (n=2), SDH-intact GISTs (n=20) and SDH-deficient GISTs (n=12) (n values indicate number of biologically-independent tumors).

Article Snippet: Chromatin immunoprecipitation was performed with 30 ul of monoclonal rabbit CTCF antibody, clone D31H2, (Cell Signaling 3418).

Techniques: HiChIP, Binding Assay, ChIP-sequencing, Expressing, Control, Methylation, Methylation Sequencing

Journal: Nature

Article Title: Altered chromosomal topology drives oncogenic programs in SDH-deficient GISTs

doi: 10.1038/s41586-019-1668-3

Figure Lengend Snippet: (a) Traces depict H3K27ac ChIP-seq signal for normal stomach muscle (NSM) and GISTs of the indicated subtype over the FGF/ANO1 locus. (b) Traces depict H3K27ac ChIP-seq signal for NSM and GISTs of the indicated subtype over the PDGFRA/KIT locus. Genes are indicated in blue, and superenhancer locations are indicated by green bars. For (a,b), traces are representative of 11 KIT-mutant and 6 SDH-deficient tumors with similar results. (c) Traces depict CTCF binding over the FGF insulator in normal stomach muscle (NSM) and GIST clinical specimens. (d) Plot depicts CTCF ChIP-seq signal over the strongest CTCF peak in the FGF insulator in normal stomach muscle (NSM, n=4), and KIT mutant (n=11), PDGFRA mutant (n=2) and SDH-deficient GISTs (n=6). (e) Traces depict CTCF binding over the KIT insulator in normal stomach muscle (NSM) and GIST clinical specimens. (f) Plot depicts CTCF ChIP-seq signal over the strongest CTCF peak in the KIT insulator in normal stomach muscle (NSM, n=4), and KIT mutant (n=11), PDGFRA mutant (n=2) and SDH-deficient GISTs (n=6). For (d) and (f), P-values indicate significance of CTCF loss in SDH-deficient GIST, as determined by Walt test (via DEseq2 ). All n values represent number of biologically independent clinical specimens.

Article Snippet: Chromatin immunoprecipitation was performed with 30 ul of monoclonal rabbit CTCF antibody, clone D31H2, (Cell Signaling 3418).

Techniques: ChIP-sequencing, Mutagenesis, Binding Assay

Journal: Scientific Reports

Article Title: Methods for Scarless, Selection-Free Generation of Human Cells and Allele-Specific Functional Analysis of Disease-Associated SNPs and Variants of Uncertain Significance

doi: 10.1038/s41598-017-15407-4

Figure Lengend Snippet: Allele-specific functional analysis of risk SNP rs6983267 within heterozygous HCT-116 clones using KASP genotyping technology. ( a ) Overview of the allele-specific applications of KASP genotyping: allele genotyping of nuclease-modified clones, relative allele-specific expression of RNA transcripts harboring a heterozygous mutation and relative allele-specific binding affinity of DNA binding proteins that bind on or in the vicinity of a heterozygous mutation. ( b ) Diagram of heterozygous 8q24 risk locus with transcription factor TCF7L2 (red) and insulating protein CTCF (purple) with binding motifs depicted as black boxes. Risk SNP is located immediately adjacent to the core TCF7L2 binding motif (TCAAAG). ( c ) KASP fluorescence ratio output of TCF7L2 and CTCF binding at 8q24 locus between G allele and T allele in heterozygous clones (n = 3) run in duplicate with input genomic DNA (orange), immunoprecipitated DNA for TCF7L2 (red), CTCF (purple) and IgG (gray) and no DNA template controls (black). CTCF IPed DNA samples cluster with Input DNA while TCF7L2 IPed DNA samples create a separate cluster favoring the G allele compared to Input DNA. ( d ) Allelic ratios of G (green) and T (blue) alleles in percentage of total fluorescence for input control, TCF7L2 and CTCF, quantifying G-allele allelic preference of TCF7L2 compared to CTCF relative to Input DNA control. Errors bars show standard deviation.

Article Snippet: For each ChIP, 3 volumes of IP Dilution Buffer (16.7 mM Tris-HCl, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton × 100, 0.01% SDS, 1x Protease Inhibitors; pH 8.0) was added to 20ug of chromatin and 2ug of antibody (TCF7L2 (D31H2); CTCF (C48H11), Cell Signaling Technology, Inc.).

Techniques: Functional Assay, Clone Assay, Modification, Expressing, Mutagenesis, Binding Assay, DNA Binding Assay, Fluorescence, Immunoprecipitation, Control, Standard Deviation

Journal: Annals of the Rheumatic Diseases

Article Title: Preferential association of a functional variant in complement receptor 2 with antibodies to double-stranded DNA

doi: 10.1136/annrheumdis-2014-205584

Figure Lengend Snippet: The ENCyclopedia Of DNA Elements (ENCODE) Project data surrounding rs1876453. (A) The first exon and 5′ end of the first intron of the CR2 gene. The 5’ untranslated region (5′ UTR) is shown before the methionine start codon, in green. These data are derived from the University of California Santa Cruz (UCSC) Genes Track. (B) The location of rs1876453 (highlighted in yellow) and previously reported systemic lupus erythematosus-associated rs3813946 (in blue font). These data are derived from the Common Single-Nucleotide Polymorphisms (SNP) (138) Track (ft.ncbi.nih.gov/snp). (C) DNaseI hypersensitive sites in the GM12878 Epstein–Barr virus (EBV)-transformed B cell line and in primary CD20+ B cells derived by DNase-seq. These data are derived from the UCSC Uniform DNaseI HS Track. Signal values are shown as grayscale-coloured items where higher signal values correspond to darker-coloured blocks. Primary B cells contain an additional hypersensitivity site that overlies rs1876453. (D) Histone marks surrounding rs1876453, as determined by chromatin immunoprecipitation (ChIP)-seq. These data were derived from the Layered H3K4Me3, H3K4Me1 and H3K27Ac Tracks. The H3K4Me3 histone mark is associated with poised or active promoters, the H3K4me1 histone mark is associated with enhancers and with DNA regions downstream of transcription sites and the H3K27Ac histone mark may enhance transcription by blocking the spread of the repressive histone mark H3K27Me3. Data shown are for the GM12878 EBV-transformed B cell line. (E) Transcription factor binding sites determined by ChIP-seq are shown as grey boxes that encompass the peaks of transcription factor occupancy, with the darkness of the box proportional to the maximal signal strength observed in any cell line. The name to the left of the box is the transcription factor, and includes in parentheses the antibody used for ChIP. The letters to the right of the box indicate the cell lines in which a signal is detected, with the darkness of the letter proportional to the signal strength in the cell line. Data are derived from the Transcription Factor ChIP Track. CCCTC-binding factor (CTCF) binding was seen in multiple EBV-transformed B cell lines (G, g) as well as a variety of other cell lines. (F) CTCF binding to primary CD20+ B cells by ChIP-seq. Peak occupancy lies over exon 1 and the 5′ UTR. Data are derived from the Broad Histone Track. (G) Transcription levels for several cell types assayed by high-throughput sequencing of polyadenylated RNA (RNA-seq). Each cell line is associated with a particular colour; the GM12878 cell line is shown in pink. This figure was obtained from the UCSC Genome Browser (Human Feb 2009 (GRCh37/hg19) Assembly; http://genome.ucsc.edu ).

Article Snippet: For competition and blocking experiments, NE were pre-incubated with unlabelled oligonucleotide or anti-CCCTC-binding factor (CTCF) (sc-15914; Santa Cruz Biotechnology) for 10 or 30 min on ice.

Techniques: Derivative Assay, Virus, Transformation Assay, Chromatin Immunoprecipitation, ChIP-sequencing, Blocking Assay, Binding Assay, Next-Generation Sequencing, RNA Sequencing

Journal: Annals of the Rheumatic Diseases

Article Title: Preferential association of a functional variant in complement receptor 2 with antibodies to double-stranded DNA

doi: 10.1136/annrheumdis-2014-205584

Figure Lengend Snippet: Allelic differences in complex formation at rs1876453. (A) Protein-DNA complexes (indicated by arrows; A–D) were formed with oligonucleotides including either the minor A or major G allele in the absence or presence of K562 (Lanes 1–10), Reh (Lanes 11–20) and Ramos (Lanes 21–30) nuclear extracts. Specificity and binding affinity of the protein-DNA complexes were demonstrated by the addition of 15-molar to 60-molar excess of unlabelled oligonucleotides. UB represents unbound control. (B) Anti-CTCF (CCCTC-binding factor) antibody was included during the formation of protein-DNA complexes to determine whether CTCF was involved in forming these complexes. Data shown are representative of at least three independent experiments.

Article Snippet: For competition and blocking experiments, NE were pre-incubated with unlabelled oligonucleotide or anti-CCCTC-binding factor (CTCF) (sc-15914; Santa Cruz Biotechnology) for 10 or 30 min on ice.

Techniques: Binding Assay, Control

Journal: Annals of the Rheumatic Diseases

Article Title: Preferential association of a functional variant in complement receptor 2 with antibodies to double-stranded DNA

doi: 10.1136/annrheumdis-2014-205584

Figure Lengend Snippet: CCCTC-binding factor (CTCF) interacts with CR2 intron 1 in vivo and demonstrates differential affinity for rs1876453 alleles. (A) Chromatin immunoprecipitation performed using an antibody specific for CTCF yielded allele-specific enrichment of the region surrounding rs1876453 from Epstein–Barr virus (EBV)-transformed B cells homozygous for the major or minor allele at rs1876453. The qPCR products were visualised by ethidium bromide staining of a 1.8% agarose gel and sized using a PCR marker (New England Biolabs). A non-specific IgG control (IgG) and a control without antibody (No Ab) were included to measure background enrichment. Decreasing amounts of enrichment were observed with serially diluted input samples (Lanes 5–8; 13–16). NTC, no template control. (B) A representative qPCR amplification plot. (C) The percentage enrichment at the CR2 promoter was determined by quantification against the standard curve. CTCF enrichment was normalised to the background enrichment generated by a non-specific IgG. (D) CTCF enrichment normalised to the minor allele at rs1876453. (E) CTCF transcript abundance for each homozygous cell line after normalisation to β-actin. (F) Transcript abundance of CR1 relative to CR2 in each homozygous cell line, with each transcript normalised to β-actin. Data shown are the mean±SEM for three independent experiments.

Article Snippet: For competition and blocking experiments, NE were pre-incubated with unlabelled oligonucleotide or anti-CCCTC-binding factor (CTCF) (sc-15914; Santa Cruz Biotechnology) for 10 or 30 min on ice.

Techniques: Binding Assay, In Vivo, Chromatin Immunoprecipitation, Virus, Transformation Assay, Staining, Agarose Gel Electrophoresis, Marker, Control, Amplification, Generated