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: Nature

Article Title: CTCF-promoted RNA polymerase II pausing links DNA methylation to splicing

doi: 10.1038/nature10442

Figure Lengend Snippet: a, CTCF ChIP in murine splenocytes and quantitative PCR (qPCR) relative to rabbit Ig control ChIP (n = 2). b, Cell-surface staining for CD45RA (exon 4-containing) and CD45RB (exon 5-containing) isoforms and total CD45 (pan) in parental BL41 B cells (RBhigh), cell-culture-derived CD45RB bimodal cells, and CD45RB low (RBlow) cells sorted from the bimodal BL41 population. c, GAPDH-normalized qRT–PCR data from RBhigh and RBlow cells using the indicated junction-spanning primers (n = 3). d, CTCF ChIP in BJAB, RBhigh and RBlow cells and qPCR for CD45 exons and introns (n = 3–6). All graphs show mean values ± standard deviation (s.d.). P = two-tailed Student’s t test comparing the indicated samples.

Article Snippet: Human CTCF recombinant protein was obtained from Abnova (catalogue no. H00010664-P01, batch no. 0991020–2).

Techniques: Real-time Polymerase Chain Reaction, Control, Staining, Cell Culture, Derivative Assay, Quantitative RT-PCR, Standard Deviation, Two Tailed Test

Journal: Nature

Article Title: CTCF-promoted RNA polymerase II pausing links DNA methylation to splicing

doi: 10.1038/nature10442

Figure Lengend Snippet: a, Cell-surface CD45RB isoform and total CD45 expression in cells transduced with short hairpin RNA (shRNA)against CTCF (CTCF-sh3 and/or sh-4) or control shRNA against red fluorescent protein (RFP). b, c, qRT–PCR in CTCF-depleted RBlow (b) and BJAB cells (c) from a to detect CD45 (left) and CTCF (right) mRNA levels (n = 3). Graphs show mean values ± s.d. P, two-tailed Student’s t test.

Article Snippet: Human CTCF recombinant protein was obtained from Abnova (catalogue no. H00010664-P01, batch no. 0991020–2).

Techniques: Expressing, Transduction, shRNA, Control, Quantitative RT-PCR, Two Tailed Test

Journal: Nature

Article Title: CTCF-promoted RNA polymerase II pausing links DNA methylation to splicing

doi: 10.1038/nature10442

Figure Lengend Snippet: a, Alternative exons were classified on the basis of the relative location of an exclusive CTCF peak within 1 kb of the exon. b, Difference in the mean exon inclusion level between bimodal BL41 cells transduced with shRNA against CTCF versus shRFP-transduced cells (from Fig. 2a) for exons with CTCF peak in upstream (blue) or downstream regions (red) but not in the exon body and for exons with no CTCF binding (black). The mean ± s.e.m. for each class of exons is plotted against increasing Bayes factor thresholds. *P < 0.05, **P < 0.01, ***P < 0.001, Wilcoxon rank sum test for differences in exon inclusion at the different thresholds. c, Same as b for BJAB shCTCF compared with wild-type BJAB cells (from Fig. 2a). d, Normalized CD4+ T cell RNA pol II read signal centred on the alternative exon or the corresponding downstream CTCF peak summit.

Article Snippet: Human CTCF recombinant protein was obtained from Abnova (catalogue no. H00010664-P01, batch no. 0991020–2).

Techniques: Transduction, shRNA, Binding Assay

Journal: Nature

Article Title: CTCF-promoted RNA polymerase II pausing links DNA methylation to splicing

doi: 10.1038/nature10442

Figure Lengend Snippet: a, RNA pol II ChIP and qPCR relative to mouse Ig control IP (n = 3). b, CTCF ChIP in RBhigh cells transduced with shRNA against CTCF versus shRFP-transduced cells and qPCR relative to rabbit Ig control IP (n = 2). c, RNA pol II ChIP of RBhigh cells from b and qPCR relative to mouse Ig control IP (n = 2). d, In vitro transcription with a DNA oligo incorporating a CTCF binding site at position 26 relative to elongation complex assembly. Recombinant CTCF and TFIIS protein were introduced as indicated, with variable effects on pausing at adenine 21 (A21). e, Representation of CD45 minigenes with wild-type (I3-I7) or mutated exon 5 CTCF binding site (I3-I7*CTCF), used in f–j. f, CTCF-ChIP in NIH3T3 and CHO cells transfected with the CD45 minigenes and qPCR relative to rabbit Ig control IP. Error bars represent standard error of the mean (s.e.m.) (n = 3). g–i, qRT–PCR from minigene-transfected HEK293, NIH3T3 and CHO cells to detect the junctions of exons 4/5 (g), 5/6 (h) and 4/6 (i) relative to exon 6 (n = 3). j, RNA pol II ChIP in CHO cells transfected with the CD45 minigenes and qPCR relative to mouse Ig control IP (n = 3). Unless indicated otherwise, graphs show mean values ± s.d. P, two-tailed Student’s t test.

Article Snippet: Human CTCF recombinant protein was obtained from Abnova (catalogue no. H00010664-P01, batch no. 0991020–2).

Techniques: Control, Transduction, shRNA, In Vitro, Binding Assay, Recombinant, Transfection, Quantitative RT-PCR, Two Tailed Test

Journal: Nature

Article Title: CTCF-promoted RNA polymerase II pausing links DNA methylation to splicing

doi: 10.1038/nature10442

Figure Lengend Snippet: a, Methylated DNA immunoprecipitation (MedIP) in B cell line genomic DNA and qPCR relative to input (n = 5). b, Representative CD45 isoform expression in primary peripheral human CD3+ T cells sorted on the basis of cell-surface CD45RB and CD45RO. c, MedIP and qPCR relative to input in sorted primary human CD3+ T cells (n = 6, compiled from two donors). d, CTCF-ChIP and qPCR relative to rabbit Ig control IP, in sorted primary CD3+ T cells (n = 2). e, MedIP and qPCR relative to input in BL41 RBlow cells transduced with shRNA against DNMT1 versus shRFP-transduced cells (n = 3). f, CTCF ChIP in cells from e and qPCR relative to rabbit Ig control IP (n = 3). g, Cell-surface CD45RB expression in cells from e. h, RNA pol II ChIP and qPCR in cells from e relative to mouse Ig control IP (n = 3). Unless indicated otherwise, graphs show mean values ± s.d. P, two-tailed Student’s t test.

Article Snippet: Human CTCF recombinant protein was obtained from Abnova (catalogue no. H00010664-P01, batch no. 0991020–2).

Techniques: Methylation, Immunoprecipitation, Methylated DNA Immunoprecipitation, Expressing, Control, Transduction, shRNA, Two Tailed Test