Journal: PLoS Genetics

Article Title: Role of CTCF Protein in Regulating FMR1 Locus Transcription

doi: 10.1371/journal.pgen.1003601

Figure Lengend Snippet: A schematic outline of CTCF binding sites spanning the FMR1 locus (white dot = unmethylated CpG; black dot = methylated CpG). Triangles indicate CTCF binding sites. Promoter, exon 1, intron 2 and methylation boundary (indicated as MB) sites had been previously described ; the site present in the upstream methylated region (indicated as MR) was identified through the database available online . The transcription start site is reported as +1, as referred to Genbank L29074.

Article Snippet: For a relative quantification of each transcript, the following pre-developed TaqMan assays (Applied Biosystems) were used: CTCF (Hs00902008_m1), GAPDH (402869), FMR1 (Hs00233632_m1).

Techniques: Binding Assay, Methylation

Journal: PLoS Genetics

Article Title: Role of CTCF Protein in Regulating FMR1 Locus Transcription

doi: 10.1371/journal.pgen.1003601

Figure Lengend Snippet: ChIP assay of CTCF binding on FMR1 promoter and exon 1 in WT, UFM and FXS cell lines, both lymphoblasts ( A ) and fibroblasts ( B ). Box-plots indicate the mean of at least 10 independent immunoprecipitations for each lymphoblastoid cell line and 3 for fibroblasts. For each region analyzed the levels of CTCF binding in WT and UFM lines was significantly higher compared to FXS lines (p = 0.0001 for lymphoblasts and p<0.05 for fibroblasts). ChIP assay of CTCF binding on intron 2 ( C ) and methylation boundary region (MB) ( D ) in WT, UFM and FXS fibroblasts. The level of CTCF binding in WT and UFM lines is significantly higher compared to FXS lines (p = 0.0001) for both regions analyzed. Box-plots indicate the mean of three independent experiments for the MB binding site and one for the intron 2 site. Note that the amount of IP-DNA (ng) is indicated in logarithmic scale. In all ChIP experiments negative controls were performed by IgG immunoprecipitation and no template control (not shown).

Article Snippet: For a relative quantification of each transcript, the following pre-developed TaqMan assays (Applied Biosystems) were used: CTCF (Hs00902008_m1), GAPDH (402869), FMR1 (Hs00233632_m1).

Techniques: Binding Assay, Methylation, Immunoprecipitation, Control

Journal: PLoS Genetics

Article Title: Role of CTCF Protein in Regulating FMR1 Locus Transcription

doi: 10.1371/journal.pgen.1003601

Figure Lengend Snippet: ChIP assay of CTCF binding to FMR1 methylation boundary, promoter and exon 1 region after a 7-day treatment with [1 µM] 5-azadC on the S1 (FXS) lymphoblastoid cell line with 450 CGGs. After the pharmacological treatment we observed 25% of FMR1 transcriptional reactivation. Box plots represent the amount of DNA bound by CTCF in untreated WT lymphoblasts, untreated FXS (FXSut) and FXS treated with 5-azadC (FXSazadC) cell lines. Note that the amount of IP-DNA (ng) is indicated in logarithmic scale. ChIP experiments included negative controls performed by IgG immunoprecipitation and no template control (not shown).

Article Snippet: For a relative quantification of each transcript, the following pre-developed TaqMan assays (Applied Biosystems) were used: CTCF (Hs00902008_m1), GAPDH (402869), FMR1 (Hs00233632_m1).

Techniques: Binding Assay, Methylation, Immunoprecipitation, Control

Journal: PLoS Genetics

Article Title: Role of CTCF Protein in Regulating FMR1 Locus Transcription

doi: 10.1371/journal.pgen.1003601

Figure Lengend Snippet: ( A ) Western blotting analysis of CTCF, visualized with ECL kit. Protein extracts from untreated (UT) and siRNA-treated WT and UFM fibroblasts were probed with an anti-CTCF rabbit polyclonal antibody (top panel) and one against GAPDH (bottom panel). After CTCF depletion a major reduction of the corresponding protein is visible. ( B ) Relative quantification through RT-PCR of CTCF and FMR1 sense and antisense transcripts in those knock-down experiments in which CTCF depletion is followed by FMR1 transcript reduction both in WT (upper panel) and UFM (bottom panel) fibroblasts. FMR1 sense transcript is reduced to around 50% in both cell lines with a consistent (about 80%) reduction of CTCF mRNA levels. Depletion of FMR1 - AS1 (80 and 60% in WT and UFM cells, respectively) is also observed and directly correlates with CTCF reduction in both cell lines. The two cell lines were also transfected with a scramble siRNA (scRNA) without any modifications of CTCF transcript levels. Histograms represent mean and standard deviation of 10 independent knock-down experiments for UFM and WT.

Article Snippet: For a relative quantification of each transcript, the following pre-developed TaqMan assays (Applied Biosystems) were used: CTCF (Hs00902008_m1), GAPDH (402869), FMR1 (Hs00233632_m1).

Techniques: Western Blot, Quantitative Proteomics, Reverse Transcription Polymerase Chain Reaction, Knockdown, Transfection, Standard Deviation

Journal: PLoS Genetics

Article Title: Role of CTCF Protein in Regulating FMR1 Locus Transcription

doi: 10.1371/journal.pgen.1003601

Figure Lengend Snippet: ChIP assay demonstrates the decrease of CTCF binding on FMR1 promoter and exon 1 in WT fibroblasts after CTCF knock-down and FMR1 reduction. Box-plots indicate the mean of at least three independent experiments, the corresponding standard error and standard deviation (thin lines). For both regions analyzed the level of CTCF binding in untreated WT (UT) is significantly higher respect to cells treated with siRNA against CTCF (siRNA) (p = 0.0003 for promoter region; p = 0.0001 for exon1). Note that the amount of IP-DNA (ng) is indicated on a logarithmic scale. ChIP experiments included negative controls performed by IgG immunoprecipitation and no template control (not shown).

Article Snippet: For a relative quantification of each transcript, the following pre-developed TaqMan assays (Applied Biosystems) were used: CTCF (Hs00902008_m1), GAPDH (402869), FMR1 (Hs00233632_m1).

Techniques: Binding Assay, Knockdown, Standard Deviation, Immunoprecipitation, Control

Journal: PLoS Genetics

Article Title: Role of CTCF Protein in Regulating FMR1 Locus Transcription

doi: 10.1371/journal.pgen.1003601

Figure Lengend Snippet: ChIP results after CTCF knock-down without FMR1 transcript reduction.

Article Snippet: For a relative quantification of each transcript, the following pre-developed TaqMan assays (Applied Biosystems) were used: CTCF (Hs00902008_m1), GAPDH (402869), FMR1 (Hs00233632_m1).

Techniques: Knockdown, Methylation

Journal: PLoS Genetics

Article Title: Role of CTCF Protein in Regulating FMR1 Locus Transcription

doi: 10.1371/journal.pgen.1003601

Figure Lengend Snippet: CTCF overexpression in WT, UFM and FXS cell lines after transfection with a commercial vector containing the open reading frame of variant 1 of CTCF . Quantitative RT-PCR showed a strong increase of CTCF mRNA after 48 and 120 hours from transfection ( A ), while levels of FMR1 transcription remained substantially unchanged ( B ). The levels of CTCF transcription in the untreated cells were arbitrarily set at 1 as well as those of FMR1 transcript in WT and UFM fibroblasts, while those of FMR1 -mRNA in FXS cells were set at 0. Bars indicate standard deviation.

Article Snippet: For a relative quantification of each transcript, the following pre-developed TaqMan assays (Applied Biosystems) were used: CTCF (Hs00902008_m1), GAPDH (402869), FMR1 (Hs00233632_m1).

Techniques: Over Expression, Transfection, Plasmid Preparation, Variant Assay, Quantitative RT-PCR, Standard Deviation

Journal: PLoS Genetics

Article Title: Role of CTCF Protein in Regulating FMR1 Locus Transcription

doi: 10.1371/journal.pgen.1003601

Figure Lengend Snippet: Bisulfite sequencing of 82 CpGs within the 5′-UTR of FMR1 gene after CTCF knock-down in WT ( A ) and UFM ( B ) fibroblasts. Every line corresponds to bisulfite sequencing of an individual cell. Black and white squares correspond to methylated and unmethylated CpG sites, respectively. In this experiment the FMR1 transcriptional reduction was around 30% with a residual 20% of CTCF transcript. In spite of FMR1 transcriptional reduction (indicated as siRNA), there was no methylation spreading towards active FMR1 promoter, that remained unmethylated as in an untreated control (UT). Note that CpG pairs between 45 and 54 are within the promoter region. Black bars indicate CTCF binding sites in the MB and in the promoter region.

Article Snippet: For a relative quantification of each transcript, the following pre-developed TaqMan assays (Applied Biosystems) were used: CTCF (Hs00902008_m1), GAPDH (402869), FMR1 (Hs00233632_m1).

Techniques: Methylation Sequencing, Knockdown, Methylation, Control, Binding Assay

Journal: PLoS Genetics

Article Title: Role of CTCF Protein in Regulating FMR1 Locus Transcription

doi: 10.1371/journal.pgen.1003601

Figure Lengend Snippet: ChIP analysis of H3-K4 ( A ) and H3-K9 ( B ) methylation in WT fibroblasts after CTCF knock-down with FMR1 reduction. Each box-plot depicts the amount (ng) of IP-DNA in promoter and exon 1 regions in control (UT) and siRNA transfected (siRNA) cells. The levels of H3-K4 methylation were significantly reduced in both regions (p<0.05), while those of H3-K9 were increased particularly in the promoter. Box-plots indicate the mean of at least three independent experiments, the corresponding standard error and standard deviation (thin lines). ChIP experiments included negative controls performed by IgG immunoprecipitation and no template control (not shown).

Article Snippet: For a relative quantification of each transcript, the following pre-developed TaqMan assays (Applied Biosystems) were used: CTCF (Hs00902008_m1), GAPDH (402869), FMR1 (Hs00233632_m1).

Techniques: Methylation, Knockdown, Control, Transfection, Standard Deviation, Immunoprecipitation

Journal: PLoS ONE

Article Title: A Common Polymorphism within the IGF2 Imprinting Control Region Is Associated with Parent of Origin Specific Effects in Infantile Hemangiomas

doi: 10.1371/journal.pone.0113168

Figure Lengend Snippet: Fig 2A: 29 patients were genotyped via direct sequencing of blood samples for a known polymorphism within the core CTCF BS6 sequence (rs10732516.) All homozygous genotypes could be deduced from this information alone. Fig 2B: All samples (heterozygotes and homozygotes) were subjected to bisulfite conversion and quantitative methylation sensitive pyrosequencing. Methylation occurs only on the paternal chromosome for CTCF BS6. In normal tissue, such as patient matched control blood, this assay is capable of isolating the genotype of the paternal chromosome. As thymidine cannot be methylated, those individuals with a paternal T at rs10732516 were not methylated at CpG#5. Paternal C carrying individuals were methylated at CpG#5. Thus, the maternal and paternal contribution to CTCFBS6 can be deduced. This assay sidesteps the need for directly sequencing parents’ DNA and eliminates the potential ambiguity ensuing from hetrozygous parents. Note: The methylation values of this assay are subject to primer bias, Tost et al (25 . ) This is evident by the 3 distinct groupings of methylation levels, which are artifactual.

Article Snippet: The assays were: IGF2—assay number Hs00171254_m1, H19—assay number Hs00399294_g1, CTCF—assay number Hs00198081_m1, and BORIS—assay number Hs00540744_m1.

Techniques: Sequencing, Methylation, Control

Journal: PLoS ONE

Article Title: A Common Polymorphism within the IGF2 Imprinting Control Region Is Associated with Parent of Origin Specific Effects in Infantile Hemangiomas

doi: 10.1371/journal.pone.0113168

Figure Lengend Snippet: Fig 3A: IGF2 mRNA levels were approximately 6X lower in the involuting samples compared to their proliferating counterparts. Proliferating vs. involuted p = .02, Proliferating vs. quiescent p = .0081, Quiescent vs. involuting p = .01. Wilcoxian Rank Sum Test. Error bars represent standard deviation. Fig 3B: %CTCF changes significantly according to clinical stage. Prolif vs. quiescent p = .01, quiescent vs. invol p = .006, prolif vs. invol p = .63, Wilcoxian two sample test. Error bars represent standard deviation. Note: All samples in the IGF2 analysis were included in the %CTCF analysis, with additional samples.

Article Snippet: The assays were: IGF2—assay number Hs00171254_m1, H19—assay number Hs00399294_g1, CTCF—assay number Hs00198081_m1, and BORIS—assay number Hs00540744_m1.

Techniques: Standard Deviation

Journal: PLoS ONE

Article Title: A Common Polymorphism within the IGF2 Imprinting Control Region Is Associated with Parent of Origin Specific Effects in Infantile Hemangiomas

doi: 10.1371/journal.pone.0113168

Figure Lengend Snippet: (A) Using the two samples with the lowest %CTCF (420 and 418 days as common points (Purple)) two curves with high correlation to age can be appreciated. %CTCF steadily decreases as lesions age until approximately 400 days (red and purple points), then CTCF once again increases compared to BORIS (purple and blue points). This roughly correlates with the transition from proliferative to quiescent lesions. (B) CSUM of %CTCF demonstrates statistically significant variation about the mean of %CTCF according to age. For a full explanation of the CSUM data and commensurate change point analysis, please see and the online reference in the section.

Article Snippet: The assays were: IGF2—assay number Hs00171254_m1, H19—assay number Hs00399294_g1, CTCF—assay number Hs00198081_m1, and BORIS—assay number Hs00540744_m1.

Techniques:

Journal: PLoS ONE

Article Title: A Common Polymorphism within the IGF2 Imprinting Control Region Is Associated with Parent of Origin Specific Effects in Infantile Hemangiomas

doi: 10.1371/journal.pone.0113168

Figure Lengend Snippet: To our knowledge, these data represent the first demonstration of the potentially antagonistic effects of CTCF and BORIS on a target gene’s transcription through a continuous curve. P = .0004 ANCOVA Model. Age effect was not significant in the model p = .241. The %CTCF is a stronger statistical predictor of IGF2 expression than BORIS alone .0004 vs. .0028 respectively. (N = 15)

Article Snippet: The assays were: IGF2—assay number Hs00171254_m1, H19—assay number Hs00399294_g1, CTCF—assay number Hs00198081_m1, and BORIS—assay number Hs00540744_m1.

Techniques: Expressing

Journal: PLoS ONE

Article Title: A Common Polymorphism within the IGF2 Imprinting Control Region Is Associated with Parent of Origin Specific Effects in Infantile Hemangiomas

doi: 10.1371/journal.pone.0113168

Figure Lengend Snippet: Twenty samples with 4 blots depicted, demonstrates 4 Stages of CTCF and BORIS Expression. 6–1: A low concentration of anti-CTCF (1:10,000) demonstrates the complete spectrum of CTCF expression with increases early (210 to 418) and late (760 to 987) in protein expression. (Note, a testes negative control was included as well as a venous malformation denoted as “VM.”) 6–2 through 6–4 were probed with 1:5000 concentration of anti-CTCF that more clearly demonstrates the early rise in CTCF that occurs after 367 days. 6–2 suggests an early increase in BORIS with precipitous downregulation after 244 days. 6–3 and 6–4 expand this critical age range demonstrating a period from 244 days to 367 where BORIS is downregulated but CTCF is not yet upregulated. Note: samples marked with an asterisk were also subjected to CTCF and BORIS rtPCR.

Article Snippet: The assays were: IGF2—assay number Hs00171254_m1, H19—assay number Hs00399294_g1, CTCF—assay number Hs00198081_m1, and BORIS—assay number Hs00540744_m1.

Techniques: Expressing, Concentration Assay, Negative Control, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: A Common Polymorphism within the IGF2 Imprinting Control Region Is Associated with Parent of Origin Specific Effects in Infantile Hemangiomas

doi: 10.1371/journal.pone.0113168

Figure Lengend Snippet: The Western analysis suggests 4 stages of CTCF and BORIS (see , panels 1 to 4) each stage leading to higher levels of CTCF expression relative to BORIS. These interval changes in protein expression closely correlate with clinical stage. Furthermore, blue bars represent the 95% confidence intervals of the two change points identified by quantitative rtPCR. These data show remarkable agreement reinforcing the idea that relative CTCF and BORIS expression levels closely mirror the clinical stage of the lesions tested. Of note, the CTCF and BORIS protein data also suggest a molecular basis for a late proliferative stage.

Article Snippet: The assays were: IGF2—assay number Hs00171254_m1, H19—assay number Hs00399294_g1, CTCF—assay number Hs00198081_m1, and BORIS—assay number Hs00540744_m1.

Techniques: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: A Common Polymorphism within the IGF2 Imprinting Control Region Is Associated with Parent of Origin Specific Effects in Infantile Hemangiomas

doi: 10.1371/journal.pone.0113168

Figure Lengend Snippet: Fig 8A: The antagonism of CTCF and BORIS relative to IGF2 production is demonstrated by calculating the difference between the two. Here IGF2 transcription correlates positively with higher relative levels of BORIS compared to CTCF. However, two curves are apparent. These are separated by the paternally contributed allele at CTCF BS6. Paternal C samples lay on a steeper curve than the paternal T counterparts (P = .05.) Note: Involuted samples were not considered in the analysis as they make similarly low levels of IGF2 regardless of paternal genotype. There is no clear relationship when samples are sorted by maternal genotype . Fig 8B: H19 positively correlates with CTCF transcript levels alone according to maternal genotype: Maternal C samples lie on a steeper curve than their maternal T counterparts (p = 0.0129, ANCOVA). There is no clear relationship when samples are sorted by paternal genotype .

Article Snippet: The assays were: IGF2—assay number Hs00171254_m1, H19—assay number Hs00399294_g1, CTCF—assay number Hs00198081_m1, and BORIS—assay number Hs00540744_m1.

Techniques:

Journal: PLoS ONE

Article Title: A Common Polymorphism within the IGF2 Imprinting Control Region Is Associated with Parent of Origin Specific Effects in Infantile Hemangiomas

doi: 10.1371/journal.pone.0113168

Figure Lengend Snippet: Fig 9A: Increased CTCF transcript level correlates with demethylation of the H19 Promoter. Those samples with the highest CTCF expression were the least methylated ranging from 34% to 14%. However, demethylation of the H19 promoter did not correlate strictly with H19 transcript expression . Fig 9B and 9C —The H19 promoter (see Fig 9D) is hypomethylated, demonstrated by bisultife converted pyrosequencing (9B) and methylation sensitive restriction digest with southern hybridization (9C.) 25 IH samples, and 13 matched blood controls were subjected to bisulfite converted pyrosequencing. 13 IH samples and 13 matched blood controls were subjected to southern analysis with methylation sensitive Pst1 digestion. Two representative gels show, 5 IH samples, a Beckwith-Weidman positive control and a 50% methylated normal control. Fig 9D : sequence showing the H19 promoter—CpG#4 of the bisulfite sequencing test corresponds to the CCCGGG Pst1 digestion site of the Southern analysis. Other CpG’s tested are in bold. This CpG is in close proximity to the transcription start site of H19 (blue arrow) and an overlapping putative CTCF binding site identified by positional weight matrix analysis.

Article Snippet: The assays were: IGF2—assay number Hs00171254_m1, H19—assay number Hs00399294_g1, CTCF—assay number Hs00198081_m1, and BORIS—assay number Hs00540744_m1.

Techniques: Expressing, Methylation, Hybridization, Positive Control, Control, Sequencing, Methylation Sequencing, Binding Assay

Journal: Nature Communications

Article Title: CTCF-mediated chromatin looping in EGR2 regulation and SUZ12 recruitment critical for peripheral myelination and repair

doi: 10.1038/s41467-020-17955-2

Figure Lengend Snippet: a Western blots for CTCF, MBP, MPZ, and EGR2 in proliferating and differentiated rat SC cultures. GAPDH served as a loading control. n = 2 independent experiments. b Relative qPCR expression of Ctcf , Mbp , Mpz , and Egr2 in proliferating and differentiated rat SC cultures. Data are presented as means ± SEM., *** P < 0.001, n = 3 independent experiments; two-tailed unpaired Student’s t -test, P ( Ctcf) = 0.00021, P ( Mbp) = 2.8E-05, P ( Mpz) = 1.7E-06, P ( Egr2) = 3.9E-05. c Colocalization of CTCF with SOX10 in SC nuclei from mice at P7, P14, and P62 evaluated by immunofluorescence labeling. Representative images are shown. n = 3 nerve tissues at each time point. Arrows indicate SOX10 + /CTCF + SCs; arrowheads indicate SOX10 + /CTCF − SCs. Scale bars: 50 μm. d The percentage of CTCF + nuclei in SCs (SOX10 + ) in sciatic nerves from P7, P14, and P62 mice. n = 3 control tissues at each time point. Data are presented as means ± SEM., * P < 0.05, ** P < 0.01; n = 3 nerve tissues at each time point; one-way ANOVA with multiple comparisons test. P (P14) = 0.0392, P (P62) = 0.0052. e Relative qPCR expression of Ctcf in mouse sciatic nerves at various developmental stages. Data are presented as means ± SEM., ** P < 0.01, *** P < 0.001; n = 3 nerve tissues at each time point; one-way ANOVA with multiple comparisons test, P (P7) = 0.0067, P (P10) = 0.0004, P (P21) = 0.1503, P (P60) = 0.0077. Source data are provided as a Source Data file.

Article Snippet: We used antibodies against CTCF (rabbit, Cell Signaling Technology, 3417 S, 1:1000), MBP (goat, Santa Cruz Biotechnology, sc-13914, 1:500), MPZ (rabbit, Abcam, ab31851, 1:1000), KROX20/EGR2 (rabbit, Santa Cruz Biotechnology, sc-20690, 1:200 or Guinea pig, provided by Michael Wegner, 1:1000), SUZ12 (rabbit, Cell Signaling Technology, 3737 S, 1:1000), EED (rabbit, Millipore, 17-10034, 1:1000), EZH2 (rabbit, Cell Signaling Technology, 5246 P, 1:1000), H3 (rabbit, Cell Signaling Technology, 4499 S, 1:1000), H3K27me3 (rabbit, Cell Signaling Technology, 9733 S, 1:1000), H3K27me2/3 (mouse, Active motif, 39536, 1:1000), H3K36me3 (rabbit, Abcam, ab9050, 1:1000), H3K4me1 (mouse, Active motif, 39635, 1:1000), H3K27ac (rabbit, Cell Signaling Technology, 4353 S, 1:1000), and GAPDH (mouse, Millipore, MAB374, 1:5000).

Techniques: Western Blot, Control, Expressing, Two Tailed Test, Immunofluorescence, Labeling

Journal: Nature Communications

Article Title: CTCF-mediated chromatin looping in EGR2 regulation and SUZ12 recruitment critical for peripheral myelination and repair

doi: 10.1038/s41467-020-17955-2

Figure Lengend Snippet: a qRT-PCR analysis of Ctcf , Sox10 , Egr2 , and Mpz expression in rat SCs transfected with control nontargeting siRNA and si Ctcf for 24 h and induced to differentiate for 9 h. n = 3 independent experiments, P ( Ctcf ) = 3.03E-05, P ( Sox10 ) = 0.0433, P ( Egr2 ) = 0.000107, P ( Mpz ) = 0.000293. b–d Rat SCs were transfected with control siRNA or si Ctcf for 24 h and induced to differentiate for 9 h and CTCF- ( b ), EGR2- and OCT6-positive ( c ) cells were visualized by immunofluorescence microscopy and d quantified; n = 3 independent experiments. Arrows indicate CTCF + or EGR2 + /OCT6 + SCs. Scale bars: 50 µm. n = 3 independent experiments, P (EGR2) = 0.00069, P (OCT6) = 0.99. e Western blots for CTCF and EGR2 in co-cultures of rat DRGs and SCs treated with control siRNA or si Ctcf . GAPDH served as a loading control. n = 4 independent experiments. f Rat SCs treated with control siRNA or si Ctcf were seeded onto rat DRGs. After 10 days, co-cultures were immunostained for MBP and neurofilament-M. Images are representative of n = 4 independent experiments. Scale bars: 100 μm. g Quantification of the number of MBP + segments per mm 2 of area in myelinating co-cultures of DRGs and SCs treated with control siRNA or si Ctcf . n = 4 independent experiments, P = 0.0068. h Western blots for CTCF in rat Schwann cells induced to differentiate following transfection with control or CTCF expression vectors. n = 2 independent experiments. i qRT-PCR quantification of differentiation regulators and negative regulators in rat SCs induced to differentiate following transfection with control or CTCF expression vectors. n = 3 independent experiments, P ( Egr2 ) = 0.0012, P ( Cnp ) = 0.00068, P ( Mbp ) = 0.011, P ( Mpz ) = 2.9E-05, P ( Pmp22 ) = 6.7E-05, P ( Sox2 ) = 0.00026, P ( Hes1 ) = 0.028, P ( Mki67 ) = 0.00024. Data are presented as means ± SEM., * P < 0.05, ** P < 0.01, *** P < 0.001, two-tailed unpaired Student’s t -test. Source data are provided as a Source Data file.

Article Snippet: We used antibodies against CTCF (rabbit, Cell Signaling Technology, 3417 S, 1:1000), MBP (goat, Santa Cruz Biotechnology, sc-13914, 1:500), MPZ (rabbit, Abcam, ab31851, 1:1000), KROX20/EGR2 (rabbit, Santa Cruz Biotechnology, sc-20690, 1:200 or Guinea pig, provided by Michael Wegner, 1:1000), SUZ12 (rabbit, Cell Signaling Technology, 3737 S, 1:1000), EED (rabbit, Millipore, 17-10034, 1:1000), EZH2 (rabbit, Cell Signaling Technology, 5246 P, 1:1000), H3 (rabbit, Cell Signaling Technology, 4499 S, 1:1000), H3K27me3 (rabbit, Cell Signaling Technology, 9733 S, 1:1000), H3K27me2/3 (mouse, Active motif, 39536, 1:1000), H3K36me3 (rabbit, Abcam, ab9050, 1:1000), H3K4me1 (mouse, Active motif, 39635, 1:1000), H3K27ac (rabbit, Cell Signaling Technology, 4353 S, 1:1000), and GAPDH (mouse, Millipore, MAB374, 1:5000).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Control, Immunofluorescence, Microscopy, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: CTCF-mediated chromatin looping in EGR2 regulation and SUZ12 recruitment critical for peripheral myelination and repair

doi: 10.1038/s41467-020-17955-2

Figure Lengend Snippet: a Excised exon 8 of the floxed Ctcf allele by Dhh-Cre . b Co-labeling of CTCF with SOX10 in control and mutant sciatic nerves at P7 ( n = 3 animals/genotype). Arrows indicate SOX10 + /CTCF + SCs. Scale bars: 50 μm. c The percentage of CTCF + nuclei in SCs (SOX10 + ) from control and Ctcf cKO sciatic nerves at P7. n = 3 animals/genotype, P = 1.73E-05. d Survival curves of control and Ctcf cKO mice. n = 25 for control and n = 23 for Ctcf cKO mice, *** P < 0.001. e Representative photographs of sciatic nerves from P13 control and Ctcf cKO mice. n = 3 animals/genotype. f Immunofluorescence labeling of MBP (red) in P7 control and Ctcf cKO sciatic nerves. n = 3 animals/genotype. Scale bars: 50 μm. g The mRNA levels of myelin-related genes in P7 control and Ctcf cKO sciatic nerves. n = 6 animals/genotype. P ( Prx ) = 1.9E-08, P ( Mbp ) = 2.0E-08, P ( Mpz ) = 8.5E-09. h, i Ultrastructure of control and Ctcf cKO sciatic nerves at ( h ) P1 and P7 and at ( i ) 8 weeks. n = 3 animals/genotype. Arrows and arrowheads indicate immature SCs and unsorted axons, respectively. Scale bars: 4 μm. j A diagram showing the tamoxifen (TAM) administration scheme. k Immunofluorescent labeling of CTCF (green) nuclei in control and Ctcf iKO sciatic nerves at P14. Scale bars: 50 μm. n = 3 animals/genotype. l EM images of P14 sciatic nerves from control and Ctcf iKO mice. n = 4 animals/genotype. Arrow indicates myelin membrane. Scale bars: 4 μm, and 1 μm in the inset on the right panel. m Myelinated axon numbers 10 −4 μm −2 sections of P14 sciatic nerves from control and Ctcf iKO mice. n = 4 animals/genotype, P = 0.0006. Data are presented as means ± SEM., *** P < 0.001; Statistical analyses performed using two-tailed unpaired Student’s t -test; Log-rank test used for survival curve. Source data are provided as a Source Data file.

Article Snippet: We used antibodies against CTCF (rabbit, Cell Signaling Technology, 3417 S, 1:1000), MBP (goat, Santa Cruz Biotechnology, sc-13914, 1:500), MPZ (rabbit, Abcam, ab31851, 1:1000), KROX20/EGR2 (rabbit, Santa Cruz Biotechnology, sc-20690, 1:200 or Guinea pig, provided by Michael Wegner, 1:1000), SUZ12 (rabbit, Cell Signaling Technology, 3737 S, 1:1000), EED (rabbit, Millipore, 17-10034, 1:1000), EZH2 (rabbit, Cell Signaling Technology, 5246 P, 1:1000), H3 (rabbit, Cell Signaling Technology, 4499 S, 1:1000), H3K27me3 (rabbit, Cell Signaling Technology, 9733 S, 1:1000), H3K27me2/3 (mouse, Active motif, 39536, 1:1000), H3K36me3 (rabbit, Abcam, ab9050, 1:1000), H3K4me1 (mouse, Active motif, 39635, 1:1000), H3K27ac (rabbit, Cell Signaling Technology, 4353 S, 1:1000), and GAPDH (mouse, Millipore, MAB374, 1:5000).

Techniques: Labeling, Control, Mutagenesis, Immunofluorescence, Membrane, Two Tailed Test

Journal: Nature Communications

Article Title: CTCF-mediated chromatin looping in EGR2 regulation and SUZ12 recruitment critical for peripheral myelination and repair

doi: 10.1038/s41467-020-17955-2

Figure Lengend Snippet: a Volcano plot of transcriptome profiles of control and Ctcf cKO sciatic nerves ( n = 2 animals/genotype). Red and blue dots represent significantly downregulated and upregulated genes in Ctcf cKO nerves compared to the control, respectively ( P < 0.05, fold-change > 1.5). b Heatmap of representative genes and their categories differentially expressed in control and Ctcf cKO sciatic nerves ( n = 2 animals/genotype). c , d Bar plots of gene ontology analysis of genes c downregulated and d upregulated genes in Ctcf cKO sciatic nerves compared with control nerves. Each dot (connected by lines) represents the gene count of the corresponding biological function categories. n = 2 independent tissues/genotype. e qPCR analysis of genes related to SC development that are decreased (left) and increased (right) in Ctcf cKO sciatic nerves relative to control. f GSEA enrichment scores for myelin sheath (left) and lipid biosynthetic process (right) gene sets in control and Ctcf cKO sciatic nerves. g GSEA enrichment scores for cell-cycle gene sets in control and Ctcf cKO sciatic nerves. Data are presented as means ± SEM., *** P < 0.001, ** P < 0.01, * P < 0.05, n = 3 animals/genotype; two-tailed unpaired Student’s t -test, P (Prx) = 2.6e-05, P (Mbp) = 4.9E-05, P (Mpz) = 5.3E-06, P (Hmgcr) = 0.0014, P (Egr2) = 8.6E-05, P (Itgb1) = 0.008, P (Itgb3bp) = 0.00022, P (Itgb5) = 0.0021, P (Itgb8) = 0.00017, P (Ccnd1) = 7.4E-05, P (Ccng1) = 5.1E-05, P (Ccno) = 0.0004, P (Cdc7) = 6.4E-05, P (Cdk5r2) = 1.6E-05, P (Ccnb1) = 3.2E-05, P (Notch1) = 0.00102, P (Hes5) = 0.028, P (Id2) = 0.23, P (Id4) = 3.3E-05. Source data are provided as a Source Data file.

Article Snippet: We used antibodies against CTCF (rabbit, Cell Signaling Technology, 3417 S, 1:1000), MBP (goat, Santa Cruz Biotechnology, sc-13914, 1:500), MPZ (rabbit, Abcam, ab31851, 1:1000), KROX20/EGR2 (rabbit, Santa Cruz Biotechnology, sc-20690, 1:200 or Guinea pig, provided by Michael Wegner, 1:1000), SUZ12 (rabbit, Cell Signaling Technology, 3737 S, 1:1000), EED (rabbit, Millipore, 17-10034, 1:1000), EZH2 (rabbit, Cell Signaling Technology, 5246 P, 1:1000), H3 (rabbit, Cell Signaling Technology, 4499 S, 1:1000), H3K27me3 (rabbit, Cell Signaling Technology, 9733 S, 1:1000), H3K27me2/3 (mouse, Active motif, 39536, 1:1000), H3K36me3 (rabbit, Abcam, ab9050, 1:1000), H3K4me1 (mouse, Active motif, 39635, 1:1000), H3K27ac (rabbit, Cell Signaling Technology, 4353 S, 1:1000), and GAPDH (mouse, Millipore, MAB374, 1:5000).

Techniques: Control, Two Tailed Test

Journal: Journal of Biological Chemistry

Article Title: CTCF Promotes Muscle Differentiation by Modulating the Activity of Myogenic Regulatory Factors

doi: 10.1074/jbc.m110.164574

Figure Lengend Snippet: FIGURE 1. Ctcf expression in developing mouse and zebrafish. A, Ctcf expression pattern in mouse. Whole mount in situ hybridization shows widely distributed Ctcf in mouse at E10.5. Ctcf was enriched in the first branchial arches (*) at E10.5. At E12, Ctcf was observed in brain (br), jaw, facial muscles, and limbs (blackarrows).AtE13,Ctcflocalizedpreferentiallyinbrainandwasbarelydetectedinintercostals(arrowhead)andlimbmusculature(arrow).Close-upsofboxed areasarepresented.B,ctcfexpressionpatterninzebrafish.ctcfwaspresentinsomitesofzebrafishembryosat10–11,14–15,and16–17hpf.In26-hpfembryos, the levels of ctcf were greatly reduced, and by 36 hpf, ctcf was present only in distal somites (boxed area), whereas in 72-hfp embryos ctcf was restricted to brain and gut (arrow). The arrowheads indicate somites. C, flat whole mount zebrafish embryos showing expression of ctcf (upper panel) and myod (lower panel) in somites. D, Ctcf, MyoD, Mrf4, and Myogenin are similarly up-regulated during myogenic differentiation. qPCR on C2C12 myoblasts (MB) and at days 2–8 (D2–D8) of differentiation. The values were normalized against -actin, which was used as endogenous control. The data are presented as the means S.D. of three biological replicates.

Article Snippet: 24 h later, the cells were transfected with 0, 1, 2, 5, and 10 pm of CTCF siRNA (Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitrogen).

Techniques: Expressing, In Situ Hybridization, Muscles, Control

Journal: Journal of Biological Chemistry

Article Title: CTCF Promotes Muscle Differentiation by Modulating the Activity of Myogenic Regulatory Factors

doi: 10.1074/jbc.m110.164574

Figure Lengend Snippet: FIGURE 2. CTCF regulates myogenic differentiation. A, C2C12 cells were transiently transfected with an empty vector () or a CTCF cDNA (). RT-PCR and Western blots (WB) were performed at 1.5 days post-transfection, except for the -SG blot, which corresponds to cells at post-transfection day 3 (D3). Gapdh and RNA polymerase II are endogenous controls in the RT-PCR and Western blot, respectively. B, qPCR of MyoD and myogenin (Myog) on CTCF-overexpressing cells.*,p0.05.C,CTCFenhancesthemyogenicpotentialofMyoD.10T1/2cellsweretransfectedwithcDNAsencodingCTCForMyc-MyoD.MHC-positivecells were detected by immunofluorescence after 7 days post-transfection. The nuclei were counterstained with DAPI, and MHC-positive cells were counted. The right panel shows the increase in MHC-positive cells transfected with MyoD plus CTCF versus MyoD. D, CTCF is required for myogenic gene expression. Upper panel, Western blot showing decreased expression of CTCF but not serum response factor (SRF). -Actin is the loading control. Lower panel, CTCF, MyoD, and Myogenin levels in CTCF-deficient cells (siCTCF; red bars) as compared with control cells (blue bars). The analysis was performed at days 2–5 of differentiation (D2–D5). The data are presented as the means S.D. E, CTCF knockdown impairs C2C12 cells differentiation. C2C12 cells in which CTCF was knocked down by stable transfection of a shRNA (siCTCF) presented limited MHC expression as compared with cells transfected with a control shRNA. The right panel shows quantification of MHC signal in several fields across three independent differentiations by ImageJ software. *, p 0.05 as determined by t test.

Article Snippet: 24 h later, the cells were transfected with 0, 1, 2, 5, and 10 pm of CTCF siRNA (Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitrogen).

Techniques: Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Gene Expression, Expressing, Control, Knockdown, Stable Transfection, shRNA, Software

Journal: Journal of Biological Chemistry

Article Title: CTCF Promotes Muscle Differentiation by Modulating the Activity of Myogenic Regulatory Factors

doi: 10.1074/jbc.m110.164574

Figure Lengend Snippet: FIGURE 3. CTCF physically interacts with MyoD. A, extracts with in vitro synthesized V5-tagged full-length CTCF (FL), the CTCF amino-terminal (N-ter), zinc finger-containing (ZF), or the carboxyl-terminal (C-ter) domains were mixed with Myc-MyoD for co-immunoprecipitations (IP). The tagged CTCF versions were identified by Western blot (WB) using an anti-V5 antibody. Inputs for MyoD and the CTCF proteins are shown in the middle and bottom panels, respectively. The numbers indicate kDa. In lane 1, Myc-MyoD was added as negative control. B, endogenous CTCF and MyoD co-immunopre- cipitate in C2C12 cell extracts. CTCF and MyoD were revealed in immunopre- cipitates obtained with anti-MyoD and anti-CTCF antibodies (-MyoD and -CTCF, respectively) by Western blot (WB). The bottom panels show inputs. An anti-Myc (-myc) antibody was used as a negative control.

Article Snippet: 24 h later, the cells were transfected with 0, 1, 2, 5, and 10 pm of CTCF siRNA (Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitrogen).

Techniques: In Vitro, Synthesized, Western Blot, Negative Control

Journal: Journal of Biological Chemistry

Article Title: CTCF Promotes Muscle Differentiation by Modulating the Activity of Myogenic Regulatory Factors

doi: 10.1074/jbc.m110.164574

Figure Lengend Snippet: FIGURE 4. CTCF activates muscle-specific gene expression and modulates the transcriptional activity of MyoD and myogenin. A, CTCF regulates the -SG full-length promoter. C2C12 cells stably expressing luciferase (LUC) under the full-length -SG promoter were transfected with CTCF. Luciferase activity was assessed 48 h post-transfection (top panel). In this and further experiments, the values are given as the means S.D. from three independent experiments performed in duplicate. The same C2C12 cell line was transfected with anti-CTCF siRNA and induced to differentiation. The middle panel shows -SG promoter activity in CTCF siRNA-transfected cells. Efficient depletion of CTCF was shown by Western blot (bottom panel). -Actin was used as loading control. B, CTCF regulatesthe-SGpromoterinaMyoD-dependentmanner.Thefull-length-SGpromoterwastransientlyco-transfectedwithCTCFwithorwithoutMyoDinto 10T1/2 fibroblasts. The data represent the means S.D. The p values were obtained by t test (n 3). C, CTCF-mediated activation of the Myogenin promoter depends on MyoD-binding sites. Luciferase reporter vectors containing the WT or a mutated version of the Myogenin promoter in which two E-boxes were ablated(E-mut)weretransfectedalone()orwithincreasingamountsofCTCFinC2C12cells.D,CTCFisrequiredforMyoD-andmyogenin-coordinatedactivity on the -SG core promoter. The -SG core promoter or the Myogenin promoter were transiently co-transfected with an empty vector (pCI) or with the indicated combinations of cDNAs encoding CTCF, MyoD, or Myogenin (Myog) in 10T1/2 fibroblasts. The data represent the means S.D. The p values were obtained by t test (n 3). E, CTCF facilitates MyoD binding to the -SG core promoter in vivo. The -SG core promoter was amplified by qPCR from chromatin immuno- precipitated with antibodies (AB) recognizing MyoD, CTCF, and acetylated histone H3 (acH3). Chromatin was obtained from 10T1/2 cells transfected with cDNAs encoding CTCF or MyoD, alone, or combined. F, CTCF without the zinc finger domain cannot recruit Myod to the -SG core promoter. MyoD- and CTCF-associated chromatin from 10T1/2 cells transfected with the full-length CTCF (CTCF-WT) or a CTCF mutant lacking the zinc finger domain (CTCF-ZF) was analyzed by qPCR. G, chromatin immunoprecipitated with anti-MyoD, CTCF, or PollII was subjected to qPCR amplifying the -SG, or the serum response factor (SRF) core promoters (CP). Chromatin was obtained from C2C12 cells transfected with a control siRNA or an anti-CTCF siRNA (CTCF siRNA). The data in E–G were normalized against inputs, are presented as relative enrichment over levels obtained with beads precipitation, and represent the means S.D. of at least two biological replicates.

Article Snippet: 24 h later, the cells were transfected with 0, 1, 2, 5, and 10 pm of CTCF siRNA (Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitrogen).

Techniques: Gene Expression, Activity Assay, Stable Transfection, Expressing, Luciferase, Transfection, Western Blot, Control, Activation Assay, Binding Assay, Plasmid Preparation, In Vivo, Amplification, Mutagenesis, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: CTCF Promotes Muscle Differentiation by Modulating the Activity of Myogenic Regulatory Factors

doi: 10.1074/jbc.m110.164574

Figure Lengend Snippet: FIGURE 5. Ctcf knockdown affects myogenic development in zebrafish embryos. A, Western blot analyses show the absence of Ctcf in zebrafish embryos injected with morpholino oligonucleotides complementary to ctcf translation start codon (ATG) or the exon 1/intron 1 boundary (SP). B, lateral views of zebrafish embryos injected with morpholino oligonucleotides complementary to the ctcf translation start codon (ATG). The embryos were grouped in class 1 or class 2. c1 presented mild somite disorganization at 24 and 48 hpf as compared with noninjected control embryos (ct). Severe somite disorganization was observed at 24 hpf in c2 embryos. The scale bars represent 200 m. C, confocal micrographs showing MHC and actin signals in somites from c1 and c2 morphants at 24 and 48 hpf. Scale bars, 50 m. D, transmission electron microscopy images show decreased myofibers (arrowheads) in c2 morphants (ATG), as compared with control (ct) embryos. E, in situ hybridization shows decreased myogenin in c1 and c2 morphants at 24 hpf. F, injection of CTCF mRNA partially rescued the abnormal phenotype of ctcf morphants. The percentage of embryos with normal somite shape and absence of curved tail obtained after injection of ATG alone or with CTCF mRNA is shown. G, lateral views of 32 hpf embryos injected with a control morpholino (CM), ATG morpholino (ATG) alone, or co-injected with CTCF mRNA (ATG CTCF mRNA). H, in situ hybridization against myogenin on 48-hpf embryos injected with a control morpholino (CM), ATG morpholino alone (ATG), or ATG co-injected with CTCF mRNA (ATG CTCF mRNA). The left and right panels show lateral and dorsal views, respectively. Superior oblique,lateralrectusocular,levatorarcuspalatini(whitebracket),transversusventralis(openarrowhead),lowerjaw(whitearrowhead),pectoralfin(arrow),and hypaxial musculature (black arrowhead) are indicated.

Article Snippet: 24 h later, the cells were transfected with 0, 1, 2, 5, and 10 pm of CTCF siRNA (Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitrogen).

Techniques: Knockdown, Western Blot, Injection, Control, Transmission Assay, Electron Microscopy, In Situ Hybridization

Journal: Journal of Biological Chemistry

Article Title: CTCF Promotes Muscle Differentiation by Modulating the Activity of Myogenic Regulatory Factors

doi: 10.1074/jbc.m110.164574

Figure Lengend Snippet: FIGURE 7. Model of CTCF involvement in myogenic differentiation. CTCF stimulates expression of Wnt11, which in turn favors MRF expression and muscle-specificgeneactivation.DownstreamactivationofWnt11,CTCFstim- ulates muscle-specific gene expression (large red arrow) by interaction with MRFs, which reinforce myogenic differentiation by a feed forward loop (solid black arrow). Upon the absence of CTCF (represented as CTCF in dotted line), the interaction of MyoD with muscle promoters is disfavored, leading to inef- ficacy in the contribution of Myogenin (Myog) to the enhancement of MyoD- activated gene expression (MyoD separated from Myog). Therefore, the absence of CTCF results in defective myogenesis caused by deregulation of WNT signaling and disruption of the MRFs-mediated feed-forward loop (bro- ken arrows) leading to altered muscle-specific transcription (small red arrow).

Article Snippet: 24 h later, the cells were transfected with 0, 1, 2, 5, and 10 pm of CTCF siRNA (Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitrogen).

Techniques: Expressing, Gene Expression, Disruption