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: International Journal of Molecular Sciences

Article Title: Investigation of the Role of Pituitary Adenylate Cyclase-Activating Peptide (PACAP) and Its Type 1 (PAC1) Receptor in Uterine Contractility during Endometritis in Pigs

doi: 10.3390/ijms23105467

Figure Lengend Snippet: Relative pituitary adenylate cyclase-activating peptide receptor (PAC1R) mRNA transcript abundances in the myometrial layer of gilts from the control (CON), saline (SAL) and E. coli ( E. coli ) groups, estimated by real-time PCR. Relative PAC1R mRNA transcript abundances are expressed as the mean ± SEM of ratios in relation to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Article Snippet: To block the non-specific bindings, membranes were incubated with 5% fat-free dry milk in a TBS-T buffer at 21 °C for 1.5 h. They were then incubated at 4 °C for 18 h with primary PAC1 receptor polyclonal rabbit antibody (dilution: 1:1000, cat. no. CBS-PA207627, Cusabio Biotech Co.).

Techniques: Control, Saline, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Investigation of the Role of Pituitary Adenylate Cyclase-Activating Peptide (PACAP) and Its Type 1 (PAC1) Receptor in Uterine Contractility during Endometritis in Pigs

doi: 10.3390/ijms23105467

Figure Lengend Snippet: Relative pituitary adenylate cyclase-activating peptide receptor (PAC1R) protein abundances in the myometrial layer of gilts from the control (CON), saline (SAL) and E. coli ( E. coli ) groups, estimated by Western blot analysis. The relative PAC1R protein abundances are expressed as the mean ± SEM of ratios in relation to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The blot with representative bands for each group is presented in . * p < 0.05, *** p < 0.001 compared between groups.

Article Snippet: To block the non-specific bindings, membranes were incubated with 5% fat-free dry milk in a TBS-T buffer at 21 °C for 1.5 h. They were then incubated at 4 °C for 18 h with primary PAC1 receptor polyclonal rabbit antibody (dilution: 1:1000, cat. no. CBS-PA207627, Cusabio Biotech Co.).

Techniques: Control, Saline, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Investigation of the Role of Pituitary Adenylate Cyclase-Activating Peptide (PACAP) and Its Type 1 (PAC1) Receptor in Uterine Contractility during Endometritis in Pigs

doi: 10.3390/ijms23105467

Figure Lengend Snippet: Representative pictures show pituitary adenylate cyclase-activating peptide receptor (PAC1R) immunostaining in the myometrial layer of gilts from the control (CON), saline (SAL) and E. coli ( E. coli ) groups. Positive immunoreaction to PAC1R is visible in muscle cells and arteries (endothelium, muscle layer) of the myometrium of the control ( A ), saline-injected ( B ) and inflamed ( C ) uteri. Negative control (NC) for PAC1R ( D ) was obtained by omitting the primary antibody. MMC—myometrial muscle cells; A—artery. The scale bar of each image is 50 µm in length.

Article Snippet: To block the non-specific bindings, membranes were incubated with 5% fat-free dry milk in a TBS-T buffer at 21 °C for 1.5 h. They were then incubated at 4 °C for 18 h with primary PAC1 receptor polyclonal rabbit antibody (dilution: 1:1000, cat. no. CBS-PA207627, Cusabio Biotech Co.).

Techniques: Immunostaining, Control, Saline, Injection, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Investigation of the Role of Pituitary Adenylate Cyclase-Activating Peptide (PACAP) and Its Type 1 (PAC1) Receptor in Uterine Contractility during Endometritis in Pigs

doi: 10.3390/ijms23105467

Figure Lengend Snippet: Influence of pituitary adenylate cyclase-activating peptide (PACAP) alone ( A , C ) and PACAP receptor (PAC1R) antagonist with PACAP ( B , D ) on the contractile amplitude in the myometrium ( A , B ) and endometrium/myometrium ( C , D ) strips of gilts from the CON (grey bars), SAL (hatched bars) and E. coli (black bars) groups. Results were calculated for five gilts in each group. The actions of the antagonist (a dose of 10 − 6 M) and particular PACAP doses are depicted as percentage (mean ± SEM) changes from the basal (pre-treatment period) amplitude taken as 100% (horizontal lines). * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the basal value in each group; A p < 0.05, AA p <0.01, AAA p <0.001 between the CON and E. coli groups for the same treatment; BB p < 0.01, BBB p < 0.001 between the SAL and E. coli groups for the same treatment; C p < 0.05 between the CON and SAL groups for the same treatment; # p <0.05, ## p < 0.01, ### p < 0.001 between the antagonist with PACAP action versus PACAP action alone for the same group/tissue/PACAP dose.

Article Snippet: To block the non-specific bindings, membranes were incubated with 5% fat-free dry milk in a TBS-T buffer at 21 °C for 1.5 h. They were then incubated at 4 °C for 18 h with primary PAC1 receptor polyclonal rabbit antibody (dilution: 1:1000, cat. no. CBS-PA207627, Cusabio Biotech Co.).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Investigation of the Role of Pituitary Adenylate Cyclase-Activating Peptide (PACAP) and Its Type 1 (PAC1) Receptor in Uterine Contractility during Endometritis in Pigs

doi: 10.3390/ijms23105467

Figure Lengend Snippet: Influence of pituitary adenylate cyclase-activating peptide (PACAP) alone ( A , C ) and PACAP receptor (PAC1R) antagonist with PACAP ( B , D ) on the contractile frequency in the myometria ( A , B ) and endometrium/myometrium ( C , D ) strips of gilts from the CON (grey bars), SAL (hatched bars) and E. coli (black bars) groups. Results were calculated for five gilts in each group. The actions of the antagonist (a dose of 10 − 6 M) and particular PACAP doses are depicted as the percentage (mean ± SEM) change from the basal (pre-treatment period) frequency, taken as 100% (horizontal lines). * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the basal value in each group; A p < 0.05, AAA p < 0.001 compared between the CON and E. coli groups for the same treatment; B p < 0.05, BBB p < 0.001 compared between the SAL and E. coli groups for the same treatment; # p < 0.05, ## p < 0.01, ### p < 0.001 compared between the antagonist with PACAP action versus PACAP action alone for the same group/tissue/PACAP dose.

Article Snippet: To block the non-specific bindings, membranes were incubated with 5% fat-free dry milk in a TBS-T buffer at 21 °C for 1.5 h. They were then incubated at 4 °C for 18 h with primary PAC1 receptor polyclonal rabbit antibody (dilution: 1:1000, cat. no. CBS-PA207627, Cusabio Biotech Co.).

Techniques:

Journal: Nucleic Acids Research

Article Title: Systematic screening of CTCF binding partners identifies that BHLHE40 regulates CTCF genome-wide distribution and long-range chromatin interactions

doi: 10.1093/nar/gkaa705

Figure Lengend Snippet: The top 50 factors ranked by maximum overlap ratio between these factors and CTCF. The overlap ratio of each factor with CTCF was calculated by using the overlapped CTCF binding sites divided by the total number of CTCF binding sites. Each dot represents a ChIP-seq result for each factor in one of the ENCODE cell lines.

Article Snippet: The following antibodies were used in this study: mouse anti-ACTIN antibody (Abcam, ab3280), rabbit anti-CTCF antibody (Millipore, 07-729), anti-BIOTIN HRP-linked antibody (Cell Signaling Technology, #7075) for western blot, rabbit anti-BHLHE40 antibody (Novus, NB100-1800) for western blot and ChIP experiments, Flag M2 beads (Sigma, M8823) for Flag co-IP, Dynabeads M-280 Streptavidin (Thermo Fisher Scientific, 11205D) for BIOTIN ChIP-seq, rabbit anti-CTCF antibody (Active Motif, 61311) for CTCF HiChIP.

Techniques: Binding Assay, ChIP-sequencing

Journal: Nucleic Acids Research

Article Title: Systematic screening of CTCF binding partners identifies that BHLHE40 regulates CTCF genome-wide distribution and long-range chromatin interactions

doi: 10.1093/nar/gkaa705

Figure Lengend Snippet: Identification of human super conserved CTCF (hscCTCF) binding sites. ( A ) Bar plot showing the distribution of conserved CTCF binding sites derived from four CTCF ChIA-PET datasets in 99 CTCF ChIP-seq datasets. ( B ) The genomic distribution of hscCTCF binding sites and genome-wide CTCF binding sites for the indicated cell lines. Genomic features are color-coded in the legend bar. The x-axis shows the cumulative percentage of genomic occupancy of each feature. ( C ) The top 50 protein factors ranked by each factor's maximum overlap ratio with hscCTCF sites. The overlap ratio for each factor with hscCTCF was calculated by using the overlapped hscCTCF binding sites divided by total hscCTCF binding sites. Each dot represents a ChIP dataset. ( D ) Scatter plots showing the relationship between the factor overlap ratio generated using hscCTCF binding sites and the ratio generated using the top 30 000, 40 000, 50 000 and 60 000 CTCF binding sites.

Article Snippet: The following antibodies were used in this study: mouse anti-ACTIN antibody (Abcam, ab3280), rabbit anti-CTCF antibody (Millipore, 07-729), anti-BIOTIN HRP-linked antibody (Cell Signaling Technology, #7075) for western blot, rabbit anti-BHLHE40 antibody (Novus, NB100-1800) for western blot and ChIP experiments, Flag M2 beads (Sigma, M8823) for Flag co-IP, Dynabeads M-280 Streptavidin (Thermo Fisher Scientific, 11205D) for BIOTIN ChIP-seq, rabbit anti-CTCF antibody (Active Motif, 61311) for CTCF HiChIP.

Techniques: Binding Assay, Derivative Assay, ChIA Pet Assay, ChIP-sequencing, Genome Wide, Generated

Journal: Nucleic Acids Research

Article Title: Systematic screening of CTCF binding partners identifies that BHLHE40 regulates CTCF genome-wide distribution and long-range chromatin interactions

doi: 10.1093/nar/gkaa705

Figure Lengend Snippet: BHLHE40 influences the genomic binding of CTCF. ( A ) Bar plot showing shRNA knockdown efficiency assessed by RT-qPCR. Results are from three biological replicates. Data are represented as mean ± SEM. *** P <0.001. P -value is calculated using two-tailed Student's t test. ( B ) The number of CTCF peaks in control shRNA and BHLHE40-depleted HeLa-S3 cells. ( C ) Scatter plot showing the CTCF binding difference in control shRNA and BHLHE40-depleted HeLa-S3 cells. ( D ) Immunoprecipitation from HeLa-S3 nuclear extracts with FLAG antibody for FLAG-tagged CTCF. Bound proteins were resolved on SDS-PAGE and detected by western blotting for the indicated antigens. ( E ) Western blot results verifying the overexpression of BIOTIN-tagged BHLHE40 in HeLa-S3 cells. ( F ) Detection of the interaction between BHLHE40 and CTCF by BIOTIN IP experiments using soluble nuclear extracts of BIOTIN-tagged BHLHE40 HeLa-S3 cells. ( G ) Normalized tag density heatmap for BHLHE40 binding sites with corresponded CTCF binding sites. 2 kb regions are shown centered on the midpoints of the BHLHE40 peaks. ( H ) Screenshot from the WashU epigenome browser showing a BHLHE40/CTCF overlap binding sites at the promoter of LRRC58 . ChIP-qPCR results (lower bar charts) show the decrease of BHLHE40 and CTCF enrichment following sh BHLHE40 treatment. Data are from three biological replicates and represented as mean ± SEM. * P <0.05. P -value is calculated by using two-tailed Student's t test.

Article Snippet: The following antibodies were used in this study: mouse anti-ACTIN antibody (Abcam, ab3280), rabbit anti-CTCF antibody (Millipore, 07-729), anti-BIOTIN HRP-linked antibody (Cell Signaling Technology, #7075) for western blot, rabbit anti-BHLHE40 antibody (Novus, NB100-1800) for western blot and ChIP experiments, Flag M2 beads (Sigma, M8823) for Flag co-IP, Dynabeads M-280 Streptavidin (Thermo Fisher Scientific, 11205D) for BIOTIN ChIP-seq, rabbit anti-CTCF antibody (Active Motif, 61311) for CTCF HiChIP.

Techniques: Binding Assay, shRNA, Knockdown, Quantitative RT-PCR, Two Tailed Test, Control, Immunoprecipitation, SDS Page, Western Blot, Over Expression, ChIP-qPCR

Journal: Nucleic Acids Research

Article Title: Systematic screening of CTCF binding partners identifies that BHLHE40 regulates CTCF genome-wide distribution and long-range chromatin interactions

doi: 10.1093/nar/gkaa705

Figure Lengend Snippet: BHLHE40 depletion reduces CTCF-mediated chromatin loops. ( A ) Bar chart showing the number of CTCF loops with different loop strength in control shRNA and BHLHE40 shRNA-depleted HeLa-S3 cells. ( B ) Boxplot showing fold-change distribution of loop strength in different groups of CTCF loop anchors, which are classified by the fold change of CTCF binding strength between control shRNA and BHLHE40 shRNA-depleted HeLa-S3 cells. P value was calculated using Wilcoxon rank sum test. ( C ) The position relationship between differential CTCF loops resulted by BHLHE40 depletion and putative EP loops. Category 1 represents CTCF loops containing EP loops. Category 2 represents EP loops containing CTCF loops. Category 3 represents CTCF loops intersecting with EP loops. Category 4 represents that CTCF loops are the same as EP loops. Category 5 represents that CTCF loops do not intersect with EP loops. ( D ) Screenshot from the WashU epigenome browser showing the change of CTCF loops between control shRNA and BHLHE40 shRNA-depleted HeLa-S3 cells. The tracks of DNase, H3K4me1, H3K4me3 and H3K27ac ChIP-seq data were downloaded from the Roadmap Epigenome project . Chromatin interaction heatmaps were shown in 5 kb resolution. Significantly differential loops were marked with red asterisk. ( E ) Distribution of the expression fold change in different groups of genes classified by the position relationship between CTCF loops and EP loops. P value was calculated using Kruskal-Wallis test.

Article Snippet: The following antibodies were used in this study: mouse anti-ACTIN antibody (Abcam, ab3280), rabbit anti-CTCF antibody (Millipore, 07-729), anti-BIOTIN HRP-linked antibody (Cell Signaling Technology, #7075) for western blot, rabbit anti-BHLHE40 antibody (Novus, NB100-1800) for western blot and ChIP experiments, Flag M2 beads (Sigma, M8823) for Flag co-IP, Dynabeads M-280 Streptavidin (Thermo Fisher Scientific, 11205D) for BIOTIN ChIP-seq, rabbit anti-CTCF antibody (Active Motif, 61311) for CTCF HiChIP.

Techniques: Control, shRNA, Binding Assay, ChIP-sequencing, Expressing

Journal:

Article Title: The Binding Sites for the Chromatin Insulator Protein CTCF Map to DNA Methylation-Free Domains Genome-Wide

doi: 10.1101/gr.2408304

Figure Lengend Snippet: Immunofluorescent analysis of CTCF and HP1β in murine lung fibroblast cells. Colocalization of CTCF (Cy-3) and HP1β (FITC) clusters within the nuclei (DAPI) was seen after double immunostaining using rabbit anti-CTCF and goat anti-HP1β antibodies. Magnification ×1000. From left to right, images show merged CTCF/HP1/DAPI, CTCF (red), HP1 (green), DAPI (blue).

Article Snippet: ChIP DNA was prepared from fetal mouse liver by using either an affinity-purified rabbit antibody against the N-terminal portion of CTCF or a rabbit antibody against MBD2 (kind gift from Dr. A. Wolffe, Sangamo, Inc.).

Techniques: Double Immunostaining

Journal:

Article Title: The Binding Sites for the Chromatin Insulator Protein CTCF Map to DNA Methylation-Free Domains Genome-Wide

doi: 10.1101/gr.2408304

Figure Lengend Snippet: The insulator trap assay. (A) Schematic maps of the various constructs used in the classical insulator study. Symbols explained at the bottom of the panel. Each construct is linked to its performance in the enhancer-blocking assays, which were normalized to RNA input and episome copy number. The SV40 enhancer-driven expression of the pREPH19A construct was assigned a value of 100 whereas all other samples were normalized relative to this value. The mean deviation of three different experiments is indicated for each vector construct. (B) Schematic maps of the different pREPtox vectors. Cerise circle: the position of the SV40 enhancer. Green square: the H19 promoter. Pink and red blocks: the different inserts from clones (indicated by its original number) and H19 ICR, respectively. The numbers of the surviving clones were estimated from a colony count assay. (C) Outline of the strategy of the toxin-A assay and its application in microarray analysis of the CTCF target-site library. (D) An example of hybridization with input library sequences, affinity-purified (with recombinant CTCF) CTCF target sites, and the selection of clones with enhancer-blocking properties. (E) Presents scatter plot analyses of insulator strength, determined from the microarray analysis, and in vitro binding patterns, broken down into different sequence categories of the CTCF library. (F) Shows a scatter plot analysis between the insulator/in vitro binding ratios and DNA content of the corresponding spots of the microarrays, as determined by oligo hybridization.

Article Snippet: ChIP DNA was prepared from fetal mouse liver by using either an affinity-purified rabbit antibody against the N-terminal portion of CTCF or a rabbit antibody against MBD2 (kind gift from Dr. A. Wolffe, Sangamo, Inc.).

Techniques: TRAP Assay, Construct, Blocking Assay, Expressing, Plasmid Preparation, Clone Assay, Microarray, Hybridization, Affinity Purification, Recombinant, Selection, In Vitro, Binding Assay, Sequencing

Journal:

Article Title: The Binding Sites for the Chromatin Insulator Protein CTCF Map to DNA Methylation-Free Domains Genome-Wide

doi: 10.1101/gr.2408304

Figure Lengend Snippet: Cross-referencing methylation status with CTCF occupancy. (A) shows that an antibody against 5-methylcytidine immunopurifies only the methylated paternal H19 ICR allele if the maternally inherited allele is of the wild type. Conversely, when the mutated H19 ICR allele is inherited maternally (labeled 142* and unable to interact with CTCF in vivo while displaying massive de novo methylation; Pant et al. 2003), both alleles are brought down as determined by using PCR primers spanning CTCF target site #3 and a diagnostic EcoRV site (Pant et al. 2003). (B,C) Scatterplot analyses comparing CTCF in vivo occupancy/insulator strength vs. single CpG methylation (B, using an antibody against methylated cytidine) and clustered (C, using an antibody against MBD2) CpG methylation states in mouse fetal liver.

Article Snippet: ChIP DNA was prepared from fetal mouse liver by using either an affinity-purified rabbit antibody against the N-terminal portion of CTCF or a rabbit antibody against MBD2 (kind gift from Dr. A. Wolffe, Sangamo, Inc.).

Techniques: Methylation, Labeling, In Vivo, Diagnostic Assay, CpG Methylation Assay

Journal: eLife

Article Title: eRNA profiling uncovers the enhancer landscape of oesophageal adenocarcinoma and reveals new deregulated pathways

doi: 10.7554/eLife.80840

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit polyclonal anti-CTCF , Merck-Millipore , 07-729 , 0.5 μg/2–4 × 10 5 cells.

Techniques: Transfection, Plasmid Preparation, Control, Recombinant, Sequencing, Amplification, Luciferase, SYBR Green Assay, Concentration Assay, Software, Staining, Cell Culture