ctcf antibody  (Cell Signaling Technology Inc)

 
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    Name:
    CTCF Antibody
    Description:
    CCCTC binding factor CTCF and its paralog the Brother of the Regulator of Imprinted Sites BORIS are highly conserved transcription factors that regulate transcriptional activation and repression insulator function and imprinting control regions ICRs 1 4 Although they have divergent amino and carboxy termini both proteins contain 11 conserved zinc finger domains that work in combination to bind the same DNA elements 1 CTCF is ubiquitously expressed and contributes to transcriptional regulation of cell growth regulated genes including c myc p19 ARF p16 INK4A BRCA1 p53 p27 E2F1 and TERT 1 CTCF also binds to and is required for the enhancer blocking activity of all known insulator elements and ICRs including the H19 IgF2 Prader Willi Angelman syndrome and Inactive X Specific Transcript XIST anti sense loci 5 7 CTCF DNA binding is sensitive to DNA methylation a mark that determines selection of the imprinted allele maternal vs paternal 1 The various functions of CTCF are regulated by at least two different post translational modifications Poly ADP ribosyl ation of CTCF is required for insulator function 8 Phosphorylation of Ser612 by protein kinase CK2 facilitates a switch of CTCF from a transcriptional repressor to an activator at the c myc promoter 9 CTCF mutations or deletions have been found in many breast prostate and Wilms tumors 10 11 Expression of BORIS is restricted to spermatocytes and is mutually exclusive of CTCF 3 In cells expressing BORIS promoters of X linked cancer testis antigens like MAGE 1A are demethylated and activated but methylated and inactive in CTCF expressing somatic cells 12 Like other testis specific proteins BORIS is abnormally expressed in different cancers such as breast cancer and has a greater affinity than CTCF for DNA binding sites detracting from CTCF s potential tumor suppressing activity 1 3 13 14
    Catalog Number:
    2899
    Price:
    None
    Category:
    Primary Antibodies
    Source:
    Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the carboxy terminus of the human CTCF protein. Antibodies are purified by protein A and peptide affinity chromatography.
    Reactivity:
    Human Mouse Rat Monkey
    Applications:
    Western Blot, Immunoprecipitation, Immunofluorescence, Chromatin Immunoprecipitation
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc ctcf antibody
    Analysis of the rs4919742-associated chromatin loops. Guide RNAs targeting regions encompassing <t>CTCF</t> site 4 (the PCa risk-associated CTCF site), CTCF site 5, and/or CTCF site 6 (or the empty guide RNA vector as a control) were introduced into <t>22Rv1</t> prostate cancer cells, along with Cas9. Cell pools were harvested, and KRT78 expression was analyzed by RT-qPCR. Shown within the blue bars is the fold change in KRT78 expression in the pools that received guide RNAs vs the vector control. The yellow X indicates which CTCF site has been deleted; the size of each deletion can be found in Additional file 5 : Table S4
    CCCTC binding factor CTCF and its paralog the Brother of the Regulator of Imprinted Sites BORIS are highly conserved transcription factors that regulate transcriptional activation and repression insulator function and imprinting control regions ICRs 1 4 Although they have divergent amino and carboxy termini both proteins contain 11 conserved zinc finger domains that work in combination to bind the same DNA elements 1 CTCF is ubiquitously expressed and contributes to transcriptional regulation of cell growth regulated genes including c myc p19 ARF p16 INK4A BRCA1 p53 p27 E2F1 and TERT 1 CTCF also binds to and is required for the enhancer blocking activity of all known insulator elements and ICRs including the H19 IgF2 Prader Willi Angelman syndrome and Inactive X Specific Transcript XIST anti sense loci 5 7 CTCF DNA binding is sensitive to DNA methylation a mark that determines selection of the imprinted allele maternal vs paternal 1 The various functions of CTCF are regulated by at least two different post translational modifications Poly ADP ribosyl ation of CTCF is required for insulator function 8 Phosphorylation of Ser612 by protein kinase CK2 facilitates a switch of CTCF from a transcriptional repressor to an activator at the c myc promoter 9 CTCF mutations or deletions have been found in many breast prostate and Wilms tumors 10 11 Expression of BORIS is restricted to spermatocytes and is mutually exclusive of CTCF 3 In cells expressing BORIS promoters of X linked cancer testis antigens like MAGE 1A are demethylated and activated but methylated and inactive in CTCF expressing somatic cells 12 Like other testis specific proteins BORIS is abnormally expressed in different cancers such as breast cancer and has a greater affinity than CTCF for DNA binding sites detracting from CTCF s potential tumor suppressing activity 1 3 13 14
    https://www.bioz.com/result/ctcf antibody/product/Cell Signaling Technology Inc
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    ctcf antibody - by Bioz Stars, 2021-09
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    Images

    1) Product Images from "CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops"

    Article Title: CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops

    Journal: Genome Biology

    doi: 10.1186/s13059-018-1531-0

    Analysis of the rs4919742-associated chromatin loops. Guide RNAs targeting regions encompassing CTCF site 4 (the PCa risk-associated CTCF site), CTCF site 5, and/or CTCF site 6 (or the empty guide RNA vector as a control) were introduced into 22Rv1 prostate cancer cells, along with Cas9. Cell pools were harvested, and KRT78 expression was analyzed by RT-qPCR. Shown within the blue bars is the fold change in KRT78 expression in the pools that received guide RNAs vs the vector control. The yellow X indicates which CTCF site has been deleted; the size of each deletion can be found in Additional file 5 : Table S4
    Figure Legend Snippet: Analysis of the rs4919742-associated chromatin loops. Guide RNAs targeting regions encompassing CTCF site 4 (the PCa risk-associated CTCF site), CTCF site 5, and/or CTCF site 6 (or the empty guide RNA vector as a control) were introduced into 22Rv1 prostate cancer cells, along with Cas9. Cell pools were harvested, and KRT78 expression was analyzed by RT-qPCR. Shown within the blue bars is the fold change in KRT78 expression in the pools that received guide RNAs vs the vector control. The yellow X indicates which CTCF site has been deleted; the size of each deletion can be found in Additional file 5 : Table S4

    Techniques Used: Plasmid Preparation, Expressing, Quantitative RT-PCR

    Experimental workflow for functional investigation of PCa risk-associated CTCF sites. Phase 1: Plasmids encoding guide RNAs that target sequences on each side of a PCa risk-associated CTCF site were introduced into the PCa cell line 22Rv1 along with a Cas9 expression vector (see the “ Methods ” section for details). The resultant cell pool was analyzed to determine deletion efficiency (red slashes represent alleles in each cell that harbor a CTCF site deletion). Single cells were then selected and expanded into clonal populations for RNA-seq analysis. Phase 2: After identifying the gene most responsive (within a ± 1-Mb window) to deletion of the region encompassing a risk-associated CTCF site, plasmids encoding guide RNAs that target the risk-associated CTCF anchor region and/or the regions encompassing the CTCF sites looped to the risk CTCF site and a Cas9 expression plasmid were introduced into 22Rv1 cells; cell pools were analyzed by PCR to check deletion frequency and by RT-qPCR to measure expression of the target gene
    Figure Legend Snippet: Experimental workflow for functional investigation of PCa risk-associated CTCF sites. Phase 1: Plasmids encoding guide RNAs that target sequences on each side of a PCa risk-associated CTCF site were introduced into the PCa cell line 22Rv1 along with a Cas9 expression vector (see the “ Methods ” section for details). The resultant cell pool was analyzed to determine deletion efficiency (red slashes represent alleles in each cell that harbor a CTCF site deletion). Single cells were then selected and expanded into clonal populations for RNA-seq analysis. Phase 2: After identifying the gene most responsive (within a ± 1-Mb window) to deletion of the region encompassing a risk-associated CTCF site, plasmids encoding guide RNAs that target the risk-associated CTCF anchor region and/or the regions encompassing the CTCF sites looped to the risk CTCF site and a Cas9 expression plasmid were introduced into 22Rv1 cells; cell pools were analyzed by PCR to check deletion frequency and by RT-qPCR to measure expression of the target gene

    Techniques Used: Functional Assay, Expressing, Plasmid Preparation, RNA Sequencing Assay, Polymerase Chain Reaction, Quantitative RT-PCR

    Analysis of the rs12144978-associated chromatin loops. Guide RNAs targeting regions encompassing CTCF site 1 (the PCa risk-associated CTCF site), CTCF site 2, and/or CTCF site 3 (or the empty guide RNA vector as a control) were introduced into 22Rv1 prostate cancer cells, along with Cas9. Cell pools were harvested, and KCNN3 expression was analyzed by RT-qPCR. Shown within the blue bars is the fold change in KCNN3 expression in the pools that received guide RNAs vs. the vector control. The yellow X indicates which CTCF site has been deleted; the size of each deletion can be found in Additional file 5 : Table S4
    Figure Legend Snippet: Analysis of the rs12144978-associated chromatin loops. Guide RNAs targeting regions encompassing CTCF site 1 (the PCa risk-associated CTCF site), CTCF site 2, and/or CTCF site 3 (or the empty guide RNA vector as a control) were introduced into 22Rv1 prostate cancer cells, along with Cas9. Cell pools were harvested, and KCNN3 expression was analyzed by RT-qPCR. Shown within the blue bars is the fold change in KCNN3 expression in the pools that received guide RNAs vs. the vector control. The yellow X indicates which CTCF site has been deleted; the size of each deletion can be found in Additional file 5 : Table S4

    Techniques Used: Plasmid Preparation, Expressing, Quantitative RT-PCR

    Experimental and analytical steps used to identify PCa risk-associated regulatory elements involved in chromatin loops. Step (1): The subset of 2,181 fine-mapped PCa-associated SNPs that overlap a DNase hypersensitive site was identified. Step (2): H3K27Ac and CTCF ChIP-seq was performed in duplicate in two normal (PrEC and RWPE-1) and five cancer (RWPE-2, 22Rv1, C4-2B, LNCaP, and VCaP) prostate cell lines; data was collected plus or minus DHT for 22Rv1 and LNCaP cells, for a total of 18 datasets for each mark (36 ChIP-seq samples). The SNPs in open chromatin sites (i.e., those that are contained within a DHS site) were then subdivided into those that overlap a H3K27Ac or a CTCF site in prostate cells; the number of PCa-associated SNPs associated with the H3K27Ac or CTCF sites is shown. Step (3): The PCa risk-associated H3K27Ac and CTCF sites were overlapped with Hi-C looping data, and the subset of each type of site involved in chromatin loops was identified; the number of PCa-associated SNPs associated with the H3K27Ac or CTCF sites involved in looping is shown
    Figure Legend Snippet: Experimental and analytical steps used to identify PCa risk-associated regulatory elements involved in chromatin loops. Step (1): The subset of 2,181 fine-mapped PCa-associated SNPs that overlap a DNase hypersensitive site was identified. Step (2): H3K27Ac and CTCF ChIP-seq was performed in duplicate in two normal (PrEC and RWPE-1) and five cancer (RWPE-2, 22Rv1, C4-2B, LNCaP, and VCaP) prostate cell lines; data was collected plus or minus DHT for 22Rv1 and LNCaP cells, for a total of 18 datasets for each mark (36 ChIP-seq samples). The SNPs in open chromatin sites (i.e., those that are contained within a DHS site) were then subdivided into those that overlap a H3K27Ac or a CTCF site in prostate cells; the number of PCa-associated SNPs associated with the H3K27Ac or CTCF sites is shown. Step (3): The PCa risk-associated H3K27Ac and CTCF sites were overlapped with Hi-C looping data, and the subset of each type of site involved in chromatin loops was identified; the number of PCa-associated SNPs associated with the H3K27Ac or CTCF sites involved in looping is shown

    Techniques Used: Chromatin Immunoprecipitation, Hi-C

    Identification and classification of H3K27Ac ( a ) and CTCF ( b ) sites in prostate cells. H3K27Ac and CTCF ChIP-seq was performed in duplicate for each cell line; for 22Rv1 and LNCaP cells, ChIP-seq was performed in duplicate in the presence or absence of DHT. Peaks were called for individual datasets using MACS2 and the ENCODE3 pipeline, then peaks present in both replicates were identified (high confidence peaks) and used for further analysis (see Additional file 3 : Table S2). The location of the peaks was classified using the HOMER annotatePeaks.pl program and the Gencode V19 database. The fraction of high confidence peaks in each category is shown on the Y axis, with the number of peaks in each category for each individual cell line and/or treatment shown within each bar
    Figure Legend Snippet: Identification and classification of H3K27Ac ( a ) and CTCF ( b ) sites in prostate cells. H3K27Ac and CTCF ChIP-seq was performed in duplicate for each cell line; for 22Rv1 and LNCaP cells, ChIP-seq was performed in duplicate in the presence or absence of DHT. Peaks were called for individual datasets using MACS2 and the ENCODE3 pipeline, then peaks present in both replicates were identified (high confidence peaks) and used for further analysis (see Additional file 3 : Table S2). The location of the peaks was classified using the HOMER annotatePeaks.pl program and the Gencode V19 database. The fraction of high confidence peaks in each category is shown on the Y axis, with the number of peaks in each category for each individual cell line and/or treatment shown within each bar

    Techniques Used: Chromatin Immunoprecipitation

    PCa risk SNPs associated with CTCF sites and chromatin loops. Each row represents one of the 93 SNPs that are associated with both a DHS site and a CTCF peak in normal or tumor prostate cells (Additional file 4 : Table S3). The location of each SNP was classified using the Gencode V19 database. “Others” represents mostly intergenic regions. To identify the subset of CTCF-associated risk SNPs located in an anchor point of a loop, chromatin loops were identified using Hi-C data from normal RWPE-1 prostate cells [ 26 ] or 22Rv1 and C4-2B prostate tumor cells (Rhie et al., in preparation); Hi-C [ 25 ] and cohesin HiChIP data [ 27 ] from GM12878 was also used
    Figure Legend Snippet: PCa risk SNPs associated with CTCF sites and chromatin loops. Each row represents one of the 93 SNPs that are associated with both a DHS site and a CTCF peak in normal or tumor prostate cells (Additional file 4 : Table S3). The location of each SNP was classified using the Gencode V19 database. “Others” represents mostly intergenic regions. To identify the subset of CTCF-associated risk SNPs located in an anchor point of a loop, chromatin loops were identified using Hi-C data from normal RWPE-1 prostate cells [ 26 ] or 22Rv1 and C4-2B prostate tumor cells (Rhie et al., in preparation); Hi-C [ 25 ] and cohesin HiChIP data [ 27 ] from GM12878 was also used

    Techniques Used: Hi-C, HiChIP

    2) Product Images from "Contribution of CTCF binding to transcriptional activity at the HOXA locus in NPM1-mutant AML cells"

    Article Title: Contribution of CTCF binding to transcriptional activity at the HOXA locus in NPM1-mutant AML cells

    Journal: bioRxiv

    doi: 10.1101/2020.02.17.952390

    CTCF defines chromatin boundaries in AML samples and normal CD34 + cells with HOXA gene expression. A. Top track shows CTCF ChIP-seq from a NPM1 -mutant primary AML sample. Tracks highlighted in yellow show ChIP-seq for H3K4me3 from primary samples with high HOXA expression, including AML samples with MLL rearrangements (green) and NPM1 mutations (purple), and primary CD34 + hematopoietic stem/progenitor cells (HSPCs) purified from normal donor bone marrow samples (gray; GSE104579). Tracks highlighted in blue show H3K27me3 ChIP-seq from the same set of samples. B. H3K4me3 and H3K27me3 ChIP-seq from primary samples with no HOXA expression, including AML samples with t(8;21) creating the RUNX1-RUNX1T1 fusion, and normal promyelocytes (CD14-, CD15+, CD16 low) and neutrophils (CD14-, CD15+, CD16 high) from healthy donor individuals. Dashed box indicates the region of dynamic chromatin that correlates with HOXA gene cluster expression.
    Figure Legend Snippet: CTCF defines chromatin boundaries in AML samples and normal CD34 + cells with HOXA gene expression. A. Top track shows CTCF ChIP-seq from a NPM1 -mutant primary AML sample. Tracks highlighted in yellow show ChIP-seq for H3K4me3 from primary samples with high HOXA expression, including AML samples with MLL rearrangements (green) and NPM1 mutations (purple), and primary CD34 + hematopoietic stem/progenitor cells (HSPCs) purified from normal donor bone marrow samples (gray; GSE104579). Tracks highlighted in blue show H3K27me3 ChIP-seq from the same set of samples. B. H3K4me3 and H3K27me3 ChIP-seq from primary samples with no HOXA expression, including AML samples with t(8;21) creating the RUNX1-RUNX1T1 fusion, and normal promyelocytes (CD14-, CD15+, CD16 low) and neutrophils (CD14-, CD15+, CD16 high) from healthy donor individuals. Dashed box indicates the region of dynamic chromatin that correlates with HOXA gene cluster expression.

    Techniques Used: Expressing, Chromatin Immunoprecipitation, Mutagenesis, Purification

    Targeted deletions eliminate CTCF binding in the NPM1 -mutant OCI-AML3 cell line. A. RNA-seq expression of HOXA and HOXB genes in OCI-AML3 cells, which display the canonical mutant NPM1 -associated HOXA/HOXB expression phenotype. Also shown are the MLL -rearranged MOLM13 cell line that expresses only HOXA genes, and the RUNX1-RUNX1T1 -containing Kasumi-1 cell line that has low HOXA and HOXB gene expression. B. ChIP-seq data from OCI-AML3 cells for CTCF (black), H3K4me3 (highlighted in yellow) and H3K27me3 (highlighted in blue), which show conserved CTCF binding sites and distinct regions of active (H3K4me3) and repressed (H3K27me3) chromatin. C-E. Targeted deletions that disrupt CTCF binding in OCI-AML3 cells at sites CBSA6/7 (in C), CBSA7/9 (in D), and CBSA10 (in E). Bottom panels show allele pairs from homozygous or compound heterozygous deletion mutants at each site; top panels show CTCF ChIP-seq signal from these mutant cell lines (multi-colored lines) compared to wild type OCI-AML3 cells (in gray). F-H. Normalized CTCF ChIP-seq for all CTCF peaks from deletion mutants (Y axis) vs. wild type OCI-AML3 cells, showing dramatically reduced CTCF ChIP-seq signal in deletion mutants at all three sites, with the exception of clones A101 and A091.31, which only partially eliminates CTCF signal at site CBSA6/7. I. CTCF ChIP-seq tracks from double (in purple) and triple mutants (in blue), generated via sequential targeted deletion experiments. CTCF ChIP-seq from wild type OCI-AML3 cells is shown in black at the top for reference.
    Figure Legend Snippet: Targeted deletions eliminate CTCF binding in the NPM1 -mutant OCI-AML3 cell line. A. RNA-seq expression of HOXA and HOXB genes in OCI-AML3 cells, which display the canonical mutant NPM1 -associated HOXA/HOXB expression phenotype. Also shown are the MLL -rearranged MOLM13 cell line that expresses only HOXA genes, and the RUNX1-RUNX1T1 -containing Kasumi-1 cell line that has low HOXA and HOXB gene expression. B. ChIP-seq data from OCI-AML3 cells for CTCF (black), H3K4me3 (highlighted in yellow) and H3K27me3 (highlighted in blue), which show conserved CTCF binding sites and distinct regions of active (H3K4me3) and repressed (H3K27me3) chromatin. C-E. Targeted deletions that disrupt CTCF binding in OCI-AML3 cells at sites CBSA6/7 (in C), CBSA7/9 (in D), and CBSA10 (in E). Bottom panels show allele pairs from homozygous or compound heterozygous deletion mutants at each site; top panels show CTCF ChIP-seq signal from these mutant cell lines (multi-colored lines) compared to wild type OCI-AML3 cells (in gray). F-H. Normalized CTCF ChIP-seq for all CTCF peaks from deletion mutants (Y axis) vs. wild type OCI-AML3 cells, showing dramatically reduced CTCF ChIP-seq signal in deletion mutants at all three sites, with the exception of clones A101 and A091.31, which only partially eliminates CTCF signal at site CBSA6/7. I. CTCF ChIP-seq tracks from double (in purple) and triple mutants (in blue), generated via sequential targeted deletion experiments. CTCF ChIP-seq from wild type OCI-AML3 cells is shown in black at the top for reference.

    Techniques Used: Binding Assay, Mutagenesis, RNA Sequencing Assay, Expressing, Chromatin Immunoprecipitation, Clone Assay, Generated

    CTCF binding is not required to maintain gene expression or chromatin boundaries in the HOXA gene cluster. A. qPCR for HOXA9 in single mutants with heterozygous or homozygous deletions at CBSA6/7, CBSA7/9, or CBSA10. HOXA9 expression from the Kasumi-1 cell line is shown in blue as a no- HOXA9 expressing control. B. qPCR for HOXA9 in double and triple mutants at the CTCF binding sites indicated. Kasumi-1 cells are included in blue as in panel A. C-F. RNA-seq expression of all HOXA genes in single mutants (C-E) and triple mutants (D). Expression level is shown in log2 transcripts per million (TPM). G. ChIP-seq for H3K4me3 and H3K27me3 in deletion mutants lacking CTCF at site CBSA7/9 (in red; N=2). Mean ChIP-seq signal from wild type OCI-AML3 cells (N=2) is shown in gray. H. Mean ChIP-seq for H3K4me3 and H3K27me3 in triple mutants (N=2), with ChIP-seq from wild type cells shown in gray, as in panel G.
    Figure Legend Snippet: CTCF binding is not required to maintain gene expression or chromatin boundaries in the HOXA gene cluster. A. qPCR for HOXA9 in single mutants with heterozygous or homozygous deletions at CBSA6/7, CBSA7/9, or CBSA10. HOXA9 expression from the Kasumi-1 cell line is shown in blue as a no- HOXA9 expressing control. B. qPCR for HOXA9 in double and triple mutants at the CTCF binding sites indicated. Kasumi-1 cells are included in blue as in panel A. C-F. RNA-seq expression of all HOXA genes in single mutants (C-E) and triple mutants (D). Expression level is shown in log2 transcripts per million (TPM). G. ChIP-seq for H3K4me3 and H3K27me3 in deletion mutants lacking CTCF at site CBSA7/9 (in red; N=2). Mean ChIP-seq signal from wild type OCI-AML3 cells (N=2) is shown in gray. H. Mean ChIP-seq for H3K4me3 and H3K27me3 in triple mutants (N=2), with ChIP-seq from wild type cells shown in gray, as in panel G.

    Techniques Used: Binding Assay, Expressing, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Chromatin Immunoprecipitation

    3) Product Images from "Active enhancers strengthen TAD insulation by bRNA mediated CTCF enrichment at the TAD boundaries"

    Article Title: Active enhancers strengthen TAD insulation by bRNA mediated CTCF enrichment at the TAD boundaries

    Journal: bioRxiv

    doi: 10.1101/2021.07.13.452118

    RNA augment the recruitment of CTCF at the TAD boundary: A. The browser shot showing the CTCF cut and run peaks at INK4a/ARF TAD with and without RNaseA treatment. B. qRT-PCRs showing the levels of; sense and anti-sense non-coding RNA at B1and B2 CTCF sites and of snoRNA upon B1 and B2 RNA knockdown in chromatin associated RNA fraction . C. qRT-PCRs showing the fold change in sense and anti-sense RNAs from B1 and B2 CTCF sites upon Flag-CTCF over expression. Last two bars show the increase in CTCF levels. D. TADs around INK4a/ARF TAD and position of arrows shows the CTCF sites at upstream and downstream TAD boundaries where CTCF occupancy was interrogated. E. Immunoblotting with CTCF and Histone H3 on soluble nucleoplasm and chromatin bound fractions of the nucleus treated with or without RNaseA F. CTCF cut Run signal at CTCF sites within different categories of boundaries. Error bars denote SEM from three biological replicates. p -values were calculated by Student’s two-tailed unpaired t -test in B and C. **** p
    Figure Legend Snippet: RNA augment the recruitment of CTCF at the TAD boundary: A. The browser shot showing the CTCF cut and run peaks at INK4a/ARF TAD with and without RNaseA treatment. B. qRT-PCRs showing the levels of; sense and anti-sense non-coding RNA at B1and B2 CTCF sites and of snoRNA upon B1 and B2 RNA knockdown in chromatin associated RNA fraction . C. qRT-PCRs showing the fold change in sense and anti-sense RNAs from B1 and B2 CTCF sites upon Flag-CTCF over expression. Last two bars show the increase in CTCF levels. D. TADs around INK4a/ARF TAD and position of arrows shows the CTCF sites at upstream and downstream TAD boundaries where CTCF occupancy was interrogated. E. Immunoblotting with CTCF and Histone H3 on soluble nucleoplasm and chromatin bound fractions of the nucleus treated with or without RNaseA F. CTCF cut Run signal at CTCF sites within different categories of boundaries. Error bars denote SEM from three biological replicates. p -values were calculated by Student’s two-tailed unpaired t -test in B and C. **** p

    Techniques Used: Over Expression, Two Tailed Test

    RNA augments the recruitment of CTCF at the TAD boundary: A. Immunoblotting with CTCF, Histone H3 and GAPDH on soluble nucleoplasm and chromatin bound fractions of the nucleus treated with or without RNaseA (Left panel). The right panel shows the quantified loss of CTCF from chromatin fractions from two replicates. B. Normalised CTCF cut and run signal in control and RNAse A treated conditions plotted at equally scaled TADs of 50kb length each and 25kb flanks . C. TAD structure at INK4a/ARF locus on 9p21 region ( Rao et al., 2014 ). The loop domain (Blue bar) is overlaid by the position of CTCF peaks, CTCF motif orientation, H3K4me3, H3K27ac (enhancers), mNET-seq tracks and gene annotations. The highlighted region shows the B1, B2 and B3 CTCF sites on 3’ boundary and super enhancer with position of enhancer E8 marked by an arrow. D. qRT-PCRs showing the levels of sense and anti-sense non-coding RNA at B1 CTCF site upon B1 RNA knockdown E. qRT-PCRs showing the levels of sense and anti-sense RNA at B2 CTCF site upon B2 RNA knockdown. F. CTCF ChIP enrichment before and after shRNA mediated knockdown of sense and anti-sense RNA at B1 CTCF sites. The bars show the CTCF on three CTCF sites (B1, B2 and B3) at 3’ boundary, on -T1 (5’ boundary) of INK4a/ARF TAD, -T2 on adjacent TAD boundary upstream, +T1 (on adjacent TAD boundary downstream) and +T2 the boundary of following TAD downstream. G. CTCF ChIP enrichment before and after shRNA mediated knockdown of sense and anti-sense RNA at B2 CTCF sites. The bars show the CTCF on three CTCF sites (B1, B2 and B3) at 3’ boundary, on -T1 (5’ boundary) of INK4a/ARF TAD, -T2 on adjacent TAD boundary upstream, +T1 (on adjacent TAD boundary downstream) and +T2 the boundary of following TAD downstream. Error bars denote SEM from three biological replicates. p -values were calculated by Student’s two-tailed unpaired t -test in D, E, F and G. ** p
    Figure Legend Snippet: RNA augments the recruitment of CTCF at the TAD boundary: A. Immunoblotting with CTCF, Histone H3 and GAPDH on soluble nucleoplasm and chromatin bound fractions of the nucleus treated with or without RNaseA (Left panel). The right panel shows the quantified loss of CTCF from chromatin fractions from two replicates. B. Normalised CTCF cut and run signal in control and RNAse A treated conditions plotted at equally scaled TADs of 50kb length each and 25kb flanks . C. TAD structure at INK4a/ARF locus on 9p21 region ( Rao et al., 2014 ). The loop domain (Blue bar) is overlaid by the position of CTCF peaks, CTCF motif orientation, H3K4me3, H3K27ac (enhancers), mNET-seq tracks and gene annotations. The highlighted region shows the B1, B2 and B3 CTCF sites on 3’ boundary and super enhancer with position of enhancer E8 marked by an arrow. D. qRT-PCRs showing the levels of sense and anti-sense non-coding RNA at B1 CTCF site upon B1 RNA knockdown E. qRT-PCRs showing the levels of sense and anti-sense RNA at B2 CTCF site upon B2 RNA knockdown. F. CTCF ChIP enrichment before and after shRNA mediated knockdown of sense and anti-sense RNA at B1 CTCF sites. The bars show the CTCF on three CTCF sites (B1, B2 and B3) at 3’ boundary, on -T1 (5’ boundary) of INK4a/ARF TAD, -T2 on adjacent TAD boundary upstream, +T1 (on adjacent TAD boundary downstream) and +T2 the boundary of following TAD downstream. G. CTCF ChIP enrichment before and after shRNA mediated knockdown of sense and anti-sense RNA at B2 CTCF sites. The bars show the CTCF on three CTCF sites (B1, B2 and B3) at 3’ boundary, on -T1 (5’ boundary) of INK4a/ARF TAD, -T2 on adjacent TAD boundary upstream, +T1 (on adjacent TAD boundary downstream) and +T2 the boundary of following TAD downstream. Error bars denote SEM from three biological replicates. p -values were calculated by Student’s two-tailed unpaired t -test in D, E, F and G. ** p

    Techniques Used: Chromatin Immunoprecipitation, shRNA, Two Tailed Test

    4) Product Images from "Proper orientation of CTCF sites in Cer is required for normal Jκ-distal and proximal Vκ gene usage"

    Article Title: Proper orientation of CTCF sites in Cer is required for normal Jκ-distal and proximal Vκ gene usage

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1800785

    Cer CTCF orientation regulates Vκ rearrangement. (A) Vκ rearrangement assay comparing Cer deletion clones to WT 445.3 cells. Three g1g2 biallelic Cer deletion clones (#1-#3) (navy), a g3g5 biallelic Cer 1 st CTCF site deletion clone (red) and two g4g6 biallelic Cer 2 nd CTCF site deletion clones (brown) were assessed. Guide locations are shown on left side. cDNA was obtained at 48 hours after STI571 stimulation for qPCR analysis of rearrangement. For bar graph, Vκ genes are listed at the bottom. Bars above the black vertical line indicate higher expression relative to WT whereas bars below the black vertical line indicate lower expression. Data for WT, g1g2 deletion clones and 1 st CTCF deletion clone represent two independent Rag1 transductions and three independent STI571 treatments. Data from the 2 nd CTCF deletion clones represents one Rag1 transduction and two independent STI571 treatments. (B) Vκ rearrangement assay comparing the three biallelic g1g2 deletion clones and the two biallelic g1g2 inversion clones (#1 and #2) to WT for cDNA. Green bars indicate inversion clones. Data for WT, all deletion clones and inversion clone #1 represents 3 independent Rag1 transductions and 4 independent STI571 treatments. Data for inversion clone #2 represents 2 independent Rag1 transductions and 3 independent STI571 treatments. Error bars represent SEM.
    Figure Legend Snippet: Cer CTCF orientation regulates Vκ rearrangement. (A) Vκ rearrangement assay comparing Cer deletion clones to WT 445.3 cells. Three g1g2 biallelic Cer deletion clones (#1-#3) (navy), a g3g5 biallelic Cer 1 st CTCF site deletion clone (red) and two g4g6 biallelic Cer 2 nd CTCF site deletion clones (brown) were assessed. Guide locations are shown on left side. cDNA was obtained at 48 hours after STI571 stimulation for qPCR analysis of rearrangement. For bar graph, Vκ genes are listed at the bottom. Bars above the black vertical line indicate higher expression relative to WT whereas bars below the black vertical line indicate lower expression. Data for WT, g1g2 deletion clones and 1 st CTCF deletion clone represent two independent Rag1 transductions and three independent STI571 treatments. Data from the 2 nd CTCF deletion clones represents one Rag1 transduction and two independent STI571 treatments. (B) Vκ rearrangement assay comparing the three biallelic g1g2 deletion clones and the two biallelic g1g2 inversion clones (#1 and #2) to WT for cDNA. Green bars indicate inversion clones. Data for WT, all deletion clones and inversion clone #1 represents 3 independent Rag1 transductions and 4 independent STI571 treatments. Data for inversion clone #2 represents 2 independent Rag1 transductions and 3 independent STI571 treatments. Error bars represent SEM.

    Techniques Used: Clone Assay, Real-time Polymerase Chain Reaction, Expressing, Transduction

    5) Product Images from "Contribution of CTCF binding to transcriptional activity at the HOXA locus in NPM1-mutant AML cells"

    Article Title: Contribution of CTCF binding to transcriptional activity at the HOXA locus in NPM1-mutant AML cells

    Journal: bioRxiv

    doi: 10.1101/2020.02.17.952390

    CTCF defines chromatin boundaries in AML samples and normal CD34 + cells with HOXA gene expression. A. Top track shows CTCF ChIP-seq from a NPM1 -mutant primary AML sample. Tracks highlighted in yellow show ChIP-seq for H3K4me3 from primary samples with high HOXA expression, including AML samples with MLL rearrangements (green) and NPM1 mutations (purple), and primary CD34 + hematopoietic stem/progenitor cells (HSPCs) purified from normal donor bone marrow samples (gray; GSE104579). Tracks highlighted in blue show H3K27me3 ChIP-seq from the same set of samples. B. H3K4me3 and H3K27me3 ChIP-seq from primary samples with no HOXA expression, including AML samples with t(8;21) creating the RUNX1-RUNX1T1 fusion, and normal promyelocytes (CD14-, CD15+, CD16 low) and neutrophils (CD14-, CD15+, CD16 high) from healthy donor individuals. Dashed box indicates the region of dynamic chromatin that correlates with HOXA gene cluster expression.
    Figure Legend Snippet: CTCF defines chromatin boundaries in AML samples and normal CD34 + cells with HOXA gene expression. A. Top track shows CTCF ChIP-seq from a NPM1 -mutant primary AML sample. Tracks highlighted in yellow show ChIP-seq for H3K4me3 from primary samples with high HOXA expression, including AML samples with MLL rearrangements (green) and NPM1 mutations (purple), and primary CD34 + hematopoietic stem/progenitor cells (HSPCs) purified from normal donor bone marrow samples (gray; GSE104579). Tracks highlighted in blue show H3K27me3 ChIP-seq from the same set of samples. B. H3K4me3 and H3K27me3 ChIP-seq from primary samples with no HOXA expression, including AML samples with t(8;21) creating the RUNX1-RUNX1T1 fusion, and normal promyelocytes (CD14-, CD15+, CD16 low) and neutrophils (CD14-, CD15+, CD16 high) from healthy donor individuals. Dashed box indicates the region of dynamic chromatin that correlates with HOXA gene cluster expression.

    Techniques Used: Expressing, Chromatin Immunoprecipitation, Mutagenesis, Purification

    Targeted deletions eliminate CTCF binding in the NPM1 -mutant OCI-AML3 cell line. A. RNA-seq expression of HOXA and HOXB genes in OCI-AML3 cells, which display the canonical mutant NPM1 -associated HOXA/HOXB expression phenotype. Also shown are the MLL -rearranged MOLM13 cell line that expresses only HOXA genes, and the RUNX1-RUNX1T1 -containing Kasumi-1 cell line that has low HOXA and HOXB gene expression. B. ChIP-seq data from OCI-AML3 cells for CTCF (black), H3K4me3 (highlighted in yellow) and H3K27me3 (highlighted in blue), which show conserved CTCF binding sites and distinct regions of active (H3K4me3) and repressed (H3K27me3) chromatin. C-E. Targeted deletions that disrupt CTCF binding in OCI-AML3 cells at sites CBSA6/7 (in C), CBSA7/9 (in D), and CBSA10 (in E). Bottom panels show allele pairs from homozygous or compound heterozygous deletion mutants at each site; top panels show CTCF ChIP-seq signal from these mutant cell lines (multi-colored lines) compared to wild type OCI-AML3 cells (in gray). F-H. Normalized CTCF ChIP-seq for all CTCF peaks from deletion mutants (Y axis) vs. wild type OCI-AML3 cells, showing dramatically reduced CTCF ChIP-seq signal in deletion mutants at all three sites, with the exception of clones A101 and A091.31, which only partially eliminates CTCF signal at site CBSA6/7. I. CTCF ChIP-seq tracks from double (in purple) and triple mutants (in blue), generated via sequential targeted deletion experiments. CTCF ChIP-seq from wild type OCI-AML3 cells is shown in black at the top for reference.
    Figure Legend Snippet: Targeted deletions eliminate CTCF binding in the NPM1 -mutant OCI-AML3 cell line. A. RNA-seq expression of HOXA and HOXB genes in OCI-AML3 cells, which display the canonical mutant NPM1 -associated HOXA/HOXB expression phenotype. Also shown are the MLL -rearranged MOLM13 cell line that expresses only HOXA genes, and the RUNX1-RUNX1T1 -containing Kasumi-1 cell line that has low HOXA and HOXB gene expression. B. ChIP-seq data from OCI-AML3 cells for CTCF (black), H3K4me3 (highlighted in yellow) and H3K27me3 (highlighted in blue), which show conserved CTCF binding sites and distinct regions of active (H3K4me3) and repressed (H3K27me3) chromatin. C-E. Targeted deletions that disrupt CTCF binding in OCI-AML3 cells at sites CBSA6/7 (in C), CBSA7/9 (in D), and CBSA10 (in E). Bottom panels show allele pairs from homozygous or compound heterozygous deletion mutants at each site; top panels show CTCF ChIP-seq signal from these mutant cell lines (multi-colored lines) compared to wild type OCI-AML3 cells (in gray). F-H. Normalized CTCF ChIP-seq for all CTCF peaks from deletion mutants (Y axis) vs. wild type OCI-AML3 cells, showing dramatically reduced CTCF ChIP-seq signal in deletion mutants at all three sites, with the exception of clones A101 and A091.31, which only partially eliminates CTCF signal at site CBSA6/7. I. CTCF ChIP-seq tracks from double (in purple) and triple mutants (in blue), generated via sequential targeted deletion experiments. CTCF ChIP-seq from wild type OCI-AML3 cells is shown in black at the top for reference.

    Techniques Used: Binding Assay, Mutagenesis, RNA Sequencing Assay, Expressing, Chromatin Immunoprecipitation, Clone Assay, Generated

    CTCF binding is not required to maintain gene expression or chromatin boundaries in the HOXA gene cluster. A. qPCR for HOXA9 in single mutants with heterozygous or homozygous deletions at CBSA6/7, CBSA7/9, or CBSA10. HOXA9 expression from the Kasumi-1 cell line is shown in blue as a no- HOXA9 expressing control. B. qPCR for HOXA9 in double and triple mutants at the CTCF binding sites indicated. Kasumi-1 cells are included in blue as in panel A. C-F. RNA-seq expression of all HOXA genes in single mutants (C-E) and triple mutants (D). Expression level is shown in log2 transcripts per million (TPM). G. ChIP-seq for H3K4me3 and H3K27me3 in deletion mutants lacking CTCF at site CBSA7/9 (in red; N=2). Mean ChIP-seq signal from wild type OCI-AML3 cells (N=2) is shown in gray. H. Mean ChIP-seq for H3K4me3 and H3K27me3 in triple mutants (N=2), with ChIP-seq from wild type cells shown in gray, as in panel G.
    Figure Legend Snippet: CTCF binding is not required to maintain gene expression or chromatin boundaries in the HOXA gene cluster. A. qPCR for HOXA9 in single mutants with heterozygous or homozygous deletions at CBSA6/7, CBSA7/9, or CBSA10. HOXA9 expression from the Kasumi-1 cell line is shown in blue as a no- HOXA9 expressing control. B. qPCR for HOXA9 in double and triple mutants at the CTCF binding sites indicated. Kasumi-1 cells are included in blue as in panel A. C-F. RNA-seq expression of all HOXA genes in single mutants (C-E) and triple mutants (D). Expression level is shown in log2 transcripts per million (TPM). G. ChIP-seq for H3K4me3 and H3K27me3 in deletion mutants lacking CTCF at site CBSA7/9 (in red; N=2). Mean ChIP-seq signal from wild type OCI-AML3 cells (N=2) is shown in gray. H. Mean ChIP-seq for H3K4me3 and H3K27me3 in triple mutants (N=2), with ChIP-seq from wild type cells shown in gray, as in panel G.

    Techniques Used: Binding Assay, Expressing, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Chromatin Immunoprecipitation

    6) Product Images from "Contribution of CTCF binding to transcriptional activity at the HOXA locus in NPM1-mutant AML cells"

    Article Title: Contribution of CTCF binding to transcriptional activity at the HOXA locus in NPM1-mutant AML cells

    Journal: Leukemia

    doi: 10.1038/s41375-020-0856-3

    Targeted deletions eliminate CTCF binding in the NPM1 -mutant OCI-AML3 cell line. A. RNA-seq expression of HOXA and HOXB genes in OCI-AML3 cells, which display the canonical mutant NPM1 -associated HOXA/HOXB expression phenotype. Also shown are the MLL -rearranged MOLM13 cell line that expresses only HOXA genes, and the RUNX1-RUNX1T1 -containing Kasumi-1 cell line that has low HOXA and HOXB gene expression. B. ChIP-seq data from OCI-AML3 cells for CTCF (black), H3K4me3 (highlighted in yellow) and H3K27me3 (highlighted in blue), which show conserved CTCF binding sites and distinct regions of active (H3K4me3) and repressed (H3K27me3) chromatin. C-E. Targeted deletions that disrupt CTCF binding in OCI-AML3 cells at sites CBSA6/7 (in C), CBSA7/9 (in D), and CBSA10 (in E). Bottom panels show allele pairs from homozygous or compound heterozygous deletion mutants at each site; top panels show CTCF ChIP-seq signal from these mutant cell lines (multi-colored lines) compared to wild type OCI-AML3 cells (in gray). F-H. CTCF ChIP-seq signal (log 2 normalized read counts) for all CTCF peaks from deletion mutants (Y axis) vs. wild type OCI-AML3 cells, showing dramatically reduced CTCF ChIP-seq signal in deletion mutants at all three sites, with the exception of clones A101 and A091.31, which only partially eliminates CTCF signal at site CBSA6/7. I. CTCF ChIP-seq tracks from double (in purple) and triple mutants (in blue), generated via sequential targeted deletion experiments. CTCF ChIP-seq from wild type OCI-AML3 cells is shown in black at the top for reference.
    Figure Legend Snippet: Targeted deletions eliminate CTCF binding in the NPM1 -mutant OCI-AML3 cell line. A. RNA-seq expression of HOXA and HOXB genes in OCI-AML3 cells, which display the canonical mutant NPM1 -associated HOXA/HOXB expression phenotype. Also shown are the MLL -rearranged MOLM13 cell line that expresses only HOXA genes, and the RUNX1-RUNX1T1 -containing Kasumi-1 cell line that has low HOXA and HOXB gene expression. B. ChIP-seq data from OCI-AML3 cells for CTCF (black), H3K4me3 (highlighted in yellow) and H3K27me3 (highlighted in blue), which show conserved CTCF binding sites and distinct regions of active (H3K4me3) and repressed (H3K27me3) chromatin. C-E. Targeted deletions that disrupt CTCF binding in OCI-AML3 cells at sites CBSA6/7 (in C), CBSA7/9 (in D), and CBSA10 (in E). Bottom panels show allele pairs from homozygous or compound heterozygous deletion mutants at each site; top panels show CTCF ChIP-seq signal from these mutant cell lines (multi-colored lines) compared to wild type OCI-AML3 cells (in gray). F-H. CTCF ChIP-seq signal (log 2 normalized read counts) for all CTCF peaks from deletion mutants (Y axis) vs. wild type OCI-AML3 cells, showing dramatically reduced CTCF ChIP-seq signal in deletion mutants at all three sites, with the exception of clones A101 and A091.31, which only partially eliminates CTCF signal at site CBSA6/7. I. CTCF ChIP-seq tracks from double (in purple) and triple mutants (in blue), generated via sequential targeted deletion experiments. CTCF ChIP-seq from wild type OCI-AML3 cells is shown in black at the top for reference.

    Techniques Used: Binding Assay, Mutagenesis, RNA Sequencing Assay, Expressing, Chromatin Immunoprecipitation, Clone Assay, Generated

    CTCF defines chromatin boundaries in AML samples and normal CD34 + cells with HOXA gene expression. A. Top track shows CTCF ChIP-seq from a NPM1 -mutant primary AML sample. Tracks highlighted in yellow show ChIP-seq for H3K4me3 from primary samples with high HOXA expression, including AML samples with MLL rearrangements (green; N=3) and NPM1 mutations (purple; N=3), and primary CD34 + hematopoietic stem/progenitor cells (HSPCs) purified from normal donor bone marrow samples (gray; N=2, from GSE104579). Tracks highlighted in blue show H3K27me3 ChIP-seq from the same set of samples. B. H3K4me3 and H3K27me3 ChIP-seq from primary samples with no HOXA expression, including AML samples with t(8;21) creating the RUNX1-RUNX1T1 fusion (blue; N=2), and normal promyelocytes (CD14-, CD15+, CD16 low; magenta, N=2) and neutrophils (CD14-, CD15+, CD16 high; cyan, N=2) from healthy donor individuals. Dashed box indicates the region of dynamic chromatin that correlates with HOXA gene cluster expression.
    Figure Legend Snippet: CTCF defines chromatin boundaries in AML samples and normal CD34 + cells with HOXA gene expression. A. Top track shows CTCF ChIP-seq from a NPM1 -mutant primary AML sample. Tracks highlighted in yellow show ChIP-seq for H3K4me3 from primary samples with high HOXA expression, including AML samples with MLL rearrangements (green; N=3) and NPM1 mutations (purple; N=3), and primary CD34 + hematopoietic stem/progenitor cells (HSPCs) purified from normal donor bone marrow samples (gray; N=2, from GSE104579). Tracks highlighted in blue show H3K27me3 ChIP-seq from the same set of samples. B. H3K4me3 and H3K27me3 ChIP-seq from primary samples with no HOXA expression, including AML samples with t(8;21) creating the RUNX1-RUNX1T1 fusion (blue; N=2), and normal promyelocytes (CD14-, CD15+, CD16 low; magenta, N=2) and neutrophils (CD14-, CD15+, CD16 high; cyan, N=2) from healthy donor individuals. Dashed box indicates the region of dynamic chromatin that correlates with HOXA gene cluster expression.

    Techniques Used: Expressing, Chromatin Immunoprecipitation, Mutagenesis, Purification

    7) Product Images from "Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding"

    Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16394-3

    NUP153 is critical for CTCF and cohesin binding at the IEG cis -regulatory sites and across the IEG loci during the paused state. NUP153, CTCF, and SMC3 occupancy across the JUN (left) and EGR1 (right) genetic elements were examined by ChIP real-time PCR at the indicated time points in control and NUP153 KD HeLa cells. Position of PCR primers are denoted as numbers in the schematics (see Supplementary Data 10 for primer sequences). NUP153 binding at JUN , minus EGF (control vs KD; TSS, *** p = 0.0000; GB, ** p = 0.0035; TTS, ** p = 0.0011; +14.6 kb, ** p = 0.0023), 15 min EGF (control vs KD; promoter, ** p = 0.0047; TSS, * p = 0.0368; GB, *** p = 0.0003; TTS, *** p = 0.0004; +14.6 kb, * p = 0.0301). NUP153 binding at EGR1 , minus EGF (control vs KD; −15.3 kb, * p = 0.0298; −1.2 kb, * p = 0.0323; −0.9 kb, ** p = 0.0039; promoter, * p = 0.0469; TTS, * p = 0.0406), 15 min EGF (control vs KD; −15.5 kb, * p = 0.0299; −15.3 kb, ** p = 0.0068; −1.2 kb, *** p = 0.0006; −0.9 kb, ** p = 0.0086; promoter, *** p = 0.0006; TSS, ** p = 0.0078; TTS, ** p = 0.0085). CTCF binding at JUN , minus EGF (control vs KD; GB, ** p = 0.0047; +14.6 kb, * p = 0.0231). CTCF binding at EGR1 , minus EGF (control vs KD; −15.3 kb, ** p = 0.0017; −1.2 kb, ** p = 0.0010; −0.9 kb, * p = 0.0291), 15 min EGF (control vs KD; −15.3 kb, * p = 0.0316). SMC3 binding at JUN , minus EGF (control vs KD; −1.3 kb, ** p = 0.0076; promoter, ** p = 0.0032; TSS, ** p = 0.0037; GB, * p = 0.0413; +14.6 kb, * p = 0.0158), 15 min EGF (control vs KD; −1 kb, * p = 0.0154; TSS, ** p = 0.0029; GB, ** p = 0.0036; +14.6 kb, * p = 0.0245). SMC3 binding at EGR1 , minus EGF (control vs KD; −15.5 kb, *** p = 0.0001; −15.3 kb, * p = 0.0309), 15 min EGF (control vs KD; −15.3 kb, *** p = 0.0004; −1.2 kb, * p = 0.0355; −0.9 kb, * p = 0.0419; TTS, *** p = 0.0003). Data shown are percent (%) of input. Values are mean ± standard deviation. Two-tailed Student’s t -test, n ≥ 3 independent experiments. Nt, nucleotide. Source data are provided as a Source Data file.
    Figure Legend Snippet: NUP153 is critical for CTCF and cohesin binding at the IEG cis -regulatory sites and across the IEG loci during the paused state. NUP153, CTCF, and SMC3 occupancy across the JUN (left) and EGR1 (right) genetic elements were examined by ChIP real-time PCR at the indicated time points in control and NUP153 KD HeLa cells. Position of PCR primers are denoted as numbers in the schematics (see Supplementary Data 10 for primer sequences). NUP153 binding at JUN , minus EGF (control vs KD; TSS, *** p = 0.0000; GB, ** p = 0.0035; TTS, ** p = 0.0011; +14.6 kb, ** p = 0.0023), 15 min EGF (control vs KD; promoter, ** p = 0.0047; TSS, * p = 0.0368; GB, *** p = 0.0003; TTS, *** p = 0.0004; +14.6 kb, * p = 0.0301). NUP153 binding at EGR1 , minus EGF (control vs KD; −15.3 kb, * p = 0.0298; −1.2 kb, * p = 0.0323; −0.9 kb, ** p = 0.0039; promoter, * p = 0.0469; TTS, * p = 0.0406), 15 min EGF (control vs KD; −15.5 kb, * p = 0.0299; −15.3 kb, ** p = 0.0068; −1.2 kb, *** p = 0.0006; −0.9 kb, ** p = 0.0086; promoter, *** p = 0.0006; TSS, ** p = 0.0078; TTS, ** p = 0.0085). CTCF binding at JUN , minus EGF (control vs KD; GB, ** p = 0.0047; +14.6 kb, * p = 0.0231). CTCF binding at EGR1 , minus EGF (control vs KD; −15.3 kb, ** p = 0.0017; −1.2 kb, ** p = 0.0010; −0.9 kb, * p = 0.0291), 15 min EGF (control vs KD; −15.3 kb, * p = 0.0316). SMC3 binding at JUN , minus EGF (control vs KD; −1.3 kb, ** p = 0.0076; promoter, ** p = 0.0032; TSS, ** p = 0.0037; GB, * p = 0.0413; +14.6 kb, * p = 0.0158), 15 min EGF (control vs KD; −1 kb, * p = 0.0154; TSS, ** p = 0.0029; GB, ** p = 0.0036; +14.6 kb, * p = 0.0245). SMC3 binding at EGR1 , minus EGF (control vs KD; −15.5 kb, *** p = 0.0001; −15.3 kb, * p = 0.0309), 15 min EGF (control vs KD; −15.3 kb, *** p = 0.0004; −1.2 kb, * p = 0.0355; −0.9 kb, * p = 0.0419; TTS, *** p = 0.0003). Data shown are percent (%) of input. Values are mean ± standard deviation. Two-tailed Student’s t -test, n ≥ 3 independent experiments. Nt, nucleotide. Source data are provided as a Source Data file.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Standard Deviation, Two Tailed Test

    NUP153 interacts with CTCF and cohesin. a Silver stain showing proteins that immunoprecipitated (IP) with FLAG-NUP153. b Co-IP shows FLAG-NUP153 interaction with CTCF, and cohesin subunits, SMC3, SMC1A, and RAD21. NUP153 was pulled down using anti-FLAG antibody. c Schematic showing steps of chromatin fractionation assay in HeLa cells. d NUP153 was detected in the nuclear insoluble fraction (P2) along with CTCF and cohesin. NUP153 detected in the chromatin-associated soluble fraction (S3) following micrococcal nuclease (MNase) treatment of P1 fraction. Ppt, precipitate; Sup, supernatant; Nucleoporin 62, NUP62; loading controls: α-TUBULIN (cytoplasm), Histone H3 (chromatin). Experiments were repeated twice. Source data are provided as a Source Data file.
    Figure Legend Snippet: NUP153 interacts with CTCF and cohesin. a Silver stain showing proteins that immunoprecipitated (IP) with FLAG-NUP153. b Co-IP shows FLAG-NUP153 interaction with CTCF, and cohesin subunits, SMC3, SMC1A, and RAD21. NUP153 was pulled down using anti-FLAG antibody. c Schematic showing steps of chromatin fractionation assay in HeLa cells. d NUP153 was detected in the nuclear insoluble fraction (P2) along with CTCF and cohesin. NUP153 detected in the chromatin-associated soluble fraction (S3) following micrococcal nuclease (MNase) treatment of P1 fraction. Ppt, precipitate; Sup, supernatant; Nucleoporin 62, NUP62; loading controls: α-TUBULIN (cytoplasm), Histone H3 (chromatin). Experiments were repeated twice. Source data are provided as a Source Data file.

    Techniques Used: Silver Staining, Immunoprecipitation, Co-Immunoprecipitation Assay, Fractionation

    NUP153 influences transcription and binding of CTCF and cohesin at bivalent genes. a Scatter plot showing expression levels of transcripts in log2[CPM] scale in control and NUP153 KD-1 mouse ES cells. Blue points denote all differentially expressed genes ( n = 711) and orange points denote differentially expressed bivalent genes (Supplementary Data 6 and 9 ). b Table showing number of differentially expressed genes that associate with all, Group I and Group II CTCF-positive TSS (top). Plots showing number of differentially regulated NUP153-positive and NUP153-negative genes that associate with Group I and II CTCF-positive TSS (bottom) (see also Supplementary Data 8 ). c NUP153 DamID-Seq, CTCF, cohesin, H3K4me3, and H3K27me3 ChIP-Seq, and RNA-Seq tracks are shown for two NUP153-positive Group I genes, Rtn4rl1 (left panel) and Calb2 (right panel) in control (WT) and NUP153 KD ES cells. Rtn4rl1 shows transcriptional upregulation and Calb2 shown transcriptional downregulation. d NUP153 DamID-Seq, CTCF, cohesin, H3K4me3, and H3K27me3 ChIP-Seq tracks are shown for a 145–150 kb region for the HoxA and HoxC loci in control (WT) and NUP153 KD mouse ES cells as indicated. Arrows point to regions where CTCF or SMC3 binding are altered in NUP153 KD mouse ES cells. CTCF sites labeled with asterisk (*) denote CTCF sites that have been reported to regulate transcription at the Hox loci by mediating the formation of TADs 48 , 70 . The 2D heat map shows the interaction frequency in mouse ES cells 44 . Hi-C data was aligned to the mm9 genome showing HoxA cluster residing in a TAD boundary and HoxC cluster in a TAD as published 44 . H3K4me3 and H3K27me3 (ref. 40 ) and CBP/P300 (ref. 43 ) ChIP-Seq data were previously published. CPM, counts per million.
    Figure Legend Snippet: NUP153 influences transcription and binding of CTCF and cohesin at bivalent genes. a Scatter plot showing expression levels of transcripts in log2[CPM] scale in control and NUP153 KD-1 mouse ES cells. Blue points denote all differentially expressed genes ( n = 711) and orange points denote differentially expressed bivalent genes (Supplementary Data 6 and 9 ). b Table showing number of differentially expressed genes that associate with all, Group I and Group II CTCF-positive TSS (top). Plots showing number of differentially regulated NUP153-positive and NUP153-negative genes that associate with Group I and II CTCF-positive TSS (bottom) (see also Supplementary Data 8 ). c NUP153 DamID-Seq, CTCF, cohesin, H3K4me3, and H3K27me3 ChIP-Seq, and RNA-Seq tracks are shown for two NUP153-positive Group I genes, Rtn4rl1 (left panel) and Calb2 (right panel) in control (WT) and NUP153 KD ES cells. Rtn4rl1 shows transcriptional upregulation and Calb2 shown transcriptional downregulation. d NUP153 DamID-Seq, CTCF, cohesin, H3K4me3, and H3K27me3 ChIP-Seq tracks are shown for a 145–150 kb region for the HoxA and HoxC loci in control (WT) and NUP153 KD mouse ES cells as indicated. Arrows point to regions where CTCF or SMC3 binding are altered in NUP153 KD mouse ES cells. CTCF sites labeled with asterisk (*) denote CTCF sites that have been reported to regulate transcription at the Hox loci by mediating the formation of TADs 48 , 70 . The 2D heat map shows the interaction frequency in mouse ES cells 44 . Hi-C data was aligned to the mm9 genome showing HoxA cluster residing in a TAD boundary and HoxC cluster in a TAD as published 44 . H3K4me3 and H3K27me3 (ref. 40 ) and CBP/P300 (ref. 43 ) ChIP-Seq data were previously published. CPM, counts per million.

    Techniques Used: Binding Assay, Expressing, Chromatin Immunoprecipitation, RNA Sequencing Assay, Labeling, Hi-C

    8) Product Images from "X-inactive-specific transcript of peripheral blood cells is regulated by exosomal Jpx and acts as a biomarker for female patients with hepatocellular carcinoma"

    Article Title: X-inactive-specific transcript of peripheral blood cells is regulated by exosomal Jpx and acts as a biomarker for female patients with hepatocellular carcinoma

    Journal: Therapeutic Advances in Medical Oncology

    doi: 10.1177/1758834017731052

    Effects of exosomal Jpx on X-inactive-specific transcript expression by evicting CTCF. (A) Diagram of coculture between Hela cells and exosomes from QGY-7703 cells transfected with Jpx expression plasmid or empty vector. (B) Relative Jpx and X-inactive-specific transcript (Xist) expressions in Hela cells treated with exosomes from QGY-7703 cells transfected with Jpx expression plasmid or empty vector. (C) Luciferase reporter plasmids containing Xist promoter with CTCF binding sites were constructed. (D) Relative luciferase activities were analyzed after reporter plasmid and CTCF expression plasmid were transfected into Hela cells treated with the corresponding exosomes. (E) Chromatin immunoprecipitation (ChIP) quantitative polymerase chain reaction (qPCR) analysis was used to evaluate the effects of exosomal Jpx on CTCF binding at the Xist promoter. CTCF antibody (left) and normal rabbit immunoglobulin G (IgG) (right) were used. The data were normalized to the value of qPCR for Xist promoter amplification of Input DNA from the same cells. * p
    Figure Legend Snippet: Effects of exosomal Jpx on X-inactive-specific transcript expression by evicting CTCF. (A) Diagram of coculture between Hela cells and exosomes from QGY-7703 cells transfected with Jpx expression plasmid or empty vector. (B) Relative Jpx and X-inactive-specific transcript (Xist) expressions in Hela cells treated with exosomes from QGY-7703 cells transfected with Jpx expression plasmid or empty vector. (C) Luciferase reporter plasmids containing Xist promoter with CTCF binding sites were constructed. (D) Relative luciferase activities were analyzed after reporter plasmid and CTCF expression plasmid were transfected into Hela cells treated with the corresponding exosomes. (E) Chromatin immunoprecipitation (ChIP) quantitative polymerase chain reaction (qPCR) analysis was used to evaluate the effects of exosomal Jpx on CTCF binding at the Xist promoter. CTCF antibody (left) and normal rabbit immunoglobulin G (IgG) (right) were used. The data were normalized to the value of qPCR for Xist promoter amplification of Input DNA from the same cells. * p

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Luciferase, Binding Assay, Construct, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification

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    Article Title: Genomic Space of MGMT in Human Glioma Revisited: Novel Motifs, Regulatory RNAs, NRF1, 2, and CTCF Involvement in Gene Expression
    Article Snippet: .. Briefly, the SF-188 GBM cells were exposed to 1% formaldehyde in the medium without serum for 10 min, followed by lysis, DNA shearing, and protein/DNA Immunoprecipitation using CTCF antibodies (Cell Signaling Technology, Danvers, MA, USA). ..

    Immunoprecipitation:

    Article Title: Genomic Space of MGMT in Human Glioma Revisited: Novel Motifs, Regulatory RNAs, NRF1, 2, and CTCF Involvement in Gene Expression
    Article Snippet: .. Briefly, the SF-188 GBM cells were exposed to 1% formaldehyde in the medium without serum for 10 min, followed by lysis, DNA shearing, and protein/DNA Immunoprecipitation using CTCF antibodies (Cell Signaling Technology, Danvers, MA, USA). ..

    Article Title: Tissue context determines the penetrance of regulatory DNA variation
    Article Snippet: .. Eighty microliter of CTCF antibody (Cell Signaling 2899S) was conjugated to M-280 Dynabeads (Invitrogen 11204D) for 6 h at 4 °C, followed by overnight immunoprecipitation. ..

    ChIP-sequencing:

    Article Title: Comprehensive epigenomic profiling of human alveolar epithelial differentiation identifies key epigenetic states and transcription factor co-regulatory networks for maintenance of distal lung identity
    Article Snippet: .. Generation of ChIP-seq and FAIRE from primary human epithelial cells Chromatin immunoprecipitation (ChIP) was performed using antibodies (Abs) against H3K27Ac (Cat # 39133, Active Motif, Carlsbad CA), H3K4me1 (pAb-037-050) and H3K79me2 (pAb-051-050) from Diagenode (Denville NJ), CTCF (Cat #2899, Cell Signaling, Danvers MA), H3K27me3 (#07-449) and H3K9/14Ac (#06-599) from Millipore (Burlington, MA) and the Imprint Ultra Chromatin Immunoprecipitation Kit (Sigma-Aldrich, St Louis MO). ..

    Chromatin Immunoprecipitation:

    Article Title: Comprehensive epigenomic profiling of human alveolar epithelial differentiation identifies key epigenetic states and transcription factor co-regulatory networks for maintenance of distal lung identity
    Article Snippet: .. Generation of ChIP-seq and FAIRE from primary human epithelial cells Chromatin immunoprecipitation (ChIP) was performed using antibodies (Abs) against H3K27Ac (Cat # 39133, Active Motif, Carlsbad CA), H3K4me1 (pAb-037-050) and H3K79me2 (pAb-051-050) from Diagenode (Denville NJ), CTCF (Cat #2899, Cell Signaling, Danvers MA), H3K27me3 (#07-449) and H3K9/14Ac (#06-599) from Millipore (Burlington, MA) and the Imprint Ultra Chromatin Immunoprecipitation Kit (Sigma-Aldrich, St Louis MO). ..

    Incubation:

    Article Title: Mapping Human Transient Transcriptomes Using Single Nucleotide Resolution 4sU Sequencing (SNU-Seq)
    Article Snippet: .. The remaining sample was incubated overnight, rotating at 4° C with antibody; anti-H3K4me3 (MERCK, 05-745R), anti-H3K27ac (Millipore, 07-360), anti-CTCF (CST, 3418S), anti-H2AV Drosophila Ab (Active Motif, 39715) and PE Mouse IgG2b, Isotype Ctrl Antibody (Biolegend, 400313). ..

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    Cell Signaling Technology Inc anti ctcf rabbit monoclonal antibody
    Transcriptional profiling of genes altered with <t>CTCF</t> knockdown. a Heat map of differentially expressed (DE) transcripts following 5 days of CTCF <t>shRNA</t> induction (Dox) versus uninduced vehicle control (vehicle). CTCF knockdown in HPECE6/E7 leads to 1308 significantly altered gene transcripts (FDR
    Anti Ctcf Rabbit Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti ctcf
    Foxg1Cre -mediated deletion of <t>Ctcf</t> results in a massive increase in apoptosis. A , Table of genotypes obtained during the embryonic and postnatal periods. Ratios at each time point were analyzed using a χ 2 test. Het, Ctcf flox/WT ;Foxg1-cre +/− . B , Dark-field images of control and Ctcf Foxg1-cre embryos at E13.5. (Please note limbs were taken for genotyping). C , H E staining of E13.5 sagittal cryosections demonstrates complete loss of cortex (Ctx), hippocampal hem (H), basal ganglia (BG), lens (L), and anterior retina (AR), but not the posterior retina (PR), in Ctcf Foxg1-cre embryos. D , Dark-field images of control and Ctcf Foxg1-cre embryos at E11.5. The dashed circle outlines the telencephalon, which is visibly reduced in size in the Ctcf Foxg1-cre embryos compared to littermate controls. E , Immunodetection of CTCF (red) in E11.5 sagittal cryosections confirms specific loss of CTCF expression in the forebrain neuroepithelium of Ctcf Foxg1-cre embryos. F , Pregnant females were subjected to a 1 h BrdU pulse before being killed. Immunodetection of BrdU in E11.5 control and Ctcf Foxg1-cre cortical neuroepithelium is shown. G , BrdU + cells were counted and expressed as a percentage of the total number of DAPI + cells ( n = 3). H , Immunodetection of activated <t>caspase-3</t> (red) in control and Ctcf Foxg1-cre cortical neuroepithelium at E11.5. I , AC3 + cells were counted and expressed as a percentage of the total number of DAPI + cells ( n = 3). J , TUNEL (green) detection in E11.5 control and Ctcf Foxg1-cre cortical neuroepithelium. Error bars represent the SEM. Original magnification: C , 25×; E , 50×; F , H , J , 200×. Scale bars: C , top, 1 mm; bottom, 400 μm; E , 200 μm; F , H , 50 μm; J , 100 μm.
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    Cell Signaling Technology Inc anti ctcf
    Topology of the EGFR locus in unamplified and amplified glioblastoma. ( a ) (Top) HiC contact map of unamplified glioblastoma model cell line G567. (Middle) <t>CTCF</t> ChlP-seq track of unamplified glioblastoma model cell line GSC23. The red and green arrows denote the orientation of the CTCF motif. (Bottom) Schematic of the boundaries of the loop domain. ( b ) 4C-seq profiles anchored from near the EGFR transcription start site (dotted line) showing interactions to the EGFR enhancers (boxed) for GSC23 (unamplified). Red arcs denote regions with strong distal interactions with EGFR at a 12 kb window thresholded at a 0.12 interaction frequency. <t>H3K27ac</t> ChIP-seq tracks accompany the 4C-seq data. The 4C-seq tracks consist of a main trend of the chromatin interactions based on a 5 kb window and domainogram colored by interaction frequency. Right side depicts a model for the toplogy of the locus. ( c ) Same as (b) for GBM3565 ( EGFR .
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    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

    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

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

    Techniques: shRNA

    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

    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

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

    Techniques: Methylation, Immunoprecipitation, Methylated DNA Immunoprecipitation, Chromatin Immunoprecipitation, shRNA, Western Blot

    Foxg1Cre -mediated deletion of Ctcf results in a massive increase in apoptosis. A , Table of genotypes obtained during the embryonic and postnatal periods. Ratios at each time point were analyzed using a χ 2 test. Het, Ctcf flox/WT ;Foxg1-cre +/− . B , Dark-field images of control and Ctcf Foxg1-cre embryos at E13.5. (Please note limbs were taken for genotyping). C , H E staining of E13.5 sagittal cryosections demonstrates complete loss of cortex (Ctx), hippocampal hem (H), basal ganglia (BG), lens (L), and anterior retina (AR), but not the posterior retina (PR), in Ctcf Foxg1-cre embryos. D , Dark-field images of control and Ctcf Foxg1-cre embryos at E11.5. The dashed circle outlines the telencephalon, which is visibly reduced in size in the Ctcf Foxg1-cre embryos compared to littermate controls. E , Immunodetection of CTCF (red) in E11.5 sagittal cryosections confirms specific loss of CTCF expression in the forebrain neuroepithelium of Ctcf Foxg1-cre embryos. F , Pregnant females were subjected to a 1 h BrdU pulse before being killed. Immunodetection of BrdU in E11.5 control and Ctcf Foxg1-cre cortical neuroepithelium is shown. G , BrdU + cells were counted and expressed as a percentage of the total number of DAPI + cells ( n = 3). H , Immunodetection of activated caspase-3 (red) in control and Ctcf Foxg1-cre cortical neuroepithelium at E11.5. I , AC3 + cells were counted and expressed as a percentage of the total number of DAPI + cells ( n = 3). J , TUNEL (green) detection in E11.5 control and Ctcf Foxg1-cre cortical neuroepithelium. Error bars represent the SEM. Original magnification: C , 25×; E , 50×; F , H , J , 200×. Scale bars: C , top, 1 mm; bottom, 400 μm; E , 200 μm; F , H , 50 μm; J , 100 μm.

    Journal: The Journal of Neuroscience

    Article Title: Dual Effect of CTCF Loss on Neuroprogenitor Differentiation and Survival

    doi: 10.1523/JNEUROSCI.3769-13.2014

    Figure Lengend Snippet: Foxg1Cre -mediated deletion of Ctcf results in a massive increase in apoptosis. A , Table of genotypes obtained during the embryonic and postnatal periods. Ratios at each time point were analyzed using a χ 2 test. Het, Ctcf flox/WT ;Foxg1-cre +/− . B , Dark-field images of control and Ctcf Foxg1-cre embryos at E13.5. (Please note limbs were taken for genotyping). C , H E staining of E13.5 sagittal cryosections demonstrates complete loss of cortex (Ctx), hippocampal hem (H), basal ganglia (BG), lens (L), and anterior retina (AR), but not the posterior retina (PR), in Ctcf Foxg1-cre embryos. D , Dark-field images of control and Ctcf Foxg1-cre embryos at E11.5. The dashed circle outlines the telencephalon, which is visibly reduced in size in the Ctcf Foxg1-cre embryos compared to littermate controls. E , Immunodetection of CTCF (red) in E11.5 sagittal cryosections confirms specific loss of CTCF expression in the forebrain neuroepithelium of Ctcf Foxg1-cre embryos. F , Pregnant females were subjected to a 1 h BrdU pulse before being killed. Immunodetection of BrdU in E11.5 control and Ctcf Foxg1-cre cortical neuroepithelium is shown. G , BrdU + cells were counted and expressed as a percentage of the total number of DAPI + cells ( n = 3). H , Immunodetection of activated caspase-3 (red) in control and Ctcf Foxg1-cre cortical neuroepithelium at E11.5. I , AC3 + cells were counted and expressed as a percentage of the total number of DAPI + cells ( n = 3). J , TUNEL (green) detection in E11.5 control and Ctcf Foxg1-cre cortical neuroepithelium. Error bars represent the SEM. Original magnification: C , 25×; E , 50×; F , H , J , 200×. Scale bars: C , top, 1 mm; bottom, 400 μm; E , 200 μm; F , H , 50 μm; J , 100 μm.

    Article Snippet: Primary antibodies used were as follows: rabbit anti-CTCF (1:400; Cell Signaling Technology), rabbit anti-cleaved caspase 3 (AC3; Asp175; 1:400; Cell Signaling Technology), mouse anti-BrdU (1:50; BD Biosciences), rabbit anti-PUMA (1:200; Cell Signaling Technology), rabbit anti-TBR2 (1:200; Abcam), goat anti-SOX2 (1:400; Santa Cruz Biotechnology), goat anti-PAX6 (1:200; Santa Cruz Biotechnology), rabbit anti-SOX2 (1:100; Millipore Bioscience Research Reagents), rabbit anti-Ki67 (1:200; Abcam), rabbit anti-TBR1 (1:200; Abcam), mouse anti-SATB2 (1:200; Abcam), and rabbit anti-CTIP2 (1:200; Abcam).

    Techniques: Staining, Immunodetection, Expressing, TUNEL Assay

    NestinCre -mediated deletion of CTCF results in activation of caspase-mediated apoptosis. A , Table of genotype ratios obtained during the embryonic period. Ratios at each time point were analyzed by a χ 2 test. B , Immunodetection of CTCF in E12.5 control and Ctcf Nes-cre coronal forebrain sections. C , Immunodetection of CTCF in E14 control and Ctcf Nes-cre coronal forebrain sections. D , Quantification of AC3 immunostaining in E12.5, E14, and E15.5 forebrain tissue ( n = 3). AC3 + cells were counted and expressed per unit area (square millimeter). E , Immunodetection of AC3 in E15.5 control and Ctcf Nes-cre basal ganglia. F , Immunodetection of AC3 in control and Ctcf Nes-cre at 4 DIV. Het, Ctcf flox/WT ;Nestin-cre ; Ctx, cortex; BG, basal ganglia; H, hippocampal hem. Error bars represent the SEM. Original magnification: B , C , 50×; D , 100×; E , 200×. Scale bars: B , 220 μm; C , 300 μm; E , 100 μm; F , 25 μm.

    Journal: The Journal of Neuroscience

    Article Title: Dual Effect of CTCF Loss on Neuroprogenitor Differentiation and Survival

    doi: 10.1523/JNEUROSCI.3769-13.2014

    Figure Lengend Snippet: NestinCre -mediated deletion of CTCF results in activation of caspase-mediated apoptosis. A , Table of genotype ratios obtained during the embryonic period. Ratios at each time point were analyzed by a χ 2 test. B , Immunodetection of CTCF in E12.5 control and Ctcf Nes-cre coronal forebrain sections. C , Immunodetection of CTCF in E14 control and Ctcf Nes-cre coronal forebrain sections. D , Quantification of AC3 immunostaining in E12.5, E14, and E15.5 forebrain tissue ( n = 3). AC3 + cells were counted and expressed per unit area (square millimeter). E , Immunodetection of AC3 in E15.5 control and Ctcf Nes-cre basal ganglia. F , Immunodetection of AC3 in control and Ctcf Nes-cre at 4 DIV. Het, Ctcf flox/WT ;Nestin-cre ; Ctx, cortex; BG, basal ganglia; H, hippocampal hem. Error bars represent the SEM. Original magnification: B , C , 50×; D , 100×; E , 200×. Scale bars: B , 220 μm; C , 300 μm; E , 100 μm; F , 25 μm.

    Article Snippet: Primary antibodies used were as follows: rabbit anti-CTCF (1:400; Cell Signaling Technology), rabbit anti-cleaved caspase 3 (AC3; Asp175; 1:400; Cell Signaling Technology), mouse anti-BrdU (1:50; BD Biosciences), rabbit anti-PUMA (1:200; Cell Signaling Technology), rabbit anti-TBR2 (1:200; Abcam), goat anti-SOX2 (1:400; Santa Cruz Biotechnology), goat anti-PAX6 (1:200; Santa Cruz Biotechnology), rabbit anti-SOX2 (1:100; Millipore Bioscience Research Reagents), rabbit anti-Ki67 (1:200; Abcam), rabbit anti-TBR1 (1:200; Abcam), mouse anti-SATB2 (1:200; Abcam), and rabbit anti-CTIP2 (1:200; Abcam).

    Techniques: Activation Assay, Immunodetection, Immunostaining

    Topology of the EGFR locus in unamplified and amplified glioblastoma. ( a ) (Top) HiC contact map of unamplified glioblastoma model cell line G567. (Middle) CTCF ChlP-seq track of unamplified glioblastoma model cell line GSC23. The red and green arrows denote the orientation of the CTCF motif. (Bottom) Schematic of the boundaries of the loop domain. ( b ) 4C-seq profiles anchored from near the EGFR transcription start site (dotted line) showing interactions to the EGFR enhancers (boxed) for GSC23 (unamplified). Red arcs denote regions with strong distal interactions with EGFR at a 12 kb window thresholded at a 0.12 interaction frequency. H3K27ac ChIP-seq tracks accompany the 4C-seq data. The 4C-seq tracks consist of a main trend of the chromatin interactions based on a 5 kb window and domainogram colored by interaction frequency. Right side depicts a model for the toplogy of the locus. ( c ) Same as (b) for GBM3565 ( EGFR .

    Journal: Cell

    Article Title: Functional enhancers shape extrachromosomal oncogene amplifications

    doi: 10.1016/j.cell.2019.10.039

    Figure Lengend Snippet: Topology of the EGFR locus in unamplified and amplified glioblastoma. ( a ) (Top) HiC contact map of unamplified glioblastoma model cell line G567. (Middle) CTCF ChlP-seq track of unamplified glioblastoma model cell line GSC23. The red and green arrows denote the orientation of the CTCF motif. (Bottom) Schematic of the boundaries of the loop domain. ( b ) 4C-seq profiles anchored from near the EGFR transcription start site (dotted line) showing interactions to the EGFR enhancers (boxed) for GSC23 (unamplified). Red arcs denote regions with strong distal interactions with EGFR at a 12 kb window thresholded at a 0.12 interaction frequency. H3K27ac ChIP-seq tracks accompany the 4C-seq data. The 4C-seq tracks consist of a main trend of the chromatin interactions based on a 5 kb window and domainogram colored by interaction frequency. Right side depicts a model for the toplogy of the locus. ( c ) Same as (b) for GBM3565 ( EGFR .

    Article Snippet: Samples were sheared in 1 ml milliTUBEs on the Covaris model S2 AFA focused ultrasonicator, duty factor 2%, intensity 4, 200 cycles/burst, for 6 min. Our ChlP-seq method using 8 μg rabbit anti-H3K27ac (Abeam #4729) or 10 μL of anti-CTCF (Cell Signaling #3418) was that of Schmidt et al. ( ) but using PCRCIean Dx paramagnetic beads (ALINE #C-1003) instead of column purifications and using adapters that we made following Illumina bar codes.

    Techniques: Amplification, Chromatin Immunoprecipitation

    Analysis of the rs4919742-associated chromatin loops. Guide RNAs targeting regions encompassing CTCF site 4 (the PCa risk-associated CTCF site), CTCF site 5, and/or CTCF site 6 (or the empty guide RNA vector as a control) were introduced into 22Rv1 prostate cancer cells, along with Cas9. Cell pools were harvested, and KRT78 expression was analyzed by RT-qPCR. Shown within the blue bars is the fold change in KRT78 expression in the pools that received guide RNAs vs the vector control. The yellow X indicates which CTCF site has been deleted; the size of each deletion can be found in Additional file 5 : Table S4

    Journal: Genome Biology

    Article Title: CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops

    doi: 10.1186/s13059-018-1531-0

    Figure Lengend Snippet: Analysis of the rs4919742-associated chromatin loops. Guide RNAs targeting regions encompassing CTCF site 4 (the PCa risk-associated CTCF site), CTCF site 5, and/or CTCF site 6 (or the empty guide RNA vector as a control) were introduced into 22Rv1 prostate cancer cells, along with Cas9. Cell pools were harvested, and KRT78 expression was analyzed by RT-qPCR. Shown within the blue bars is the fold change in KRT78 expression in the pools that received guide RNAs vs the vector control. The yellow X indicates which CTCF site has been deleted; the size of each deletion can be found in Additional file 5 : Table S4

    Article Snippet: Five micrograms of CTCF antibody (Active Motif #61311) was used to precipitate 20 μg chromatin for 22Rv1, PrEC, RWPE-2, VCaP (rep1) cells, and 10 ul CTCF antibody (Cell Signaling #3418S) were used to precipitate 20 μg chromatin for LNCaP, C4-2B, RWPE-1, VCaP (rep2) cells.

    Techniques: Plasmid Preparation, Expressing, Quantitative RT-PCR

    Experimental workflow for functional investigation of PCa risk-associated CTCF sites. Phase 1: Plasmids encoding guide RNAs that target sequences on each side of a PCa risk-associated CTCF site were introduced into the PCa cell line 22Rv1 along with a Cas9 expression vector (see the “ Methods ” section for details). The resultant cell pool was analyzed to determine deletion efficiency (red slashes represent alleles in each cell that harbor a CTCF site deletion). Single cells were then selected and expanded into clonal populations for RNA-seq analysis. Phase 2: After identifying the gene most responsive (within a ± 1-Mb window) to deletion of the region encompassing a risk-associated CTCF site, plasmids encoding guide RNAs that target the risk-associated CTCF anchor region and/or the regions encompassing the CTCF sites looped to the risk CTCF site and a Cas9 expression plasmid were introduced into 22Rv1 cells; cell pools were analyzed by PCR to check deletion frequency and by RT-qPCR to measure expression of the target gene

    Journal: Genome Biology

    Article Title: CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops

    doi: 10.1186/s13059-018-1531-0

    Figure Lengend Snippet: Experimental workflow for functional investigation of PCa risk-associated CTCF sites. Phase 1: Plasmids encoding guide RNAs that target sequences on each side of a PCa risk-associated CTCF site were introduced into the PCa cell line 22Rv1 along with a Cas9 expression vector (see the “ Methods ” section for details). The resultant cell pool was analyzed to determine deletion efficiency (red slashes represent alleles in each cell that harbor a CTCF site deletion). Single cells were then selected and expanded into clonal populations for RNA-seq analysis. Phase 2: After identifying the gene most responsive (within a ± 1-Mb window) to deletion of the region encompassing a risk-associated CTCF site, plasmids encoding guide RNAs that target the risk-associated CTCF anchor region and/or the regions encompassing the CTCF sites looped to the risk CTCF site and a Cas9 expression plasmid were introduced into 22Rv1 cells; cell pools were analyzed by PCR to check deletion frequency and by RT-qPCR to measure expression of the target gene

    Article Snippet: Five micrograms of CTCF antibody (Active Motif #61311) was used to precipitate 20 μg chromatin for 22Rv1, PrEC, RWPE-2, VCaP (rep1) cells, and 10 ul CTCF antibody (Cell Signaling #3418S) were used to precipitate 20 μg chromatin for LNCaP, C4-2B, RWPE-1, VCaP (rep2) cells.

    Techniques: Functional Assay, Expressing, Plasmid Preparation, RNA Sequencing Assay, Polymerase Chain Reaction, Quantitative RT-PCR

    Analysis of the rs12144978-associated chromatin loops. Guide RNAs targeting regions encompassing CTCF site 1 (the PCa risk-associated CTCF site), CTCF site 2, and/or CTCF site 3 (or the empty guide RNA vector as a control) were introduced into 22Rv1 prostate cancer cells, along with Cas9. Cell pools were harvested, and KCNN3 expression was analyzed by RT-qPCR. Shown within the blue bars is the fold change in KCNN3 expression in the pools that received guide RNAs vs. the vector control. The yellow X indicates which CTCF site has been deleted; the size of each deletion can be found in Additional file 5 : Table S4

    Journal: Genome Biology

    Article Title: CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops

    doi: 10.1186/s13059-018-1531-0

    Figure Lengend Snippet: Analysis of the rs12144978-associated chromatin loops. Guide RNAs targeting regions encompassing CTCF site 1 (the PCa risk-associated CTCF site), CTCF site 2, and/or CTCF site 3 (or the empty guide RNA vector as a control) were introduced into 22Rv1 prostate cancer cells, along with Cas9. Cell pools were harvested, and KCNN3 expression was analyzed by RT-qPCR. Shown within the blue bars is the fold change in KCNN3 expression in the pools that received guide RNAs vs. the vector control. The yellow X indicates which CTCF site has been deleted; the size of each deletion can be found in Additional file 5 : Table S4

    Article Snippet: Five micrograms of CTCF antibody (Active Motif #61311) was used to precipitate 20 μg chromatin for 22Rv1, PrEC, RWPE-2, VCaP (rep1) cells, and 10 ul CTCF antibody (Cell Signaling #3418S) were used to precipitate 20 μg chromatin for LNCaP, C4-2B, RWPE-1, VCaP (rep2) cells.

    Techniques: Plasmid Preparation, Expressing, Quantitative RT-PCR

    Experimental and analytical steps used to identify PCa risk-associated regulatory elements involved in chromatin loops. Step (1): The subset of 2,181 fine-mapped PCa-associated SNPs that overlap a DNase hypersensitive site was identified. Step (2): H3K27Ac and CTCF ChIP-seq was performed in duplicate in two normal (PrEC and RWPE-1) and five cancer (RWPE-2, 22Rv1, C4-2B, LNCaP, and VCaP) prostate cell lines; data was collected plus or minus DHT for 22Rv1 and LNCaP cells, for a total of 18 datasets for each mark (36 ChIP-seq samples). The SNPs in open chromatin sites (i.e., those that are contained within a DHS site) were then subdivided into those that overlap a H3K27Ac or a CTCF site in prostate cells; the number of PCa-associated SNPs associated with the H3K27Ac or CTCF sites is shown. Step (3): The PCa risk-associated H3K27Ac and CTCF sites were overlapped with Hi-C looping data, and the subset of each type of site involved in chromatin loops was identified; the number of PCa-associated SNPs associated with the H3K27Ac or CTCF sites involved in looping is shown

    Journal: Genome Biology

    Article Title: CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops

    doi: 10.1186/s13059-018-1531-0

    Figure Lengend Snippet: Experimental and analytical steps used to identify PCa risk-associated regulatory elements involved in chromatin loops. Step (1): The subset of 2,181 fine-mapped PCa-associated SNPs that overlap a DNase hypersensitive site was identified. Step (2): H3K27Ac and CTCF ChIP-seq was performed in duplicate in two normal (PrEC and RWPE-1) and five cancer (RWPE-2, 22Rv1, C4-2B, LNCaP, and VCaP) prostate cell lines; data was collected plus or minus DHT for 22Rv1 and LNCaP cells, for a total of 18 datasets for each mark (36 ChIP-seq samples). The SNPs in open chromatin sites (i.e., those that are contained within a DHS site) were then subdivided into those that overlap a H3K27Ac or a CTCF site in prostate cells; the number of PCa-associated SNPs associated with the H3K27Ac or CTCF sites is shown. Step (3): The PCa risk-associated H3K27Ac and CTCF sites were overlapped with Hi-C looping data, and the subset of each type of site involved in chromatin loops was identified; the number of PCa-associated SNPs associated with the H3K27Ac or CTCF sites involved in looping is shown

    Article Snippet: Five micrograms of CTCF antibody (Active Motif #61311) was used to precipitate 20 μg chromatin for 22Rv1, PrEC, RWPE-2, VCaP (rep1) cells, and 10 ul CTCF antibody (Cell Signaling #3418S) were used to precipitate 20 μg chromatin for LNCaP, C4-2B, RWPE-1, VCaP (rep2) cells.

    Techniques: Chromatin Immunoprecipitation, Hi-C

    Identification and classification of H3K27Ac ( a ) and CTCF ( b ) sites in prostate cells. H3K27Ac and CTCF ChIP-seq was performed in duplicate for each cell line; for 22Rv1 and LNCaP cells, ChIP-seq was performed in duplicate in the presence or absence of DHT. Peaks were called for individual datasets using MACS2 and the ENCODE3 pipeline, then peaks present in both replicates were identified (high confidence peaks) and used for further analysis (see Additional file 3 : Table S2). The location of the peaks was classified using the HOMER annotatePeaks.pl program and the Gencode V19 database. The fraction of high confidence peaks in each category is shown on the Y axis, with the number of peaks in each category for each individual cell line and/or treatment shown within each bar

    Journal: Genome Biology

    Article Title: CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops

    doi: 10.1186/s13059-018-1531-0

    Figure Lengend Snippet: Identification and classification of H3K27Ac ( a ) and CTCF ( b ) sites in prostate cells. H3K27Ac and CTCF ChIP-seq was performed in duplicate for each cell line; for 22Rv1 and LNCaP cells, ChIP-seq was performed in duplicate in the presence or absence of DHT. Peaks were called for individual datasets using MACS2 and the ENCODE3 pipeline, then peaks present in both replicates were identified (high confidence peaks) and used for further analysis (see Additional file 3 : Table S2). The location of the peaks was classified using the HOMER annotatePeaks.pl program and the Gencode V19 database. The fraction of high confidence peaks in each category is shown on the Y axis, with the number of peaks in each category for each individual cell line and/or treatment shown within each bar

    Article Snippet: Five micrograms of CTCF antibody (Active Motif #61311) was used to precipitate 20 μg chromatin for 22Rv1, PrEC, RWPE-2, VCaP (rep1) cells, and 10 ul CTCF antibody (Cell Signaling #3418S) were used to precipitate 20 μg chromatin for LNCaP, C4-2B, RWPE-1, VCaP (rep2) cells.

    Techniques: Chromatin Immunoprecipitation

    PCa risk SNPs associated with CTCF sites and chromatin loops. Each row represents one of the 93 SNPs that are associated with both a DHS site and a CTCF peak in normal or tumor prostate cells (Additional file 4 : Table S3). The location of each SNP was classified using the Gencode V19 database. “Others” represents mostly intergenic regions. To identify the subset of CTCF-associated risk SNPs located in an anchor point of a loop, chromatin loops were identified using Hi-C data from normal RWPE-1 prostate cells [ 26 ] or 22Rv1 and C4-2B prostate tumor cells (Rhie et al., in preparation); Hi-C [ 25 ] and cohesin HiChIP data [ 27 ] from GM12878 was also used

    Journal: Genome Biology

    Article Title: CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops

    doi: 10.1186/s13059-018-1531-0

    Figure Lengend Snippet: PCa risk SNPs associated with CTCF sites and chromatin loops. Each row represents one of the 93 SNPs that are associated with both a DHS site and a CTCF peak in normal or tumor prostate cells (Additional file 4 : Table S3). The location of each SNP was classified using the Gencode V19 database. “Others” represents mostly intergenic regions. To identify the subset of CTCF-associated risk SNPs located in an anchor point of a loop, chromatin loops were identified using Hi-C data from normal RWPE-1 prostate cells [ 26 ] or 22Rv1 and C4-2B prostate tumor cells (Rhie et al., in preparation); Hi-C [ 25 ] and cohesin HiChIP data [ 27 ] from GM12878 was also used

    Article Snippet: Five micrograms of CTCF antibody (Active Motif #61311) was used to precipitate 20 μg chromatin for 22Rv1, PrEC, RWPE-2, VCaP (rep1) cells, and 10 ul CTCF antibody (Cell Signaling #3418S) were used to precipitate 20 μg chromatin for LNCaP, C4-2B, RWPE-1, VCaP (rep2) cells.

    Techniques: Hi-C, HiChIP