Journal: Genome Research

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

doi: 10.1101/gr.136101.111

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

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

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

Journal: Genome Research

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

doi: 10.1101/gr.136101.111

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

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

Techniques: Binding Assay, ChIP-sequencing

Journal: Genome Research

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

doi: 10.1101/gr.136101.111

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

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

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

Journal: Genome Research

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

doi: 10.1101/gr.136101.111

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

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

Techniques: Methylation, Genome Wide

Journal: Genome Research

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

doi: 10.1101/gr.136101.111

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

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

Techniques: Methylation, Methylation Sequencing, Genome Wide

Journal: bioRxiv

Article Title: TET-catalyzed 5-carboxylcytosine promotes CTCF binding to suboptimal sequences genome-wide

doi: 10.1101/480525

Figure Lengend Snippet: (A) CTCF ChIP-seq peaks in WT and Tdg-/- mESCs were assigned motif scores through the CTCFBSDB 2.0 algorithm and segregated into high, mid and low motif bins. (B) Box and whisker diagram of maximum motif score distribution in common vs. Tdg-/- induced CTCF peaks. (C) Tdg-/- mESC 5caC meDIP-seq within empirically determined CTCF peaks, segregated by motif score. (D) CpG dinucleotide frequency within Tdg-/- mESC CTCF peaks, segregated by motif score. (E) Tdg-/- mESC RNA pol II ChIP-seq density within common and Tdg-/- induced CTCF peaks relative to randomly permutated (shuffled) control peaks. (F) Tdg-/- mESC Pol II ChIP-seq in common and Tdg-/- induced CTCF sites as in (E), segregated on the basis of overlapping 5caC. (G) Tdg-/- mESC pol II ChIP-seq at 5caC-rich (+) and poor (-) regions not associated with proximal CTCF binding.

Article Snippet: The following antibodies were used for ChIP: anti-CTCF (CST, #3418), anti-RNA pol II (Millipore, #05-623), monoclonal rabbit isotype control IgG (DA1E) (CST, #3900), normal rabbit polyclonal control IgG (CST, #2729).

Techniques: ChIP-sequencing, Whisker Assay, Methylated DNA Immunoprecipitation, Binding Assay

Journal: bioRxiv

Article Title: TET-catalyzed 5-carboxylcytosine promotes CTCF binding to suboptimal sequences genome-wide

doi: 10.1101/480525

Figure Lengend Snippet: (A) Naïve CD4+ T cells were isolated from human peripheral blood through immunomagnetic enrichment and subjected to genome-wide analyses. (B) Distribution of maximum motif scores at CTCF peaks detected in naïve T cells. Peaks were segregated into high, mid, and low bins corresponding to scores within the top quartile, middle quartiles, and bottom quartile, respectively. (C) pG dinucleotide frequency within CD4+ T CTCF peaks, segregated by motif score. (D-E) 5caC meDIP-seq (D) and RNA pol II ChIP-seq (E) within determined CD4+ T CTCF peaks, segregated by motif score.

Article Snippet: The following antibodies were used for ChIP: anti-CTCF (CST, #3418), anti-RNA pol II (Millipore, #05-623), monoclonal rabbit isotype control IgG (DA1E) (CST, #3900), normal rabbit polyclonal control IgG (CST, #2729).

Techniques: Isolation, Genome Wide, Methylated DNA Immunoprecipitation, ChIP-sequencing

Journal: bioRxiv

Article Title: TET-catalyzed 5-carboxylcytosine promotes CTCF binding to suboptimal sequences genome-wide

doi: 10.1101/480525

Figure Lengend Snippet: (A) Schematic of EMSA probes derived from the human CD45 gene. Point mutations were incorporated into a “strong” CBS within exon 5 to generate an analogous “weak” CBS probe. A region devoid of CTCF binding in vivo within exon 6 was selected as a “motif-free” control. CTCFBSDB 2.0 motif scores are shown. Bold sequences within the exon 5 probes show locations of CpGs and red text indicates mutated residues. (B) EMSA with affinity-purified CTCF and radiolabeled PCR-generated 72mer or commercially-synthesized 41mer DNA probes. Unlabeled strong (S) or weak (W) motif cold-competitor DNA added to indicated lanes; 10x and 100x molar excess used for 72mer and 41mer, respectively. Super-shift performed with α-CTCF antibody. Quantification of bound probe indicated as % shift below figure. (C) EMSA using affinity-purified CTCF and motif-free PCR-generated CD45 exon 6 probes of varying carboxylation status; 10x molar excess of unlabeled CD45 exon 5 72mer used as cold-competitor where indicated. (D) Titration EMSA performed using 41mer CD45 exon 5 DNA probes (3.76 nM); P indicates lane containing probe alone, CTCF concentration: 342.3-0.0418 nM. E) Saturation binding curves, derived from EMSAs in (D), and summary chart of apparent KD values for each 41mer probe; markers on curves indicate mean of two (Strong Cyt.) or three (Strong 5caC, Weak 5caC) independent replicates and error bars indicate standard deviation.

Article Snippet: The following antibodies were used for ChIP: anti-CTCF (CST, #3418), anti-RNA pol II (Millipore, #05-623), monoclonal rabbit isotype control IgG (DA1E) (CST, #3900), normal rabbit polyclonal control IgG (CST, #2729).

Techniques: Derivative Assay, Binding Assay, In Vivo, Affinity Purification, Generated, Synthesized, Titration, Concentration Assay, Standard Deviation

Journal: Nature

Article Title: CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning

doi: 10.1038/s41586-020-2578-0

Figure Lengend Snippet: a, Diagram of the CTCF-degron. b, Left: Targeting strategy for introducing in-frame mAID-GFP sequences into both Ctcf alleles. Cas9/sgRNAs and southern blotting probe are indicated. B: BglII. Right: Southern blotting confirmation of correctly targeted alleles ( n =3 biological repeats with similar results). c, d, Southern blotting confirmation of correctly targeted Rosa26 alleles in CTCF-degron ( c , n =3 biological repeats with similar results) and OsTir1-expressing ( d , left, n =2 biological repeats with similar results) lines following OsTir1-V5 targeting strategy shown in c . Western blotting on nuclear extracts of G1-arresed OsTir1-expressing cells with correctly targeted Rosa26 alleles confirms the constitutive expression of OsTir1-V5 protein ( d , right, n =2 biological repeats with similar results). Histone H3 was used as a loading control. e, Representative GFP signal-based flow-cytometry plots for CTCF-degron cells that are non-treated (NT) or treated with IAA (+IAA) for 6h (top) followed by 4-day STI-571 treatment (without or with IAA) (bottom). The parental cells were used as GFP negative controls. Three biological repeats with similar results. f, Fucci cell cycle assay of CTCF-degron cells without (NT) or with IAA treatment followed by 4-day STI-571 treatment. Representative flow-cytometry plots and average percentage ± s.d. of cells arrested in G1-stage at indicated condition ( n =3 biologically independent samples) are shown. The parental cells without Fucci were used as negative controls. g, Western blotting on nuclear extracts of G1-arrested cells as indicated using indicated antibodies ( n =3 biological repeats with similar results). h, Time-course cell viability assay for parental and IAA-treated CTCF-degron cells, following STI-571 treatment for G1-arrest (+STI). Average percentage ± s.d. of viable cells for each timepoint and under each condition was shown ( n =3 biologically independent samples). p values were calculated using unpaired two-tailed t -test. See Methods.

Article Snippet: The targeting constructs used for introducing the in-frame mAID sequences into mouse endogenous Ctcf locus (pEN84, Addgene #86230) and introducing the OsTir1-V5 expression cassette into endogenous Rosa26 locus (pEN114, Addgene #92143) were described previously .

Techniques: Southern Blot, Expressing, Western Blot, Control, Flow Cytometry, Cell Cycle Assay, Viability Assay, Two Tailed Test

Journal: Nature

Article Title: CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning

doi: 10.1038/s41586-020-2578-0

Figure Lengend Snippet: a, Diagram of joining between RAG-mediated breaks at initiating bona fide J H 4-RSS (blue triangle) and cryptic RSSs, mostly represented by CAC motifs (green triangles), across Igh locus, upon down-modulation of CTCF/CBEs (indicated by light purple arrowheads). Deletional and inversional joining is denoted by red (+) and blue (−) curves, respectively. b, Pooled HTGTS junction profiles across highlighted V H domains for deletional and inversional joining in RAG2-complemented, IAA-treated and G1-arrested parental ( n =4 biological repeats with similar results) and CTCF-degron ( n =6 biological repeats with similar results) cells. For presentation, all libraries from parental or CTCF-degron cells were pooled and normalized to 3,819 total off-target junctions. Note that the high-level deletional cryptic RSS joining signals (red bars) within IGCR1-J H region are off-scale on the y -axis for viewing of relatively lower level deletional cryptic RSS joining signals across the V H locus. The low-level, but reproducible, inversional joining events (blue bars) within IGCR1-J H region involve cryptic RSSs predominantly at IGCR1 and D H 3–2 locales, as described . The vast majority of deletional joining events across the V H locus are reproducible among replicates and involve, at a minimum the CAC of an RSS. Almost all of the handful of the very low-level inversional joining events interspersed across V H locus are not reproducible among replicates consistent with being background events. c, Average frequencies ± s.d. of deletional (left) and inversional (right) joining events within indicated regions in RAG2-complemented, IAA-treated and G1-arrested parental ( n =4 biological repeats with similar results) and CTCF-degron ( n =6 biological repeats with similar results) cells. Each of the biological library replicates was normalized to 415 off-target junctions for statistical analysis. p values were calculated using unpaired two-tailed t -test. NA: not applicable. See Methods.

Article Snippet: The targeting constructs used for introducing the in-frame mAID sequences into mouse endogenous Ctcf locus (pEN84, Addgene #86230) and introducing the OsTir1-V5 expression cassette into endogenous Rosa26 locus (pEN114, Addgene #92143) were described previously .

Techniques: Two Tailed Test

Journal: Nature

Article Title: CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning

doi: 10.1038/s41586-020-2578-0

Figure Lengend Snippet: a , Average 3C-HTGTS signal counts ± s.e.m. across the four V H domains in G1-arrested parental (top, n =3) and IAA-treated CTCF-degron (middle, n =3) v-Abl cells and RAG2-deficient mouse BM pro-B cells (bottom, n =2). In all panels, “n” indicates biological repeats. Spearman’s correlation coefficient (r) and p values determined by two-sided Spearman’s correlation test between data in middle and bottom panels are: r=0.86, p <2.2×10 -16 . Related low-level RC interactions across the parental cell V H locus and those of BM pro-B cells (r=0.63) and IAA-treated CTCF-degron cells (r=0.74) suggest low-level Igh loop extrusion in parental cells. b - d , Average V H utilization frequencies ± s.d. in RAG2-complemented, IAA-treated and G1-arrested parental ( b , n =4), CTCF-degron ( c , n =6) v-Abl cells and BM pro-B cells ( d , n =6). Average percentage ± s.d. of V H DJ H and DJ H rearrangements are indicated. Spearman’s correlation coefficient (r) and p values by two-sided Spearman’s correlation test between data in panels c and d are r=0.76, p<2.2×10 -16 . See and . e , Average utilization frequencies ± s.d. of indicated V H s in RAG2-complemented, G1-arrested parental ( n =4) and IAA-treated CTCF-degron ( n =6) v-Abl cells. Zoom-in profiles of indicated assays (mean ± s.e.m) for ±10kb regions of indicated V H s in G1-arrested parental ( n =3 for each assay) and IAA-treated CTCF-degron ( n =3 for each assay) lines along with putative CBE motif and orientation are presented. High-level signals for some V H s are off-scale on the y -axis for viewing low-level V H signals. See , for on-scale profiles. Other details are in and . p values were calculated using unpaired two-tailed t -test. V H s with decreased utilization upon substantial CTCF depletion are marked in blue. Detailed data for all V H s are shown in .

Article Snippet: The targeting constructs used for introducing the in-frame mAID sequences into mouse endogenous Ctcf locus (pEN84, Addgene #86230) and introducing the OsTir1-V5 expression cassette into endogenous Rosa26 locus (pEN114, Addgene #92143) were described previously .

Techniques: Two Tailed Test

Journal: Nature

Article Title: CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning

doi: 10.1038/s41586-020-2578-0

Figure Lengend Snippet: a, Average signal counts ± s.e.m. of GRO-seq from three biologically independent experiments across 3’ part of Igh locus as diagramed at the top in G1-arrested parental and IAA-treated CTCF-degron cells are plotted. S/AS: sense/anti-sense transcription. b, Average 3C-HTGTS signal counts ± s.e.m. from three independent experiments with RC-based baits (red asterisk) across 3’ part of Igh locus as diagramed at the top in G1-arrested parental and IAA-treated CTCF-degron cells are plotted. c, Scatter plots of transcriptome-wide GRO-seq counts in G1-arrested parental ( x axis) and IAA-treated CTCF-degron ( y axis) cells. Three biologically independent repeats with similar results. Representative known requisite genes for V(D)J recombination and chromatin interaction are highlighted by red circles and blue arrows. Spearman’s correlation coefficient (r) and p values determined by two-sided Spearman’s correlation test are presented. d, Scatter plots of normalized GRO-seq read counts ( x axis) and 3C-HTGTS junction counts ( y axis) calculated from regions within ±5kb of highly-utilized V H s across four indicated V H domains in IAA-treated CTCF-degron cells. Data were normalized and quantified among three biologically independent experiments for each assay. Spearman’s correlation coefficient (r) and p values determined by two-sided Spearman’s correlation test are presented. See Methods for details.

Article Snippet: The targeting constructs used for introducing the in-frame mAID sequences into mouse endogenous Ctcf locus (pEN84, Addgene #86230) and introducing the OsTir1-V5 expression cassette into endogenous Rosa26 locus (pEN114, Addgene #92143) were described previously .

Techniques:

Journal: Nature

Article Title: CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning

doi: 10.1038/s41586-020-2578-0

Figure Lengend Snippet: a, b, Average signal counts ± s.e.m. of CTCF ( a ) and Rad21 ( b ) ChIP-seq from three biologically independent experiments across 3’ part of Igh locus as diagramed at the top in G1-arrested parental and IAA-treated CTCF-degron cells are plotted. “<”/”>”: orientation of indicated CBEs. c, d, CTCF ( c ) and Rad21 ( d ) ChIP-seq signal across all peaks genome-wide called in G1-arrested parental cells. See legend for other details. Three biologically independent repeats with similar results. IAA-treated CTCF-degron cells had greatly diminished CTCF and Rad21 chromatin occupancy, but, also had residual binding at some CBEs genome-wide. See Methods for details. Notably, CTCF binding increased at the RC and at a subset transcribed V H s ( , ) upon CTCF depletion. While increases in apparent CTCF-binding to the non-CBE-containing RC and also transcribed V H s upon CTCF depletion could be considered surprising, it may likely occur indirectly due to cohesin-mediated loop extrusion-driven dynamic associations of the RC dynamic anchor with residual-CTCF-bound CBEs across Igh.

Article Snippet: The targeting constructs used for introducing the in-frame mAID sequences into mouse endogenous Ctcf locus (pEN84, Addgene #86230) and introducing the OsTir1-V5 expression cassette into endogenous Rosa26 locus (pEN114, Addgene #92143) were described previously .

Techniques: ChIP-sequencing, Genome Wide, Binding Assay

Journal: Nature

Article Title: CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning

doi: 10.1038/s41586-020-2578-0

Figure Lengend Snippet: a-h, HTGTS-V(D)J-seq analysis showing average utilization frequencies ± s.d. of V H segments across four highlighted V H domains in RAG2-complemented and G1-arrested parental ( a , b ), OsTir1-expressing ( , d ), CTCF-tagged ( e , f ), and CTCF-degron ( g , h ) cells without or with IAA treatment. n =4 ( a - f ) or n =6 ( g , h ) biologically independent repeats with similar results. Utilization of V H s presented in b - h are compared to those in NT parental cells shown in a , with red, blue, and grey bars indicating V H s with significantly increased, decreased, and unchanged utilization, respectively. To facilitate direct comparisons, the HTGTS-V(D)J-seq data for the IAA-treated parental and CTCF-degron cells shown in b and h are the same as those presented in and , respectively, with different y -axis scales. p values were calculated using unpaired two-tailed t -test (*: p ≤ 0.05, **: p ≤ 0.01, and ***: p ≤ 0.001). See for exact p values. See Methods for details.

Article Snippet: The targeting constructs used for introducing the in-frame mAID sequences into mouse endogenous Ctcf locus (pEN84, Addgene #86230) and introducing the OsTir1-V5 expression cassette into endogenous Rosa26 locus (pEN114, Addgene #92143) were described previously .

Techniques: Expressing, Two Tailed Test

Journal: Nature

Article Title: CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning

doi: 10.1038/s41586-020-2578-0

Figure Lengend Snippet: a, Schematic of the entire murine Igh locus with details as shown in a . b, The four V H domains are highlighted different colors which are maintained in related figures. c-e, Average signal counts ± s.e.m. of CTCF ( c ) and Rad21 ( d ) ChIP-seq and GRO-seq ( e ) from three biologically independent experiments across the four highlighted V H domains in G1-arrested parental and IAA-treated CTCF-degron cells are plotted. S/AS: sense/anti-sense transcription. See , and for the plots across other parts of Igh locus. V H s for comparisons in are highlighted. f-i, Box-and-Whisker plots are presented with median, upper and lower quartiles and whiskers showing 1.5x interquartile range for CTCF ( f ) and Rad21 ( g ) ChIP-seq, GRO-seq ( h ), and 3C-HTGTS ( i ) signal counts within indicated V H domains in G1-arrested parental (“Par.”) and IAA-treated CTCF-degron cells. n =3 biological repeats with similar results for each condition in f-i . p values were calculated using two-sided Mann-Whitney U test. See Methods for details. Comparison of panels ( f ) and ( h ) reveals that residual CTCF binding levels are significantly higher in the highly transcribed distal V H domain than those of the proximal domains with less overall transcription, which might reflect known roles of transcription in CTCF chromatin binding , .

Article Snippet: The targeting constructs used for introducing the in-frame mAID sequences into mouse endogenous Ctcf locus (pEN84, Addgene #86230) and introducing the OsTir1-V5 expression cassette into endogenous Rosa26 locus (pEN114, Addgene #92143) were described previously .

Techniques: ChIP-sequencing, Whisker Assay, MANN-WHITNEY, Comparison, Binding Assay

Journal: Genome Biology

Article Title: Auxin-inducible degron 2 system deciphers functions of CTCF domains in transcriptional regulation

doi: 10.1186/s13059-022-02843-3

Figure Lengend Snippet: The AID2 system promoted rapid CTCF degradation, reduced cellular toxicity, and genome-wide loss of CTCF binding and chromatin looping. a Diagram illustrating how the AID2 system works. The 5-Ph-IAA auxin analog binds to the miniAID tag and recruits the SCF complex by binding to the OsTIR1(F74G) protein. b Flow cytometry analysis was performed on CTCF AID2 cells following 10 μM 5-Ph-IAA treatment over a time course from 0 to 24 h. RFP fluorescence represents exogenous expression from lentiviral integration of the pCDH-MND-OsTIR1(F74G)-P2A-EGFP AID2 -EF1a-RFP construct. Endogenous CTCF was expressed in frame with a miniAID tag and mClover3 (CTCF AIDmClover3 ), and exogenous EGFP from the pCDH-MND-OsTIR1(F74G)-P2A-EGFP AID2 -EF1a-RFP construct was expressed in frame with a miniAID tag (EGFP AID2 ). The fluorescence of both CTCF AIDmClover3 and EGFP AID2 was detected in the mCLover3 panel. c Immunoblot analysis from SEM WT , SEM OsTIR1(F74G) , CTCF AID1 , and CTCF AID2 cells following 24 h of DMSO or 1 μM 5-Ph-IAA treatment. The lower CTCF band represents untagged endogenous CTCF (CTCF-WT). The higher CTCF band represents CTCF AIDmClover3 (CTCF-AID). GAPDH was included as a loading control. d Immunoblot analysis of CTCF AID2 cells treated with AID2 conditions over a 4-day time course. CTCF antibody was used to detect CTCF AIDmClover3 . e Immunoblot analysis of CTCF AID2 cells treated with AID1 conditions over a 4-day time course. CTCF antibody was used to detect CTCF AIDmClover3 . f Growth assay of CTCF AID2 cells treated with AID2 conditions over 4 days. Samples were set up in triplicate and cell counts for each replicate were collected daily. Bars represent cell numbers (millions). g Growth assay of CTCF AID2 cells treated with AID1 conditions over 4 days. Samples were set up in triplicate and cell counts for each replicate were collected daily. Bars represent cell numbers (millions). h Cell cycle analysis was performed on samples from the 24-h time points from f and g and plotted for cell fraction percentage according to cell cycle phase. i Cell cycle analysis was performed on samples from the 72-h time points from f and g and plotted for cell fraction percentage according to cell cycle phase. j Homer motif enrichment analysis illustrated the overall distribution of motifs identified in CTCF ChIP-seq of CTCF AID2 cells without auxin treatment. Enriched CTCF motifs were shown as red circles. k Principal component analysis from CTCF ChIP-seq of CTCF AID2 cells with (2 replicates) or without (3 replicates) 10 μM 5-Ph-IAA treatment for 24 h. l Genomic heatmap centered at reproducible CTCF peak summits from CTCF AID2 cells with (2 replicates) or without (3 replicates) 10 μM 5-Ph-IAA treatment for 24 h. m Aggregate peak analysis (APA) plots from CTCF HiChIP of CTCF AID2 cells with or without 10 μM 5-Ph-IAA treatment for 6 h (2 replicates each, merged after confirmation of reproducibility). 7220 loops were called in CTCF HiChIP without 5-Ph-IAA. The x / y -axis was centered at loop anchors spanning from − 10 to + 10 windows (window size 10 kb), and the z -axis was normalized aggregated contact frequency

Article Snippet: Lysates were run on an SDS-PAGE (Thermo Fisher Scientific) gel and transferred to a PVDF membrane according to the manufacturer’s protocols (Bio-Rad) at 100 V for 1 h. After blocking incubation with 5% non-fat milk in TBS-T (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween-20) for 1 h at room temperature, membranes were incubated with antibodies against GAPDH (Thermo Fisher Scientific, AM4300, 1:10,000), AID (MBL, M3-214–3, 1:2000), OsTIR1 (MBL, PD048, 1:1000), or CTCF (Santa Cruz, sc-271514, 1:200) at 4 °C overnight with gentle shaking.

Techniques: Genome Wide, Binding Assay, Flow Cytometry, Fluorescence, Expressing, Construct, Western Blot, Control, Growth Assay, Cell Cycle Assay, ChIP-sequencing, HiChIP

Journal: Genome Biology

Article Title: Auxin-inducible degron 2 system deciphers functions of CTCF domains in transcriptional regulation

doi: 10.1186/s13059-022-02843-3

Figure Lengend Snippet: AID2 improved gene expression analysis by SLAM-seq. a Schematic diagram illustrating the SLAM-seq experiment. b Unsupervised data dimensionality reduction by PCA of the top 3000 most variably expressed nascent transcripts (summarized at the gene level) as determined by median absolute deviation. SLAM-seq quantified nascent gene expression from CTCF AID1 and CTCF. AID2 cells that underwent AID1 or AID2 auxin treatment conditions over a 24-h time course. Time 0 samples were untreated. RNA was collected following 0, 2, 6, 12, and 24 h of AID1 treatment and after 0, 2, 4, 6, 12, and 24 h of AID2 treatment. Samples for each time point were performed in duplicate. c Venn diagrams highlight the comparison of T > C converted reads (termed nascent and colored red) and all or total reads (termed steady-state and colored blue) of DE genes identified in each corresponding AID1 or AID2 auxin depletion SLAM-seq time-course experiment. DE genes determined in both nascent and steady states are colored purple. Genes passing thresholds of p value ≤ 0.05 and |log 2 (FC)|≥ 1 were determined to be differentially expressed. T0 = no AID treatment; T2 = 2-h AID treatment; T4 = 4-h AID treatment; T6 = 6-h AID treatment; T12 = 12-h AID treatment; T24 = 24-h AID treatment. d Gene set enrichment analysis of nascent transcripts summarized at the gene level corresponding to AID1 or AID2 auxin depletion SLAM-seq time-course experiments. Up-regulation and down-regulation by normalized enrichment score (NES) of hallmark gene sets compared to T0 are denoted in red and blue, respectively. Circle size indicates the false discovery rate (FDR)

Article Snippet: Lysates were run on an SDS-PAGE (Thermo Fisher Scientific) gel and transferred to a PVDF membrane according to the manufacturer’s protocols (Bio-Rad) at 100 V for 1 h. After blocking incubation with 5% non-fat milk in TBS-T (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween-20) for 1 h at room temperature, membranes were incubated with antibodies against GAPDH (Thermo Fisher Scientific, AM4300, 1:10,000), AID (MBL, M3-214–3, 1:2000), OsTIR1 (MBL, PD048, 1:1000), or CTCF (Santa Cruz, sc-271514, 1:200) at 4 °C overnight with gentle shaking.

Techniques: Gene Expression, Comparison