Journal: International Journal of Molecular Sciences

Article Title: DNA G-Quadruplexes Contribute to CTCF Recruitment

doi: 10.3390/ijms22137090

Figure Lengend Snippet: G-quadruplex (G4) structures and their presumed role in CTCF positioning. ( A ) Schematic representation of G4s. ( B ) Summary of the major factors that affects CTCF binding to genomic DNA. Dashed red lines indicate presumed links that are verified in this study.

Article Snippet: Recombinant mouse CTCF with polyhistidine (His) tag was purchased from MyBioSource, San Diego, CA, USA.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: DNA G-Quadruplexes Contribute to CTCF Recruitment

doi: 10.3390/ijms22137090

Figure Lengend Snippet: CTCF positioning relative to G4 sites in pyridostatin (PDS)-treated and non-treated cells. ( A ) Summary of the CTCF ChIP-seq data: Pearson correlation between ChIP-seq data sets obtained for PDS-treated (PDS+) and non-treated (PDS−) K562 cells (two biological repeats). ( B ) Relative distance between PDS+ and PDS- CTCF ChIP-seq peaks. ( C ) Venn diagram summarizing the overlap between PDS+/− CTCF peaks and the previously reported CTCF ChIP-seq peaks. ( D ) Metaplots illustrating G4-seq and BG4 peak distribution relative to PDS+ or PDS-(control) CTCF peaks. ( E ) Summary of CTCF peak intersections with G4 sites. ( F ) Relative distances between CTCF peaks and G4 sites.

Article Snippet: Recombinant mouse CTCF with polyhistidine (His) tag was purchased from MyBioSource, San Diego, CA, USA.

Techniques: ChIP-sequencing, Control

Journal: International Journal of Molecular Sciences

Article Title: DNA G-Quadruplexes Contribute to CTCF Recruitment

doi: 10.3390/ijms22137090

Figure Lengend Snippet: CTCF occupancy at G4 sites in PDS-treated and non-treated cells. ( A ) Genome browser snapshots for VEGFA, USP24, STAT3, VPS4A, MYC, BDNF and SHANK promoter regions/gene bodies harboring G4 motifs, G4-seq peaks, G4 ChIP-seq (BG4) peaks and/or CTCF-bound sites in pyridostatin (PDS)-treated and non-treated K562 cells. Black arrows mark sites analyzed in ChIP-qPCR assays. The CTCF-positive G4-negative site in USP24 was used as a reference, and the CTCF-negative G4-negative site in VPS4A was used as a negative control. Red arrows mark sites predicted to form stable G4s and i-motifs that were used in the binding assays. ( B ) ChIP-qPCR results. The histograms illustrate normalized CTCF occupancy at G4-prone (MYC, VEGFA, and STAT3) and non-G4 (VPS4A) sites relative to the control site (USP24) in non-treated (blue) K562 cells and those treated with PDS (red) or PhenDC3 (grey).

Article Snippet: Recombinant mouse CTCF with polyhistidine (His) tag was purchased from MyBioSource, San Diego, CA, USA.

Techniques: ChIP-sequencing, ChIP-qPCR, Negative Control, Binding Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: DNA G-Quadruplexes Contribute to CTCF Recruitment

doi: 10.3390/ijms22137090

Figure Lengend Snippet: Analysis of CTCF-G4 interactions. ( A ) In silico verification of G4-CTCF binding. The best binding energy conformation of the complex obtained by docking MYC-G4 (red) to CTCF is shown. The complex with the consensus duplex (blue) is shown for comparison. In the middle panel, the surface of MYC-G4-bound CTCF is colored according to the electrostatic potential: from negative (blue) to positive (red). ( B ) Circular dichroism spectra of the 2 µM G4 (red) and iM (black) solutions in 140 mM potassium-phosphate buffer, pH 6.7, supplemented with 10 mM NaCl, obtained at 22 °C. ( C ) Microscale thermophoresis (MST)-based analysis of CTCF interactions the G4s (red), respective i-motifs (black) and the control duplexes (blue). ( D ) Comparison of G4 motif and i-motif intersections with CTCF-bound sites in K562 cells.

Article Snippet: Recombinant mouse CTCF with polyhistidine (His) tag was purchased from MyBioSource, San Diego, CA, USA.

Techniques: In Silico, Binding Assay, Comparison, Circular Dichroism, Microscale Thermophoresis, Control

Journal: International Journal of Molecular Sciences

Article Title: DNA G-Quadruplexes Contribute to CTCF Recruitment

doi: 10.3390/ijms22137090

Figure Lengend Snippet: Verification of G4 contribution to the link between CpG islands (CGIs) and CTCF recruitment. ( A ) Hypothetical schemes summarizing the link between G4s, CTCF and CGI methylation: G4s are enriched in CGIs and supposedly protect CGIs from methylation by inhibiting DNMT1, which is favorable for CTCF binding. ( B ) Intersections of CTCF peaks with various subsets of CGIs and BG4 peaks. ( C ) Analysis of average CpG methylation levels in CpG-containing BG4 peaks, CTCF peaks, CGIs and their intersections in K562 cells.

Article Snippet: Recombinant mouse CTCF with polyhistidine (His) tag was purchased from MyBioSource, San Diego, CA, USA.

Techniques: Methylation, Binding Assay, CpG Methylation Assay

Journal: International Journal of Molecular Sciences

Article Title: DNA G-Quadruplexes Contribute to CTCF Recruitment

doi: 10.3390/ijms22137090

Figure Lengend Snippet: Search for G4-binding chromatin modulators that may affects CTCF positioning. ( A ) A heatmap summarizing G4 binding affinity (while, low; red, high) of HMG proteins in previous assays and hypothetical schemes summarizing the link between G4s, HMG and CTCF: the presumed recruitment of HMGN1 and HMGN3 to G4-prone sites induces chromatin decondensation by the disruption of internucleosome contacts and activation of histone acetyltransferase (HAT). Recruitment of HMGB2 prevents CTCF aggregation. ( B ) Intersection table for HMGN3 ChIP-seq, G4-seq and G4 ChIP-seq (BG4) peaks in K562 cells. The values equal to the number of intersections. Data in parentheses represent the number of G4-seq/BG4 peaks intersecting HMGN3 peaks with flanks (±200 bp). ( C ) Whole-genome analysis of the intersections between G4-seq peaks and HMG protein-bound sites and significance of the G4-HMG correlations (Monte-Carlo simulation results). Red boxes indicate the true number of G4-seq-overlapping HMG protein peaks. The boxplots refer to the randomized peaks.

Article Snippet: Recombinant mouse CTCF with polyhistidine (His) tag was purchased from MyBioSource, San Diego, CA, USA.

Techniques: Binding Assay, Disruption, Activation Assay, ChIP-sequencing

Journal: International Journal of Molecular Sciences

Article Title: DNA G-Quadruplexes Contribute to CTCF Recruitment

doi: 10.3390/ijms22137090

Figure Lengend Snippet: G4-HMG protein interactions and their possible contributions to CTCF positioning. ( A ) Microscale thermophoresis (MST)-based analysis of HMG proteins interactions with representative sequences that comply with enriched motifs (G4s and duplexes), the control hairpin and single-stranded oligonucleotides. Conditions: 50 nM labeled protein, 0–20 µM oligonucleotide, 140 mM potassium-phosphate buffer, pH 7.2 (6.7 for the i-motif N1-3). ( B ) Venn diagrams illustrating the overlaps between G4-ChIP-seq (BG4) peaks, HMGN3-bound sites, and CTCF-bound sites in K562 (left); G4-seq peaks, HMGB2-bound sites, and CTCF-bound sites in IMR-90; and G4-seq peaks, HMGN1-bound sites, and CTCF-bound sites in CD4+ T-cells.

Article Snippet: Recombinant mouse CTCF with polyhistidine (His) tag was purchased from MyBioSource, San Diego, CA, USA.

Techniques: Microscale Thermophoresis, Control, Labeling, ChIP-sequencing

Journal: Nucleic Acids Research

Article Title: Transcriptional plasticity through differential assembly of a multiprotein activation complex

doi: 10.1093/nar/gkq257

Figure Lengend Snippet: Saccharomyces cerevisiae strains used in this study

Article Snippet: The 32 P-labeled DNA fragments indicated were incubated with 0, 50 and 200 ng of a recombinant derivative of Cbf1 containing the bHLH domain fused to a polyhistidine tag ( his Cbf1 BD ).

Techniques:

Journal: Nucleic Acids Research

Article Title: Transcriptional plasticity through differential assembly of a multiprotein activation complex

doi: 10.1093/nar/gkq257

Figure Lengend Snippet: PDC6 activation in response to Cd 2+ is controlled by Met4 and its cofactors. ( A ) mRNA levels for PDC6 in mutant strains. A WT, and the indicated mutants were grown to early log phase in YPD medium and exposed to 0.5 mM Cd 2+ . Total RNA was prepared from samples taken before and at different time points after addition of Cd 2+ . RNA was quantified by RT followed by real-time PCR. Values were normalized to 25S ribosomal RNA and represent the average of two independent experiments. To facilitate comparison, the maximum value was set up to 100 and other values were expressed in proportion. Error bars indicate average deviations. ( B ) Association of Met4 and its cofactors with PDC6 promoter. Isogenic strains expressing Myc-tagged Met4, Cbf1 or Met32 were grown and exposed to Cd 2+ as in (A). Aliquots were crosslinked with formaldehyde at the time points indicated following Cd 2+ addition and subjected to ChIP using antibodies to Myc or to Met28. DNA fragments were quantified by real time PCR using primer pairs specific for PDC6 upstream region (from −455 to −306 relative to ATG), and primer pairs specific for IME2 ORF as a negative control. Occupancy levels correspond to the fraction of PDC6 promoter immunoprecipitates relative to the fraction IME2 ORF immunoprecipitates. Values represent the average of two independent experiments, and error bars indicate average deviations.

Article Snippet: The 32 P-labeled DNA fragments indicated were incubated with 0, 50 and 200 ng of a recombinant derivative of Cbf1 containing the bHLH domain fused to a polyhistidine tag ( his Cbf1 BD ).

Techniques: Activation Assay, Mutagenesis, Real-time Polymerase Chain Reaction, Comparison, Expressing, Negative Control

Journal: Nucleic Acids Research

Article Title: Transcriptional plasticity through differential assembly of a multiprotein activation complex

doi: 10.1093/nar/gkq257

Figure Lengend Snippet: In vitro association of Cbf1 with PDC6 promoter. ( A ) Schematic of PDC6 showing the DNA fragments used in the gel shift assay. ‘M’ boxes represent the CTGTGGC motifs. ‘T’ boxes represent the TATA elements. ( B ) Gel shift assays with large DNA fragments. The 32 P-labeled DNA fragments indicated were incubated with 0, 50 and 200 ng of a recombinant derivative of Cbf1 containing the bHLH domain fused to a polyhistidine tag ( his Cbf1 BD ). Fragments were resolved by 5% polyacrylamide gel electrophoresis and visualized by PhosphorImager analysis. The MET16 fragment covers positions from –126 to –270 and contains TCACGTG. ( C ) Gel shift assays with 40 bp DNA fragments. The 32 P-labeled DNA fragments indicated were incubated with 0, 200, 100, 50 and 25 ng of his Cbf1 BD (only 0 and 200 ng in the case of mut PDC6 ). The PDC6 fragment covers positions from –350 to –389 and contains the TCACGTT sequence. The MET16 fragment covers positions from –155 to –194 and contains the TCACGTG sequence. mut PDC6 contains TC CA GTT instead of TCACGTT.

Article Snippet: The 32 P-labeled DNA fragments indicated were incubated with 0, 50 and 200 ng of a recombinant derivative of Cbf1 containing the bHLH domain fused to a polyhistidine tag ( his Cbf1 BD ).

Techniques: In Vitro, Gel Shift, Labeling, Incubation, Recombinant, Polyacrylamide Gel Electrophoresis, Sequencing

Journal: Nucleic Acids Research

Article Title: Transcriptional plasticity through differential assembly of a multiprotein activation complex

doi: 10.1093/nar/gkq257

Figure Lengend Snippet: Replacement of Met4 bZIP by Cbf1 bHLH. ( A ) Model scheme for association to MET promoters of Met4 and a Met4-Cbf1 chimera containing residues 1–590 of Met4 fused to residues 210–351 of Cbf1. ‘Act’ represents Met4 activation domain and ‘Int’ represents Met4 interaction domain with Met31/32. The three parallel bars represent known protein–protein and protein–DNA interactions. ( B ) Transcription analysis in WT and cbf1Δ cells expressing the Met4-Cbf1 derivative described in (A) from MET4 endogenous promoter. Cells were cultivated, exposed to cadmium, and RNA levels were quantified by RT real-time PCR as in A. For comparison, the maximum level for each gene was set to 100. Error bars indicate average deviations from two independent experiments.

Article Snippet: The 32 P-labeled DNA fragments indicated were incubated with 0, 50 and 200 ng of a recombinant derivative of Cbf1 containing the bHLH domain fused to a polyhistidine tag ( his Cbf1 BD ).

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Comparison

Journal: Nucleic Acids Research

Article Title: Transcriptional plasticity through differential assembly of a multiprotein activation complex

doi: 10.1093/nar/gkq257

Figure Lengend Snippet: Association of Met4, Cbf1 and Met32 with promoters in response to Cd 2+ . Isogenic strains expressing Myc-tagged versions of Met4, Cbf1 or Met32 were grown to early log phase in YPD medium and exposed to 0.5 mM Cd 2+ . ChIPs were performed on samples crosslinked with formaldehyde at the time points indicated using Myc antibodies. DNA fragments were quantified by real-time PCR using primers for the indicated promoters and for IME2 ORF as a control. Occupancy levels were calculated as in B but are given for each gene as a percentage of the maximum value obtained in the time course. Error bars indicate average deviations from two independent experiments.

Article Snippet: The 32 P-labeled DNA fragments indicated were incubated with 0, 50 and 200 ng of a recombinant derivative of Cbf1 containing the bHLH domain fused to a polyhistidine tag ( his Cbf1 BD ).

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

Journal: Nucleic Acids Research

Article Title: Transcriptional plasticity through differential assembly of a multiprotein activation complex

doi: 10.1093/nar/gkq257

Figure Lengend Snippet: Transcriptional regulation of PDC6 in S. cerevisiae relatives. ( A ) Sequence alignment. PDC6 orthologs in S. cerevisiae (YGR087C), S. paradoxus (spar6-g11.1), S. bayanus (sbayc642-g17.1) and C. glabrata (CAGL02937) were aligned with Clustal W. Fully conserved positions are marked by an asterisk. Positions conserved among S. cerevisiae and at least one other species are marked in blue. Positions conserved among several species except S. cerevisiae are marked in green. Positions occupied by different nucleotides are marked in orange. Binding sites for Met31/32 and Cbf1 are marked by red boxes. ( B ) Saccharomyces cerevisiae (W303-1A), S. paradoxus (CLIB228), S. bayanus (CLIB181) and C. glabrata (CLIB298) strains were grown to early log phase in YPD medium and exposed to 0.5 mM Cd 2+ . Total RNA was extracted from samples taken just before (−) or 120 min (+) after the addition of cadmium. RNA levels for PDC6 and MET3 orthologs were quantified by RT real-time PCR and normalized to 25S ribosomal RNA. Values represent the average of three independent experiments and error bars indicate average deviations.

Article Snippet: The 32 P-labeled DNA fragments indicated were incubated with 0, 50 and 200 ng of a recombinant derivative of Cbf1 containing the bHLH domain fused to a polyhistidine tag ( his Cbf1 BD ).

Techniques: Sequencing, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Transcriptional plasticity through differential assembly of a multiprotein activation complex

doi: 10.1093/nar/gkq257

Figure Lengend Snippet: Models for Met4 recruitment to MET ( A ) versus PDC6 ( B ) promoters. Met4 recruitment involves direct interactions with Cbf1 (through the bZIP domain) and Met31 or Met32 (through the ‘Int’ domain). Met28 associates with Met4 bZIP and participates in Met4 recruitment through stabilization of the Met4–Cbf1–DNA complex (see text for details and references). The question marks at the extremity of the arrows originating from Met4 bZIP indicate that, even though direct interactions with promoter DNA are likely [see ref. ], the precise points of contact remain to be established. At PDC6 promoter, Met31 and Met32 bind to two distinct sites. Met4 recruitment to this promoter requires interaction with Met32 specifically, and Met31 participates by assisting Met32 association with its binding site. The width of the arrows pointing toward Cbf1 and Met31/32 DNA binding sites translate the strength of the interaction, which differs between MET and PDC6 promoters.

Article Snippet: The 32 P-labeled DNA fragments indicated were incubated with 0, 50 and 200 ng of a recombinant derivative of Cbf1 containing the bHLH domain fused to a polyhistidine tag ( his Cbf1 BD ).

Techniques: Binding Assay