Journal: Cells

Article Title: Transcriptomic Response to 1,25-Dihydroxyvitamin D in Human Fibroblasts with or without a Functional Vitamin D Receptor (VDR): Novel Target Genes and Insights into VDR Basal Transcriptional Activity

doi: 10.3390/cells8040318

Figure Lengend Snippet: Top-ranking up- and down- regulated 1,25D-activated VDR target genes in human fibroblasts (CO).

Article Snippet: First-strand cDNA was generated using SuperScript III reverse transcriptase (Thermo Fisher) and quantitative PCR performed in a StepOnePlus Real-Time PCR System (Thermo Fisher) using the following inventoried TaqMan® assays (Thermo Fisher): CYP24A1 Hs00167999 _m1, TGFB2 Hs00234244_m1, RASSF2 Hs00248129_m1, CILP Hs00173647_m1, STC1 Hs00174970_m1, SPP1 Hs00959010_m1, GALNT12 Hs00226436_m1, HBEGF Hs00181813_m1, EFNB2 Hs00187950_m1, and EGR1 Hs00152928_m1.

Techniques:

Journal: Cells

Article Title: Transcriptomic Response to 1,25-Dihydroxyvitamin D in Human Fibroblasts with or without a Functional Vitamin D Receptor (VDR): Novel Target Genes and Insights into VDR Basal Transcriptional Activity

doi: 10.3390/cells8040318

Figure Lengend Snippet: Quantitative RT-PCR validation of differential gene expression findings.

Article Snippet: First-strand cDNA was generated using SuperScript III reverse transcriptase (Thermo Fisher) and quantitative PCR performed in a StepOnePlus Real-Time PCR System (Thermo Fisher) using the following inventoried TaqMan® assays (Thermo Fisher): CYP24A1 Hs00167999 _m1, TGFB2 Hs00234244_m1, RASSF2 Hs00248129_m1, CILP Hs00173647_m1, STC1 Hs00174970_m1, SPP1 Hs00959010_m1, GALNT12 Hs00226436_m1, HBEGF Hs00181813_m1, EFNB2 Hs00187950_m1, and EGR1 Hs00152928_m1.

Techniques: Quantitative RT-PCR, Biomarker Discovery, Gene Expression, Quantitative Proteomics, Microarray

Journal:

Article Title: DNA/DNA hybridization to microarrays reveals gene-specific differences between closely related microbial genomes

doi: 10.1073/pnas.171178898

Figure Lengend Snippet: Strains used in DNA:DNA microarray hybridization analysis

Article Snippet: 184 ATCC 8073 Butter AF387349 * AF387354 * S. woodyi MS32 ATCC 51908, TS Persian Gulf seawater AF003548 AF014944 Environmental isolate W3 18-1 Pacific Ocean marine sediments (630 m, 5–6 cm in core) AF387350 * AF387355 * Environmental isolate PV-4 Pacific Ocean seawater, Naha Vents, Hawaii AF387348 * AF387352 * E. coli K-12 ATCC 10798 Human feces AE000452 AE000447 Open in a separate window Type strains (TS) indicated.

Techniques: Microarray, Hybridization

Journal: PLoS ONE

Article Title: Mesenchymal stromal cell-derived extracellular vesicles reduce lung inflammation and damage in nonclinical acute lung injury: Implications for COVID-19

doi: 10.1371/journal.pone.0259732

Figure Lengend Snippet: (A) Antibody microarray particle analysis of tetraspanin proteins. (B) Electrochemiluminescent multiplexed immunoassay detection of tetraspanin proteins. (C) Western blot analysis of MSC-EV proteins flotillin-1, Annexin-2, Syntenin-1, MHC-I and contaminating proteins MHC-II and calreticulin. (D) Immunogold cryo-transmission electron microscopy imaging for CD63 and phosphatidylserine. Atomic force microscopy for MSC-EV morphology and size. (E) Nanoparticle tracking for MSC-EV size and concentration. (F) MSC-EV particle density correlated to phospholipid content. Error bars are SEM of the mean. *p ≤0.05 vs CD63. Scale bar denotes 100 nm.

Article Snippet: Lysates were volume loaded and separated on a 12% Bolt Bis-Tris Gel and probed using Abcam antibodies against flotillin-1 (ab133497 at 1:10,000 dilution), Annexin-2 (ab41803 at 1:1000 dilution), Syntenin-1 (ab19903 at 1:1000 dilution), MHC-I (ab110645 at 1:1000 dilution), MHC-II (157210 at 1:10000 dilution), and Calreticulin (ab92516 at 1:1000 dilution).

Techniques: Microarray, Western Blot, Transmission Assay, Electron Microscopy, Imaging, Microscopy, Concentration Assay

Journal: Nature Communications

Article Title: Context-specific regulation of surface and soluble IL7R expression by an autoimmune risk allele

doi: 10.1038/s41467-019-12393-1

Figure Lengend Snippet: LPS induces profound monocyte IL7R cell surface expression. a Representative flow cytometry plots from live gated positively selected CD14 monocytes from one individual where cells were incubated for 24 h alone (top panel) or in presence of LPS (bottom panels). b Violin plot demonstrating significant induction of IL7R + monocytes in paired cultures of positively selected monocytes (Monocyte IL7R + post LPS- median: 23.8, min: 6.5, max: 55.8, IQR: 15.6–34.9%; n = 84, paired t -test). c Comparative effects of LPS on IL7R + counts across CD14 + monocytes, CD4 + and CD8 + T cells, and CD56 + NK cells from the same PBMC cultures, untreated or with LPS for 24 h (PBMC IL7R + post LPS-median: 29.9%, min: 5.9, max: 66.40, IQR: 22.3–40.0; n = 103, paired t -test). d Pearson correlation analyses were performed between indicated cells and treatments on number of IL7R + cells. *** P < 0.001, ** P < 0.01. e Comparative induction of IL7R + monocytes from 6 individuals with monocytes treated for 24 h with either the TLR7 agonist Imiquimod, TLR1/2 agonist Pam3CysK4 or TLR4 agonist LPS (paired t -tests). Error bars show mean, IQR, min and max. f Array derived RNA expression of TNF at 2 h LPS assayed versus RNA expression of IL7R from monocytes from same individuals at 24 h LPS. g Comparative incubation of monocytes alone ( n = 69), with LPS or with LPS + anti-TNF monoclonal antibody (Infliximab). Incubation with TNF antagonist significantly reduces LPS induced IL7R + monocyte counts (paired t -test). h Violin plots of responses across cell types of PBMCs treated with TNF alone leads to significant induction of IL7R + monocytes and significantly reduces CD4 and CD8 T cell IL7R + positivity ( n = 78, paired t -test). Source data are provided as a Source Data file

Article Snippet: Plates were blocked with 5% BSA for 1 h, washed, and cell supernatants added for 2 h. Bound sIL7R was detected with 12.5 ng/ml biotinylated goat anti-human CD127 polyclonal Ab (R&D Systems) for 1 h, followed by a 30 min incubation with streptavidin-HRP and a 15 min incubation with TMB peroxidase substrate (Thermo Fisher).

Techniques: Expressing, Flow Cytometry, Incubation, Derivative Assay, RNA Expression

Journal: Nature Communications

Article Title: Context-specific regulation of surface and soluble IL7R expression by an autoimmune risk allele

doi: 10.1038/s41467-019-12393-1

Figure Lengend Snippet: rs6897932 specifically regulates monocyte cell surface IL7R levels. a Microarray data demonstrating eQTL to IL7R noted at rs931555 after both 2 and 24 h treatment with LPS (2 h LPS: n = 261, 24 h LPS n = 322). b Local association plot for monocyte IL7R after exposure to LPS from positively selected monocytes demonstrates peak association to rs6897932 ( n = 84). c Batch corrected log values for surface IL7R from positively selected monocytes demonstrating significant effect of rs6897932 carriage after stimulation in comparison to baseline monocytes ( n = 84, ANOVA for linear model). d Surface IL7R in PBMCs across cell subsets after exposure to LPS. ( n = 87, Note a significant effect of rs6897932 on surface IL7R is only observed in CD14 + monocytes, ANOVA for linear model). e As per d but PBMCs treated with TNF for 24 h ( n = 62). Error bars show mean, IQR, min, and max. Source data are provided as a Source Data file

Article Snippet: Plates were blocked with 5% BSA for 1 h, washed, and cell supernatants added for 2 h. Bound sIL7R was detected with 12.5 ng/ml biotinylated goat anti-human CD127 polyclonal Ab (R&D Systems) for 1 h, followed by a 30 min incubation with streptavidin-HRP and a 15 min incubation with TMB peroxidase substrate (Thermo Fisher).

Techniques: Microarray, Comparison

Journal: Nature Communications

Article Title: Context-specific regulation of surface and soluble IL7R expression by an autoimmune risk allele

doi: 10.1038/s41467-019-12393-1

Figure Lengend Snippet: Monocyte-derived soluble IL7R is regulated by rs6897932 carriage and DDX39A expression. a ELISA of sIL7R performed on supernatants of monocytes treated with LPS for 24 h. Samples were randomly chosen from original eQTL dataset and genotypes revealed post-hoc – a significant association was observed at rs6897932 ( n = 161, ANOVA for linear model). b ELISA data from a showing additional effect of rs931555 genotype on sIL7R (linear model). c Correlation analysis performed between 15421 microarray probes and soluble IL7R ( y -axis) identified DDX39A expression ( x -axis) to be significantly anti-correlated with soluble IL7R levels, supporting evidence for its regulation of soluble IL7R. Trend lines indicate correlation by allele, with rho and P -value for all data ( n = 161, Spearman rank analysis). d ELISA of sIL7R from isolated primary CD14 + and CD4 + cells cultured for 24 h either alone or in the presence of LPS, LPS + IL-7 and CD3/CD28 activating beads. ( n = 6). e ELISA of sIL7R from isolated primary CD14 + cultured for 24 h either alone, in the presence of TNF or in the presence of LPS ( n = 5, ANOVA). Error bars show mean, IQR, min and max. Source data are provided as a Source Data file

Article Snippet: Plates were blocked with 5% BSA for 1 h, washed, and cell supernatants added for 2 h. Bound sIL7R was detected with 12.5 ng/ml biotinylated goat anti-human CD127 polyclonal Ab (R&D Systems) for 1 h, followed by a 30 min incubation with streptavidin-HRP and a 15 min incubation with TMB peroxidase substrate (Thermo Fisher).

Techniques: Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay, Microarray, Isolation, Cell Culture

Journal: Nature Communications

Article Title: Context-specific regulation of surface and soluble IL7R expression by an autoimmune risk allele

doi: 10.1038/s41467-019-12393-1

Figure Lengend Snippet: Monocyte surface IL7R is functional, activating multiple transcriptional pathways. a Violin plot demonstrating monocytes from PBMC cultures in the untreated state, after exposure to LPS and after exposure to LPS with recombinant IL-7. IL-7 leads to marked downregulation of IL7R on LPS stimulated monocytes, indicative sensitivity to exogenous cytokine (paired t -test). b Volcano plot of mRNA from RNAseq experiments of 8 paired monocyte samples either treated with LPS alone for 24 h or with LPS with additional IL-7 added for the last 2 h of culture. Treatment leads to widespread differential transcript expression with the most significant 20 transcripts labelled. c Example boxplots of genes differentially regulated by recombinant IL-7 in monocytes (linear model). d t-SNE plot of single-cell real-time quantitative PCR (RT-qPCR) data from 10 individuals (1218 cells) either untreated or in the presence LPS showing expression of membrane-bound IL7R ( IL7R mb), soluble IL7R (s IL7R ), CLEC4E, TNF, CUX1, AKT1, STAB1, CD163, CSF1R, CCL5, DDX39A, DDX39B, MALAT1, LTB, IFNB1, CD14, FCGR3B, LYZ, and ACTB . e Density plots of single-cell RT-qPCR expression of sIL7R and mbIL7R normalized expression in untreated and LPS treated monocytes according to rs6897932 genotype. Kolmogorov Smirnov test. f Violin plots of single-cell RT-qPCR expression of MALAT1 in LPS stimulated monocytes either alone or in the presence of recombinant human IL-7 according to rs6897932 genotype, t -test

Article Snippet: Plates were blocked with 5% BSA for 1 h, washed, and cell supernatants added for 2 h. Bound sIL7R was detected with 12.5 ng/ml biotinylated goat anti-human CD127 polyclonal Ab (R&D Systems) for 1 h, followed by a 30 min incubation with streptavidin-HRP and a 15 min incubation with TMB peroxidase substrate (Thermo Fisher).

Techniques: Functional Assay, Recombinant, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Membrane

Journal: Nature Communications

Article Title: Context-specific regulation of surface and soluble IL7R expression by an autoimmune risk allele

doi: 10.1038/s41467-019-12393-1

Figure Lengend Snippet: IL7R + monocytes form a distinct subset detectable in synovial fluid. a Representative flow cytometry of IL7R staining of spondyloarthritis matched patient PBMC (left) and SFMC (middle isotype control, right) gated on CD3 - CD14 + as per sequential gating strategy shown in Supplementary Fig. . b Results from 4 patients demonstrating comparative monocyte IL7R staining in matched PBMC and SFMC using flow cytometry. c t-SNE clustering of monocytes from single-cell RNA sequencing of spondyloarthritis patient SFMC showing 5 clusters ( n = 3). d Single-cell monocyte expression of IL7R, LTB, CD14, CCL5, IL32 , and TCF7 overlaid on t-SNE clustering in c . e Volcano plot of genes significantly upregulated in the SFMC monocyte cluster 4 compared to all other SFMC monocyte clusters with top 20 genes annotated. Wilcoxon rank sum test. f Heatmap of top 10 genes from the each of the five SFMC monocyte clusters identified in c

Article Snippet: Plates were blocked with 5% BSA for 1 h, washed, and cell supernatants added for 2 h. Bound sIL7R was detected with 12.5 ng/ml biotinylated goat anti-human CD127 polyclonal Ab (R&D Systems) for 1 h, followed by a 30 min incubation with streptavidin-HRP and a 15 min incubation with TMB peroxidase substrate (Thermo Fisher).

Techniques: Flow Cytometry, Staining, Control, RNA Sequencing, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Angiogenic patterning by STEEL, an endothelial-enriched long noncoding RNA

doi: 10.1073/pnas.1715182115

Figure Lengend Snippet: Gene regulation in endothelial cells by STEEL RNA. (A) RT-qPCR of STEEL knockdown by two independent siRNA sequences. (B) Volcano plot representing mRNAs regulated by STEEL knockdown in HUVECs and HMVECs from microarray. Red indicates differentially regulated genes. (C–F) RT-qPCR of HOXD1 and HOXD3 mRNA with STEEL knockdown. (D) Western blot of HOXD1 and HOXD3 protein with STEEL knockdown. (E) RT-qPCR of KLF2 and eNOS mRNA with STEEL knockdown. (F) Western blot of KLF2 and eNOS protein with STEEL knockdown. (G) RT-qPCR of KLF2 and eNOS hnRNA (pre-mRNA) with STEEL knockdown. (H) RT-qPCR of RNA Pol II ChIP at KLF2 and eNOS promoters. (I) Promoter luciferase-reporter assays with STEEL knockdown. (J) CHART-qPCR with STEEL knockdown. (K and L) ChIP-qPCR of the core histone, histone H3, and the activating epigenetic mark histone 3 lysine 4 trimethyl (H3K4me3) at the KLF2 and eNOS promoters after STEEL siRNA knockdown in HUVECs. *P < 0.05. Error bars represent SEM.

Article Snippet: Custom lncRNA microarrays on Arraystar Human lncRNA Microarray (Nimblegen 12 × 135 K array platform, 23,155 lncRNAs, 19,484 protein-coding genes) and Human lncRNA Microarray V2.0 (Agilent 8 × 60 K array platform, 33,045 lncRNA probes, 30,215 protein-coding mRNA probes) were used for cell specificity and STEEL knockdown experiments, respectively.

Techniques: Quantitative RT-PCR, Microarray, Western Blot, Luciferase

Journal: Nucleic Acids Research

Article Title: Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells

doi: 10.1093/nar/gkt504

Figure Lengend Snippet: Genome-wide p53 binding after various treatments. ( A ) Representative western blot of p53 levels in U2OS cytoplasmic, nuclear or whole-cell extracts from untreated (NT) cells, or after 24 h treatment with 0.1% DMSO, 0.6 µg/ml DXR or 10 µM Nutlin. Lamin B and actin Hsp90, lamin B and actin are loading controls. ( B ) Number of p53 binding sites identified in U2OS 24 h after treatment with DMSO, DXR or Nutlin. ( C ) UCSC Genome Browser shots summarizing p53 ChIP-Seq and Input control data for known p53 target genes after various treatments. The Y axis corresponds to the number of reads. The same scale is used for all examples presented. ( D ) Genomic distribution of p53 binding sites relative to genic regions after the various treatments: TSS, 5 kb upstream through the first intron; Intragenic, second exon through 3′UTR; and Intergenic, everything else.

Article Snippet: Chromatin was sheared to 100–300 bp using a Bioruptor sonicator (Diagenode, Denville, NJ) for 30 min at high power, 30 s ON, 30 s OFF. p53 (DO-1) antibody (12 μg) or normal mouse immunoglobulin G (IgG) (3 μg) (Santa Cruz) was bound to 200 μl Dynabeads Protein G magnetic beads (Invitrogen) by incubation at 4°C in 5 mg/ml of bovine serum albumin for 6 h. Five percent of the chromatin was saved as input.

Techniques: Genome Wide, Binding Assay, Western Blot, ChIP-sequencing, Control

Journal: Nucleic Acids Research

Article Title: Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells

doi: 10.1093/nar/gkt504

Figure Lengend Snippet: Motif analysis and distribution of p53-binding sites. ( A ) Consensus p53-binding motifs identified from the top 1000 p53-binding sites for each treatment. ( B ) Characterization of p53-binding sites based on the type of sequence bound: zero spacer, consensus motif containing two decamers with no spacer sequence as shown in (A); 1–15 spacer, two decamers with a spacer sequence of length 1–15 nt; half site, one decamer; no motif, no evidence of zero spacer, 1–15 spacer, or a half site. ( C ) Frequency of p53-binding sites containing zero spacer (red), 1–15 spacer (blue), and half sites (green). ( D ) Box-plot depicting distribution of ChIP-over-Input fold enrichment for p53-binding sites containing zero spacer, 1–15 spacer, half site and no motif. Asterisk indicates statistically significant ( P < 10 −7 ).

Article Snippet: Chromatin was sheared to 100–300 bp using a Bioruptor sonicator (Diagenode, Denville, NJ) for 30 min at high power, 30 s ON, 30 s OFF. p53 (DO-1) antibody (12 μg) or normal mouse immunoglobulin G (IgG) (3 μg) (Santa Cruz) was bound to 200 μl Dynabeads Protein G magnetic beads (Invitrogen) by incubation at 4°C in 5 mg/ml of bovine serum albumin for 6 h. Five percent of the chromatin was saved as input.

Techniques: Binding Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells

doi: 10.1093/nar/gkt504

Figure Lengend Snippet: Gene expression analysis following various treatments. ( A ) Number of genes with altered expression following DXR or Nutlin treatment as compared with the control NT or DMSO controls, respectively. ( B ) Heat map showing gene expression changes based on microarray analysis of cells after 24 h of DXR, DMSO or Nutlin treatment. DXR and DMSO changes were assessed in relation to NT, and Nutlin changes were assessed in relation to DMSO. The KEGG/GO terms shown are non-redundant terms manually selected among the most significantly enriched categories for each of the bracketed clusters. The full lists of terms are available in Supplementary Data Set 5 . ( C ) KEGG pathways and GO biological processes enriched among all the differentially expressed genes in (A) after various treatments. ( D ) Breakdown of expression changes of genes with a p53-binding site near the TSS or ( E ) in the intragenic region. ( F ) Number of genes that were bound by p53 near a TSS and were differentially expressed after DXR or Nutlin treatment.

Article Snippet: Chromatin was sheared to 100–300 bp using a Bioruptor sonicator (Diagenode, Denville, NJ) for 30 min at high power, 30 s ON, 30 s OFF. p53 (DO-1) antibody (12 μg) or normal mouse immunoglobulin G (IgG) (3 μg) (Santa Cruz) was bound to 200 μl Dynabeads Protein G magnetic beads (Invitrogen) by incubation at 4°C in 5 mg/ml of bovine serum albumin for 6 h. Five percent of the chromatin was saved as input.

Techniques: Gene Expression, Expressing, Control, Microarray, Binding Assay

Journal: Nucleic Acids Research

Article Title: Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells

doi: 10.1093/nar/gkt504

Figure Lengend Snippet: Correlation between p53 binding, changes in gene expression and histone modifications H3K4me3 and H3K27me3 at 24 h after DXR, Nutlin or DMSO treatments. The heat map represents fold changes in mRNA levels quantified using qRT-PCR, as well as fold changes in p53, H3K4me3 and H3K27me3 occupancy quantified using ChIP-qPCR (see also Supplementary Figures S1D and S2A–C ). Presented are results for 36 genes that were bound by p53 and two genes ( GAPDH and MYOD1 ) that were not bound by p53. Genes are clustered into groups depending on whether the p53 target site contains a consensus p53-binding motif containing two decamers with no spacer (zero spacer), two decamers with a spacer sequence (spacers), or one decamer (half site) or no motif. Fold changes for gene expression were normalized to β-2-microglobulin, whereas p53, H3K4me3 and H3K27me3 ChIP-qPCRs were normalized to the negative controls GAPDH, MYOD1 and GAPDH, respectively. ( A ) DXR/NT, ( B ) Nutlin/DMSO and ( C ) DMSO/NT.

Article Snippet: Chromatin was sheared to 100–300 bp using a Bioruptor sonicator (Diagenode, Denville, NJ) for 30 min at high power, 30 s ON, 30 s OFF. p53 (DO-1) antibody (12 μg) or normal mouse immunoglobulin G (IgG) (3 μg) (Santa Cruz) was bound to 200 μl Dynabeads Protein G magnetic beads (Invitrogen) by incubation at 4°C in 5 mg/ml of bovine serum albumin for 6 h. Five percent of the chromatin was saved as input.

Techniques: Binding Assay, Gene Expression, Quantitative RT-PCR, ChIP-qPCR, Sequencing

Journal: Nucleic Acids Research

Article Title: Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells

doi: 10.1093/nar/gkt504

Figure Lengend Snippet: Validation of new p53 direct target genes. Real-time PCR validation of newly identified putative p53 targets in U2OS cells using shRNA-mediated silencing of p53 expression. Parental U2OS cells treated with ( A ) no shRNA, ( B ) scramble (control) shRNA, ( C ) p53shRNA 3755 or ( D ) p53shRNA 3756. mRNA levels were quantified in U2OS cells 24 h after NT, DMSO, DXR or Nutlin treatment using qRT-PCR and normalized to GAPDH . mRNA levels are shown as fold change after treatment relative to the NT control. Expression of the CDKN1A gene was used as a positive control. Shown are averages of three independent experiments. The dashed line corresponds to a 2-fold change in expression. Error bars represent standard deviation (SD).

Article Snippet: Chromatin was sheared to 100–300 bp using a Bioruptor sonicator (Diagenode, Denville, NJ) for 30 min at high power, 30 s ON, 30 s OFF. p53 (DO-1) antibody (12 μg) or normal mouse immunoglobulin G (IgG) (3 μg) (Santa Cruz) was bound to 200 μl Dynabeads Protein G magnetic beads (Invitrogen) by incubation at 4°C in 5 mg/ml of bovine serum albumin for 6 h. Five percent of the chromatin was saved as input.

Techniques: Biomarker Discovery, Real-time Polymerase Chain Reaction, shRNA, Expressing, Control, Quantitative RT-PCR, Positive Control, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells

doi: 10.1093/nar/gkt504

Figure Lengend Snippet: Proposed models for p53-DNA interaction at half-sites (1 decamer) and full sites (two decamers) with and without a spacer. The minimal p53-binding unit is a half site composed of the consensus decamer RRRCWWGYYY (except for the NT control) based on binding to half sites (decamers) and full sites (two decamers) with a spacer. The binding models assume that p53 exists as a dimer and tetramers are formed following p53 dimer binding to a target sequence. p53-bound half sites as well as full sites containing a spacer are much less frequent across the genome than full sites with zero spacer. ( A ) Efficient tetramer binding at full sites with zero spacer. Strong interactions between dimers and target sequences can occur when the dimers bind to a pair of decamers with no spacer. The dark grey ovals indicates possible changes in p53 (such as configuration) that enable the strong binding. ( B ) Binding to a half site, which is much less efficient than to a full site with no spacer, could be due to weaker interactions of the decamer sequence with just one p53 dimer or with a dimer of dimers, where one dimer makes contact with the decamer sequence, whereas the second dimer interacts non-specifically with nearby DNA. ( C ) Binding to a full site with a spacer is proposed to occur through p53-decamer interactions that are described for the half sites in (B) with the additional possibility of weak interactions between the two bound p53 dimers (lower left panel).

Article Snippet: Chromatin was sheared to 100–300 bp using a Bioruptor sonicator (Diagenode, Denville, NJ) for 30 min at high power, 30 s ON, 30 s OFF. p53 (DO-1) antibody (12 μg) or normal mouse immunoglobulin G (IgG) (3 μg) (Santa Cruz) was bound to 200 μl Dynabeads Protein G magnetic beads (Invitrogen) by incubation at 4°C in 5 mg/ml of bovine serum albumin for 6 h. Five percent of the chromatin was saved as input.

Techniques: Binding Assay, Control, Sequencing