dnase i  (Worthington Biochemical)

Journal: Molecular cell

Article Title: Catalytic-Independent Functions of PARP-1 Determine Sox2 Pioneer Activity at Intractable Genomic Loci

doi: 10.1016/j.molcel.2017.01.017

Figure Lengend Snippet: PARP-1 stabilizes Sox2 binding to nucleosome in vitro A) In vitro nucleosome binding assays show PARP-1-dependent binding of Sox2 to nucleosomes, but not naked DNA. Ten nM of biotin-labeled 601 NPE DNA (207 bp) with or without a Sox motif (Sox+ or Sox−, respectively), or the same DNA assembled into a mononucleosome (Nuc), was immobilized on streptavidin beads and incubated ± recombinant Sox2 (10 nM) and PARP-1 (10 nM), as indicated. After washing, the bound proteins were analyzed by Western blotting. B) In vitro DNase I footprinting assays show PARP-1-dependent binding of Sox2 to nucleosomes, but not naked DNA. Left, Footprinting assay with naked 3x 601 NPE DNA containing a Sox motif. Right, Footprinting assay with a trinucleosome containing a Sox motif located at the dyad axis of the middle nucleosome. Addition of 10 nM WT PARP-1, but not a DNA binding domain mutant PARP-1 (DBDmut), enhances Sox2 (25 nM) binding, as indicated. PARP-1 binding to the nucleosome is also evident. C) Quantification of the DNase I footprinting assays shown in panel C. The bands marked with a red asterisk in panel B were quantified using a phosphorimager and ImageJ software. Each bar represents the mean plus the SEM, n = 3. Asterisks indicate significant differences versus the control (−Sox2) (Student’s t test, ** p

Article Snippet: For these assays (see ), mononucleosomes corresponding to 375 ng of DNA were incubated with 300 ng of Sox2 protein with or without 600 ng of His-tagged human PARP-1 in 50 μL of binding buffer (25 mM HEPES pH 7.5, 100 mM KCl, 20% glycerol, 0.1% NP-40, 10 μM ZnSO4 , 1 mM DTT, 1x complete protease inhibitor cocktail) for 1 hour at 30°C, followed by the addition of 5 U of DNaseI (Worthington; DPFF grade, diluted in 50 μL of digestion buffer containing 10 mM MgCl2 and 5 mM CaCl2 ) and incubated at 25°C for 5 min.

Techniques: Binding Assay, In Vitro, Labeling, Incubation, Recombinant, Western Blot, Footprinting, Mutagenesis, Software

Journal: Molecular cell

Article Title: Catalytic-Independent Functions of PARP-1 Determine Sox2 Pioneer Activity at Intractable Genomic Loci

doi: 10.1016/j.molcel.2017.01.017

Figure Lengend Snippet: PARP-1 is required for maintaining transcriptional program in embryonic stem cells A) Western blots showing the relative levels of three pluripotency factors (Sox2, Oct4, and Nanog) and PARP-1 in WT and Parp1 −/− mESCs. Actin is used as an internal loading control. B) Effect of Parp1 knockout on gene expression in mESCs as determined by RNA-seq. The heatmap shows the relative expression levels of genes whose expression significantly (FDR

Article Snippet: For these assays (see ), mononucleosomes corresponding to 375 ng of DNA were incubated with 300 ng of Sox2 protein with or without 600 ng of His-tagged human PARP-1 in 50 μL of binding buffer (25 mM HEPES pH 7.5, 100 mM KCl, 20% glycerol, 0.1% NP-40, 10 μM ZnSO4 , 1 mM DTT, 1x complete protease inhibitor cocktail) for 1 hour at 30°C, followed by the addition of 5 U of DNaseI (Worthington; DPFF grade, diluted in 50 μL of digestion buffer containing 10 mM MgCl2 and 5 mM CaCl2 ) and incubated at 25°C for 5 min.

Techniques: Western Blot, Knock-Out, Expressing, RNA Sequencing Assay

Journal: Molecular cell

Article Title: Catalytic-Independent Functions of PARP-1 Determine Sox2 Pioneer Activity at Intractable Genomic Loci

doi: 10.1016/j.molcel.2017.01.017

Figure Lengend Snippet: PARP-1 is required for the binding of Sox2 to a subset of its genomic sites in mESCs A) Genome browser tracks of Sox2 ChIP-seq data around the Nanog and Pou5f1 (encoding Oct4) genes in WT and Parp1 −/− mESCs. B) Left and middle , Heatmaps of Sox2 ChIP-seq signals in WT and Parp1 −/− mESCs centered on the Sox2 binding sites (± 2 kb) and ordered top to bottom by signal intensity. Right , PARP-1 ChIP-seq signals in WT mESCs associated with the corresponding Sox2 binding sites. PARP-1-dependent Sox2 binding sites are defined as those sites whose ChIP-seq signals are significantly decreased upon PARP-1 knockout (p-value

Article Snippet: For these assays (see ), mononucleosomes corresponding to 375 ng of DNA were incubated with 300 ng of Sox2 protein with or without 600 ng of His-tagged human PARP-1 in 50 μL of binding buffer (25 mM HEPES pH 7.5, 100 mM KCl, 20% glycerol, 0.1% NP-40, 10 μM ZnSO4 , 1 mM DTT, 1x complete protease inhibitor cocktail) for 1 hour at 30°C, followed by the addition of 5 U of DNaseI (Worthington; DPFF grade, diluted in 50 μL of digestion buffer containing 10 mM MgCl2 and 5 mM CaCl2 ) and incubated at 25°C for 5 min.

Techniques: Binding Assay, Chromatin Immunoprecipitation, Knock-Out, Significance Assay

Journal: Molecular cell

Article Title: Catalytic-Independent Functions of PARP-1 Determine Sox2 Pioneer Activity at Intractable Genomic Loci

doi: 10.1016/j.molcel.2017.01.017

Figure Lengend Snippet: PARP-1 colocalizes with Sox2 genome-wide A) Top, Summary of the known roles of PARP-1 protein and PARylation activity in embryonic stem cells. Bottom, Summary of known changes in Sox2 protein and PARylation levels, total cellular PARylation levels during embryonic stem cell differentiation. B) The level of PARP-1-mediated PARylation is very low in undifferentiated mESCs and increases upon differentiation. Western blots of poly(ADP-ribose) (PAR) and PARP-1 showing their relative levels in mESCs during a 9 day time course of differentiation upon LIF removal. C) Distribution of significant peaks of PARP-1 binding from ChIP-seq across genomic features in mESCs. D) Distribution of significant peaks of PARP-1 binding relative to the TSSs of all RefSeq genes. E) PARP-1 colocalizes with Sox2 genome-wide. Correlation matrix of genome-wide enrichment for chromatin- and transcription-related factors from ChIP-seq data in mESCs, organized and ordered using hierarchical clustering. F) Left , Heatmap of high confidence Sox2 peaks in mESCs from ChIP-seq data (n = 16,042; p-value

Article Snippet: For these assays (see ), mononucleosomes corresponding to 375 ng of DNA were incubated with 300 ng of Sox2 protein with or without 600 ng of His-tagged human PARP-1 in 50 μL of binding buffer (25 mM HEPES pH 7.5, 100 mM KCl, 20% glycerol, 0.1% NP-40, 10 μM ZnSO4 , 1 mM DTT, 1x complete protease inhibitor cocktail) for 1 hour at 30°C, followed by the addition of 5 U of DNaseI (Worthington; DPFF grade, diluted in 50 μL of digestion buffer containing 10 mM MgCl2 and 5 mM CaCl2 ) and incubated at 25°C for 5 min.

Techniques: Genome Wide, Activity Assay, Cell Differentiation, Western Blot, Binding Assay, Chromatin Immunoprecipitation