mnase  (Worthington Biochemical)


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    Structured Review

    Worthington Biochemical mnase
    Expression of mutant <t>H2B</t> in yeast destabilizes nucleosomes, deregulates gene expression and reduces nucleosome occupancy at the PHO5 promoter. WT or E79K H2B (analogous to human H2B-E76K) was expressed in S. Cerevisiae. (A) Yeast cells expressing H2B-E79K are temperature sensitive. Limiting dilutions of yeast expressing WT, E79A, E79Q or E79K were plated and incubated at 30°C or 37°C. Cell growth was evaluated after 1 day. (B) Yeast doubling time is significantly increased in cells expressing E79K-H2B at 37°C. (C) Time course of <t>MNase</t> sensitivity from spheroplasted yeast grown in rich media. M, marker. (D) Chromatin pellets were extracted with increasing concentrations of salt as indicated. Immuno-blotting of the soluble fraction was performed with antibody to H4. (E) Cells expressing WT, E79Q or E79K H2B were maintained in either rich media (YPDA) or phosphate-free media and expression of the phosphate-inducible PHO5 gene was measured by RT-PCR. (F) Nucleosome scanning assay of the PHO5 promoter from cells expressing either WT or E79K grown in rich media. Chromatin was digested with MNase, mononucleosomal DNA was purified and MNase protection was determined by qPCR. H2B occupancy at −2 nucleosome position of PHO5 is reduced in E79K cells as indicated by the arrow.
    Mnase, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 92/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "A Mutation in Histone H2B Represents A New Class Of Oncogenic Driver"

    Article Title: A Mutation in Histone H2B Represents A New Class Of Oncogenic Driver

    Journal: Cancer discovery

    doi: 10.1158/2159-8290.CD-19-0393

    Expression of mutant H2B in yeast destabilizes nucleosomes, deregulates gene expression and reduces nucleosome occupancy at the PHO5 promoter. WT or E79K H2B (analogous to human H2B-E76K) was expressed in S. Cerevisiae. (A) Yeast cells expressing H2B-E79K are temperature sensitive. Limiting dilutions of yeast expressing WT, E79A, E79Q or E79K were plated and incubated at 30°C or 37°C. Cell growth was evaluated after 1 day. (B) Yeast doubling time is significantly increased in cells expressing E79K-H2B at 37°C. (C) Time course of MNase sensitivity from spheroplasted yeast grown in rich media. M, marker. (D) Chromatin pellets were extracted with increasing concentrations of salt as indicated. Immuno-blotting of the soluble fraction was performed with antibody to H4. (E) Cells expressing WT, E79Q or E79K H2B were maintained in either rich media (YPDA) or phosphate-free media and expression of the phosphate-inducible PHO5 gene was measured by RT-PCR. (F) Nucleosome scanning assay of the PHO5 promoter from cells expressing either WT or E79K grown in rich media. Chromatin was digested with MNase, mononucleosomal DNA was purified and MNase protection was determined by qPCR. H2B occupancy at −2 nucleosome position of PHO5 is reduced in E79K cells as indicated by the arrow.
    Figure Legend Snippet: Expression of mutant H2B in yeast destabilizes nucleosomes, deregulates gene expression and reduces nucleosome occupancy at the PHO5 promoter. WT or E79K H2B (analogous to human H2B-E76K) was expressed in S. Cerevisiae. (A) Yeast cells expressing H2B-E79K are temperature sensitive. Limiting dilutions of yeast expressing WT, E79A, E79Q or E79K were plated and incubated at 30°C or 37°C. Cell growth was evaluated after 1 day. (B) Yeast doubling time is significantly increased in cells expressing E79K-H2B at 37°C. (C) Time course of MNase sensitivity from spheroplasted yeast grown in rich media. M, marker. (D) Chromatin pellets were extracted with increasing concentrations of salt as indicated. Immuno-blotting of the soluble fraction was performed with antibody to H4. (E) Cells expressing WT, E79Q or E79K H2B were maintained in either rich media (YPDA) or phosphate-free media and expression of the phosphate-inducible PHO5 gene was measured by RT-PCR. (F) Nucleosome scanning assay of the PHO5 promoter from cells expressing either WT or E79K grown in rich media. Chromatin was digested with MNase, mononucleosomal DNA was purified and MNase protection was determined by qPCR. H2B occupancy at −2 nucleosome position of PHO5 is reduced in E79K cells as indicated by the arrow.

    Techniques Used: Expressing, Mutagenesis, Incubation, Marker, Reverse Transcription Polymerase Chain Reaction, Purification, Real-time Polymerase Chain Reaction

    The E76K mutation in H2B destabilizes the histone octamer and fails to protect the nucleosome from nuclease treatment in vitro . (A) The H2B-E76K mutant was unable to form stable octamers in vitro . Recombinant human histones (H2A, H2B, H3, and H4) were mixed and histone octamers resolved from (H3/H4) 2 tetramers, H2A/H2B dimers and free histones by gel filtration chromatography. (B) Nucleosomes were reconstituted by mixing equimolar amounts of DNA (147bp) and octamers or in the case of E76K of tetramers and dimers (1:2 molar ratio) and resolved by Native PAGE. Nucleosomes containing H2B-E76K and E76Q have an altered migration pattern, intermediate between a tetrasome and a WT nucleosome. (C) Micrococcal nuclease (MNase) sensitivity assay performed on nucleosomes made with WT, E76Q and E76K H2B mutants shows more rapid digestion of E76K containing nucleosomes than those with WT H2B. A time course by gel (left) and densitometry quantification (right) of intact nucleosomes following MNase treatment. (D) The MNase susceptibility of E76K nucleosomes is distinct from nucleosomes formed only with tetrasomes.
    Figure Legend Snippet: The E76K mutation in H2B destabilizes the histone octamer and fails to protect the nucleosome from nuclease treatment in vitro . (A) The H2B-E76K mutant was unable to form stable octamers in vitro . Recombinant human histones (H2A, H2B, H3, and H4) were mixed and histone octamers resolved from (H3/H4) 2 tetramers, H2A/H2B dimers and free histones by gel filtration chromatography. (B) Nucleosomes were reconstituted by mixing equimolar amounts of DNA (147bp) and octamers or in the case of E76K of tetramers and dimers (1:2 molar ratio) and resolved by Native PAGE. Nucleosomes containing H2B-E76K and E76Q have an altered migration pattern, intermediate between a tetrasome and a WT nucleosome. (C) Micrococcal nuclease (MNase) sensitivity assay performed on nucleosomes made with WT, E76Q and E76K H2B mutants shows more rapid digestion of E76K containing nucleosomes than those with WT H2B. A time course by gel (left) and densitometry quantification (right) of intact nucleosomes following MNase treatment. (D) The MNase susceptibility of E76K nucleosomes is distinct from nucleosomes formed only with tetrasomes.

    Techniques Used: Mutagenesis, In Vitro, Recombinant, Filtration, Chromatography, Clear Native PAGE, Migration, Sensitive Assay

    H2B-E76K fundamentally alters chromatin structure and dynamics. (A) Micrococcal nuclease (MNase) assay with nuclei of MCF10A cells stably expressing either WT or H2B-E76K demonstrate that E76K expression significantly increases sensitivity to MNase. Digest efficiency was visualized by agarose gel. (B) Fluorescence recovery after photobleaching (FRAP) analysis demonstrates that H2B-E76K has significantly faster chromatin dynamics than WT. FRAP analysis was carried out after induction of either WT or E76K mutant H2B-GFP fusions for 5 or 35 days in MCF10A cells. Dashed lines represent cells expressing H2B-E76K, solid lines represent data from cells expressing inducible GFP-tagged WT H2B (n = 10 cells). (C) Representative FRAP assay pre-bleach, bleach and post-bleach images of nuclei expressing GFP-tagged WT or H2B-E76K indicate faster fluorescent recovery in cells expressing E76K. (D) FRAP analysis of histone H2A-GFP dynamics in MCF10A cells expressing only H2A-GFP (n = 20 cells) or both H2A-GFP and a mutant H2B E76K mCherry fusion (n = 25 cells) with standard deviation envelopes. Inset demonstrates co-expression of H2A GFP and E76K mCherry. (E) Representative pre-bleach, bleach and post-bleach images of H2A GFP in WT MCF10A cells or in cells co-expressing mCherry tagged H2B-E76K. (F) 100nm particles injected into the nucleus had significantly increased mean square displacement (MSD) over time in MCF10A cells stably expressing exogenous H2B-E76K (red) compared to cells expressing WT H2B (blue). N=68 (WT) and N=67 (E76K) cells were analyzed. Error bars represent SEM.
    Figure Legend Snippet: H2B-E76K fundamentally alters chromatin structure and dynamics. (A) Micrococcal nuclease (MNase) assay with nuclei of MCF10A cells stably expressing either WT or H2B-E76K demonstrate that E76K expression significantly increases sensitivity to MNase. Digest efficiency was visualized by agarose gel. (B) Fluorescence recovery after photobleaching (FRAP) analysis demonstrates that H2B-E76K has significantly faster chromatin dynamics than WT. FRAP analysis was carried out after induction of either WT or E76K mutant H2B-GFP fusions for 5 or 35 days in MCF10A cells. Dashed lines represent cells expressing H2B-E76K, solid lines represent data from cells expressing inducible GFP-tagged WT H2B (n = 10 cells). (C) Representative FRAP assay pre-bleach, bleach and post-bleach images of nuclei expressing GFP-tagged WT or H2B-E76K indicate faster fluorescent recovery in cells expressing E76K. (D) FRAP analysis of histone H2A-GFP dynamics in MCF10A cells expressing only H2A-GFP (n = 20 cells) or both H2A-GFP and a mutant H2B E76K mCherry fusion (n = 25 cells) with standard deviation envelopes. Inset demonstrates co-expression of H2A GFP and E76K mCherry. (E) Representative pre-bleach, bleach and post-bleach images of H2A GFP in WT MCF10A cells or in cells co-expressing mCherry tagged H2B-E76K. (F) 100nm particles injected into the nucleus had significantly increased mean square displacement (MSD) over time in MCF10A cells stably expressing exogenous H2B-E76K (red) compared to cells expressing WT H2B (blue). N=68 (WT) and N=67 (E76K) cells were analyzed. Error bars represent SEM.

    Techniques Used: Stable Transfection, Expressing, Agarose Gel Electrophoresis, Fluorescence, Mutagenesis, FRAP Assay, Standard Deviation, Injection

    2) Product Images from "Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis"

    Article Title: Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis

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

    doi: 10.1073/pnas.0909927107

    Serum DNase1 degrades NETs. Human neutrophils were activated to form NETs and incubated in the indicated conditions before measurement of the digested and released NET DNA using the fluorescencent DNA dye Picogreen. ( A ) In the absence of serum (open bars) NETs are stable for at least 90 h in vitro. NET degradation with MNase for 10 min at each time point (black bars) represents the total NETs recovered. ( B ) Serum-mediated degradation of NETs is concentration and ( C ) time dependent when exposed to 10% serum, suggesting an enzymatic activity (open bars are medium controls). ( D ) Activated neutrophils that formed NETs were incubated in media with 10% serum for the indicated time points, fixed, and immunostained for myeloperoxidase (green) and histones (red). DNA (blue) was stained with Draq5. (Scale bar, 10 μm.) ( E ) Serum NET degradation requires calcium. NETs were incubated with 10% serum for 6 h. EGTA, a calcium chelator, inhibited degradation. Calcium, but not magnesium ions, restored the NET-degrading activity. ( F ) Inhibition of NET degradation by G-actin, a specific inhibitor of DNase1, is dose dependent. NETs were incubated with 10% serum for 6 h in the presence of the indicated concentrations of G-actin, and NET degradation was measured as described. ( G ) To control for the specificity of G-actin for DNase1, we incubated NETs with 20 μM of G-actin with either commercially available purified DNase1 or MNase to digest the NETs. G-actin blocked degradation by DNase1 but not MNase. ( H ) NETs were incubated with serum as described above and with the indicated concentrations of polyclonal anti-DNase1 (black bars) or irrelevant control Abs (open bars). Inhibition of NET degradation by anti-DNase1 Abs was specific and dose dependent. ( I ) Anti-DNase1 antibodies are specific. We incubated NETs in the presence of 40 μg/mL of anti-DNase1 Abs and incubated with purified DNase1 or MNase. The data shown are representative of experiments performed in triplicate and are presented as mean ± SD.
    Figure Legend Snippet: Serum DNase1 degrades NETs. Human neutrophils were activated to form NETs and incubated in the indicated conditions before measurement of the digested and released NET DNA using the fluorescencent DNA dye Picogreen. ( A ) In the absence of serum (open bars) NETs are stable for at least 90 h in vitro. NET degradation with MNase for 10 min at each time point (black bars) represents the total NETs recovered. ( B ) Serum-mediated degradation of NETs is concentration and ( C ) time dependent when exposed to 10% serum, suggesting an enzymatic activity (open bars are medium controls). ( D ) Activated neutrophils that formed NETs were incubated in media with 10% serum for the indicated time points, fixed, and immunostained for myeloperoxidase (green) and histones (red). DNA (blue) was stained with Draq5. (Scale bar, 10 μm.) ( E ) Serum NET degradation requires calcium. NETs were incubated with 10% serum for 6 h. EGTA, a calcium chelator, inhibited degradation. Calcium, but not magnesium ions, restored the NET-degrading activity. ( F ) Inhibition of NET degradation by G-actin, a specific inhibitor of DNase1, is dose dependent. NETs were incubated with 10% serum for 6 h in the presence of the indicated concentrations of G-actin, and NET degradation was measured as described. ( G ) To control for the specificity of G-actin for DNase1, we incubated NETs with 20 μM of G-actin with either commercially available purified DNase1 or MNase to digest the NETs. G-actin blocked degradation by DNase1 but not MNase. ( H ) NETs were incubated with serum as described above and with the indicated concentrations of polyclonal anti-DNase1 (black bars) or irrelevant control Abs (open bars). Inhibition of NET degradation by anti-DNase1 Abs was specific and dose dependent. ( I ) Anti-DNase1 antibodies are specific. We incubated NETs in the presence of 40 μg/mL of anti-DNase1 Abs and incubated with purified DNase1 or MNase. The data shown are representative of experiments performed in triplicate and are presented as mean ± SD.

    Techniques Used: Incubation, In Vitro, Concentration Assay, Activity Assay, Staining, Inhibition, Purification

    Inhibitory mechanisms of NET degradation. ( A ) A subset of SLE sera contains DNase1 inhibitor(s). NETs incubated with sera from healthy donors ( n = 5) or SLE patients who did not degrade NETs ( n = 22) were spiked with exogenous DNase1 or MNase, and then we quantified NET degradation. Degradation of NETs by healthy sera was unaffected by the addition of the exogenous nucleases. The SLE nondegrader sera fell into two groups: in group 1, addition of MNase but not DNase1 fully restored NET degradation activity, suggesting the presence of specific DNase1 inhibitor(s). In group 2, neither DNase1 nor MNase completely restored NET degradation, suggesting a mechanism of NET protection. *** P
    Figure Legend Snippet: Inhibitory mechanisms of NET degradation. ( A ) A subset of SLE sera contains DNase1 inhibitor(s). NETs incubated with sera from healthy donors ( n = 5) or SLE patients who did not degrade NETs ( n = 22) were spiked with exogenous DNase1 or MNase, and then we quantified NET degradation. Degradation of NETs by healthy sera was unaffected by the addition of the exogenous nucleases. The SLE nondegrader sera fell into two groups: in group 1, addition of MNase but not DNase1 fully restored NET degradation activity, suggesting the presence of specific DNase1 inhibitor(s). In group 2, neither DNase1 nor MNase completely restored NET degradation, suggesting a mechanism of NET protection. *** P

    Techniques Used: Incubation, Activity Assay

    3) Product Images from "DNA methyltransferase 3b preferentially associates with condensed chromatin"

    Article Title: DNA methyltransferase 3b preferentially associates with condensed chromatin

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq870

    Dnmt3b is enriched in histone H1-containing chromatin fractions. ( A ) Dnmt3b, Dnmt3a2, Dnmt3a, Dnmt3L and Oct3/4 expression during ES cell differentiation. Equal amounts of protein (10 µg) from undifferentiated ES cells (Day 0) and differentiated ES cells (from Day 1 to 10) were analyzed by immunoblot using the indicated antibodies. ( B ) Subcellular distribution of Dnmt3b, Dnmt3a2 and Dnmt3L. ES cells were sequentially extracted to obtain cytoplasmic, chromatin and nuclear matrix fractions. Equal amounts of fractionated protein (10 µg) were separated by SDS–PAGE and then analyzed by immunoblot using the indicated antibodies. Fractionation efficiency was confirmed by immunoblot analysis using anti-PCNA (cytoplasm) and anti-lamin B1 (nuclear matrix) antibodies, and by Coomassie Brilliant Blue (CBB) staining of core histones (chromatin). ( C ) Schematic representation of chromatin fractionation. Isolated ES cell nuclei (N) were digested with 5, 20 or 80 U of MNase/mg DNA. Digested nuclei were fractionated into three fractions: transcriptionally active (S1), transcriptionally inactive (S2) and nuclear matrix-containing (P). ( D ) DNA samples from nuclear fractions N, S1, S2 and P. Equivalent amounts of DNA from each fraction were separated by 1.5% agarose gel electrophoresis in 1× TAE followed by SYBR-Green I staining. ( E ) Nuclear aliquots corresponding to 600 ng of DNA from N, S1, S2 and P fractions were separated by SDS–PAGE and then analyzed by immunoblot using the indicated antibodies.
    Figure Legend Snippet: Dnmt3b is enriched in histone H1-containing chromatin fractions. ( A ) Dnmt3b, Dnmt3a2, Dnmt3a, Dnmt3L and Oct3/4 expression during ES cell differentiation. Equal amounts of protein (10 µg) from undifferentiated ES cells (Day 0) and differentiated ES cells (from Day 1 to 10) were analyzed by immunoblot using the indicated antibodies. ( B ) Subcellular distribution of Dnmt3b, Dnmt3a2 and Dnmt3L. ES cells were sequentially extracted to obtain cytoplasmic, chromatin and nuclear matrix fractions. Equal amounts of fractionated protein (10 µg) were separated by SDS–PAGE and then analyzed by immunoblot using the indicated antibodies. Fractionation efficiency was confirmed by immunoblot analysis using anti-PCNA (cytoplasm) and anti-lamin B1 (nuclear matrix) antibodies, and by Coomassie Brilliant Blue (CBB) staining of core histones (chromatin). ( C ) Schematic representation of chromatin fractionation. Isolated ES cell nuclei (N) were digested with 5, 20 or 80 U of MNase/mg DNA. Digested nuclei were fractionated into three fractions: transcriptionally active (S1), transcriptionally inactive (S2) and nuclear matrix-containing (P). ( D ) DNA samples from nuclear fractions N, S1, S2 and P. Equivalent amounts of DNA from each fraction were separated by 1.5% agarose gel electrophoresis in 1× TAE followed by SYBR-Green I staining. ( E ) Nuclear aliquots corresponding to 600 ng of DNA from N, S1, S2 and P fractions were separated by SDS–PAGE and then analyzed by immunoblot using the indicated antibodies.

    Techniques Used: Expressing, Cell Differentiation, SDS Page, Fractionation, Staining, Isolation, Agarose Gel Electrophoresis, SYBR Green Assay

    Dnmt3b associates with nuclease-resistant heterochromatin and the association requires higher-order chromatin structure. ( A ) DNA from nuclei digested with 5 or 80 U MNase/mg DNA. Equivalent amounts of DNA were separated by 1.5% agarose gel electrophoresis in 1× TAE and then visualized with SYBR-green I. ( B ) Detection of chromatin in FLAG-Dnmt3b immunoprecipitates. Nuclear extracts were digested with 5 or 80 U MNase/mg DNA, and subjected to immunoprecipitation using an anti-FLAG antibody in the presence of 75 mM NaCl. FLAG-Dnmt3b immunoprecipitates were analyzed by immunoblot using an anti-histone H3 antibody to confirm the interaction between Dnmt3b and chromatin. Input represents 4% of the nuclear extract used in the assay. ( C ) Dnmt3b co-sedimented with histone H1-containing poly-nucleosomes on a sucrose gradient. S2 fractions were subjected to 10–30% (w/v) sucrose gradient sedimentation. Equivalent aliquots of each DNA fraction and protein sample were analyzed by agarose gel electrophoresis and immunoblot using the indicated antibodies, respectively. Core histones in each fraction were visualized by CBB staining.
    Figure Legend Snippet: Dnmt3b associates with nuclease-resistant heterochromatin and the association requires higher-order chromatin structure. ( A ) DNA from nuclei digested with 5 or 80 U MNase/mg DNA. Equivalent amounts of DNA were separated by 1.5% agarose gel electrophoresis in 1× TAE and then visualized with SYBR-green I. ( B ) Detection of chromatin in FLAG-Dnmt3b immunoprecipitates. Nuclear extracts were digested with 5 or 80 U MNase/mg DNA, and subjected to immunoprecipitation using an anti-FLAG antibody in the presence of 75 mM NaCl. FLAG-Dnmt3b immunoprecipitates were analyzed by immunoblot using an anti-histone H3 antibody to confirm the interaction between Dnmt3b and chromatin. Input represents 4% of the nuclear extract used in the assay. ( C ) Dnmt3b co-sedimented with histone H1-containing poly-nucleosomes on a sucrose gradient. S2 fractions were subjected to 10–30% (w/v) sucrose gradient sedimentation. Equivalent aliquots of each DNA fraction and protein sample were analyzed by agarose gel electrophoresis and immunoblot using the indicated antibodies, respectively. Core histones in each fraction were visualized by CBB staining.

    Techniques Used: Agarose Gel Electrophoresis, SYBR Green Assay, Immunoprecipitation, Sedimentation, Staining

    Dnmt3b preferentially binds to histone H1-assembled chromatin. ( A ) 12-mer nucleosomal arrays (70 nM nucleosome cores) were incubated with various amounts of histone H1 (0, 70, 140 or 210 nM). Binding of histone H1 to reconstituted chromatin was analyzed by 0.7% agarose gel electrophoresis. ( B ) MNase digestion of histone H1-containing 12-mer nucleosomal arrays. The reconstituted chromatin shown in A was digested with increasing amounts of MNase (0.141, 0.281, 0.562 and 1.125 U) as indicated by the triangles above the lanes. Products of digestion were labeled with [γ- 32 P]ATP and analyzed by native PAGE. Arrows indicate the positions of core particles (CP) and chromatosomes (CH). ( C ) Analysis of the protein component of the nucleosomal arrays. Reconstituted chromatin, as shown in A, was precipitated with MgCl 2 , and subjected to 15–25% SDS–PAGE and visualized by SYPRO-orange staining ( Supplementary Figure S6 ). ( D ) Data in C and Supplementary Figure S6 were quantified. The relative intensity of histone H1 to nucleosomes is shown as a solid line. Data represent the means with SD from three independent experiments. ( E ) The data in Supplementary Figure S7 were quantified, and are presented as a bar graph. Dnmt3b preferentially bound to histone H1-containing reconstituted 12-mers. Protein-bound DNA is expressed as a percentage of the input DNA. Data represent the means with SD of three or six independent experiments. The binding affinity of GST-Dnmt3b to 12-mer nucleosomal arrays (H1 = 0) was tested six times, while binding to the other three DNA templates was tested three times. Asterisk indicates a P- value of
    Figure Legend Snippet: Dnmt3b preferentially binds to histone H1-assembled chromatin. ( A ) 12-mer nucleosomal arrays (70 nM nucleosome cores) were incubated with various amounts of histone H1 (0, 70, 140 or 210 nM). Binding of histone H1 to reconstituted chromatin was analyzed by 0.7% agarose gel electrophoresis. ( B ) MNase digestion of histone H1-containing 12-mer nucleosomal arrays. The reconstituted chromatin shown in A was digested with increasing amounts of MNase (0.141, 0.281, 0.562 and 1.125 U) as indicated by the triangles above the lanes. Products of digestion were labeled with [γ- 32 P]ATP and analyzed by native PAGE. Arrows indicate the positions of core particles (CP) and chromatosomes (CH). ( C ) Analysis of the protein component of the nucleosomal arrays. Reconstituted chromatin, as shown in A, was precipitated with MgCl 2 , and subjected to 15–25% SDS–PAGE and visualized by SYPRO-orange staining ( Supplementary Figure S6 ). ( D ) Data in C and Supplementary Figure S6 were quantified. The relative intensity of histone H1 to nucleosomes is shown as a solid line. Data represent the means with SD from three independent experiments. ( E ) The data in Supplementary Figure S7 were quantified, and are presented as a bar graph. Dnmt3b preferentially bound to histone H1-containing reconstituted 12-mers. Protein-bound DNA is expressed as a percentage of the input DNA. Data represent the means with SD of three or six independent experiments. The binding affinity of GST-Dnmt3b to 12-mer nucleosomal arrays (H1 = 0) was tested six times, while binding to the other three DNA templates was tested three times. Asterisk indicates a P- value of

    Techniques Used: Incubation, Binding Assay, Agarose Gel Electrophoresis, Labeling, Clear Native PAGE, SDS Page, Staining

    Related Articles

    In Vivo:

    Article Title: Tolerance to lipopolysaccharide promotes an enhanced neutrophil extracellular traps formation leading to a more efficient bacterial clearance in mice
    Article Snippet: .. In vivo, NET contribution was determined by i.p. inoculation of microccocal nuclease (µccal, 100 U/mouse, prepared in PBS ×1 with 5 mM Cl2 Mg and 5 mM Cl2 Ca; Worthington, Lakewood, NJ, USA) or µccal plus EDTA (10 mM) in order to inactivate µccal activity, 10 min before i.p. bacterial challenge. ..

    Purification:

    Article Title: Hierarchical regulation of the genome: global changes in nucleosome organization potentiate genome response
    Article Snippet: .. MNase cleavage and purification of mononucleosomal and subnucleosomal DNA iSLK.219 nuclei were digested for 5 min at 37°C with a titration of MNase: 4 units/mL, 2 units/mL, and 1 unit/mL of MNase (Worthington Biochemical Corp.) in MNase cleavage buffer (4 mM MgCl2 , 5 mM KCl, 50 mM Tris-Cl (pH 7.4), 1 mM CaCl2 , 12.5% glycerol). ..

    Concentration Assay:

    Article Title: DNA methyltransferase 3b preferentially associates with condensed chromatin
    Article Snippet: .. The nuclear pellets were suspended in NIB at a concentration of 1.5 mg/ml DNA, pre-incubated for 10 min at 30°C and treated with increasing amounts of MNase (5, 20 or 80 U/mg DNA, Worthington) for 10 min at 30°C. .. The supernatant (S1 fraction) was collected and the pellets were resuspended in 2 mM EDTA, and then incubated for 10 min on ice.

    Incubation:

    Article Title: Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis
    Article Snippet: .. Wells containing NETs were incubated with 1 U/mL MNase or DNase1 (both from Worthington) for 10 min or 10% human serum for 6 h. We then added 2 mM EDTA to stop nuclease activity and collected the culture supernatants. .. Picogreen (Invitrogen), a DNA fluorescent DNA dye, was added, and the DNA content was quantified by fluorescence spectrometry ( ).

    Activity Assay:

    Article Title: Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis
    Article Snippet: .. Wells containing NETs were incubated with 1 U/mL MNase or DNase1 (both from Worthington) for 10 min or 10% human serum for 6 h. We then added 2 mM EDTA to stop nuclease activity and collected the culture supernatants. .. Picogreen (Invitrogen), a DNA fluorescent DNA dye, was added, and the DNA content was quantified by fluorescence spectrometry ( ).

    Article Title: Tolerance to lipopolysaccharide promotes an enhanced neutrophil extracellular traps formation leading to a more efficient bacterial clearance in mice
    Article Snippet: .. In vivo, NET contribution was determined by i.p. inoculation of microccocal nuclease (µccal, 100 U/mouse, prepared in PBS ×1 with 5 mM Cl2 Mg and 5 mM Cl2 Ca; Worthington, Lakewood, NJ, USA) or µccal plus EDTA (10 mM) in order to inactivate µccal activity, 10 min before i.p. bacterial challenge. ..

    Titration:

    Article Title: Hierarchical regulation of the genome: global changes in nucleosome organization potentiate genome response
    Article Snippet: .. MNase cleavage and purification of mononucleosomal and subnucleosomal DNA iSLK.219 nuclei were digested for 5 min at 37°C with a titration of MNase: 4 units/mL, 2 units/mL, and 1 unit/mL of MNase (Worthington Biochemical Corp.) in MNase cleavage buffer (4 mM MgCl2 , 5 mM KCl, 50 mM Tris-Cl (pH 7.4), 1 mM CaCl2 , 12.5% glycerol). ..

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    Worthington Biochemical mnase
    RSC Maintains Open NFRs in Lowly Expressed Genes but Is Not Necessary for an Acute Transcriptional Response (A) Experiment outline (see Figure 2 A). (B) RNA fold change during Sth1 depletion and recovery. RNA level was normalized with K. lactis spike-in. Each row is a gene (5,529 genes), and each column is a sample. Heatmap is normalized to expression level prior to auxin addition (also mid-log). The levels of genes at this time are shown by the orange and purple columns. (C) NFR width per RNA level. NFR width per RNA percentile in each sample (Loess smoothed) (top). Percentage of NFRs that closed in the presence of auxin for 0.5 hr (orange line) and 2 hr (yellow line) out of the NFRs that were open in steady state, per RNA percentile at the same time point (bottom). (D) Stress experiment outline. Yeast cells were grown to mid-log in YPD. Auxin was added for 20 min, followed by salt addition (0.4 M KCl); samples were taken in time course and were subjected to <t>MNase-seq</t> and RNA sequencing (RNA-seq). Control samples without auxin or without KCL were performed. (E) Heatmap of RNA fold change in three treatments: auxin only, salt only, and both salt and auxin. RNA levels are normalized per library. 2,322 clustered genes that change in response to the treatments are shown as fold change with respect to the matching expression at T = 0. Time points are indicated in the experiment outline (A). (F) Metagene of subsets of stress-induced genes showing a typical response of chromatin structure to salt induction in time points in three treatments: auxin only, KCl only, and both KCl and auxin. Genes are positioned according to the nucleosome +1 center at T = 0. Black arrows mark location of changes. See also Figure S4 .
    Mnase, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 92/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RSC Maintains Open NFRs in Lowly Expressed Genes but Is Not Necessary for an Acute Transcriptional Response (A) Experiment outline (see Figure 2 A). (B) RNA fold change during Sth1 depletion and recovery. RNA level was normalized with K. lactis spike-in. Each row is a gene (5,529 genes), and each column is a sample. Heatmap is normalized to expression level prior to auxin addition (also mid-log). The levels of genes at this time are shown by the orange and purple columns. (C) NFR width per RNA level. NFR width per RNA percentile in each sample (Loess smoothed) (top). Percentage of NFRs that closed in the presence of auxin for 0.5 hr (orange line) and 2 hr (yellow line) out of the NFRs that were open in steady state, per RNA percentile at the same time point (bottom). (D) Stress experiment outline. Yeast cells were grown to mid-log in YPD. Auxin was added for 20 min, followed by salt addition (0.4 M KCl); samples were taken in time course and were subjected to MNase-seq and RNA sequencing (RNA-seq). Control samples without auxin or without KCL were performed. (E) Heatmap of RNA fold change in three treatments: auxin only, salt only, and both salt and auxin. RNA levels are normalized per library. 2,322 clustered genes that change in response to the treatments are shown as fold change with respect to the matching expression at T = 0. Time points are indicated in the experiment outline (A). (F) Metagene of subsets of stress-induced genes showing a typical response of chromatin structure to salt induction in time points in three treatments: auxin only, KCl only, and both KCl and auxin. Genes are positioned according to the nucleosome +1 center at T = 0. Black arrows mark location of changes. See also Figure S4 .

    Journal: Cell Reports

    Article Title: Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex

    doi: 10.1016/j.celrep.2018.12.020

    Figure Lengend Snippet: RSC Maintains Open NFRs in Lowly Expressed Genes but Is Not Necessary for an Acute Transcriptional Response (A) Experiment outline (see Figure 2 A). (B) RNA fold change during Sth1 depletion and recovery. RNA level was normalized with K. lactis spike-in. Each row is a gene (5,529 genes), and each column is a sample. Heatmap is normalized to expression level prior to auxin addition (also mid-log). The levels of genes at this time are shown by the orange and purple columns. (C) NFR width per RNA level. NFR width per RNA percentile in each sample (Loess smoothed) (top). Percentage of NFRs that closed in the presence of auxin for 0.5 hr (orange line) and 2 hr (yellow line) out of the NFRs that were open in steady state, per RNA percentile at the same time point (bottom). (D) Stress experiment outline. Yeast cells were grown to mid-log in YPD. Auxin was added for 20 min, followed by salt addition (0.4 M KCl); samples were taken in time course and were subjected to MNase-seq and RNA sequencing (RNA-seq). Control samples without auxin or without KCL were performed. (E) Heatmap of RNA fold change in three treatments: auxin only, salt only, and both salt and auxin. RNA levels are normalized per library. 2,322 clustered genes that change in response to the treatments are shown as fold change with respect to the matching expression at T = 0. Time points are indicated in the experiment outline (A). (F) Metagene of subsets of stress-induced genes showing a typical response of chromatin structure to salt induction in time points in three treatments: auxin only, KCl only, and both KCl and auxin. Genes are positioned according to the nucleosome +1 center at T = 0. Black arrows mark location of changes. See also Figure S4 .

    Article Snippet: Spheroplasts were washed, resuspended in NP buffer and treated with MNase (Micrococcal nuclease, Worthington) to generate 80% mono-nucleosomes (1 unit for 2.5 OD initial culture, 37°C, 20 minutes).

    Techniques: Expressing, RNA Sequencing Assay

    Changes in the +1 Nucleosome Position Are Reflected in TSS Usage (A) Experimental outline (as in Figure 2 A). An example of the data representation showing RNA 5′ ends (black), MNase read centers (dark red), and coverage (light red) around the TSS. (B) Nucleosome positioning and 5′ RNA ends during Sth1 depletion in CDC8 and ATG27 promoters. Dashed lines represent peak centers before and 1 hr after auxin addition. (C) 5′ RNA level at each position over the genome before and after Sth1 depletion (normalized with K. lactis spike-in). (D) Median nucleosome positioning around mRNA 5′ ends before (top) and 1 hr after (bottom) auxin addition. mRNA 5′ positions are separated to groups according to their fold change following Sth1 depletion. (E) Change in expression (1 hr/0 hr) versus change in accessibility (1 hr/0 hr) for mRNA 5′ locations that are expressed ( Figure 5 C) and accessible ( Figure S5 B) before auxin addition. See also Figure S5 .

    Journal: Cell Reports

    Article Title: Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex

    doi: 10.1016/j.celrep.2018.12.020

    Figure Lengend Snippet: Changes in the +1 Nucleosome Position Are Reflected in TSS Usage (A) Experimental outline (as in Figure 2 A). An example of the data representation showing RNA 5′ ends (black), MNase read centers (dark red), and coverage (light red) around the TSS. (B) Nucleosome positioning and 5′ RNA ends during Sth1 depletion in CDC8 and ATG27 promoters. Dashed lines represent peak centers before and 1 hr after auxin addition. (C) 5′ RNA level at each position over the genome before and after Sth1 depletion (normalized with K. lactis spike-in). (D) Median nucleosome positioning around mRNA 5′ ends before (top) and 1 hr after (bottom) auxin addition. mRNA 5′ positions are separated to groups according to their fold change following Sth1 depletion. (E) Change in expression (1 hr/0 hr) versus change in accessibility (1 hr/0 hr) for mRNA 5′ locations that are expressed ( Figure 5 C) and accessible ( Figure S5 B) before auxin addition. See also Figure S5 .

    Article Snippet: Spheroplasts were washed, resuspended in NP buffer and treated with MNase (Micrococcal nuclease, Worthington) to generate 80% mono-nucleosomes (1 unit for 2.5 OD initial culture, 37°C, 20 minutes).

    Techniques: Expressing

    Dynamics of Sth1 Depletion and Recovery Show Massive yet Reversible Disruptions in Chromatin Organization (A) Experimental outline. For depletion, auxin (IAA) was added to mid-log degron-Sth1 cells, and MNase-seq was performed at the indicated time points. For recovery, mid-log degron-Sth1 cells were incubated in the presence of auxin for 2 hr. Auxin was washed from the media, and MNase-seq was performed at the indicated time points. (B) Median MNase coverage positioned relative to the TSS (metagene) following Sth1 depletion (top) and recovery (bottom). (C) MNase read centers (lines, dark color) and coverage (shade, light color) following Sth1 depletion and recovery in the TAF6/NSA1 promoter area. Dashed lines represent the position of nucleosomes +1 and −1 center before depletion and after full recovery. (D) Distribution of NFR width (defined as the distance between the peak −/+1 nucleosomes) through sth1 depletion and recovery. (E) Average MNase coverage (metagene) before (red line) and 1 hr after (yellow line) Sth1 depletion in genes with a GRF-binding site (top) and without GRF binding but with a poly(A/T) tract (bottom). Genes were positioned relative to the GRF-binding site or poly(A/T) tract site. GRF-binding sites were obtained from Gutin et al. (2018) . (F) Distribution of NFR width throughout Sth1 depletion in the two groups as in (E). The distribution of all genes before auxin addition is shown in gray. (G) Comparison of NFR width before Sth1 depletion and after recovery. Each point is related to NFR of a gene. Genes with fuzzy +1 or −1 nucleosomes were excluded. See also Figure S2 .

    Journal: Cell Reports

    Article Title: Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex

    doi: 10.1016/j.celrep.2018.12.020

    Figure Lengend Snippet: Dynamics of Sth1 Depletion and Recovery Show Massive yet Reversible Disruptions in Chromatin Organization (A) Experimental outline. For depletion, auxin (IAA) was added to mid-log degron-Sth1 cells, and MNase-seq was performed at the indicated time points. For recovery, mid-log degron-Sth1 cells were incubated in the presence of auxin for 2 hr. Auxin was washed from the media, and MNase-seq was performed at the indicated time points. (B) Median MNase coverage positioned relative to the TSS (metagene) following Sth1 depletion (top) and recovery (bottom). (C) MNase read centers (lines, dark color) and coverage (shade, light color) following Sth1 depletion and recovery in the TAF6/NSA1 promoter area. Dashed lines represent the position of nucleosomes +1 and −1 center before depletion and after full recovery. (D) Distribution of NFR width (defined as the distance between the peak −/+1 nucleosomes) through sth1 depletion and recovery. (E) Average MNase coverage (metagene) before (red line) and 1 hr after (yellow line) Sth1 depletion in genes with a GRF-binding site (top) and without GRF binding but with a poly(A/T) tract (bottom). Genes were positioned relative to the GRF-binding site or poly(A/T) tract site. GRF-binding sites were obtained from Gutin et al. (2018) . (F) Distribution of NFR width throughout Sth1 depletion in the two groups as in (E). The distribution of all genes before auxin addition is shown in gray. (G) Comparison of NFR width before Sth1 depletion and after recovery. Each point is related to NFR of a gene. Genes with fuzzy +1 or −1 nucleosomes were excluded. See also Figure S2 .

    Article Snippet: Spheroplasts were washed, resuspended in NP buffer and treated with MNase (Micrococcal nuclease, Worthington) to generate 80% mono-nucleosomes (1 unit for 2.5 OD initial culture, 37°C, 20 minutes).

    Techniques: Incubation, Binding Assay

    Sth1-Dependent NFR Clearing Is Replication Independent (A) Experimental outline in G1-arrested cells. For depletion, yeast cells were grown to mid-log in YPD and incubated with or without alpha factor for 2 hr. At the indicated time, cells were transferred to a new tube, and Sth1 depletion was induced by auxin addition. All samples were fixed at the same time. For recovery, yeast cells were grown to mid-log in YPD and incubated with alpha-factor and auxin for 2 hr. Cells were washed and resuspended with or without alpha factor. MNase-seq was performed at the indicated time points. (B) Distribution of NFR width in time course through Sth1 depletion (top) and recovery (bottom) in G1-arrested cells (right) and in unsynchronized cells (left). (C) Density scatter of the change in NFR width for all genes through Sth1 depletion (1 hr, top) and recovery (4 hr, bottom), in G1 arrested versus unsynchronized cells. See also Figure S3 .

    Journal: Cell Reports

    Article Title: Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex

    doi: 10.1016/j.celrep.2018.12.020

    Figure Lengend Snippet: Sth1-Dependent NFR Clearing Is Replication Independent (A) Experimental outline in G1-arrested cells. For depletion, yeast cells were grown to mid-log in YPD and incubated with or without alpha factor for 2 hr. At the indicated time, cells were transferred to a new tube, and Sth1 depletion was induced by auxin addition. All samples were fixed at the same time. For recovery, yeast cells were grown to mid-log in YPD and incubated with alpha-factor and auxin for 2 hr. Cells were washed and resuspended with or without alpha factor. MNase-seq was performed at the indicated time points. (B) Distribution of NFR width in time course through Sth1 depletion (top) and recovery (bottom) in G1-arrested cells (right) and in unsynchronized cells (left). (C) Density scatter of the change in NFR width for all genes through Sth1 depletion (1 hr, top) and recovery (4 hr, bottom), in G1 arrested versus unsynchronized cells. See also Figure S3 .

    Article Snippet: Spheroplasts were washed, resuspended in NP buffer and treated with MNase (Micrococcal nuclease, Worthington) to generate 80% mono-nucleosomes (1 unit for 2.5 OD initial culture, 37°C, 20 minutes).

    Techniques: Incubation

    Induced Knockdown Screen of ATP-Dependent Chromatin Remodelers (A) An auxin-inducible degron (AID) system ( Morawska and Ulrich, 2013 ) yielding an auxin-inducible, rapid degradation of tagged chromatin remodelers. Plant hormone auxin (IAA) directly induces rapid degradation of the AID-tagged protein by mediating the interaction of a degron domain in the target protein with the substrate recognition domain of TIR1. (B) Experimental outline. AID-tagged chromatin remodeler strains were grown to mid-log in YPD. MNase-seq was performed to compare nucleosome positioning before and at two time points after auxin addition. (C) Average MNase coverage positioned relative to the transcription start site (TSS) (“metagene”) for each chromatin remodeler AID strain before and after auxin addition and in the relevant KO strains (if available) (top). Average of the change in MNase coverage before and after auxin addition (1 hr to 0 hr) positioned relative to the TSS for each chromatin remodeler AID strain (bottom). (D) Heatmaps representing the change in MNase coverage before and after auxin addition (1 hr to 0 hr) positioned relative to the TSS (in yellow) for each AID strain. Genes (rows) are sorted, in each strain, by the magnitude of changes in coverage following the depletion in the NFR area. See also Figure S1 .

    Journal: Cell Reports

    Article Title: Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex

    doi: 10.1016/j.celrep.2018.12.020

    Figure Lengend Snippet: Induced Knockdown Screen of ATP-Dependent Chromatin Remodelers (A) An auxin-inducible degron (AID) system ( Morawska and Ulrich, 2013 ) yielding an auxin-inducible, rapid degradation of tagged chromatin remodelers. Plant hormone auxin (IAA) directly induces rapid degradation of the AID-tagged protein by mediating the interaction of a degron domain in the target protein with the substrate recognition domain of TIR1. (B) Experimental outline. AID-tagged chromatin remodeler strains were grown to mid-log in YPD. MNase-seq was performed to compare nucleosome positioning before and at two time points after auxin addition. (C) Average MNase coverage positioned relative to the transcription start site (TSS) (“metagene”) for each chromatin remodeler AID strain before and after auxin addition and in the relevant KO strains (if available) (top). Average of the change in MNase coverage before and after auxin addition (1 hr to 0 hr) positioned relative to the TSS for each chromatin remodeler AID strain (bottom). (D) Heatmaps representing the change in MNase coverage before and after auxin addition (1 hr to 0 hr) positioned relative to the TSS (in yellow) for each AID strain. Genes (rows) are sorted, in each strain, by the magnitude of changes in coverage following the depletion in the NFR area. See also Figure S1 .

    Article Snippet: Spheroplasts were washed, resuspended in NP buffer and treated with MNase (Micrococcal nuclease, Worthington) to generate 80% mono-nucleosomes (1 unit for 2.5 OD initial culture, 37°C, 20 minutes).

    Techniques:

    Expression of mutant H2B in yeast destabilizes nucleosomes, deregulates gene expression and reduces nucleosome occupancy at the PHO5 promoter. WT or E79K H2B (analogous to human H2B-E76K) was expressed in S. Cerevisiae. (A) Yeast cells expressing H2B-E79K are temperature sensitive. Limiting dilutions of yeast expressing WT, E79A, E79Q or E79K were plated and incubated at 30°C or 37°C. Cell growth was evaluated after 1 day. (B) Yeast doubling time is significantly increased in cells expressing E79K-H2B at 37°C. (C) Time course of MNase sensitivity from spheroplasted yeast grown in rich media. M, marker. (D) Chromatin pellets were extracted with increasing concentrations of salt as indicated. Immuno-blotting of the soluble fraction was performed with antibody to H4. (E) Cells expressing WT, E79Q or E79K H2B were maintained in either rich media (YPDA) or phosphate-free media and expression of the phosphate-inducible PHO5 gene was measured by RT-PCR. (F) Nucleosome scanning assay of the PHO5 promoter from cells expressing either WT or E79K grown in rich media. Chromatin was digested with MNase, mononucleosomal DNA was purified and MNase protection was determined by qPCR. H2B occupancy at −2 nucleosome position of PHO5 is reduced in E79K cells as indicated by the arrow.

    Journal: Cancer discovery

    Article Title: A Mutation in Histone H2B Represents A New Class Of Oncogenic Driver

    doi: 10.1158/2159-8290.CD-19-0393

    Figure Lengend Snippet: Expression of mutant H2B in yeast destabilizes nucleosomes, deregulates gene expression and reduces nucleosome occupancy at the PHO5 promoter. WT or E79K H2B (analogous to human H2B-E76K) was expressed in S. Cerevisiae. (A) Yeast cells expressing H2B-E79K are temperature sensitive. Limiting dilutions of yeast expressing WT, E79A, E79Q or E79K were plated and incubated at 30°C or 37°C. Cell growth was evaluated after 1 day. (B) Yeast doubling time is significantly increased in cells expressing E79K-H2B at 37°C. (C) Time course of MNase sensitivity from spheroplasted yeast grown in rich media. M, marker. (D) Chromatin pellets were extracted with increasing concentrations of salt as indicated. Immuno-blotting of the soluble fraction was performed with antibody to H4. (E) Cells expressing WT, E79Q or E79K H2B were maintained in either rich media (YPDA) or phosphate-free media and expression of the phosphate-inducible PHO5 gene was measured by RT-PCR. (F) Nucleosome scanning assay of the PHO5 promoter from cells expressing either WT or E79K grown in rich media. Chromatin was digested with MNase, mononucleosomal DNA was purified and MNase protection was determined by qPCR. H2B occupancy at −2 nucleosome position of PHO5 is reduced in E79K cells as indicated by the arrow.

    Article Snippet: MNase susceptibility assays were performed on nucleosomes made with WT, E76Q and E76K H2B mutants by mixing each nucleosome (0.4 pmol) with 9.6U of MNase (Worthington) and incubating for indicated amount of time.

    Techniques: Expressing, Mutagenesis, Incubation, Marker, Reverse Transcription Polymerase Chain Reaction, Purification, Real-time Polymerase Chain Reaction

    The E76K mutation in H2B destabilizes the histone octamer and fails to protect the nucleosome from nuclease treatment in vitro . (A) The H2B-E76K mutant was unable to form stable octamers in vitro . Recombinant human histones (H2A, H2B, H3, and H4) were mixed and histone octamers resolved from (H3/H4) 2 tetramers, H2A/H2B dimers and free histones by gel filtration chromatography. (B) Nucleosomes were reconstituted by mixing equimolar amounts of DNA (147bp) and octamers or in the case of E76K of tetramers and dimers (1:2 molar ratio) and resolved by Native PAGE. Nucleosomes containing H2B-E76K and E76Q have an altered migration pattern, intermediate between a tetrasome and a WT nucleosome. (C) Micrococcal nuclease (MNase) sensitivity assay performed on nucleosomes made with WT, E76Q and E76K H2B mutants shows more rapid digestion of E76K containing nucleosomes than those with WT H2B. A time course by gel (left) and densitometry quantification (right) of intact nucleosomes following MNase treatment. (D) The MNase susceptibility of E76K nucleosomes is distinct from nucleosomes formed only with tetrasomes.

    Journal: Cancer discovery

    Article Title: A Mutation in Histone H2B Represents A New Class Of Oncogenic Driver

    doi: 10.1158/2159-8290.CD-19-0393

    Figure Lengend Snippet: The E76K mutation in H2B destabilizes the histone octamer and fails to protect the nucleosome from nuclease treatment in vitro . (A) The H2B-E76K mutant was unable to form stable octamers in vitro . Recombinant human histones (H2A, H2B, H3, and H4) were mixed and histone octamers resolved from (H3/H4) 2 tetramers, H2A/H2B dimers and free histones by gel filtration chromatography. (B) Nucleosomes were reconstituted by mixing equimolar amounts of DNA (147bp) and octamers or in the case of E76K of tetramers and dimers (1:2 molar ratio) and resolved by Native PAGE. Nucleosomes containing H2B-E76K and E76Q have an altered migration pattern, intermediate between a tetrasome and a WT nucleosome. (C) Micrococcal nuclease (MNase) sensitivity assay performed on nucleosomes made with WT, E76Q and E76K H2B mutants shows more rapid digestion of E76K containing nucleosomes than those with WT H2B. A time course by gel (left) and densitometry quantification (right) of intact nucleosomes following MNase treatment. (D) The MNase susceptibility of E76K nucleosomes is distinct from nucleosomes formed only with tetrasomes.

    Article Snippet: MNase susceptibility assays were performed on nucleosomes made with WT, E76Q and E76K H2B mutants by mixing each nucleosome (0.4 pmol) with 9.6U of MNase (Worthington) and incubating for indicated amount of time.

    Techniques: Mutagenesis, In Vitro, Recombinant, Filtration, Chromatography, Clear Native PAGE, Migration, Sensitive Assay

    H2B-E76K fundamentally alters chromatin structure and dynamics. (A) Micrococcal nuclease (MNase) assay with nuclei of MCF10A cells stably expressing either WT or H2B-E76K demonstrate that E76K expression significantly increases sensitivity to MNase. Digest efficiency was visualized by agarose gel. (B) Fluorescence recovery after photobleaching (FRAP) analysis demonstrates that H2B-E76K has significantly faster chromatin dynamics than WT. FRAP analysis was carried out after induction of either WT or E76K mutant H2B-GFP fusions for 5 or 35 days in MCF10A cells. Dashed lines represent cells expressing H2B-E76K, solid lines represent data from cells expressing inducible GFP-tagged WT H2B (n = 10 cells). (C) Representative FRAP assay pre-bleach, bleach and post-bleach images of nuclei expressing GFP-tagged WT or H2B-E76K indicate faster fluorescent recovery in cells expressing E76K. (D) FRAP analysis of histone H2A-GFP dynamics in MCF10A cells expressing only H2A-GFP (n = 20 cells) or both H2A-GFP and a mutant H2B E76K mCherry fusion (n = 25 cells) with standard deviation envelopes. Inset demonstrates co-expression of H2A GFP and E76K mCherry. (E) Representative pre-bleach, bleach and post-bleach images of H2A GFP in WT MCF10A cells or in cells co-expressing mCherry tagged H2B-E76K. (F) 100nm particles injected into the nucleus had significantly increased mean square displacement (MSD) over time in MCF10A cells stably expressing exogenous H2B-E76K (red) compared to cells expressing WT H2B (blue). N=68 (WT) and N=67 (E76K) cells were analyzed. Error bars represent SEM.

    Journal: Cancer discovery

    Article Title: A Mutation in Histone H2B Represents A New Class Of Oncogenic Driver

    doi: 10.1158/2159-8290.CD-19-0393

    Figure Lengend Snippet: H2B-E76K fundamentally alters chromatin structure and dynamics. (A) Micrococcal nuclease (MNase) assay with nuclei of MCF10A cells stably expressing either WT or H2B-E76K demonstrate that E76K expression significantly increases sensitivity to MNase. Digest efficiency was visualized by agarose gel. (B) Fluorescence recovery after photobleaching (FRAP) analysis demonstrates that H2B-E76K has significantly faster chromatin dynamics than WT. FRAP analysis was carried out after induction of either WT or E76K mutant H2B-GFP fusions for 5 or 35 days in MCF10A cells. Dashed lines represent cells expressing H2B-E76K, solid lines represent data from cells expressing inducible GFP-tagged WT H2B (n = 10 cells). (C) Representative FRAP assay pre-bleach, bleach and post-bleach images of nuclei expressing GFP-tagged WT or H2B-E76K indicate faster fluorescent recovery in cells expressing E76K. (D) FRAP analysis of histone H2A-GFP dynamics in MCF10A cells expressing only H2A-GFP (n = 20 cells) or both H2A-GFP and a mutant H2B E76K mCherry fusion (n = 25 cells) with standard deviation envelopes. Inset demonstrates co-expression of H2A GFP and E76K mCherry. (E) Representative pre-bleach, bleach and post-bleach images of H2A GFP in WT MCF10A cells or in cells co-expressing mCherry tagged H2B-E76K. (F) 100nm particles injected into the nucleus had significantly increased mean square displacement (MSD) over time in MCF10A cells stably expressing exogenous H2B-E76K (red) compared to cells expressing WT H2B (blue). N=68 (WT) and N=67 (E76K) cells were analyzed. Error bars represent SEM.

    Article Snippet: MNase susceptibility assays were performed on nucleosomes made with WT, E76Q and E76K H2B mutants by mixing each nucleosome (0.4 pmol) with 9.6U of MNase (Worthington) and incubating for indicated amount of time.

    Techniques: Stable Transfection, Expressing, Agarose Gel Electrophoresis, Fluorescence, Mutagenesis, FRAP Assay, Standard Deviation, Injection

    Chromatin-remodeling (nucleosome insertion and rearrangement) on Nanog gene promoter. ( A ) ESCs were treated with RA for 3 days, then treated with 5, 10 and 30 U of MNase for 6 min at 37°C. Extracted chromatin DNA was separated on 1.5% agarose gels followed by Southern blot hybridization with 32 P-labeled 500-bp probes specific to three regions on the Nanog promoter. Positions of probes are depicted on the map. ( B ) Restriction enzyme accessibility of Nanog promoter region in ESCs. Asterisk marks the diagnostic band indicative of each sensitive site. The results are summarized and shown on the map above these blots. Restriction sites are labeled under the map. ( C ) N1, N2, N3 and N4 PCR fragments, amplified from mononucleosomal DNA using the primers a/b (N1), c/d (N2), e/f (N3) and g/h (N4). Primer sequences were listed in Supplementary Table S1 . Genomic DNAs isolated from 1 × 10 6 cells were used for N1 amplification as control (upper panel). ESCs were transfected with siRNA specific for Brm or siRNA control. Changes in mononucleosome formation on the Nanog gene promoter were monitored (bottom panel). ( D ) Upper: nucleosome occupancy on the Nanog promoter in ESCs with or without RA treatment. The gray-highlighted region (q8–12) represents the location of the N2 nucleosome formation. Lower: analysis of nucleosome occupancy at the q8–12 regions of Nanog in control and Brm-knockdown ES cell by the MNase resistance assay. Data points represent average qPCR signals from two independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Coordinated repressive chromatin-remodeling of Oct4 and Nanog genes in RA-induced differentiation of embryonic stem cells involves RIP140

    doi: 10.1093/nar/gku092

    Figure Lengend Snippet: Chromatin-remodeling (nucleosome insertion and rearrangement) on Nanog gene promoter. ( A ) ESCs were treated with RA for 3 days, then treated with 5, 10 and 30 U of MNase for 6 min at 37°C. Extracted chromatin DNA was separated on 1.5% agarose gels followed by Southern blot hybridization with 32 P-labeled 500-bp probes specific to three regions on the Nanog promoter. Positions of probes are depicted on the map. ( B ) Restriction enzyme accessibility of Nanog promoter region in ESCs. Asterisk marks the diagnostic band indicative of each sensitive site. The results are summarized and shown on the map above these blots. Restriction sites are labeled under the map. ( C ) N1, N2, N3 and N4 PCR fragments, amplified from mononucleosomal DNA using the primers a/b (N1), c/d (N2), e/f (N3) and g/h (N4). Primer sequences were listed in Supplementary Table S1 . Genomic DNAs isolated from 1 × 10 6 cells were used for N1 amplification as control (upper panel). ESCs were transfected with siRNA specific for Brm or siRNA control. Changes in mononucleosome formation on the Nanog gene promoter were monitored (bottom panel). ( D ) Upper: nucleosome occupancy on the Nanog promoter in ESCs with or without RA treatment. The gray-highlighted region (q8–12) represents the location of the N2 nucleosome formation. Lower: analysis of nucleosome occupancy at the q8–12 regions of Nanog in control and Brm-knockdown ES cell by the MNase resistance assay. Data points represent average qPCR signals from two independent experiments.

    Article Snippet: Nuclei isolated from ESCs were digested with 5 and 30 U of MNase (Worthington, Lakewood, NJ, USA, www.worthington-biochem.com ) at 37°C for 5 min, followed by proteinase K treatment at 37°C overnight.

    Techniques: Southern Blot, Hybridization, Labeling, Diagnostic Assay, Polymerase Chain Reaction, Amplification, Isolation, Transfection, Real-time Polymerase Chain Reaction