nucleosome dna  (New England Biolabs)


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    New England Biolabs nucleosome dna
    Rap1 as a Pioneer Factor in Budding Yeast (A) Scheme of pTF function: following target search (1), the pTF invades compact chromatin (2), opens chromatin, and recruits the transcription machinery (3). (B) Domain organization of budding yeast Rap1 (above) and crystal structure of a <t>Rap1:DNA</t> complex (PDB: 3ukg ; <xref ref-type=Matot et al., 2012 ). Act, transcription activation domain; BRCT, BRCA 1 C terminus; DBD, DNA binding domain; RCT, Rap1 C terminus; Tox, toxicity region. (C) Organization of the RPL30 promoter. Gray, MNase-seq profile after Rap1 depletion ( Kubik et al., 2015 ), revealing nucleosome positions in the absence of Rap1 (black dotted circles). Plotted is nucleosome occupancy reads, normalized to 10 7 total reads. The Rap1 binding site 1 ( S1 ) (high affinity) and site 2 ( S2 ) (medium affinity) fall on the −1 nucleosome. (D) Promoter −1 nucleosome, showing Rap1 binding sites S1 and S2 (PDB: 1AOI ; Luger et al., 1997 ). The numbers indicate super helical locations (SHLs) of the nucleosomal DNA. See also Figure S1 and , , and . " width="250" height="auto" />
    Nucleosome Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Chromatin Fiber Invasion and Nucleosome Displacement by the Rap1 Transcription Factor"

    Article Title: Chromatin Fiber Invasion and Nucleosome Displacement by the Rap1 Transcription Factor

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2019.10.025

    Rap1 as a Pioneer Factor in Budding Yeast (A) Scheme of pTF function: following target search (1), the pTF invades compact chromatin (2), opens chromatin, and recruits the transcription machinery (3). (B) Domain organization of budding yeast Rap1 (above) and crystal structure of a Rap1:DNA complex (PDB: 3ukg ; <xref ref-type=Matot et al., 2012 ). Act, transcription activation domain; BRCT, BRCA 1 C terminus; DBD, DNA binding domain; RCT, Rap1 C terminus; Tox, toxicity region. (C) Organization of the RPL30 promoter. Gray, MNase-seq profile after Rap1 depletion ( Kubik et al., 2015 ), revealing nucleosome positions in the absence of Rap1 (black dotted circles). Plotted is nucleosome occupancy reads, normalized to 10 7 total reads. The Rap1 binding site 1 ( S1 ) (high affinity) and site 2 ( S2 ) (medium affinity) fall on the −1 nucleosome. (D) Promoter −1 nucleosome, showing Rap1 binding sites S1 and S2 (PDB: 1AOI ; Luger et al., 1997 ). The numbers indicate super helical locations (SHLs) of the nucleosomal DNA. See also Figure S1 and , , and . " title="... Kubik et al., 2015 ), revealing nucleosome positions in the absence of Rap1 (black dotted ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Rap1 as a Pioneer Factor in Budding Yeast (A) Scheme of pTF function: following target search (1), the pTF invades compact chromatin (2), opens chromatin, and recruits the transcription machinery (3). (B) Domain organization of budding yeast Rap1 (above) and crystal structure of a Rap1:DNA complex (PDB: 3ukg ; Matot et al., 2012 ). Act, transcription activation domain; BRCT, BRCA 1 C terminus; DBD, DNA binding domain; RCT, Rap1 C terminus; Tox, toxicity region. (C) Organization of the RPL30 promoter. Gray, MNase-seq profile after Rap1 depletion ( Kubik et al., 2015 ), revealing nucleosome positions in the absence of Rap1 (black dotted circles). Plotted is nucleosome occupancy reads, normalized to 10 7 total reads. The Rap1 binding site 1 ( S1 ) (high affinity) and site 2 ( S2 ) (medium affinity) fall on the −1 nucleosome. (D) Promoter −1 nucleosome, showing Rap1 binding sites S1 and S2 (PDB: 1AOI ; Luger et al., 1997 ). The numbers indicate super helical locations (SHLs) of the nucleosomal DNA. See also Figure S1 and , , and .

    Techniques Used: Activation Assay, Binding Assay

    Rap1 Recognizes Target Sites within Nucleosomal DNA (A) Scheme of the smTIRFM experiment to detect Rap1 binding to S1 - or S2 -containing, Alexa-Fluor-647-labeled, and immobilized DNA or nucleosomes. bt-NA, biotin-neutravidin. (B) Expression and labeling of Rap1. Lanes: (1) purified MBP-Rap1-Halo; (2) MBP cleavage; (3 and 4) before and after JF-549 labeling; and (5) purified Rap1. (C) Representative smTIRF images showing nucleosome positions in the far-red channel (left, red circles) and Rap1 binding events in the green-orange channel (right). Scale bars: 5 μm; ex, excitation wavelength; em, emission wavelength. (D) Representative fluorescence time trace of Rap1 binding events to S2 containing naked DNA, detected by JF-549 emission. The trace was fitted (red), and t dark and t bright were determined by a thresholding algorithm. (E) Cumulative histogram of Rap1 binding intervals ( t bright ) on S2 DNA fitted by a 2-exponential function y = ∑ i = 1 2 A i exp ( − t / τ o f f , i ) (solid line). For all fit results, see . (F) Specific dissociation time constants (τ off,i > 1 s) of Rap1 for S2 DNA, S1 and S2 containing mononucleosomes (MN), or nucleosomes lacking a binding site (NS), uncorrected for dye photobleaching. The width of the bars indicates the percentage of events associated with the indicated time constants (i.e., amplitudes A i of the multi-exponential fits shown in E and H). n = 4 to 5; error bars: SD. (G) Representative fluorescence time trace of Rap1 binding events to S1 (bottom) and S2 (top) containing MNs. The data were analyzed as in (D). (H) Cumulative histogram of Rap1 binding intervals ( t bright ) on S1 - and S2 -containing MNs fitted by a 3-exponential function y = ∑ i = 0 2 A i exp ( − t / τ o f f , i ) (solid line). (I) Specific on-rate constants ( k on = 1/ τ on ) for all species obtained from a single-exponential fit to cumulative histograms of t dark values and corrected for the contribution from nonspecific interactions . See also and and , , , and .
    Figure Legend Snippet: Rap1 Recognizes Target Sites within Nucleosomal DNA (A) Scheme of the smTIRFM experiment to detect Rap1 binding to S1 - or S2 -containing, Alexa-Fluor-647-labeled, and immobilized DNA or nucleosomes. bt-NA, biotin-neutravidin. (B) Expression and labeling of Rap1. Lanes: (1) purified MBP-Rap1-Halo; (2) MBP cleavage; (3 and 4) before and after JF-549 labeling; and (5) purified Rap1. (C) Representative smTIRF images showing nucleosome positions in the far-red channel (left, red circles) and Rap1 binding events in the green-orange channel (right). Scale bars: 5 μm; ex, excitation wavelength; em, emission wavelength. (D) Representative fluorescence time trace of Rap1 binding events to S2 containing naked DNA, detected by JF-549 emission. The trace was fitted (red), and t dark and t bright were determined by a thresholding algorithm. (E) Cumulative histogram of Rap1 binding intervals ( t bright ) on S2 DNA fitted by a 2-exponential function y = ∑ i = 1 2 A i exp ( − t / τ o f f , i ) (solid line). For all fit results, see . (F) Specific dissociation time constants (τ off,i > 1 s) of Rap1 for S2 DNA, S1 and S2 containing mononucleosomes (MN), or nucleosomes lacking a binding site (NS), uncorrected for dye photobleaching. The width of the bars indicates the percentage of events associated with the indicated time constants (i.e., amplitudes A i of the multi-exponential fits shown in E and H). n = 4 to 5; error bars: SD. (G) Representative fluorescence time trace of Rap1 binding events to S1 (bottom) and S2 (top) containing MNs. The data were analyzed as in (D). (H) Cumulative histogram of Rap1 binding intervals ( t bright ) on S1 - and S2 -containing MNs fitted by a 3-exponential function y = ∑ i = 0 2 A i exp ( − t / τ o f f , i ) (solid line). (I) Specific on-rate constants ( k on = 1/ τ on ) for all species obtained from a single-exponential fit to cumulative histograms of t dark values and corrected for the contribution from nonspecific interactions . See also and and , , , and .

    Techniques Used: Binding Assay, Labeling, Expressing, Purification, Fluorescence

    Chromatin Higher-Order Structure Reduces Rap1 Dwell Time (A) Scheme of DNA preparation used to introduce Rap1 target sites S1 and S2 into the central nucleosome (N6) of a chromatin fiber (CH). (B) Scheme of the smTIRFM experiment to measure Rap1 binding kinetics in a chromatin fiber context. (C) Representative fluorescence time trace of Rap1 binding events to S1 -containing chromatin arrays. The trace is fitted (red); t dark and t bright were determined by a thresholding algorithm. (D) Cumulative histogram of Rap1 binding intervals (t bright ) to chromatin fibers, containing S1 fitted by a 3-exponential function (solid line). For all fit results, see . (E) Cumulative histogram of Rap1 binding to chromatin arrays, containing S2 fitted by a 3-exponential function (solid line). (F) Specific binding time constants (τ off,i > 1 s) of Rap1 for S1 in a nucleosome (MN) versus chromatin fiber (CH) and S2 MN versus CH. The widths of the bars indicate the percentage of events associated with the indicated time constants (i.e., amplitudes A i of the multi-exponential fits shown in D and E). n = 4 to 5; error bars: SD. (G) Specific on-rate constants ( k on = 1/ τ on ) for MNs and CHs containing S1 and S2 , obtained from a single-exponential fit to cumulative histograms of t dark values and corrected for the contribution from nonspecific interactions . See also <xref ref-type=Figure S4 and , , , and . " title="... target sites S1 and S2 into the central nucleosome (N6) of a chromatin fiber (CH). (B) Scheme ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Chromatin Higher-Order Structure Reduces Rap1 Dwell Time (A) Scheme of DNA preparation used to introduce Rap1 target sites S1 and S2 into the central nucleosome (N6) of a chromatin fiber (CH). (B) Scheme of the smTIRFM experiment to measure Rap1 binding kinetics in a chromatin fiber context. (C) Representative fluorescence time trace of Rap1 binding events to S1 -containing chromatin arrays. The trace is fitted (red); t dark and t bright were determined by a thresholding algorithm. (D) Cumulative histogram of Rap1 binding intervals (t bright ) to chromatin fibers, containing S1 fitted by a 3-exponential function (solid line). For all fit results, see . (E) Cumulative histogram of Rap1 binding to chromatin arrays, containing S2 fitted by a 3-exponential function (solid line). (F) Specific binding time constants (τ off,i > 1 s) of Rap1 for S1 in a nucleosome (MN) versus chromatin fiber (CH) and S2 MN versus CH. The widths of the bars indicate the percentage of events associated with the indicated time constants (i.e., amplitudes A i of the multi-exponential fits shown in D and E). n = 4 to 5; error bars: SD. (G) Specific on-rate constants ( k on = 1/ τ on ) for MNs and CHs containing S1 and S2 , obtained from a single-exponential fit to cumulative histograms of t dark values and corrected for the contribution from nonspecific interactions . See also Figure S4 and , , , and .

    Techniques Used: Introduce, Binding Assay, Fluorescence

    Rap1 Does Not Open Nucleosome Structure (A) Scheme of experiment to detect nucleosome structure change due to Rap1 binding by FRET. (B) Nucleosome structure (PDB: 1AOI ) showing attachment points of FRET probes. (C) EMSA showing Rap1 binding to S1 and S2 nucleosomes at indicated concentration equivalents (eq.). Lanes were re-arranged for clarity. (D) Fluorescence spectra for S2 nucleosome in complex with indicated equivalents of Rap1. (E) Fluorescence spectra for S1 nucleosome in complex with indicated equivalents of Rap1. (F) FRET efficiency calculated for S2 and S1 nucleosomes as a function of equivalents added Rap1. Error bars: SD; n = 2. See also <xref ref-type=Figure S5 . " title="Rap1 Does Not Open Nucleosome Structure (A) Scheme of experiment to detect nucleosome ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Rap1 Does Not Open Nucleosome Structure (A) Scheme of experiment to detect nucleosome structure change due to Rap1 binding by FRET. (B) Nucleosome structure (PDB: 1AOI ) showing attachment points of FRET probes. (C) EMSA showing Rap1 binding to S1 and S2 nucleosomes at indicated concentration equivalents (eq.). Lanes were re-arranged for clarity. (D) Fluorescence spectra for S2 nucleosome in complex with indicated equivalents of Rap1. (E) Fluorescence spectra for S1 nucleosome in complex with indicated equivalents of Rap1. (F) FRET efficiency calculated for S2 and S1 nucleosomes as a function of equivalents added Rap1. Error bars: SD; n = 2. See also Figure S5 .

    Techniques Used: Binding Assay, Concentration Assay, Fluorescence

    Chromatin Remodeling Induced by Rap1 Invasion as Observed by smFRET (A) Scheme of chromatin DNA assembly to introduce a Rap1 site at nucleosome N6, as well as a FRET donor (Cy3B, yellow) and acceptor (Alexa Fluor 647, red) at nucleosomes N5 and N7. (B) Scheme of a smFRET-TIRF experiment. (C) Individual kinetic traces of donor (orange) and acceptor (red) fluorescence emission and FRET efficiency ( E FRET , blue) for chromatin fibers containing S2 at the indicated KCl and Rap1 concentrations. All Rap1 experiments were performed at 150 mM KCl. (D) Similar to (C) but for chromatin lacking Rap1 binding sites ( NS ). (E) Histograms of E FRET of S2 -containing chromatin fibers at the indicated KCl and Rap1 concentrations. All Rap1 experiments were performed at 150 mM KCl. Histograms were fitted by Gaussian functions, revealing a low-FRET (LF) (gray), medium-FRET (MF) (green), and high-FRET (HF) (red) population. Error bars are SEM; for the number of traces and parameters of Gaussian fits, see and . (F) Similar to (E) but for chromatin lacking Rap1 binding sites ( NS ). (G) Percentage of each FRET sub-population, LF, MF, and HF for chromatin containing S2 . Box: 25–75 percentiles; whiskers: outliers (factor 1.5); line: median; open symbol: mean. For number of experiments, see . ∗ 10 −3 > p < 10 −4 ; ∗∗ 10 −4 > p < 10 −5 ; ∗∗∗ p < 10 −5 , two-tailed Student’s t test between peak area % of LF, MF, and HF populations for S2 or NS nucleosomes (see H). (H) Similar to (G) but for chromatin lacking Rap1 binding sites ( NS ). (I) Percentage of dynamic traces for S2 and NS chromatin. Box: similar to (H). For the identification of dynamic traces, see . p: two-tailed Student’s t test; n.s.: p > 0.05. See also <xref ref-type=Figure S6 and and . " title="... DNA assembly to introduce a Rap1 site at nucleosome N6, as well as a FRET donor (Cy3B, ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Chromatin Remodeling Induced by Rap1 Invasion as Observed by smFRET (A) Scheme of chromatin DNA assembly to introduce a Rap1 site at nucleosome N6, as well as a FRET donor (Cy3B, yellow) and acceptor (Alexa Fluor 647, red) at nucleosomes N5 and N7. (B) Scheme of a smFRET-TIRF experiment. (C) Individual kinetic traces of donor (orange) and acceptor (red) fluorescence emission and FRET efficiency ( E FRET , blue) for chromatin fibers containing S2 at the indicated KCl and Rap1 concentrations. All Rap1 experiments were performed at 150 mM KCl. (D) Similar to (C) but for chromatin lacking Rap1 binding sites ( NS ). (E) Histograms of E FRET of S2 -containing chromatin fibers at the indicated KCl and Rap1 concentrations. All Rap1 experiments were performed at 150 mM KCl. Histograms were fitted by Gaussian functions, revealing a low-FRET (LF) (gray), medium-FRET (MF) (green), and high-FRET (HF) (red) population. Error bars are SEM; for the number of traces and parameters of Gaussian fits, see and . (F) Similar to (E) but for chromatin lacking Rap1 binding sites ( NS ). (G) Percentage of each FRET sub-population, LF, MF, and HF for chromatin containing S2 . Box: 25–75 percentiles; whiskers: outliers (factor 1.5); line: median; open symbol: mean. For number of experiments, see . ∗ 10 −3 > p < 10 −4 ; ∗∗ 10 −4 > p < 10 −5 ; ∗∗∗ p < 10 −5 , two-tailed Student’s t test between peak area % of LF, MF, and HF populations for S2 or NS nucleosomes (see H). (H) Similar to (G) but for chromatin lacking Rap1 binding sites ( NS ). (I) Percentage of dynamic traces for S2 and NS chromatin. Box: similar to (H). For the identification of dynamic traces, see . p: two-tailed Student’s t test; n.s.: p > 0.05. See also Figure S6 and and .

    Techniques Used: Introduce, Fluorescence, Binding Assay, Two Tailed Test

    RSC Enables Stable Rap1 Binding by Exposing Binding Sites (A) Native PAGE analysis of Rap1 binding for indicated times followed by incubation with competitor plasmid DNA (PL). L1 and L2–L5: lanes in (A) and (C) and (D). (B) Scheme of RSC remodeling assay. Note that Nap1 is not strictly required in these experiments ( <xref ref-type=Figure S7 C). (C) Native PAGE analysis of remodeling assays; MN ∗ , remodeled mononucleosome. (D) Native PAGE analysis of remodeling assays in the presence of 10 eq. of Rap1. (E) Integrated unbound nucleosome bands from (D) (n = 3; error bars SD). (F) MNase-seq results from RSC remodeling assays for 601 nucleosomes (P3_S1S2). Gray, nucleosome start position; blue, RSC remodeling for 90 min in absence of Rap1; red, RSC remodeling for 90 min in presence of 10 eq. Rap1. Shown are reads normalized to number of total reads. (G) Same as in (F) but for RPL30 nucleosomes (P3_RPL30). (H) Effect of Rap1 binding on nucleosome stability at the RPL30 promoter in yeast. Nucleosome positions were determined using qPCR after MNase digestion of chromatin. Promoters analyzed contained both Rap1 binding sites ( S1S2 ), S1 mutated (S1 mut S2), S2 mutated (S1S2 mut ), or both binding sites mutated (S1 mut S2 mut ). Data shown are for cells where Rap1 is present (Rap1+, red), Rap1 has been depleted from the nucleus for 1 h by anchor-away (Rap1−, blue), and where Rap1 has been re-introduced for 2 h following depletion by expressing a RAP1 construct from an inducible promoter (Rap1 ind, green). See also Figure S7 and . " title="... of 10 eq. of Rap1. (E) Integrated unbound nucleosome bands from (D) (n = 3; error bars ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: RSC Enables Stable Rap1 Binding by Exposing Binding Sites (A) Native PAGE analysis of Rap1 binding for indicated times followed by incubation with competitor plasmid DNA (PL). L1 and L2–L5: lanes in (A) and (C) and (D). (B) Scheme of RSC remodeling assay. Note that Nap1 is not strictly required in these experiments ( Figure S7 C). (C) Native PAGE analysis of remodeling assays; MN ∗ , remodeled mononucleosome. (D) Native PAGE analysis of remodeling assays in the presence of 10 eq. of Rap1. (E) Integrated unbound nucleosome bands from (D) (n = 3; error bars SD). (F) MNase-seq results from RSC remodeling assays for 601 nucleosomes (P3_S1S2). Gray, nucleosome start position; blue, RSC remodeling for 90 min in absence of Rap1; red, RSC remodeling for 90 min in presence of 10 eq. Rap1. Shown are reads normalized to number of total reads. (G) Same as in (F) but for RPL30 nucleosomes (P3_RPL30). (H) Effect of Rap1 binding on nucleosome stability at the RPL30 promoter in yeast. Nucleosome positions were determined using qPCR after MNase digestion of chromatin. Promoters analyzed contained both Rap1 binding sites ( S1S2 ), S1 mutated (S1 mut S2), S2 mutated (S1S2 mut ), or both binding sites mutated (S1 mut S2 mut ). Data shown are for cells where Rap1 is present (Rap1+, red), Rap1 has been depleted from the nucleus for 1 h by anchor-away (Rap1−, blue), and where Rap1 has been re-introduced for 2 h following depletion by expressing a RAP1 construct from an inducible promoter (Rap1 ind, green). See also Figure S7 and .

    Techniques Used: Binding Assay, Clear Native PAGE, Incubation, Plasmid Preparation, Expressing, Construct

    A Dynamic Model for Rap1-Mediated Promoter Chromatin Remodeling Rap1 searches chromatin (step 1) and its dynamic binding (2–25 s) to a promoter site results in local chromatin opening (step 2), where Rap1 remains dynamically bound. RSC-mediated nucleosome sliding opens the NDR and exposes the DNA containing Rap1 binding sites (step 3). The fully exposed binding sites allow stable Rap1 binding (step 4) with long residence times (free DNA τ res > 450 s) and prevent further nucleosome encroachment.
    Figure Legend Snippet: A Dynamic Model for Rap1-Mediated Promoter Chromatin Remodeling Rap1 searches chromatin (step 1) and its dynamic binding (2–25 s) to a promoter site results in local chromatin opening (step 2), where Rap1 remains dynamically bound. RSC-mediated nucleosome sliding opens the NDR and exposes the DNA containing Rap1 binding sites (step 3). The fully exposed binding sites allow stable Rap1 binding (step 4) with long residence times (free DNA τ res > 450 s) and prevent further nucleosome encroachment.

    Techniques Used: Binding Assay

    nucleosome dna  (New England Biolabs)


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    Structured Review

    New England Biolabs nucleosome dna
    Rap1 as a Pioneer Factor in Budding Yeast (A) Scheme of pTF function: following target search (1), the pTF invades compact chromatin (2), opens chromatin, and recruits the transcription machinery (3). (B) Domain organization of budding yeast Rap1 (above) and crystal structure of a <t>Rap1:DNA</t> complex (PDB: 3ukg ; <xref ref-type=Matot et al., 2012 ). Act, transcription activation domain; BRCT, BRCA 1 C terminus; DBD, DNA binding domain; RCT, Rap1 C terminus; Tox, toxicity region. (C) Organization of the RPL30 promoter. Gray, MNase-seq profile after Rap1 depletion ( Kubik et al., 2015 ), revealing nucleosome positions in the absence of Rap1 (black dotted circles). Plotted is nucleosome occupancy reads, normalized to 10 7 total reads. The Rap1 binding site 1 ( S1 ) (high affinity) and site 2 ( S2 ) (medium affinity) fall on the −1 nucleosome. (D) Promoter −1 nucleosome, showing Rap1 binding sites S1 and S2 (PDB: 1AOI ; Luger et al., 1997 ). The numbers indicate super helical locations (SHLs) of the nucleosomal DNA. See also Figure S1 and , , and . " width="250" height="auto" />
    Nucleosome Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nucleosome dna/product/New England Biolabs
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nucleosome dna - by Bioz Stars, 2024-04
    90/100 stars

    Images

    1) Product Images from "Chromatin Fiber Invasion and Nucleosome Displacement by the Rap1 Transcription Factor"

    Article Title: Chromatin Fiber Invasion and Nucleosome Displacement by the Rap1 Transcription Factor

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2019.10.025

    Rap1 as a Pioneer Factor in Budding Yeast (A) Scheme of pTF function: following target search (1), the pTF invades compact chromatin (2), opens chromatin, and recruits the transcription machinery (3). (B) Domain organization of budding yeast Rap1 (above) and crystal structure of a Rap1:DNA complex (PDB: 3ukg ; <xref ref-type=Matot et al., 2012 ). Act, transcription activation domain; BRCT, BRCA 1 C terminus; DBD, DNA binding domain; RCT, Rap1 C terminus; Tox, toxicity region. (C) Organization of the RPL30 promoter. Gray, MNase-seq profile after Rap1 depletion ( Kubik et al., 2015 ), revealing nucleosome positions in the absence of Rap1 (black dotted circles). Plotted is nucleosome occupancy reads, normalized to 10 7 total reads. The Rap1 binding site 1 ( S1 ) (high affinity) and site 2 ( S2 ) (medium affinity) fall on the −1 nucleosome. (D) Promoter −1 nucleosome, showing Rap1 binding sites S1 and S2 (PDB: 1AOI ; Luger et al., 1997 ). The numbers indicate super helical locations (SHLs) of the nucleosomal DNA. See also Figure S1 and , , and . " title="... Kubik et al., 2015 ), revealing nucleosome positions in the absence of Rap1 (black dotted ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Rap1 as a Pioneer Factor in Budding Yeast (A) Scheme of pTF function: following target search (1), the pTF invades compact chromatin (2), opens chromatin, and recruits the transcription machinery (3). (B) Domain organization of budding yeast Rap1 (above) and crystal structure of a Rap1:DNA complex (PDB: 3ukg ; Matot et al., 2012 ). Act, transcription activation domain; BRCT, BRCA 1 C terminus; DBD, DNA binding domain; RCT, Rap1 C terminus; Tox, toxicity region. (C) Organization of the RPL30 promoter. Gray, MNase-seq profile after Rap1 depletion ( Kubik et al., 2015 ), revealing nucleosome positions in the absence of Rap1 (black dotted circles). Plotted is nucleosome occupancy reads, normalized to 10 7 total reads. The Rap1 binding site 1 ( S1 ) (high affinity) and site 2 ( S2 ) (medium affinity) fall on the −1 nucleosome. (D) Promoter −1 nucleosome, showing Rap1 binding sites S1 and S2 (PDB: 1AOI ; Luger et al., 1997 ). The numbers indicate super helical locations (SHLs) of the nucleosomal DNA. See also Figure S1 and , , and .

    Techniques Used: Activation Assay, Binding Assay

    Rap1 Recognizes Target Sites within Nucleosomal DNA (A) Scheme of the smTIRFM experiment to detect Rap1 binding to S1 - or S2 -containing, Alexa-Fluor-647-labeled, and immobilized DNA or nucleosomes. bt-NA, biotin-neutravidin. (B) Expression and labeling of Rap1. Lanes: (1) purified MBP-Rap1-Halo; (2) MBP cleavage; (3 and 4) before and after JF-549 labeling; and (5) purified Rap1. (C) Representative smTIRF images showing nucleosome positions in the far-red channel (left, red circles) and Rap1 binding events in the green-orange channel (right). Scale bars: 5 μm; ex, excitation wavelength; em, emission wavelength. (D) Representative fluorescence time trace of Rap1 binding events to S2 containing naked DNA, detected by JF-549 emission. The trace was fitted (red), and t dark and t bright were determined by a thresholding algorithm. (E) Cumulative histogram of Rap1 binding intervals ( t bright ) on S2 DNA fitted by a 2-exponential function y = ∑ i = 1 2 A i exp ( − t / τ o f f , i ) (solid line). For all fit results, see . (F) Specific dissociation time constants (τ off,i > 1 s) of Rap1 for S2 DNA, S1 and S2 containing mononucleosomes (MN), or nucleosomes lacking a binding site (NS), uncorrected for dye photobleaching. The width of the bars indicates the percentage of events associated with the indicated time constants (i.e., amplitudes A i of the multi-exponential fits shown in E and H). n = 4 to 5; error bars: SD. (G) Representative fluorescence time trace of Rap1 binding events to S1 (bottom) and S2 (top) containing MNs. The data were analyzed as in (D). (H) Cumulative histogram of Rap1 binding intervals ( t bright ) on S1 - and S2 -containing MNs fitted by a 3-exponential function y = ∑ i = 0 2 A i exp ( − t / τ o f f , i ) (solid line). (I) Specific on-rate constants ( k on = 1/ τ on ) for all species obtained from a single-exponential fit to cumulative histograms of t dark values and corrected for the contribution from nonspecific interactions . See also and and , , , and .
    Figure Legend Snippet: Rap1 Recognizes Target Sites within Nucleosomal DNA (A) Scheme of the smTIRFM experiment to detect Rap1 binding to S1 - or S2 -containing, Alexa-Fluor-647-labeled, and immobilized DNA or nucleosomes. bt-NA, biotin-neutravidin. (B) Expression and labeling of Rap1. Lanes: (1) purified MBP-Rap1-Halo; (2) MBP cleavage; (3 and 4) before and after JF-549 labeling; and (5) purified Rap1. (C) Representative smTIRF images showing nucleosome positions in the far-red channel (left, red circles) and Rap1 binding events in the green-orange channel (right). Scale bars: 5 μm; ex, excitation wavelength; em, emission wavelength. (D) Representative fluorescence time trace of Rap1 binding events to S2 containing naked DNA, detected by JF-549 emission. The trace was fitted (red), and t dark and t bright were determined by a thresholding algorithm. (E) Cumulative histogram of Rap1 binding intervals ( t bright ) on S2 DNA fitted by a 2-exponential function y = ∑ i = 1 2 A i exp ( − t / τ o f f , i ) (solid line). For all fit results, see . (F) Specific dissociation time constants (τ off,i > 1 s) of Rap1 for S2 DNA, S1 and S2 containing mononucleosomes (MN), or nucleosomes lacking a binding site (NS), uncorrected for dye photobleaching. The width of the bars indicates the percentage of events associated with the indicated time constants (i.e., amplitudes A i of the multi-exponential fits shown in E and H). n = 4 to 5; error bars: SD. (G) Representative fluorescence time trace of Rap1 binding events to S1 (bottom) and S2 (top) containing MNs. The data were analyzed as in (D). (H) Cumulative histogram of Rap1 binding intervals ( t bright ) on S1 - and S2 -containing MNs fitted by a 3-exponential function y = ∑ i = 0 2 A i exp ( − t / τ o f f , i ) (solid line). (I) Specific on-rate constants ( k on = 1/ τ on ) for all species obtained from a single-exponential fit to cumulative histograms of t dark values and corrected for the contribution from nonspecific interactions . See also and and , , , and .

    Techniques Used: Binding Assay, Labeling, Expressing, Purification, Fluorescence

    Chromatin Higher-Order Structure Reduces Rap1 Dwell Time (A) Scheme of DNA preparation used to introduce Rap1 target sites S1 and S2 into the central nucleosome (N6) of a chromatin fiber (CH). (B) Scheme of the smTIRFM experiment to measure Rap1 binding kinetics in a chromatin fiber context. (C) Representative fluorescence time trace of Rap1 binding events to S1 -containing chromatin arrays. The trace is fitted (red); t dark and t bright were determined by a thresholding algorithm. (D) Cumulative histogram of Rap1 binding intervals (t bright ) to chromatin fibers, containing S1 fitted by a 3-exponential function (solid line). For all fit results, see . (E) Cumulative histogram of Rap1 binding to chromatin arrays, containing S2 fitted by a 3-exponential function (solid line). (F) Specific binding time constants (τ off,i > 1 s) of Rap1 for S1 in a nucleosome (MN) versus chromatin fiber (CH) and S2 MN versus CH. The widths of the bars indicate the percentage of events associated with the indicated time constants (i.e., amplitudes A i of the multi-exponential fits shown in D and E). n = 4 to 5; error bars: SD. (G) Specific on-rate constants ( k on = 1/ τ on ) for MNs and CHs containing S1 and S2 , obtained from a single-exponential fit to cumulative histograms of t dark values and corrected for the contribution from nonspecific interactions . See also <xref ref-type=Figure S4 and , , , and . " title="... target sites S1 and S2 into the central nucleosome (N6) of a chromatin fiber (CH). (B) Scheme ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Chromatin Higher-Order Structure Reduces Rap1 Dwell Time (A) Scheme of DNA preparation used to introduce Rap1 target sites S1 and S2 into the central nucleosome (N6) of a chromatin fiber (CH). (B) Scheme of the smTIRFM experiment to measure Rap1 binding kinetics in a chromatin fiber context. (C) Representative fluorescence time trace of Rap1 binding events to S1 -containing chromatin arrays. The trace is fitted (red); t dark and t bright were determined by a thresholding algorithm. (D) Cumulative histogram of Rap1 binding intervals (t bright ) to chromatin fibers, containing S1 fitted by a 3-exponential function (solid line). For all fit results, see . (E) Cumulative histogram of Rap1 binding to chromatin arrays, containing S2 fitted by a 3-exponential function (solid line). (F) Specific binding time constants (τ off,i > 1 s) of Rap1 for S1 in a nucleosome (MN) versus chromatin fiber (CH) and S2 MN versus CH. The widths of the bars indicate the percentage of events associated with the indicated time constants (i.e., amplitudes A i of the multi-exponential fits shown in D and E). n = 4 to 5; error bars: SD. (G) Specific on-rate constants ( k on = 1/ τ on ) for MNs and CHs containing S1 and S2 , obtained from a single-exponential fit to cumulative histograms of t dark values and corrected for the contribution from nonspecific interactions . See also Figure S4 and , , , and .

    Techniques Used: Introduce, Binding Assay, Fluorescence

    Rap1 Does Not Open Nucleosome Structure (A) Scheme of experiment to detect nucleosome structure change due to Rap1 binding by FRET. (B) Nucleosome structure (PDB: 1AOI ) showing attachment points of FRET probes. (C) EMSA showing Rap1 binding to S1 and S2 nucleosomes at indicated concentration equivalents (eq.). Lanes were re-arranged for clarity. (D) Fluorescence spectra for S2 nucleosome in complex with indicated equivalents of Rap1. (E) Fluorescence spectra for S1 nucleosome in complex with indicated equivalents of Rap1. (F) FRET efficiency calculated for S2 and S1 nucleosomes as a function of equivalents added Rap1. Error bars: SD; n = 2. See also <xref ref-type=Figure S5 . " title="Rap1 Does Not Open Nucleosome Structure (A) Scheme of experiment to detect nucleosome ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Rap1 Does Not Open Nucleosome Structure (A) Scheme of experiment to detect nucleosome structure change due to Rap1 binding by FRET. (B) Nucleosome structure (PDB: 1AOI ) showing attachment points of FRET probes. (C) EMSA showing Rap1 binding to S1 and S2 nucleosomes at indicated concentration equivalents (eq.). Lanes were re-arranged for clarity. (D) Fluorescence spectra for S2 nucleosome in complex with indicated equivalents of Rap1. (E) Fluorescence spectra for S1 nucleosome in complex with indicated equivalents of Rap1. (F) FRET efficiency calculated for S2 and S1 nucleosomes as a function of equivalents added Rap1. Error bars: SD; n = 2. See also Figure S5 .

    Techniques Used: Binding Assay, Concentration Assay, Fluorescence

    Chromatin Remodeling Induced by Rap1 Invasion as Observed by smFRET (A) Scheme of chromatin DNA assembly to introduce a Rap1 site at nucleosome N6, as well as a FRET donor (Cy3B, yellow) and acceptor (Alexa Fluor 647, red) at nucleosomes N5 and N7. (B) Scheme of a smFRET-TIRF experiment. (C) Individual kinetic traces of donor (orange) and acceptor (red) fluorescence emission and FRET efficiency ( E FRET , blue) for chromatin fibers containing S2 at the indicated KCl and Rap1 concentrations. All Rap1 experiments were performed at 150 mM KCl. (D) Similar to (C) but for chromatin lacking Rap1 binding sites ( NS ). (E) Histograms of E FRET of S2 -containing chromatin fibers at the indicated KCl and Rap1 concentrations. All Rap1 experiments were performed at 150 mM KCl. Histograms were fitted by Gaussian functions, revealing a low-FRET (LF) (gray), medium-FRET (MF) (green), and high-FRET (HF) (red) population. Error bars are SEM; for the number of traces and parameters of Gaussian fits, see and . (F) Similar to (E) but for chromatin lacking Rap1 binding sites ( NS ). (G) Percentage of each FRET sub-population, LF, MF, and HF for chromatin containing S2 . Box: 25–75 percentiles; whiskers: outliers (factor 1.5); line: median; open symbol: mean. For number of experiments, see . ∗ 10 −3 > p < 10 −4 ; ∗∗ 10 −4 > p < 10 −5 ; ∗∗∗ p < 10 −5 , two-tailed Student’s t test between peak area % of LF, MF, and HF populations for S2 or NS nucleosomes (see H). (H) Similar to (G) but for chromatin lacking Rap1 binding sites ( NS ). (I) Percentage of dynamic traces for S2 and NS chromatin. Box: similar to (H). For the identification of dynamic traces, see . p: two-tailed Student’s t test; n.s.: p > 0.05. See also <xref ref-type=Figure S6 and and . " title="... DNA assembly to introduce a Rap1 site at nucleosome N6, as well as a FRET donor (Cy3B, ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Chromatin Remodeling Induced by Rap1 Invasion as Observed by smFRET (A) Scheme of chromatin DNA assembly to introduce a Rap1 site at nucleosome N6, as well as a FRET donor (Cy3B, yellow) and acceptor (Alexa Fluor 647, red) at nucleosomes N5 and N7. (B) Scheme of a smFRET-TIRF experiment. (C) Individual kinetic traces of donor (orange) and acceptor (red) fluorescence emission and FRET efficiency ( E FRET , blue) for chromatin fibers containing S2 at the indicated KCl and Rap1 concentrations. All Rap1 experiments were performed at 150 mM KCl. (D) Similar to (C) but for chromatin lacking Rap1 binding sites ( NS ). (E) Histograms of E FRET of S2 -containing chromatin fibers at the indicated KCl and Rap1 concentrations. All Rap1 experiments were performed at 150 mM KCl. Histograms were fitted by Gaussian functions, revealing a low-FRET (LF) (gray), medium-FRET (MF) (green), and high-FRET (HF) (red) population. Error bars are SEM; for the number of traces and parameters of Gaussian fits, see and . (F) Similar to (E) but for chromatin lacking Rap1 binding sites ( NS ). (G) Percentage of each FRET sub-population, LF, MF, and HF for chromatin containing S2 . Box: 25–75 percentiles; whiskers: outliers (factor 1.5); line: median; open symbol: mean. For number of experiments, see . ∗ 10 −3 > p < 10 −4 ; ∗∗ 10 −4 > p < 10 −5 ; ∗∗∗ p < 10 −5 , two-tailed Student’s t test between peak area % of LF, MF, and HF populations for S2 or NS nucleosomes (see H). (H) Similar to (G) but for chromatin lacking Rap1 binding sites ( NS ). (I) Percentage of dynamic traces for S2 and NS chromatin. Box: similar to (H). For the identification of dynamic traces, see . p: two-tailed Student’s t test; n.s.: p > 0.05. See also Figure S6 and and .

    Techniques Used: Introduce, Fluorescence, Binding Assay, Two Tailed Test

    RSC Enables Stable Rap1 Binding by Exposing Binding Sites (A) Native PAGE analysis of Rap1 binding for indicated times followed by incubation with competitor plasmid DNA (PL). L1 and L2–L5: lanes in (A) and (C) and (D). (B) Scheme of RSC remodeling assay. Note that Nap1 is not strictly required in these experiments ( <xref ref-type=Figure S7 C). (C) Native PAGE analysis of remodeling assays; MN ∗ , remodeled mononucleosome. (D) Native PAGE analysis of remodeling assays in the presence of 10 eq. of Rap1. (E) Integrated unbound nucleosome bands from (D) (n = 3; error bars SD). (F) MNase-seq results from RSC remodeling assays for 601 nucleosomes (P3_S1S2). Gray, nucleosome start position; blue, RSC remodeling for 90 min in absence of Rap1; red, RSC remodeling for 90 min in presence of 10 eq. Rap1. Shown are reads normalized to number of total reads. (G) Same as in (F) but for RPL30 nucleosomes (P3_RPL30). (H) Effect of Rap1 binding on nucleosome stability at the RPL30 promoter in yeast. Nucleosome positions were determined using qPCR after MNase digestion of chromatin. Promoters analyzed contained both Rap1 binding sites ( S1S2 ), S1 mutated (S1 mut S2), S2 mutated (S1S2 mut ), or both binding sites mutated (S1 mut S2 mut ). Data shown are for cells where Rap1 is present (Rap1+, red), Rap1 has been depleted from the nucleus for 1 h by anchor-away (Rap1−, blue), and where Rap1 has been re-introduced for 2 h following depletion by expressing a RAP1 construct from an inducible promoter (Rap1 ind, green). See also Figure S7 and . " title="... of 10 eq. of Rap1. (E) Integrated unbound nucleosome bands from (D) (n = 3; error bars ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: RSC Enables Stable Rap1 Binding by Exposing Binding Sites (A) Native PAGE analysis of Rap1 binding for indicated times followed by incubation with competitor plasmid DNA (PL). L1 and L2–L5: lanes in (A) and (C) and (D). (B) Scheme of RSC remodeling assay. Note that Nap1 is not strictly required in these experiments ( Figure S7 C). (C) Native PAGE analysis of remodeling assays; MN ∗ , remodeled mononucleosome. (D) Native PAGE analysis of remodeling assays in the presence of 10 eq. of Rap1. (E) Integrated unbound nucleosome bands from (D) (n = 3; error bars SD). (F) MNase-seq results from RSC remodeling assays for 601 nucleosomes (P3_S1S2). Gray, nucleosome start position; blue, RSC remodeling for 90 min in absence of Rap1; red, RSC remodeling for 90 min in presence of 10 eq. Rap1. Shown are reads normalized to number of total reads. (G) Same as in (F) but for RPL30 nucleosomes (P3_RPL30). (H) Effect of Rap1 binding on nucleosome stability at the RPL30 promoter in yeast. Nucleosome positions were determined using qPCR after MNase digestion of chromatin. Promoters analyzed contained both Rap1 binding sites ( S1S2 ), S1 mutated (S1 mut S2), S2 mutated (S1S2 mut ), or both binding sites mutated (S1 mut S2 mut ). Data shown are for cells where Rap1 is present (Rap1+, red), Rap1 has been depleted from the nucleus for 1 h by anchor-away (Rap1−, blue), and where Rap1 has been re-introduced for 2 h following depletion by expressing a RAP1 construct from an inducible promoter (Rap1 ind, green). See also Figure S7 and .

    Techniques Used: Binding Assay, Clear Native PAGE, Incubation, Plasmid Preparation, Expressing, Construct

    A Dynamic Model for Rap1-Mediated Promoter Chromatin Remodeling Rap1 searches chromatin (step 1) and its dynamic binding (2–25 s) to a promoter site results in local chromatin opening (step 2), where Rap1 remains dynamically bound. RSC-mediated nucleosome sliding opens the NDR and exposes the DNA containing Rap1 binding sites (step 3). The fully exposed binding sites allow stable Rap1 binding (step 4) with long residence times (free DNA τ res > 450 s) and prevent further nucleosome encroachment.
    Figure Legend Snippet: A Dynamic Model for Rap1-Mediated Promoter Chromatin Remodeling Rap1 searches chromatin (step 1) and its dynamic binding (2–25 s) to a promoter site results in local chromatin opening (step 2), where Rap1 remains dynamically bound. RSC-mediated nucleosome sliding opens the NDR and exposes the DNA containing Rap1 binding sites (step 3). The fully exposed binding sites allow stable Rap1 binding (step 4) with long residence times (free DNA τ res > 450 s) and prevent further nucleosome encroachment.

    Techniques Used: Binding Assay

    208 bp model dna  (New England Biolabs)


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    nucleosome  (New England Biolabs)


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    New England Biolabs nucleosome
    Assembly of H2B K34ub and unmodified <t>nucleosomes.</t> ( A ) Left panel: SDS-PAGE of H2B K34ub- (lane 1) and unmodified (lane 2) histone octamers. Right panel: native PAGE of modified nucleosomes assembled on 147 bp 601 DNA using increasing amounts histones. ( B ) SDS-PAGE of faster- (lane 1) and slower- (lane 2) migrating H2B K34ub assembly products extracted from the native gel in A. The densitometric tracing of the gel lanes is shown on the right. Quantification, by ImageJ, is normalized to that of the histone H4, which is arbitrarily set as 1. ( C ) Native PAGE for samples prepared by serial dilution of 2M NaCl mixtures of 147 bp 601 DNA and modified or unmodified histones to 1 M NaCl. ( D ) Left panel: SDS-PAGE of recombinant linker histone H1 0 . Right panel: assembly of H2B K34ub modified and unmodified nucleosomes with increasing concentration of recombinant histone H1 0 .
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    1) Product Images from "Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics"

    Article Title: Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky526

    Assembly of H2B K34ub and unmodified nucleosomes. ( A ) Left panel: SDS-PAGE of H2B K34ub- (lane 1) and unmodified (lane 2) histone octamers. Right panel: native PAGE of modified nucleosomes assembled on 147 bp 601 DNA using increasing amounts histones. ( B ) SDS-PAGE of faster- (lane 1) and slower- (lane 2) migrating H2B K34ub assembly products extracted from the native gel in A. The densitometric tracing of the gel lanes is shown on the right. Quantification, by ImageJ, is normalized to that of the histone H4, which is arbitrarily set as 1. ( C ) Native PAGE for samples prepared by serial dilution of 2M NaCl mixtures of 147 bp 601 DNA and modified or unmodified histones to 1 M NaCl. ( D ) Left panel: SDS-PAGE of recombinant linker histone H1 0 . Right panel: assembly of H2B K34ub modified and unmodified nucleosomes with increasing concentration of recombinant histone H1 0 .
    Figure Legend Snippet: Assembly of H2B K34ub and unmodified nucleosomes. ( A ) Left panel: SDS-PAGE of H2B K34ub- (lane 1) and unmodified (lane 2) histone octamers. Right panel: native PAGE of modified nucleosomes assembled on 147 bp 601 DNA using increasing amounts histones. ( B ) SDS-PAGE of faster- (lane 1) and slower- (lane 2) migrating H2B K34ub assembly products extracted from the native gel in A. The densitometric tracing of the gel lanes is shown on the right. Quantification, by ImageJ, is normalized to that of the histone H4, which is arbitrarily set as 1. ( C ) Native PAGE for samples prepared by serial dilution of 2M NaCl mixtures of 147 bp 601 DNA and modified or unmodified histones to 1 M NaCl. ( D ) Left panel: SDS-PAGE of recombinant linker histone H1 0 . Right panel: assembly of H2B K34ub modified and unmodified nucleosomes with increasing concentration of recombinant histone H1 0 .

    Techniques Used: SDS Page, Clear Native PAGE, Modification, Serial Dilution, Recombinant, Concentration Assay

    Stability of H2B K34ub nucleosomes. ( A ) Native PAGE for nucleosome assembly with increasing amounts of unmodified and H2B K34ub histone octamers and their mixture at ratios indicated on top. Quantification was performed by Image J and relative abundance of each species was summarized in Table . The representative result from three repeats was presented. ( B ) H2B K34ub (top panel) or Unmodified (bottom panel) nucleosomes were assembled on nicked, positively or negatively supercoiled plasmids containing 12 copies of 177 bp 601 DNA. After assembly 601 nucleosomes were released with ScaI and resolved on native PAGE. ( C ) The nucleosome assembly containing histones of indicated ratios (top) were incubated with competitor DNA for 2 h at 26°C in a buffer containing 200 mm NaCl. ( D ) Nucleosomes assembled on 147 bp 601 DNA (as indicated on top) were incubated with or without CM-50 Sephadex.
    Figure Legend Snippet: Stability of H2B K34ub nucleosomes. ( A ) Native PAGE for nucleosome assembly with increasing amounts of unmodified and H2B K34ub histone octamers and their mixture at ratios indicated on top. Quantification was performed by Image J and relative abundance of each species was summarized in Table . The representative result from three repeats was presented. ( B ) H2B K34ub (top panel) or Unmodified (bottom panel) nucleosomes were assembled on nicked, positively or negatively supercoiled plasmids containing 12 copies of 177 bp 601 DNA. After assembly 601 nucleosomes were released with ScaI and resolved on native PAGE. ( C ) The nucleosome assembly containing histones of indicated ratios (top) were incubated with competitor DNA for 2 h at 26°C in a buffer containing 200 mm NaCl. ( D ) Nucleosomes assembled on 147 bp 601 DNA (as indicated on top) were incubated with or without CM-50 Sephadex.

    Techniques Used: Clear Native PAGE, Incubation

    Dynamics of H2B K34ub nucleosomes. ( A ) Nucleosomes assembled on 147 bp 601 DNA were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. ( B ) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with MNase at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.
    Figure Legend Snippet: Dynamics of H2B K34ub nucleosomes. ( A ) Nucleosomes assembled on 147 bp 601 DNA were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. ( B ) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with MNase at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.

    Techniques Used: Clear Native PAGE, Electrophoresis, Staining

    Eviction of histone dimer in H2B K34ub nucleosome by histone dimer acceptors. ( A ) Left panel: SDS-PAGE of NAP1. Middle, right panels: unmodified and H2B K34ub nucleosomes assembled on a 147 or 177 bp 601 DNA were post-assembly incubated with NAP1 for 2.5 h at 26 or 37°C in a buffer containing 100 mM NaCl. ( B ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA for 2.5 h at 26/37°C in a buffer containing at 100 mM NaCl. ( C ) H2B K34ub nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA at 26/37°C for 2.5 h or at 26°C for 20 h (the ‘20 h’ and ‘2.5 h’ samples were resolved in different gels).
    Figure Legend Snippet: Eviction of histone dimer in H2B K34ub nucleosome by histone dimer acceptors. ( A ) Left panel: SDS-PAGE of NAP1. Middle, right panels: unmodified and H2B K34ub nucleosomes assembled on a 147 or 177 bp 601 DNA were post-assembly incubated with NAP1 for 2.5 h at 26 or 37°C in a buffer containing 100 mM NaCl. ( B ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA for 2.5 h at 26/37°C in a buffer containing at 100 mM NaCl. ( C ) H2B K34ub nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA at 26/37°C for 2.5 h or at 26°C for 20 h (the ‘20 h’ and ‘2.5 h’ samples were resolved in different gels).

    Techniques Used: SDS Page, Incubation

    Stability of H2B K34ub- versus H2B K120ub-nucleosomes. ( A ) Left panel: SDS-PAGE of H2B K120ub and K34ub histone octamers. Right panel: unmodified and H2B K120ub/ K34ub nucleosomes assembled on 147 bp 601 DNA. ( B ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA for 2.5 h at indicated temperature/ionic conditions. ( C ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with NAP1 for 2.5 h at 26/37°C in a buffer containing at 140 mM NaCl.
    Figure Legend Snippet: Stability of H2B K34ub- versus H2B K120ub-nucleosomes. ( A ) Left panel: SDS-PAGE of H2B K120ub and K34ub histone octamers. Right panel: unmodified and H2B K120ub/ K34ub nucleosomes assembled on 147 bp 601 DNA. ( B ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA for 2.5 h at indicated temperature/ionic conditions. ( C ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with NAP1 for 2.5 h at 26/37°C in a buffer containing at 140 mM NaCl.

    Techniques Used: SDS Page, Incubation

    Effects of underlying DNA sequence on stability of H2B K120ub and K34ub nucleosomes. ( A ) Nucleosomes assembled on 146 bp 5S DNA. ( B ) Assembled nucleosomes were incubated with competitor DNA for 2.5 h at 26°C in a buffer containing at 200 mM NaCl. ( C ) Nucleosomes incubated with competitor DNA for 2.5 h at indicated temperature/ ionic conditions. ( D and E ) Nucleosomes assembled with unmodified or H2B K34ub histones, or their 1/0.57 mixture, were post-assembly incubated for 2.5 h at 26 or 37°C with (D) competitor DNA in a buffer containing at 100 mM NaCl or (E) NAP1 in a buffer containing at 150 mM NaCl. Densitometry tracing of indicated gel lanes is shown at the bottom of gel images.
    Figure Legend Snippet: Effects of underlying DNA sequence on stability of H2B K120ub and K34ub nucleosomes. ( A ) Nucleosomes assembled on 146 bp 5S DNA. ( B ) Assembled nucleosomes were incubated with competitor DNA for 2.5 h at 26°C in a buffer containing at 200 mM NaCl. ( C ) Nucleosomes incubated with competitor DNA for 2.5 h at indicated temperature/ ionic conditions. ( D and E ) Nucleosomes assembled with unmodified or H2B K34ub histones, or their 1/0.57 mixture, were post-assembly incubated for 2.5 h at 26 or 37°C with (D) competitor DNA in a buffer containing at 100 mM NaCl or (E) NAP1 in a buffer containing at 150 mM NaCl. Densitometry tracing of indicated gel lanes is shown at the bottom of gel images.

    Techniques Used: Sequencing, Incubation

    5s rdna  (New England Biolabs)


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    5srdna  (New England Biolabs)


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    New England Biolabs 5srdna
    a, Gel electrophoresis and quantitation of nucleosomal <t>5SrDNA,</t> Myh6 promoter and Neo DNA. Arrowheads: DNA-histone complex. Arrows: naked DNA. Nucleosome assembly efficiency is defined as the fraction of DNA bound to histones (arrowheads). P-value: Student’s t-test. Error bar: standard error of the mean (SEM). b-d, Quantification of amylose pull-down of MBP-D1D2 (D1D2) with nucleosomal and naked Myh6 promoter DNA ( b ), with nucleosomal Myh6 promoter, Neo , and 5SrDNA ( c ), or with nucleosomal Myh6 promoter in the presence of Mhrt779 ( d ). P-value: Student’s t-test. Error bar: SEM. e, Amylose pull-down of MBP-D1D2 and histone 3. Anti-histone 3 and anti-MBP antibodies were used for western blot analysis. f, ChIP analysis of Brg1 on chromatinized and naked Myh6 promoter in rat ventricular cardiomyocytes. GFP: green fluorescence protein control. P-value: Student’s t-test. Error bar: SEM. g, h, Luciferase reporter activity of Brg1 on naked Myh6 promoter ( g ) or of helicase-deficient Brg1 on chromatinized Myh6 promoter ( h ) in rat ventricular cardiomyocytes. ΔD1: Brg1 lacking amino acid 774–913; ΔD2: Brg1 lacking 1086–1246. GFP: green fluorescence protein control. ChIP: H-10 antibody recognizing N-terminus, non-disrupted region of Brg1. P-value: Student’s t-test. Error bar: SEM. i, j, ChIP analysis in SW13 cells of chromatinized Myh6 promoter in the presence of Mhrt779 ( i ) or helicase-deficient Brg1 ( j ). Vector: pAdd2 empty vector. Mhrt : pAdd2- Mhrt779 . P-value: Student’s t-test. Error bar: SEM. k, Schematic illustration and PCR of human MHRT. MHRT originates from MYH7 and is transcribed into MYH7. MYH7 e xons and introns are indicated. R1 and R2 are strand-specific PCR primers; F1 and R1 target MHRT and MYH7 ; F2 and R2 are specific for MHRT . l, Quantification of MHRT in human heart tissues. Ctrl: control. LVH: left ventricular hypertrophy. ICM: ischemic cardiomyopathy. IDCM: idiopathic dilated cardiomyopathy. P-value: Student’s t-test. Error bar: SEM. m, Working model of a Brg1- Mhrt negative feedback circuit in the heart. Brg1 represses Mhrt transcription, whereas Mhrt prevents Brg1 from recognizing its chromatin targets. Brg1 functions through two distinct promoter elements to bidirectionally repress Myh6 and Mhrt expression. n , Molecular model of how Brg1 binds to its genomic DNA targets. Brg1 helicase (D1D2) binds chromatinized DNA, C-terminal extension (CTE) binds histone 3 (H3), and bromodomain binds acetylated (Ac) histone 3 or 4 (H4).
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    1) Product Images from "A long non-coding RNA protects the heart from pathological hypertrophy"

    Article Title: A long non-coding RNA protects the heart from pathological hypertrophy

    Journal: Nature

    doi: 10.1038/nature13596

    a, Gel electrophoresis and quantitation of nucleosomal 5SrDNA, Myh6 promoter and Neo DNA. Arrowheads: DNA-histone complex. Arrows: naked DNA. Nucleosome assembly efficiency is defined as the fraction of DNA bound to histones (arrowheads). P-value: Student’s t-test. Error bar: standard error of the mean (SEM). b-d, Quantification of amylose pull-down of MBP-D1D2 (D1D2) with nucleosomal and naked Myh6 promoter DNA ( b ), with nucleosomal Myh6 promoter, Neo , and 5SrDNA ( c ), or with nucleosomal Myh6 promoter in the presence of Mhrt779 ( d ). P-value: Student’s t-test. Error bar: SEM. e, Amylose pull-down of MBP-D1D2 and histone 3. Anti-histone 3 and anti-MBP antibodies were used for western blot analysis. f, ChIP analysis of Brg1 on chromatinized and naked Myh6 promoter in rat ventricular cardiomyocytes. GFP: green fluorescence protein control. P-value: Student’s t-test. Error bar: SEM. g, h, Luciferase reporter activity of Brg1 on naked Myh6 promoter ( g ) or of helicase-deficient Brg1 on chromatinized Myh6 promoter ( h ) in rat ventricular cardiomyocytes. ΔD1: Brg1 lacking amino acid 774–913; ΔD2: Brg1 lacking 1086–1246. GFP: green fluorescence protein control. ChIP: H-10 antibody recognizing N-terminus, non-disrupted region of Brg1. P-value: Student’s t-test. Error bar: SEM. i, j, ChIP analysis in SW13 cells of chromatinized Myh6 promoter in the presence of Mhrt779 ( i ) or helicase-deficient Brg1 ( j ). Vector: pAdd2 empty vector. Mhrt : pAdd2- Mhrt779 . P-value: Student’s t-test. Error bar: SEM. k, Schematic illustration and PCR of human MHRT. MHRT originates from MYH7 and is transcribed into MYH7. MYH7 e xons and introns are indicated. R1 and R2 are strand-specific PCR primers; F1 and R1 target MHRT and MYH7 ; F2 and R2 are specific for MHRT . l, Quantification of MHRT in human heart tissues. Ctrl: control. LVH: left ventricular hypertrophy. ICM: ischemic cardiomyopathy. IDCM: idiopathic dilated cardiomyopathy. P-value: Student’s t-test. Error bar: SEM. m, Working model of a Brg1- Mhrt negative feedback circuit in the heart. Brg1 represses Mhrt transcription, whereas Mhrt prevents Brg1 from recognizing its chromatin targets. Brg1 functions through two distinct promoter elements to bidirectionally repress Myh6 and Mhrt expression. n , Molecular model of how Brg1 binds to its genomic DNA targets. Brg1 helicase (D1D2) binds chromatinized DNA, C-terminal extension (CTE) binds histone 3 (H3), and bromodomain binds acetylated (Ac) histone 3 or 4 (H4).
    Figure Legend Snippet: a, Gel electrophoresis and quantitation of nucleosomal 5SrDNA, Myh6 promoter and Neo DNA. Arrowheads: DNA-histone complex. Arrows: naked DNA. Nucleosome assembly efficiency is defined as the fraction of DNA bound to histones (arrowheads). P-value: Student’s t-test. Error bar: standard error of the mean (SEM). b-d, Quantification of amylose pull-down of MBP-D1D2 (D1D2) with nucleosomal and naked Myh6 promoter DNA ( b ), with nucleosomal Myh6 promoter, Neo , and 5SrDNA ( c ), or with nucleosomal Myh6 promoter in the presence of Mhrt779 ( d ). P-value: Student’s t-test. Error bar: SEM. e, Amylose pull-down of MBP-D1D2 and histone 3. Anti-histone 3 and anti-MBP antibodies were used for western blot analysis. f, ChIP analysis of Brg1 on chromatinized and naked Myh6 promoter in rat ventricular cardiomyocytes. GFP: green fluorescence protein control. P-value: Student’s t-test. Error bar: SEM. g, h, Luciferase reporter activity of Brg1 on naked Myh6 promoter ( g ) or of helicase-deficient Brg1 on chromatinized Myh6 promoter ( h ) in rat ventricular cardiomyocytes. ΔD1: Brg1 lacking amino acid 774–913; ΔD2: Brg1 lacking 1086–1246. GFP: green fluorescence protein control. ChIP: H-10 antibody recognizing N-terminus, non-disrupted region of Brg1. P-value: Student’s t-test. Error bar: SEM. i, j, ChIP analysis in SW13 cells of chromatinized Myh6 promoter in the presence of Mhrt779 ( i ) or helicase-deficient Brg1 ( j ). Vector: pAdd2 empty vector. Mhrt : pAdd2- Mhrt779 . P-value: Student’s t-test. Error bar: SEM. k, Schematic illustration and PCR of human MHRT. MHRT originates from MYH7 and is transcribed into MYH7. MYH7 e xons and introns are indicated. R1 and R2 are strand-specific PCR primers; F1 and R1 target MHRT and MYH7 ; F2 and R2 are specific for MHRT . l, Quantification of MHRT in human heart tissues. Ctrl: control. LVH: left ventricular hypertrophy. ICM: ischemic cardiomyopathy. IDCM: idiopathic dilated cardiomyopathy. P-value: Student’s t-test. Error bar: SEM. m, Working model of a Brg1- Mhrt negative feedback circuit in the heart. Brg1 represses Mhrt transcription, whereas Mhrt prevents Brg1 from recognizing its chromatin targets. Brg1 functions through two distinct promoter elements to bidirectionally repress Myh6 and Mhrt expression. n , Molecular model of how Brg1 binds to its genomic DNA targets. Brg1 helicase (D1D2) binds chromatinized DNA, C-terminal extension (CTE) binds histone 3 (H3), and bromodomain binds acetylated (Ac) histone 3 or 4 (H4).

    Techniques Used: Nucleic Acid Electrophoresis, Quantitation Assay, Western Blot, Fluorescence, Luciferase, Activity Assay, Plasmid Preparation, Expressing

    control dna  (New England Biolabs)


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    New England Biolabs control dna
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    nucleosome  (New England Biolabs)


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    New England Biolabs nucleosome
    hSWI/SNF-catalyzed octamer sliding is not inhibited by branched DNA structures within the <t>nucleosome.</t> (A) DNA fragments used for nucleosome reconstitution. The flap, hairpin, and nick substrates contain the same sequences as the 215-bp intact fragment. The positions of the branch site, relevant restriction enzyme sites, and the main region of DNA assembled into the nucleosome (oval) are shown. (B) Nucleosome sliding assay. Purified nucleosomes prepared with native, flap, hairpin, and nicked DNAs were incubated in the absence of SWI/SNF (solid bars) or with hSWI/SNF without ATP (open bars) or with ATP (hatched bars) for 15 min and then subjected to HhaI digestion for 10 min. The bar graph shows the fraction of DNA remaining uncut after HhaI digestion, normalized to the amount remaining in the controls without SWI/SNF. Data were normalized in this manner since the fraction of nucleosomes remaining undigested (inset numbers) varied slightly between nucleosome preparations. Note that the nucleosomes reconstituted with the native DNA fragment contained H2BG26C-APB and were irradiated to produce portions of the sample that either were not cross-linked (Native-X) or were cross-linked (Native+X). Experiments with native DNA template and native histones produced results identical to those for the Native-X samples (data not shown).
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    1) Product Images from "hSWI/SNF-Catalyzed Nucleosome Sliding Does Not Occur Solely via a Twist-Diffusion Mechanism"

    Article Title: hSWI/SNF-Catalyzed Nucleosome Sliding Does Not Occur Solely via a Twist-Diffusion Mechanism

    Journal:

    doi: 10.1128/MCB.22.21.7484-7490.2002

    hSWI/SNF-catalyzed octamer sliding is not inhibited by branched DNA structures within the nucleosome. (A) DNA fragments used for nucleosome reconstitution. The flap, hairpin, and nick substrates contain the same sequences as the 215-bp intact fragment. The positions of the branch site, relevant restriction enzyme sites, and the main region of DNA assembled into the nucleosome (oval) are shown. (B) Nucleosome sliding assay. Purified nucleosomes prepared with native, flap, hairpin, and nicked DNAs were incubated in the absence of SWI/SNF (solid bars) or with hSWI/SNF without ATP (open bars) or with ATP (hatched bars) for 15 min and then subjected to HhaI digestion for 10 min. The bar graph shows the fraction of DNA remaining uncut after HhaI digestion, normalized to the amount remaining in the controls without SWI/SNF. Data were normalized in this manner since the fraction of nucleosomes remaining undigested (inset numbers) varied slightly between nucleosome preparations. Note that the nucleosomes reconstituted with the native DNA fragment contained H2BG26C-APB and were irradiated to produce portions of the sample that either were not cross-linked (Native-X) or were cross-linked (Native+X). Experiments with native DNA template and native histones produced results identical to those for the Native-X samples (data not shown).
    Figure Legend Snippet: hSWI/SNF-catalyzed octamer sliding is not inhibited by branched DNA structures within the nucleosome. (A) DNA fragments used for nucleosome reconstitution. The flap, hairpin, and nick substrates contain the same sequences as the 215-bp intact fragment. The positions of the branch site, relevant restriction enzyme sites, and the main region of DNA assembled into the nucleosome (oval) are shown. (B) Nucleosome sliding assay. Purified nucleosomes prepared with native, flap, hairpin, and nicked DNAs were incubated in the absence of SWI/SNF (solid bars) or with hSWI/SNF without ATP (open bars) or with ATP (hatched bars) for 15 min and then subjected to HhaI digestion for 10 min. The bar graph shows the fraction of DNA remaining uncut after HhaI digestion, normalized to the amount remaining in the controls without SWI/SNF. Data were normalized in this manner since the fraction of nucleosomes remaining undigested (inset numbers) varied slightly between nucleosome preparations. Note that the nucleosomes reconstituted with the native DNA fragment contained H2BG26C-APB and were irradiated to produce portions of the sample that either were not cross-linked (Native-X) or were cross-linked (Native+X). Experiments with native DNA template and native histones produced results identical to those for the Native-X samples (data not shown).

    Techniques Used: Purification, Incubation, Irradiation, Produced

    hSWI/SNF remodeling of nucleosomes containing branched and nicked DNAs. (A) Analysis of hSWI/SNF remodeling of nucleosomes reconstituted with native, flap, hairpin, and nick templates by DNase I digestion. In each panel, lanes 1 show G-specific reaction markers of the native 5S DNA fragment, lanes 2 show DNase I digestion pattern of naked templates, lanes 3 are nucleosomes prior to DNase I digestion, lanes 4 show the DNase I cleavage pattern of nucleosomes incubated with hSWI/SNF without ATP, and lanes 5 show the DNase I cleavage patterns of nucleosomes incubated with hSWI/SNF and ATP for 15 min. (B) Effects of branched DNA structures on remodeling as determined by restriction enzyme accessibility assay. Glycerol-gradient-purified native, nicked, hairpin, and flap nucleosomes were incubated for the indicated times with hSWI/SNF in the presence or absence of ATP (open and solid symbols, respectively) and subjected to EcoRV digestion over a 45-min time course. The percent nucleosomes remaining uncut is plotted versus time of EcoRV digestion for native (diamonds), hairpin (squares), flap (triangles), and nicked (circles) nucleosomes.
    Figure Legend Snippet: hSWI/SNF remodeling of nucleosomes containing branched and nicked DNAs. (A) Analysis of hSWI/SNF remodeling of nucleosomes reconstituted with native, flap, hairpin, and nick templates by DNase I digestion. In each panel, lanes 1 show G-specific reaction markers of the native 5S DNA fragment, lanes 2 show DNase I digestion pattern of naked templates, lanes 3 are nucleosomes prior to DNase I digestion, lanes 4 show the DNase I cleavage pattern of nucleosomes incubated with hSWI/SNF without ATP, and lanes 5 show the DNase I cleavage patterns of nucleosomes incubated with hSWI/SNF and ATP for 15 min. (B) Effects of branched DNA structures on remodeling as determined by restriction enzyme accessibility assay. Glycerol-gradient-purified native, nicked, hairpin, and flap nucleosomes were incubated for the indicated times with hSWI/SNF in the presence or absence of ATP (open and solid symbols, respectively) and subjected to EcoRV digestion over a 45-min time course. The percent nucleosomes remaining uncut is plotted versus time of EcoRV digestion for native (diamonds), hairpin (squares), flap (triangles), and nicked (circles) nucleosomes.

    Techniques Used: Incubation, Purification

    Exo III analysis of nucleosome positioning before and after remodeling by hSWI/SNF. Glycerol-gradient-purified nucleosomes reconstituted with native (A), flap (B), hairpin (C), or nick (D) templates were incubated in the absence or presence of hSWI/SNF+ATP and then subjected to Exo III digestion for 5 min. Each panel shows, from left to right, G-specific cleavage of native 5S DNA as a marker, Exo III digestion pattern of naked template DNA, undigested nucleosomes, and Exo III digestion of nucleosomes before and after hSWI/SNF remodeling. The positions of the downstream edge of the nucleosomes before and after SWI/SNF remodeling are schematically represented on the left and right sides of the gel, respectively. The dark half ovals and the light ovals represent the major and minor nucleosome positions as determined from the Exo III data.
    Figure Legend Snippet: Exo III analysis of nucleosome positioning before and after remodeling by hSWI/SNF. Glycerol-gradient-purified nucleosomes reconstituted with native (A), flap (B), hairpin (C), or nick (D) templates were incubated in the absence or presence of hSWI/SNF+ATP and then subjected to Exo III digestion for 5 min. Each panel shows, from left to right, G-specific cleavage of native 5S DNA as a marker, Exo III digestion pattern of naked template DNA, undigested nucleosomes, and Exo III digestion of nucleosomes before and after hSWI/SNF remodeling. The positions of the downstream edge of the nucleosomes before and after SWI/SNF remodeling are schematically represented on the left and right sides of the gel, respectively. The dark half ovals and the light ovals represent the major and minor nucleosome positions as determined from the Exo III data.

    Techniques Used: Purification, Incubation, Marker

    Branched and nick DNAs reconstitute into canonical nucleosomes with conserved translational and rotational positioning. (A) Nucleosomes were reconstituted with either native or branched substrates and were analyzed by nucleoprotein gel electrophoresis (0.7% agarose, 1/2× Tris-borate-EDTA). Lanes 1 to 3 show nucleosome reconstitutions containing native, hairpin, and flap DNA, respectively. The positions of the native free DNA (N-FD), the branched free DNA (B-FD), and the nucleosome (Nuc) are indicated. (B) Hydroxyl radical footprinting analysis of nucleosomes containing native and branched DNA. Lane 1 shows the G-specific reaction of the 5S DNA. Lanes 2, 5, and 8 show the hydroxyl radical cleavage pattern of naked native, hairpin, and flap DNA prior to reconstitution, respectively. Lanes 3, 6, and 9 are native, hairpin, and flap DNA fragments prior to hydroxyl radical cleavage, respectively. Lanes 4, 7, and 10 show hydroxyl radical footprints of nucleosomes containing native, hairpin, and flap DNA, respectively. The 10-bp ladder cleavage pattern is indicated by the dots. The position of the branch on flap and hairpinsubstrates is indicated (arrow). (C) Translational positioning of native 5S DNA fragment is conserved in branched and nicked DNAs. Equal amounts of glycerol-gradient-purified nucleosomes reconstituted with either native, nicked, or branched templates were subjected to cleavage by BamHI, BbvI, EcoRV, RsaI, and HhaI for 10 min as described in Materials and Methods. The bar graph shows the percent DNA remaining uncut after the restriction enzyme digestion in each nucleosome for each site. A distribution of translational positions that accounts for the observed protections is shown below.
    Figure Legend Snippet: Branched and nick DNAs reconstitute into canonical nucleosomes with conserved translational and rotational positioning. (A) Nucleosomes were reconstituted with either native or branched substrates and were analyzed by nucleoprotein gel electrophoresis (0.7% agarose, 1/2× Tris-borate-EDTA). Lanes 1 to 3 show nucleosome reconstitutions containing native, hairpin, and flap DNA, respectively. The positions of the native free DNA (N-FD), the branched free DNA (B-FD), and the nucleosome (Nuc) are indicated. (B) Hydroxyl radical footprinting analysis of nucleosomes containing native and branched DNA. Lane 1 shows the G-specific reaction of the 5S DNA. Lanes 2, 5, and 8 show the hydroxyl radical cleavage pattern of naked native, hairpin, and flap DNA prior to reconstitution, respectively. Lanes 3, 6, and 9 are native, hairpin, and flap DNA fragments prior to hydroxyl radical cleavage, respectively. Lanes 4, 7, and 10 show hydroxyl radical footprints of nucleosomes containing native, hairpin, and flap DNA, respectively. The 10-bp ladder cleavage pattern is indicated by the dots. The position of the branch on flap and hairpinsubstrates is indicated (arrow). (C) Translational positioning of native 5S DNA fragment is conserved in branched and nicked DNAs. Equal amounts of glycerol-gradient-purified nucleosomes reconstituted with either native, nicked, or branched templates were subjected to cleavage by BamHI, BbvI, EcoRV, RsaI, and HhaI for 10 min as described in Materials and Methods. The bar graph shows the percent DNA remaining uncut after the restriction enzyme digestion in each nucleosome for each site. A distribution of translational positions that accounts for the observed protections is shown below.

    Techniques Used: Nucleic Acid Electrophoresis, Footprinting, Purification

    nucleosome  (New England Biolabs)


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    New England Biolabs nucleosome
    hSWI/SNF-catalyzed octamer sliding is not inhibited by branched DNA structures within the <t>nucleosome.</t> (A) DNA fragments used for nucleosome reconstitution. The flap, hairpin, and nick substrates contain the same sequences as the 215-bp intact fragment. The positions of the branch site, relevant restriction enzyme sites, and the main region of DNA assembled into the nucleosome (oval) are shown. (B) Nucleosome sliding assay. Purified nucleosomes prepared with native, flap, hairpin, and nicked DNAs were incubated in the absence of SWI/SNF (solid bars) or with hSWI/SNF without ATP (open bars) or with ATP (hatched bars) for 15 min and then subjected to HhaI digestion for 10 min. The bar graph shows the fraction of DNA remaining uncut after HhaI digestion, normalized to the amount remaining in the controls without SWI/SNF. Data were normalized in this manner since the fraction of nucleosomes remaining undigested (inset numbers) varied slightly between nucleosome preparations. Note that the nucleosomes reconstituted with the native DNA fragment contained H2BG26C-APB and were irradiated to produce portions of the sample that either were not cross-linked (Native-X) or were cross-linked (Native+X). Experiments with native DNA template and native histones produced results identical to those for the Native-X samples (data not shown).
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    1) Product Images from "hSWI/SNF-Catalyzed Nucleosome Sliding Does Not Occur Solely via a Twist-Diffusion Mechanism"

    Article Title: hSWI/SNF-Catalyzed Nucleosome Sliding Does Not Occur Solely via a Twist-Diffusion Mechanism

    Journal:

    doi: 10.1128/MCB.22.21.7484-7490.2002

    hSWI/SNF-catalyzed octamer sliding is not inhibited by branched DNA structures within the nucleosome. (A) DNA fragments used for nucleosome reconstitution. The flap, hairpin, and nick substrates contain the same sequences as the 215-bp intact fragment. The positions of the branch site, relevant restriction enzyme sites, and the main region of DNA assembled into the nucleosome (oval) are shown. (B) Nucleosome sliding assay. Purified nucleosomes prepared with native, flap, hairpin, and nicked DNAs were incubated in the absence of SWI/SNF (solid bars) or with hSWI/SNF without ATP (open bars) or with ATP (hatched bars) for 15 min and then subjected to HhaI digestion for 10 min. The bar graph shows the fraction of DNA remaining uncut after HhaI digestion, normalized to the amount remaining in the controls without SWI/SNF. Data were normalized in this manner since the fraction of nucleosomes remaining undigested (inset numbers) varied slightly between nucleosome preparations. Note that the nucleosomes reconstituted with the native DNA fragment contained H2BG26C-APB and were irradiated to produce portions of the sample that either were not cross-linked (Native-X) or were cross-linked (Native+X). Experiments with native DNA template and native histones produced results identical to those for the Native-X samples (data not shown).
    Figure Legend Snippet: hSWI/SNF-catalyzed octamer sliding is not inhibited by branched DNA structures within the nucleosome. (A) DNA fragments used for nucleosome reconstitution. The flap, hairpin, and nick substrates contain the same sequences as the 215-bp intact fragment. The positions of the branch site, relevant restriction enzyme sites, and the main region of DNA assembled into the nucleosome (oval) are shown. (B) Nucleosome sliding assay. Purified nucleosomes prepared with native, flap, hairpin, and nicked DNAs were incubated in the absence of SWI/SNF (solid bars) or with hSWI/SNF without ATP (open bars) or with ATP (hatched bars) for 15 min and then subjected to HhaI digestion for 10 min. The bar graph shows the fraction of DNA remaining uncut after HhaI digestion, normalized to the amount remaining in the controls without SWI/SNF. Data were normalized in this manner since the fraction of nucleosomes remaining undigested (inset numbers) varied slightly between nucleosome preparations. Note that the nucleosomes reconstituted with the native DNA fragment contained H2BG26C-APB and were irradiated to produce portions of the sample that either were not cross-linked (Native-X) or were cross-linked (Native+X). Experiments with native DNA template and native histones produced results identical to those for the Native-X samples (data not shown).

    Techniques Used: Purification, Incubation, Irradiation, Produced

    hSWI/SNF remodeling of nucleosomes containing branched and nicked DNAs. (A) Analysis of hSWI/SNF remodeling of nucleosomes reconstituted with native, flap, hairpin, and nick templates by DNase I digestion. In each panel, lanes 1 show G-specific reaction markers of the native 5S DNA fragment, lanes 2 show DNase I digestion pattern of naked templates, lanes 3 are nucleosomes prior to DNase I digestion, lanes 4 show the DNase I cleavage pattern of nucleosomes incubated with hSWI/SNF without ATP, and lanes 5 show the DNase I cleavage patterns of nucleosomes incubated with hSWI/SNF and ATP for 15 min. (B) Effects of branched DNA structures on remodeling as determined by restriction enzyme accessibility assay. Glycerol-gradient-purified native, nicked, hairpin, and flap nucleosomes were incubated for the indicated times with hSWI/SNF in the presence or absence of ATP (open and solid symbols, respectively) and subjected to EcoRV digestion over a 45-min time course. The percent nucleosomes remaining uncut is plotted versus time of EcoRV digestion for native (diamonds), hairpin (squares), flap (triangles), and nicked (circles) nucleosomes.
    Figure Legend Snippet: hSWI/SNF remodeling of nucleosomes containing branched and nicked DNAs. (A) Analysis of hSWI/SNF remodeling of nucleosomes reconstituted with native, flap, hairpin, and nick templates by DNase I digestion. In each panel, lanes 1 show G-specific reaction markers of the native 5S DNA fragment, lanes 2 show DNase I digestion pattern of naked templates, lanes 3 are nucleosomes prior to DNase I digestion, lanes 4 show the DNase I cleavage pattern of nucleosomes incubated with hSWI/SNF without ATP, and lanes 5 show the DNase I cleavage patterns of nucleosomes incubated with hSWI/SNF and ATP for 15 min. (B) Effects of branched DNA structures on remodeling as determined by restriction enzyme accessibility assay. Glycerol-gradient-purified native, nicked, hairpin, and flap nucleosomes were incubated for the indicated times with hSWI/SNF in the presence or absence of ATP (open and solid symbols, respectively) and subjected to EcoRV digestion over a 45-min time course. The percent nucleosomes remaining uncut is plotted versus time of EcoRV digestion for native (diamonds), hairpin (squares), flap (triangles), and nicked (circles) nucleosomes.

    Techniques Used: Incubation, Purification

    Exo III analysis of nucleosome positioning before and after remodeling by hSWI/SNF. Glycerol-gradient-purified nucleosomes reconstituted with native (A), flap (B), hairpin (C), or nick (D) templates were incubated in the absence or presence of hSWI/SNF+ATP and then subjected to Exo III digestion for 5 min. Each panel shows, from left to right, G-specific cleavage of native 5S DNA as a marker, Exo III digestion pattern of naked template DNA, undigested nucleosomes, and Exo III digestion of nucleosomes before and after hSWI/SNF remodeling. The positions of the downstream edge of the nucleosomes before and after SWI/SNF remodeling are schematically represented on the left and right sides of the gel, respectively. The dark half ovals and the light ovals represent the major and minor nucleosome positions as determined from the Exo III data.
    Figure Legend Snippet: Exo III analysis of nucleosome positioning before and after remodeling by hSWI/SNF. Glycerol-gradient-purified nucleosomes reconstituted with native (A), flap (B), hairpin (C), or nick (D) templates were incubated in the absence or presence of hSWI/SNF+ATP and then subjected to Exo III digestion for 5 min. Each panel shows, from left to right, G-specific cleavage of native 5S DNA as a marker, Exo III digestion pattern of naked template DNA, undigested nucleosomes, and Exo III digestion of nucleosomes before and after hSWI/SNF remodeling. The positions of the downstream edge of the nucleosomes before and after SWI/SNF remodeling are schematically represented on the left and right sides of the gel, respectively. The dark half ovals and the light ovals represent the major and minor nucleosome positions as determined from the Exo III data.

    Techniques Used: Purification, Incubation, Marker

    Branched and nick DNAs reconstitute into canonical nucleosomes with conserved translational and rotational positioning. (A) Nucleosomes were reconstituted with either native or branched substrates and were analyzed by nucleoprotein gel electrophoresis (0.7% agarose, 1/2× Tris-borate-EDTA). Lanes 1 to 3 show nucleosome reconstitutions containing native, hairpin, and flap DNA, respectively. The positions of the native free DNA (N-FD), the branched free DNA (B-FD), and the nucleosome (Nuc) are indicated. (B) Hydroxyl radical footprinting analysis of nucleosomes containing native and branched DNA. Lane 1 shows the G-specific reaction of the 5S DNA. Lanes 2, 5, and 8 show the hydroxyl radical cleavage pattern of naked native, hairpin, and flap DNA prior to reconstitution, respectively. Lanes 3, 6, and 9 are native, hairpin, and flap DNA fragments prior to hydroxyl radical cleavage, respectively. Lanes 4, 7, and 10 show hydroxyl radical footprints of nucleosomes containing native, hairpin, and flap DNA, respectively. The 10-bp ladder cleavage pattern is indicated by the dots. The position of the branch on flap and hairpinsubstrates is indicated (arrow). (C) Translational positioning of native 5S DNA fragment is conserved in branched and nicked DNAs. Equal amounts of glycerol-gradient-purified nucleosomes reconstituted with either native, nicked, or branched templates were subjected to cleavage by BamHI, BbvI, EcoRV, RsaI, and HhaI for 10 min as described in Materials and Methods. The bar graph shows the percent DNA remaining uncut after the restriction enzyme digestion in each nucleosome for each site. A distribution of translational positions that accounts for the observed protections is shown below.
    Figure Legend Snippet: Branched and nick DNAs reconstitute into canonical nucleosomes with conserved translational and rotational positioning. (A) Nucleosomes were reconstituted with either native or branched substrates and were analyzed by nucleoprotein gel electrophoresis (0.7% agarose, 1/2× Tris-borate-EDTA). Lanes 1 to 3 show nucleosome reconstitutions containing native, hairpin, and flap DNA, respectively. The positions of the native free DNA (N-FD), the branched free DNA (B-FD), and the nucleosome (Nuc) are indicated. (B) Hydroxyl radical footprinting analysis of nucleosomes containing native and branched DNA. Lane 1 shows the G-specific reaction of the 5S DNA. Lanes 2, 5, and 8 show the hydroxyl radical cleavage pattern of naked native, hairpin, and flap DNA prior to reconstitution, respectively. Lanes 3, 6, and 9 are native, hairpin, and flap DNA fragments prior to hydroxyl radical cleavage, respectively. Lanes 4, 7, and 10 show hydroxyl radical footprints of nucleosomes containing native, hairpin, and flap DNA, respectively. The 10-bp ladder cleavage pattern is indicated by the dots. The position of the branch on flap and hairpinsubstrates is indicated (arrow). (C) Translational positioning of native 5S DNA fragment is conserved in branched and nicked DNAs. Equal amounts of glycerol-gradient-purified nucleosomes reconstituted with either native, nicked, or branched templates were subjected to cleavage by BamHI, BbvI, EcoRV, RsaI, and HhaI for 10 min as described in Materials and Methods. The bar graph shows the percent DNA remaining uncut after the restriction enzyme digestion in each nucleosome for each site. A distribution of translational positions that accounts for the observed protections is shown below.

    Techniques Used: Nucleic Acid Electrophoresis, Footprinting, Purification

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  • 90
    New England Biolabs nucleosome
    Assembly of H2B K34ub and unmodified <t>nucleosomes.</t> ( A ) Left panel: SDS-PAGE of H2B K34ub- (lane 1) and unmodified (lane 2) histone octamers. Right panel: native PAGE of modified nucleosomes assembled on 147 bp 601 DNA using increasing amounts histones. ( B ) SDS-PAGE of faster- (lane 1) and slower- (lane 2) migrating H2B K34ub assembly products extracted from the native gel in A. The densitometric tracing of the gel lanes is shown on the right. Quantification, by ImageJ, is normalized to that of the histone H4, which is arbitrarily set as 1. ( C ) Native PAGE for samples prepared by serial dilution of 2M NaCl mixtures of 147 bp 601 DNA and modified or unmodified histones to 1 M NaCl. ( D ) Left panel: SDS-PAGE of recombinant linker histone H1 0 . Right panel: assembly of H2B K34ub modified and unmodified nucleosomes with increasing concentration of recombinant histone H1 0 .
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    Assembly of H2B K34ub and unmodified <t>nucleosomes.</t> ( A ) Left panel: SDS-PAGE of H2B K34ub- (lane 1) and unmodified (lane 2) histone octamers. Right panel: native PAGE of modified nucleosomes assembled on 147 bp 601 DNA using increasing amounts histones. ( B ) SDS-PAGE of faster- (lane 1) and slower- (lane 2) migrating H2B K34ub assembly products extracted from the native gel in A. The densitometric tracing of the gel lanes is shown on the right. Quantification, by ImageJ, is normalized to that of the histone H4, which is arbitrarily set as 1. ( C ) Native PAGE for samples prepared by serial dilution of 2M NaCl mixtures of 147 bp 601 DNA and modified or unmodified histones to 1 M NaCl. ( D ) Left panel: SDS-PAGE of recombinant linker histone H1 0 . Right panel: assembly of H2B K34ub modified and unmodified nucleosomes with increasing concentration of recombinant histone H1 0 .
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    a, Gel electrophoresis and quantitation of nucleosomal <t>5SrDNA,</t> Myh6 promoter and Neo DNA. Arrowheads: DNA-histone complex. Arrows: naked DNA. Nucleosome assembly efficiency is defined as the fraction of DNA bound to histones (arrowheads). P-value: Student’s t-test. Error bar: standard error of the mean (SEM). b-d, Quantification of amylose pull-down of MBP-D1D2 (D1D2) with nucleosomal and naked Myh6 promoter DNA ( b ), with nucleosomal Myh6 promoter, Neo , and 5SrDNA ( c ), or with nucleosomal Myh6 promoter in the presence of Mhrt779 ( d ). P-value: Student’s t-test. Error bar: SEM. e, Amylose pull-down of MBP-D1D2 and histone 3. Anti-histone 3 and anti-MBP antibodies were used for western blot analysis. f, ChIP analysis of Brg1 on chromatinized and naked Myh6 promoter in rat ventricular cardiomyocytes. GFP: green fluorescence protein control. P-value: Student’s t-test. Error bar: SEM. g, h, Luciferase reporter activity of Brg1 on naked Myh6 promoter ( g ) or of helicase-deficient Brg1 on chromatinized Myh6 promoter ( h ) in rat ventricular cardiomyocytes. ΔD1: Brg1 lacking amino acid 774–913; ΔD2: Brg1 lacking 1086–1246. GFP: green fluorescence protein control. ChIP: H-10 antibody recognizing N-terminus, non-disrupted region of Brg1. P-value: Student’s t-test. Error bar: SEM. i, j, ChIP analysis in SW13 cells of chromatinized Myh6 promoter in the presence of Mhrt779 ( i ) or helicase-deficient Brg1 ( j ). Vector: pAdd2 empty vector. Mhrt : pAdd2- Mhrt779 . P-value: Student’s t-test. Error bar: SEM. k, Schematic illustration and PCR of human MHRT. MHRT originates from MYH7 and is transcribed into MYH7. MYH7 e xons and introns are indicated. R1 and R2 are strand-specific PCR primers; F1 and R1 target MHRT and MYH7 ; F2 and R2 are specific for MHRT . l, Quantification of MHRT in human heart tissues. Ctrl: control. LVH: left ventricular hypertrophy. ICM: ischemic cardiomyopathy. IDCM: idiopathic dilated cardiomyopathy. P-value: Student’s t-test. Error bar: SEM. m, Working model of a Brg1- Mhrt negative feedback circuit in the heart. Brg1 represses Mhrt transcription, whereas Mhrt prevents Brg1 from recognizing its chromatin targets. Brg1 functions through two distinct promoter elements to bidirectionally repress Myh6 and Mhrt expression. n , Molecular model of how Brg1 binds to its genomic DNA targets. Brg1 helicase (D1D2) binds chromatinized DNA, C-terminal extension (CTE) binds histone 3 (H3), and bromodomain binds acetylated (Ac) histone 3 or 4 (H4).
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    New England Biolabs control dna
    a, Gel electrophoresis and quantitation of nucleosomal <t>5SrDNA,</t> Myh6 promoter and Neo DNA. Arrowheads: DNA-histone complex. Arrows: naked DNA. Nucleosome assembly efficiency is defined as the fraction of DNA bound to histones (arrowheads). P-value: Student’s t-test. Error bar: standard error of the mean (SEM). b-d, Quantification of amylose pull-down of MBP-D1D2 (D1D2) with nucleosomal and naked Myh6 promoter DNA ( b ), with nucleosomal Myh6 promoter, Neo , and 5SrDNA ( c ), or with nucleosomal Myh6 promoter in the presence of Mhrt779 ( d ). P-value: Student’s t-test. Error bar: SEM. e, Amylose pull-down of MBP-D1D2 and histone 3. Anti-histone 3 and anti-MBP antibodies were used for western blot analysis. f, ChIP analysis of Brg1 on chromatinized and naked Myh6 promoter in rat ventricular cardiomyocytes. GFP: green fluorescence protein control. P-value: Student’s t-test. Error bar: SEM. g, h, Luciferase reporter activity of Brg1 on naked Myh6 promoter ( g ) or of helicase-deficient Brg1 on chromatinized Myh6 promoter ( h ) in rat ventricular cardiomyocytes. ΔD1: Brg1 lacking amino acid 774–913; ΔD2: Brg1 lacking 1086–1246. GFP: green fluorescence protein control. ChIP: H-10 antibody recognizing N-terminus, non-disrupted region of Brg1. P-value: Student’s t-test. Error bar: SEM. i, j, ChIP analysis in SW13 cells of chromatinized Myh6 promoter in the presence of Mhrt779 ( i ) or helicase-deficient Brg1 ( j ). Vector: pAdd2 empty vector. Mhrt : pAdd2- Mhrt779 . P-value: Student’s t-test. Error bar: SEM. k, Schematic illustration and PCR of human MHRT. MHRT originates from MYH7 and is transcribed into MYH7. MYH7 e xons and introns are indicated. R1 and R2 are strand-specific PCR primers; F1 and R1 target MHRT and MYH7 ; F2 and R2 are specific for MHRT . l, Quantification of MHRT in human heart tissues. Ctrl: control. LVH: left ventricular hypertrophy. ICM: ischemic cardiomyopathy. IDCM: idiopathic dilated cardiomyopathy. P-value: Student’s t-test. Error bar: SEM. m, Working model of a Brg1- Mhrt negative feedback circuit in the heart. Brg1 represses Mhrt transcription, whereas Mhrt prevents Brg1 from recognizing its chromatin targets. Brg1 functions through two distinct promoter elements to bidirectionally repress Myh6 and Mhrt expression. n , Molecular model of how Brg1 binds to its genomic DNA targets. Brg1 helicase (D1D2) binds chromatinized DNA, C-terminal extension (CTE) binds histone 3 (H3), and bromodomain binds acetylated (Ac) histone 3 or 4 (H4).
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    Image Search Results


    Rap1 as a Pioneer Factor in Budding Yeast (A) Scheme of pTF function: following target search (1), the pTF invades compact chromatin (2), opens chromatin, and recruits the transcription machinery (3). (B) Domain organization of budding yeast Rap1 (above) and crystal structure of a Rap1:DNA complex (PDB: 3ukg ; <xref ref-type=Matot et al., 2012 ). Act, transcription activation domain; BRCT, BRCA 1 C terminus; DBD, DNA binding domain; RCT, Rap1 C terminus; Tox, toxicity region. (C) Organization of the RPL30 promoter. Gray, MNase-seq profile after Rap1 depletion ( Kubik et al., 2015 ), revealing nucleosome positions in the absence of Rap1 (black dotted circles). Plotted is nucleosome occupancy reads, normalized to 10 7 total reads. The Rap1 binding site 1 ( S1 ) (high affinity) and site 2 ( S2 ) (medium affinity) fall on the −1 nucleosome. (D) Promoter −1 nucleosome, showing Rap1 binding sites S1 and S2 (PDB: 1AOI ; Luger et al., 1997 ). The numbers indicate super helical locations (SHLs) of the nucleosomal DNA. See also Figure S1 and , , and . " width="100%" height="100%">

    Journal: Molecular Cell

    Article Title: Chromatin Fiber Invasion and Nucleosome Displacement by the Rap1 Transcription Factor

    doi: 10.1016/j.molcel.2019.10.025

    Figure Lengend Snippet: Rap1 as a Pioneer Factor in Budding Yeast (A) Scheme of pTF function: following target search (1), the pTF invades compact chromatin (2), opens chromatin, and recruits the transcription machinery (3). (B) Domain organization of budding yeast Rap1 (above) and crystal structure of a Rap1:DNA complex (PDB: 3ukg ; Matot et al., 2012 ). Act, transcription activation domain; BRCT, BRCA 1 C terminus; DBD, DNA binding domain; RCT, Rap1 C terminus; Tox, toxicity region. (C) Organization of the RPL30 promoter. Gray, MNase-seq profile after Rap1 depletion ( Kubik et al., 2015 ), revealing nucleosome positions in the absence of Rap1 (black dotted circles). Plotted is nucleosome occupancy reads, normalized to 10 7 total reads. The Rap1 binding site 1 ( S1 ) (high affinity) and site 2 ( S2 ) (medium affinity) fall on the −1 nucleosome. (D) Promoter −1 nucleosome, showing Rap1 binding sites S1 and S2 (PDB: 1AOI ; Luger et al., 1997 ). The numbers indicate super helical locations (SHLs) of the nucleosomal DNA. See also Figure S1 and , , and .

    Article Snippet: For the generation of nucleosome DNA for single-molecule experiments, a biotin containing anchor (Anchor_rev, ) was annealed to its complementary strand containing a phosphorylated 5′- BsaI overhang (P3_Anchor_fwd, ) and a 10-fold excess was added to 150-300 pmol (∼20-40 μg) of digested PCR generated DNA (P3_S1, P3_S2, P3_S2 ∗ and P3_S1S2) in 100 μl 1x T4 ligase buffer (NEB).

    Techniques: Activation Assay, Binding Assay

    Rap1 Recognizes Target Sites within Nucleosomal DNA (A) Scheme of the smTIRFM experiment to detect Rap1 binding to S1 - or S2 -containing, Alexa-Fluor-647-labeled, and immobilized DNA or nucleosomes. bt-NA, biotin-neutravidin. (B) Expression and labeling of Rap1. Lanes: (1) purified MBP-Rap1-Halo; (2) MBP cleavage; (3 and 4) before and after JF-549 labeling; and (5) purified Rap1. (C) Representative smTIRF images showing nucleosome positions in the far-red channel (left, red circles) and Rap1 binding events in the green-orange channel (right). Scale bars: 5 μm; ex, excitation wavelength; em, emission wavelength. (D) Representative fluorescence time trace of Rap1 binding events to S2 containing naked DNA, detected by JF-549 emission. The trace was fitted (red), and t dark and t bright were determined by a thresholding algorithm. (E) Cumulative histogram of Rap1 binding intervals ( t bright ) on S2 DNA fitted by a 2-exponential function y = ∑ i = 1 2 A i exp ( − t / τ o f f , i ) (solid line). For all fit results, see . (F) Specific dissociation time constants (τ off,i > 1 s) of Rap1 for S2 DNA, S1 and S2 containing mononucleosomes (MN), or nucleosomes lacking a binding site (NS), uncorrected for dye photobleaching. The width of the bars indicates the percentage of events associated with the indicated time constants (i.e., amplitudes A i of the multi-exponential fits shown in E and H). n = 4 to 5; error bars: SD. (G) Representative fluorescence time trace of Rap1 binding events to S1 (bottom) and S2 (top) containing MNs. The data were analyzed as in (D). (H) Cumulative histogram of Rap1 binding intervals ( t bright ) on S1 - and S2 -containing MNs fitted by a 3-exponential function y = ∑ i = 0 2 A i exp ( − t / τ o f f , i ) (solid line). (I) Specific on-rate constants ( k on = 1/ τ on ) for all species obtained from a single-exponential fit to cumulative histograms of t dark values and corrected for the contribution from nonspecific interactions . See also and and , , , and .

    Journal: Molecular Cell

    Article Title: Chromatin Fiber Invasion and Nucleosome Displacement by the Rap1 Transcription Factor

    doi: 10.1016/j.molcel.2019.10.025

    Figure Lengend Snippet: Rap1 Recognizes Target Sites within Nucleosomal DNA (A) Scheme of the smTIRFM experiment to detect Rap1 binding to S1 - or S2 -containing, Alexa-Fluor-647-labeled, and immobilized DNA or nucleosomes. bt-NA, biotin-neutravidin. (B) Expression and labeling of Rap1. Lanes: (1) purified MBP-Rap1-Halo; (2) MBP cleavage; (3 and 4) before and after JF-549 labeling; and (5) purified Rap1. (C) Representative smTIRF images showing nucleosome positions in the far-red channel (left, red circles) and Rap1 binding events in the green-orange channel (right). Scale bars: 5 μm; ex, excitation wavelength; em, emission wavelength. (D) Representative fluorescence time trace of Rap1 binding events to S2 containing naked DNA, detected by JF-549 emission. The trace was fitted (red), and t dark and t bright were determined by a thresholding algorithm. (E) Cumulative histogram of Rap1 binding intervals ( t bright ) on S2 DNA fitted by a 2-exponential function y = ∑ i = 1 2 A i exp ( − t / τ o f f , i ) (solid line). For all fit results, see . (F) Specific dissociation time constants (τ off,i > 1 s) of Rap1 for S2 DNA, S1 and S2 containing mononucleosomes (MN), or nucleosomes lacking a binding site (NS), uncorrected for dye photobleaching. The width of the bars indicates the percentage of events associated with the indicated time constants (i.e., amplitudes A i of the multi-exponential fits shown in E and H). n = 4 to 5; error bars: SD. (G) Representative fluorescence time trace of Rap1 binding events to S1 (bottom) and S2 (top) containing MNs. The data were analyzed as in (D). (H) Cumulative histogram of Rap1 binding intervals ( t bright ) on S1 - and S2 -containing MNs fitted by a 3-exponential function y = ∑ i = 0 2 A i exp ( − t / τ o f f , i ) (solid line). (I) Specific on-rate constants ( k on = 1/ τ on ) for all species obtained from a single-exponential fit to cumulative histograms of t dark values and corrected for the contribution from nonspecific interactions . See also and and , , , and .

    Article Snippet: For the generation of nucleosome DNA for single-molecule experiments, a biotin containing anchor (Anchor_rev, ) was annealed to its complementary strand containing a phosphorylated 5′- BsaI overhang (P3_Anchor_fwd, ) and a 10-fold excess was added to 150-300 pmol (∼20-40 μg) of digested PCR generated DNA (P3_S1, P3_S2, P3_S2 ∗ and P3_S1S2) in 100 μl 1x T4 ligase buffer (NEB).

    Techniques: Binding Assay, Labeling, Expressing, Purification, Fluorescence

    Chromatin Higher-Order Structure Reduces Rap1 Dwell Time (A) Scheme of DNA preparation used to introduce Rap1 target sites S1 and S2 into the central nucleosome (N6) of a chromatin fiber (CH). (B) Scheme of the smTIRFM experiment to measure Rap1 binding kinetics in a chromatin fiber context. (C) Representative fluorescence time trace of Rap1 binding events to S1 -containing chromatin arrays. The trace is fitted (red); t dark and t bright were determined by a thresholding algorithm. (D) Cumulative histogram of Rap1 binding intervals (t bright ) to chromatin fibers, containing S1 fitted by a 3-exponential function (solid line). For all fit results, see . (E) Cumulative histogram of Rap1 binding to chromatin arrays, containing S2 fitted by a 3-exponential function (solid line). (F) Specific binding time constants (τ off,i > 1 s) of Rap1 for S1 in a nucleosome (MN) versus chromatin fiber (CH) and S2 MN versus CH. The widths of the bars indicate the percentage of events associated with the indicated time constants (i.e., amplitudes A i of the multi-exponential fits shown in D and E). n = 4 to 5; error bars: SD. (G) Specific on-rate constants ( k on = 1/ τ on ) for MNs and CHs containing S1 and S2 , obtained from a single-exponential fit to cumulative histograms of t dark values and corrected for the contribution from nonspecific interactions . See also <xref ref-type=Figure S4 and , , , and . " width="100%" height="100%">

    Journal: Molecular Cell

    Article Title: Chromatin Fiber Invasion and Nucleosome Displacement by the Rap1 Transcription Factor

    doi: 10.1016/j.molcel.2019.10.025

    Figure Lengend Snippet: Chromatin Higher-Order Structure Reduces Rap1 Dwell Time (A) Scheme of DNA preparation used to introduce Rap1 target sites S1 and S2 into the central nucleosome (N6) of a chromatin fiber (CH). (B) Scheme of the smTIRFM experiment to measure Rap1 binding kinetics in a chromatin fiber context. (C) Representative fluorescence time trace of Rap1 binding events to S1 -containing chromatin arrays. The trace is fitted (red); t dark and t bright were determined by a thresholding algorithm. (D) Cumulative histogram of Rap1 binding intervals (t bright ) to chromatin fibers, containing S1 fitted by a 3-exponential function (solid line). For all fit results, see . (E) Cumulative histogram of Rap1 binding to chromatin arrays, containing S2 fitted by a 3-exponential function (solid line). (F) Specific binding time constants (τ off,i > 1 s) of Rap1 for S1 in a nucleosome (MN) versus chromatin fiber (CH) and S2 MN versus CH. The widths of the bars indicate the percentage of events associated with the indicated time constants (i.e., amplitudes A i of the multi-exponential fits shown in D and E). n = 4 to 5; error bars: SD. (G) Specific on-rate constants ( k on = 1/ τ on ) for MNs and CHs containing S1 and S2 , obtained from a single-exponential fit to cumulative histograms of t dark values and corrected for the contribution from nonspecific interactions . See also Figure S4 and , , , and .

    Article Snippet: For the generation of nucleosome DNA for single-molecule experiments, a biotin containing anchor (Anchor_rev, ) was annealed to its complementary strand containing a phosphorylated 5′- BsaI overhang (P3_Anchor_fwd, ) and a 10-fold excess was added to 150-300 pmol (∼20-40 μg) of digested PCR generated DNA (P3_S1, P3_S2, P3_S2 ∗ and P3_S1S2) in 100 μl 1x T4 ligase buffer (NEB).

    Techniques: Introduce, Binding Assay, Fluorescence

    Rap1 Does Not Open Nucleosome Structure (A) Scheme of experiment to detect nucleosome structure change due to Rap1 binding by FRET. (B) Nucleosome structure (PDB: 1AOI ) showing attachment points of FRET probes. (C) EMSA showing Rap1 binding to S1 and S2 nucleosomes at indicated concentration equivalents (eq.). Lanes were re-arranged for clarity. (D) Fluorescence spectra for S2 nucleosome in complex with indicated equivalents of Rap1. (E) Fluorescence spectra for S1 nucleosome in complex with indicated equivalents of Rap1. (F) FRET efficiency calculated for S2 and S1 nucleosomes as a function of equivalents added Rap1. Error bars: SD; n = 2. See also <xref ref-type=Figure S5 . " width="100%" height="100%">

    Journal: Molecular Cell

    Article Title: Chromatin Fiber Invasion and Nucleosome Displacement by the Rap1 Transcription Factor

    doi: 10.1016/j.molcel.2019.10.025

    Figure Lengend Snippet: Rap1 Does Not Open Nucleosome Structure (A) Scheme of experiment to detect nucleosome structure change due to Rap1 binding by FRET. (B) Nucleosome structure (PDB: 1AOI ) showing attachment points of FRET probes. (C) EMSA showing Rap1 binding to S1 and S2 nucleosomes at indicated concentration equivalents (eq.). Lanes were re-arranged for clarity. (D) Fluorescence spectra for S2 nucleosome in complex with indicated equivalents of Rap1. (E) Fluorescence spectra for S1 nucleosome in complex with indicated equivalents of Rap1. (F) FRET efficiency calculated for S2 and S1 nucleosomes as a function of equivalents added Rap1. Error bars: SD; n = 2. See also Figure S5 .

    Article Snippet: For the generation of nucleosome DNA for single-molecule experiments, a biotin containing anchor (Anchor_rev, ) was annealed to its complementary strand containing a phosphorylated 5′- BsaI overhang (P3_Anchor_fwd, ) and a 10-fold excess was added to 150-300 pmol (∼20-40 μg) of digested PCR generated DNA (P3_S1, P3_S2, P3_S2 ∗ and P3_S1S2) in 100 μl 1x T4 ligase buffer (NEB).

    Techniques: Binding Assay, Concentration Assay, Fluorescence

    Chromatin Remodeling Induced by Rap1 Invasion as Observed by smFRET (A) Scheme of chromatin DNA assembly to introduce a Rap1 site at nucleosome N6, as well as a FRET donor (Cy3B, yellow) and acceptor (Alexa Fluor 647, red) at nucleosomes N5 and N7. (B) Scheme of a smFRET-TIRF experiment. (C) Individual kinetic traces of donor (orange) and acceptor (red) fluorescence emission and FRET efficiency ( E FRET , blue) for chromatin fibers containing S2 at the indicated KCl and Rap1 concentrations. All Rap1 experiments were performed at 150 mM KCl. (D) Similar to (C) but for chromatin lacking Rap1 binding sites ( NS ). (E) Histograms of E FRET of S2 -containing chromatin fibers at the indicated KCl and Rap1 concentrations. All Rap1 experiments were performed at 150 mM KCl. Histograms were fitted by Gaussian functions, revealing a low-FRET (LF) (gray), medium-FRET (MF) (green), and high-FRET (HF) (red) population. Error bars are SEM; for the number of traces and parameters of Gaussian fits, see and . (F) Similar to (E) but for chromatin lacking Rap1 binding sites ( NS ). (G) Percentage of each FRET sub-population, LF, MF, and HF for chromatin containing S2 . Box: 25–75 percentiles; whiskers: outliers (factor 1.5); line: median; open symbol: mean. For number of experiments, see . ∗ 10 −3 > p < 10 −4 ; ∗∗ 10 −4 > p < 10 −5 ; ∗∗∗ p < 10 −5 , two-tailed Student’s t test between peak area % of LF, MF, and HF populations for S2 or NS nucleosomes (see H). (H) Similar to (G) but for chromatin lacking Rap1 binding sites ( NS ). (I) Percentage of dynamic traces for S2 and NS chromatin. Box: similar to (H). For the identification of dynamic traces, see . p: two-tailed Student’s t test; n.s.: p > 0.05. See also <xref ref-type=Figure S6 and and . " width="100%" height="100%">

    Journal: Molecular Cell

    Article Title: Chromatin Fiber Invasion and Nucleosome Displacement by the Rap1 Transcription Factor

    doi: 10.1016/j.molcel.2019.10.025

    Figure Lengend Snippet: Chromatin Remodeling Induced by Rap1 Invasion as Observed by smFRET (A) Scheme of chromatin DNA assembly to introduce a Rap1 site at nucleosome N6, as well as a FRET donor (Cy3B, yellow) and acceptor (Alexa Fluor 647, red) at nucleosomes N5 and N7. (B) Scheme of a smFRET-TIRF experiment. (C) Individual kinetic traces of donor (orange) and acceptor (red) fluorescence emission and FRET efficiency ( E FRET , blue) for chromatin fibers containing S2 at the indicated KCl and Rap1 concentrations. All Rap1 experiments were performed at 150 mM KCl. (D) Similar to (C) but for chromatin lacking Rap1 binding sites ( NS ). (E) Histograms of E FRET of S2 -containing chromatin fibers at the indicated KCl and Rap1 concentrations. All Rap1 experiments were performed at 150 mM KCl. Histograms were fitted by Gaussian functions, revealing a low-FRET (LF) (gray), medium-FRET (MF) (green), and high-FRET (HF) (red) population. Error bars are SEM; for the number of traces and parameters of Gaussian fits, see and . (F) Similar to (E) but for chromatin lacking Rap1 binding sites ( NS ). (G) Percentage of each FRET sub-population, LF, MF, and HF for chromatin containing S2 . Box: 25–75 percentiles; whiskers: outliers (factor 1.5); line: median; open symbol: mean. For number of experiments, see . ∗ 10 −3 > p < 10 −4 ; ∗∗ 10 −4 > p < 10 −5 ; ∗∗∗ p < 10 −5 , two-tailed Student’s t test between peak area % of LF, MF, and HF populations for S2 or NS nucleosomes (see H). (H) Similar to (G) but for chromatin lacking Rap1 binding sites ( NS ). (I) Percentage of dynamic traces for S2 and NS chromatin. Box: similar to (H). For the identification of dynamic traces, see . p: two-tailed Student’s t test; n.s.: p > 0.05. See also Figure S6 and and .

    Article Snippet: For the generation of nucleosome DNA for single-molecule experiments, a biotin containing anchor (Anchor_rev, ) was annealed to its complementary strand containing a phosphorylated 5′- BsaI overhang (P3_Anchor_fwd, ) and a 10-fold excess was added to 150-300 pmol (∼20-40 μg) of digested PCR generated DNA (P3_S1, P3_S2, P3_S2 ∗ and P3_S1S2) in 100 μl 1x T4 ligase buffer (NEB).

    Techniques: Introduce, Fluorescence, Binding Assay, Two Tailed Test

    RSC Enables Stable Rap1 Binding by Exposing Binding Sites (A) Native PAGE analysis of Rap1 binding for indicated times followed by incubation with competitor plasmid DNA (PL). L1 and L2–L5: lanes in (A) and (C) and (D). (B) Scheme of RSC remodeling assay. Note that Nap1 is not strictly required in these experiments ( <xref ref-type=Figure S7 C). (C) Native PAGE analysis of remodeling assays; MN ∗ , remodeled mononucleosome. (D) Native PAGE analysis of remodeling assays in the presence of 10 eq. of Rap1. (E) Integrated unbound nucleosome bands from (D) (n = 3; error bars SD). (F) MNase-seq results from RSC remodeling assays for 601 nucleosomes (P3_S1S2). Gray, nucleosome start position; blue, RSC remodeling for 90 min in absence of Rap1; red, RSC remodeling for 90 min in presence of 10 eq. Rap1. Shown are reads normalized to number of total reads. (G) Same as in (F) but for RPL30 nucleosomes (P3_RPL30). (H) Effect of Rap1 binding on nucleosome stability at the RPL30 promoter in yeast. Nucleosome positions were determined using qPCR after MNase digestion of chromatin. Promoters analyzed contained both Rap1 binding sites ( S1S2 ), S1 mutated (S1 mut S2), S2 mutated (S1S2 mut ), or both binding sites mutated (S1 mut S2 mut ). Data shown are for cells where Rap1 is present (Rap1+, red), Rap1 has been depleted from the nucleus for 1 h by anchor-away (Rap1−, blue), and where Rap1 has been re-introduced for 2 h following depletion by expressing a RAP1 construct from an inducible promoter (Rap1 ind, green). See also Figure S7 and . " width="100%" height="100%">

    Journal: Molecular Cell

    Article Title: Chromatin Fiber Invasion and Nucleosome Displacement by the Rap1 Transcription Factor

    doi: 10.1016/j.molcel.2019.10.025

    Figure Lengend Snippet: RSC Enables Stable Rap1 Binding by Exposing Binding Sites (A) Native PAGE analysis of Rap1 binding for indicated times followed by incubation with competitor plasmid DNA (PL). L1 and L2–L5: lanes in (A) and (C) and (D). (B) Scheme of RSC remodeling assay. Note that Nap1 is not strictly required in these experiments ( Figure S7 C). (C) Native PAGE analysis of remodeling assays; MN ∗ , remodeled mononucleosome. (D) Native PAGE analysis of remodeling assays in the presence of 10 eq. of Rap1. (E) Integrated unbound nucleosome bands from (D) (n = 3; error bars SD). (F) MNase-seq results from RSC remodeling assays for 601 nucleosomes (P3_S1S2). Gray, nucleosome start position; blue, RSC remodeling for 90 min in absence of Rap1; red, RSC remodeling for 90 min in presence of 10 eq. Rap1. Shown are reads normalized to number of total reads. (G) Same as in (F) but for RPL30 nucleosomes (P3_RPL30). (H) Effect of Rap1 binding on nucleosome stability at the RPL30 promoter in yeast. Nucleosome positions were determined using qPCR after MNase digestion of chromatin. Promoters analyzed contained both Rap1 binding sites ( S1S2 ), S1 mutated (S1 mut S2), S2 mutated (S1S2 mut ), or both binding sites mutated (S1 mut S2 mut ). Data shown are for cells where Rap1 is present (Rap1+, red), Rap1 has been depleted from the nucleus for 1 h by anchor-away (Rap1−, blue), and where Rap1 has been re-introduced for 2 h following depletion by expressing a RAP1 construct from an inducible promoter (Rap1 ind, green). See also Figure S7 and .

    Article Snippet: For the generation of nucleosome DNA for single-molecule experiments, a biotin containing anchor (Anchor_rev, ) was annealed to its complementary strand containing a phosphorylated 5′- BsaI overhang (P3_Anchor_fwd, ) and a 10-fold excess was added to 150-300 pmol (∼20-40 μg) of digested PCR generated DNA (P3_S1, P3_S2, P3_S2 ∗ and P3_S1S2) in 100 μl 1x T4 ligase buffer (NEB).

    Techniques: Binding Assay, Clear Native PAGE, Incubation, Plasmid Preparation, Expressing, Construct

    A Dynamic Model for Rap1-Mediated Promoter Chromatin Remodeling Rap1 searches chromatin (step 1) and its dynamic binding (2–25 s) to a promoter site results in local chromatin opening (step 2), where Rap1 remains dynamically bound. RSC-mediated nucleosome sliding opens the NDR and exposes the DNA containing Rap1 binding sites (step 3). The fully exposed binding sites allow stable Rap1 binding (step 4) with long residence times (free DNA τ res > 450 s) and prevent further nucleosome encroachment.

    Journal: Molecular Cell

    Article Title: Chromatin Fiber Invasion and Nucleosome Displacement by the Rap1 Transcription Factor

    doi: 10.1016/j.molcel.2019.10.025

    Figure Lengend Snippet: A Dynamic Model for Rap1-Mediated Promoter Chromatin Remodeling Rap1 searches chromatin (step 1) and its dynamic binding (2–25 s) to a promoter site results in local chromatin opening (step 2), where Rap1 remains dynamically bound. RSC-mediated nucleosome sliding opens the NDR and exposes the DNA containing Rap1 binding sites (step 3). The fully exposed binding sites allow stable Rap1 binding (step 4) with long residence times (free DNA τ res > 450 s) and prevent further nucleosome encroachment.

    Article Snippet: For the generation of nucleosome DNA for single-molecule experiments, a biotin containing anchor (Anchor_rev, ) was annealed to its complementary strand containing a phosphorylated 5′- BsaI overhang (P3_Anchor_fwd, ) and a 10-fold excess was added to 150-300 pmol (∼20-40 μg) of digested PCR generated DNA (P3_S1, P3_S2, P3_S2 ∗ and P3_S1S2) in 100 μl 1x T4 ligase buffer (NEB).

    Techniques: Binding Assay

    Assembly of H2B K34ub and unmodified nucleosomes. ( A ) Left panel: SDS-PAGE of H2B K34ub- (lane 1) and unmodified (lane 2) histone octamers. Right panel: native PAGE of modified nucleosomes assembled on 147 bp 601 DNA using increasing amounts histones. ( B ) SDS-PAGE of faster- (lane 1) and slower- (lane 2) migrating H2B K34ub assembly products extracted from the native gel in A. The densitometric tracing of the gel lanes is shown on the right. Quantification, by ImageJ, is normalized to that of the histone H4, which is arbitrarily set as 1. ( C ) Native PAGE for samples prepared by serial dilution of 2M NaCl mixtures of 147 bp 601 DNA and modified or unmodified histones to 1 M NaCl. ( D ) Left panel: SDS-PAGE of recombinant linker histone H1 0 . Right panel: assembly of H2B K34ub modified and unmodified nucleosomes with increasing concentration of recombinant histone H1 0 .

    Journal: Nucleic Acids Research

    Article Title: Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics

    doi: 10.1093/nar/gky526

    Figure Lengend Snippet: Assembly of H2B K34ub and unmodified nucleosomes. ( A ) Left panel: SDS-PAGE of H2B K34ub- (lane 1) and unmodified (lane 2) histone octamers. Right panel: native PAGE of modified nucleosomes assembled on 147 bp 601 DNA using increasing amounts histones. ( B ) SDS-PAGE of faster- (lane 1) and slower- (lane 2) migrating H2B K34ub assembly products extracted from the native gel in A. The densitometric tracing of the gel lanes is shown on the right. Quantification, by ImageJ, is normalized to that of the histone H4, which is arbitrarily set as 1. ( C ) Native PAGE for samples prepared by serial dilution of 2M NaCl mixtures of 147 bp 601 DNA and modified or unmodified histones to 1 M NaCl. ( D ) Left panel: SDS-PAGE of recombinant linker histone H1 0 . Right panel: assembly of H2B K34ub modified and unmodified nucleosomes with increasing concentration of recombinant histone H1 0 .

    Article Snippet: Assembled nucleosome (∼30 ng DNA) were digested with increasing amounts (typically 0.3/1/3/10/30/100 Kunitz units) of MNase (NEB) for 20 min at 26 or 37°C.

    Techniques: SDS Page, Clear Native PAGE, Modification, Serial Dilution, Recombinant, Concentration Assay

    Stability of H2B K34ub nucleosomes. ( A ) Native PAGE for nucleosome assembly with increasing amounts of unmodified and H2B K34ub histone octamers and their mixture at ratios indicated on top. Quantification was performed by Image J and relative abundance of each species was summarized in Table . The representative result from three repeats was presented. ( B ) H2B K34ub (top panel) or Unmodified (bottom panel) nucleosomes were assembled on nicked, positively or negatively supercoiled plasmids containing 12 copies of 177 bp 601 DNA. After assembly 601 nucleosomes were released with ScaI and resolved on native PAGE. ( C ) The nucleosome assembly containing histones of indicated ratios (top) were incubated with competitor DNA for 2 h at 26°C in a buffer containing 200 mm NaCl. ( D ) Nucleosomes assembled on 147 bp 601 DNA (as indicated on top) were incubated with or without CM-50 Sephadex.

    Journal: Nucleic Acids Research

    Article Title: Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics

    doi: 10.1093/nar/gky526

    Figure Lengend Snippet: Stability of H2B K34ub nucleosomes. ( A ) Native PAGE for nucleosome assembly with increasing amounts of unmodified and H2B K34ub histone octamers and their mixture at ratios indicated on top. Quantification was performed by Image J and relative abundance of each species was summarized in Table . The representative result from three repeats was presented. ( B ) H2B K34ub (top panel) or Unmodified (bottom panel) nucleosomes were assembled on nicked, positively or negatively supercoiled plasmids containing 12 copies of 177 bp 601 DNA. After assembly 601 nucleosomes were released with ScaI and resolved on native PAGE. ( C ) The nucleosome assembly containing histones of indicated ratios (top) were incubated with competitor DNA for 2 h at 26°C in a buffer containing 200 mm NaCl. ( D ) Nucleosomes assembled on 147 bp 601 DNA (as indicated on top) were incubated with or without CM-50 Sephadex.

    Article Snippet: Assembled nucleosome (∼30 ng DNA) were digested with increasing amounts (typically 0.3/1/3/10/30/100 Kunitz units) of MNase (NEB) for 20 min at 26 or 37°C.

    Techniques: Clear Native PAGE, Incubation

    Dynamics of H2B K34ub nucleosomes. ( A ) Nucleosomes assembled on 147 bp 601 DNA were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. ( B ) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with MNase at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.

    Journal: Nucleic Acids Research

    Article Title: Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics

    doi: 10.1093/nar/gky526

    Figure Lengend Snippet: Dynamics of H2B K34ub nucleosomes. ( A ) Nucleosomes assembled on 147 bp 601 DNA were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. ( B ) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with MNase at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.

    Article Snippet: Assembled nucleosome (∼30 ng DNA) were digested with increasing amounts (typically 0.3/1/3/10/30/100 Kunitz units) of MNase (NEB) for 20 min at 26 or 37°C.

    Techniques: Clear Native PAGE, Electrophoresis, Staining

    Eviction of histone dimer in H2B K34ub nucleosome by histone dimer acceptors. ( A ) Left panel: SDS-PAGE of NAP1. Middle, right panels: unmodified and H2B K34ub nucleosomes assembled on a 147 or 177 bp 601 DNA were post-assembly incubated with NAP1 for 2.5 h at 26 or 37°C in a buffer containing 100 mM NaCl. ( B ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA for 2.5 h at 26/37°C in a buffer containing at 100 mM NaCl. ( C ) H2B K34ub nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA at 26/37°C for 2.5 h or at 26°C for 20 h (the ‘20 h’ and ‘2.5 h’ samples were resolved in different gels).

    Journal: Nucleic Acids Research

    Article Title: Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics

    doi: 10.1093/nar/gky526

    Figure Lengend Snippet: Eviction of histone dimer in H2B K34ub nucleosome by histone dimer acceptors. ( A ) Left panel: SDS-PAGE of NAP1. Middle, right panels: unmodified and H2B K34ub nucleosomes assembled on a 147 or 177 bp 601 DNA were post-assembly incubated with NAP1 for 2.5 h at 26 or 37°C in a buffer containing 100 mM NaCl. ( B ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA for 2.5 h at 26/37°C in a buffer containing at 100 mM NaCl. ( C ) H2B K34ub nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA at 26/37°C for 2.5 h or at 26°C for 20 h (the ‘20 h’ and ‘2.5 h’ samples were resolved in different gels).

    Article Snippet: Assembled nucleosome (∼30 ng DNA) were digested with increasing amounts (typically 0.3/1/3/10/30/100 Kunitz units) of MNase (NEB) for 20 min at 26 or 37°C.

    Techniques: SDS Page, Incubation

    Stability of H2B K34ub- versus H2B K120ub-nucleosomes. ( A ) Left panel: SDS-PAGE of H2B K120ub and K34ub histone octamers. Right panel: unmodified and H2B K120ub/ K34ub nucleosomes assembled on 147 bp 601 DNA. ( B ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA for 2.5 h at indicated temperature/ionic conditions. ( C ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with NAP1 for 2.5 h at 26/37°C in a buffer containing at 140 mM NaCl.

    Journal: Nucleic Acids Research

    Article Title: Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics

    doi: 10.1093/nar/gky526

    Figure Lengend Snippet: Stability of H2B K34ub- versus H2B K120ub-nucleosomes. ( A ) Left panel: SDS-PAGE of H2B K120ub and K34ub histone octamers. Right panel: unmodified and H2B K120ub/ K34ub nucleosomes assembled on 147 bp 601 DNA. ( B ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA for 2.5 h at indicated temperature/ionic conditions. ( C ) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with NAP1 for 2.5 h at 26/37°C in a buffer containing at 140 mM NaCl.

    Article Snippet: Assembled nucleosome (∼30 ng DNA) were digested with increasing amounts (typically 0.3/1/3/10/30/100 Kunitz units) of MNase (NEB) for 20 min at 26 or 37°C.

    Techniques: SDS Page, Incubation

    Effects of underlying DNA sequence on stability of H2B K120ub and K34ub nucleosomes. ( A ) Nucleosomes assembled on 146 bp 5S DNA. ( B ) Assembled nucleosomes were incubated with competitor DNA for 2.5 h at 26°C in a buffer containing at 200 mM NaCl. ( C ) Nucleosomes incubated with competitor DNA for 2.5 h at indicated temperature/ ionic conditions. ( D and E ) Nucleosomes assembled with unmodified or H2B K34ub histones, or their 1/0.57 mixture, were post-assembly incubated for 2.5 h at 26 or 37°C with (D) competitor DNA in a buffer containing at 100 mM NaCl or (E) NAP1 in a buffer containing at 150 mM NaCl. Densitometry tracing of indicated gel lanes is shown at the bottom of gel images.

    Journal: Nucleic Acids Research

    Article Title: Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics

    doi: 10.1093/nar/gky526

    Figure Lengend Snippet: Effects of underlying DNA sequence on stability of H2B K120ub and K34ub nucleosomes. ( A ) Nucleosomes assembled on 146 bp 5S DNA. ( B ) Assembled nucleosomes were incubated with competitor DNA for 2.5 h at 26°C in a buffer containing at 200 mM NaCl. ( C ) Nucleosomes incubated with competitor DNA for 2.5 h at indicated temperature/ ionic conditions. ( D and E ) Nucleosomes assembled with unmodified or H2B K34ub histones, or their 1/0.57 mixture, were post-assembly incubated for 2.5 h at 26 or 37°C with (D) competitor DNA in a buffer containing at 100 mM NaCl or (E) NAP1 in a buffer containing at 150 mM NaCl. Densitometry tracing of indicated gel lanes is shown at the bottom of gel images.

    Article Snippet: Assembled nucleosome (∼30 ng DNA) were digested with increasing amounts (typically 0.3/1/3/10/30/100 Kunitz units) of MNase (NEB) for 20 min at 26 or 37°C.

    Techniques: Sequencing, Incubation

    a, Gel electrophoresis and quantitation of nucleosomal 5SrDNA, Myh6 promoter and Neo DNA. Arrowheads: DNA-histone complex. Arrows: naked DNA. Nucleosome assembly efficiency is defined as the fraction of DNA bound to histones (arrowheads). P-value: Student’s t-test. Error bar: standard error of the mean (SEM). b-d, Quantification of amylose pull-down of MBP-D1D2 (D1D2) with nucleosomal and naked Myh6 promoter DNA ( b ), with nucleosomal Myh6 promoter, Neo , and 5SrDNA ( c ), or with nucleosomal Myh6 promoter in the presence of Mhrt779 ( d ). P-value: Student’s t-test. Error bar: SEM. e, Amylose pull-down of MBP-D1D2 and histone 3. Anti-histone 3 and anti-MBP antibodies were used for western blot analysis. f, ChIP analysis of Brg1 on chromatinized and naked Myh6 promoter in rat ventricular cardiomyocytes. GFP: green fluorescence protein control. P-value: Student’s t-test. Error bar: SEM. g, h, Luciferase reporter activity of Brg1 on naked Myh6 promoter ( g ) or of helicase-deficient Brg1 on chromatinized Myh6 promoter ( h ) in rat ventricular cardiomyocytes. ΔD1: Brg1 lacking amino acid 774–913; ΔD2: Brg1 lacking 1086–1246. GFP: green fluorescence protein control. ChIP: H-10 antibody recognizing N-terminus, non-disrupted region of Brg1. P-value: Student’s t-test. Error bar: SEM. i, j, ChIP analysis in SW13 cells of chromatinized Myh6 promoter in the presence of Mhrt779 ( i ) or helicase-deficient Brg1 ( j ). Vector: pAdd2 empty vector. Mhrt : pAdd2- Mhrt779 . P-value: Student’s t-test. Error bar: SEM. k, Schematic illustration and PCR of human MHRT. MHRT originates from MYH7 and is transcribed into MYH7. MYH7 e xons and introns are indicated. R1 and R2 are strand-specific PCR primers; F1 and R1 target MHRT and MYH7 ; F2 and R2 are specific for MHRT . l, Quantification of MHRT in human heart tissues. Ctrl: control. LVH: left ventricular hypertrophy. ICM: ischemic cardiomyopathy. IDCM: idiopathic dilated cardiomyopathy. P-value: Student’s t-test. Error bar: SEM. m, Working model of a Brg1- Mhrt negative feedback circuit in the heart. Brg1 represses Mhrt transcription, whereas Mhrt prevents Brg1 from recognizing its chromatin targets. Brg1 functions through two distinct promoter elements to bidirectionally repress Myh6 and Mhrt expression. n , Molecular model of how Brg1 binds to its genomic DNA targets. Brg1 helicase (D1D2) binds chromatinized DNA, C-terminal extension (CTE) binds histone 3 (H3), and bromodomain binds acetylated (Ac) histone 3 or 4 (H4).

    Journal: Nature

    Article Title: A long non-coding RNA protects the heart from pathological hypertrophy

    doi: 10.1038/nature13596

    Figure Lengend Snippet: a, Gel electrophoresis and quantitation of nucleosomal 5SrDNA, Myh6 promoter and Neo DNA. Arrowheads: DNA-histone complex. Arrows: naked DNA. Nucleosome assembly efficiency is defined as the fraction of DNA bound to histones (arrowheads). P-value: Student’s t-test. Error bar: standard error of the mean (SEM). b-d, Quantification of amylose pull-down of MBP-D1D2 (D1D2) with nucleosomal and naked Myh6 promoter DNA ( b ), with nucleosomal Myh6 promoter, Neo , and 5SrDNA ( c ), or with nucleosomal Myh6 promoter in the presence of Mhrt779 ( d ). P-value: Student’s t-test. Error bar: SEM. e, Amylose pull-down of MBP-D1D2 and histone 3. Anti-histone 3 and anti-MBP antibodies were used for western blot analysis. f, ChIP analysis of Brg1 on chromatinized and naked Myh6 promoter in rat ventricular cardiomyocytes. GFP: green fluorescence protein control. P-value: Student’s t-test. Error bar: SEM. g, h, Luciferase reporter activity of Brg1 on naked Myh6 promoter ( g ) or of helicase-deficient Brg1 on chromatinized Myh6 promoter ( h ) in rat ventricular cardiomyocytes. ΔD1: Brg1 lacking amino acid 774–913; ΔD2: Brg1 lacking 1086–1246. GFP: green fluorescence protein control. ChIP: H-10 antibody recognizing N-terminus, non-disrupted region of Brg1. P-value: Student’s t-test. Error bar: SEM. i, j, ChIP analysis in SW13 cells of chromatinized Myh6 promoter in the presence of Mhrt779 ( i ) or helicase-deficient Brg1 ( j ). Vector: pAdd2 empty vector. Mhrt : pAdd2- Mhrt779 . P-value: Student’s t-test. Error bar: SEM. k, Schematic illustration and PCR of human MHRT. MHRT originates from MYH7 and is transcribed into MYH7. MYH7 e xons and introns are indicated. R1 and R2 are strand-specific PCR primers; F1 and R1 target MHRT and MYH7 ; F2 and R2 are specific for MHRT . l, Quantification of MHRT in human heart tissues. Ctrl: control. LVH: left ventricular hypertrophy. ICM: ischemic cardiomyopathy. IDCM: idiopathic dilated cardiomyopathy. P-value: Student’s t-test. Error bar: SEM. m, Working model of a Brg1- Mhrt negative feedback circuit in the heart. Brg1 represses Mhrt transcription, whereas Mhrt prevents Brg1 from recognizing its chromatin targets. Brg1 functions through two distinct promoter elements to bidirectionally repress Myh6 and Mhrt expression. n , Molecular model of how Brg1 binds to its genomic DNA targets. Brg1 helicase (D1D2) binds chromatinized DNA, C-terminal extension (CTE) binds histone 3 (H3), and bromodomain binds acetylated (Ac) histone 3 or 4 (H4).

    Article Snippet: In brief, recombinant human core histone octamer, which consist of the 2:1 mix of histone H2A/H2B dimer and histone H3.1/H4 tetramer, were mixed with purified 5SrDNA (208bp, N1202S, NEB), Neo (512bp, amplified from pST18- Neo , 1175025, Roche), Myh6 core promoter (596bp, −426 to +170) and Mhrt core promoter (a3a4, 596bp, −2290 to −1775) DNA at 2 M NaCl.

    Techniques: Nucleic Acid Electrophoresis, Quantitation Assay, Western Blot, Fluorescence, Luciferase, Activity Assay, Plasmid Preparation, Expressing