Structured Review

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Structure and stability of H3.X- and H3.Y-containing nucleosomes. (A) In silico homology model of H3.X (purple, left) and H3.Y (light blue, right) protein structures in overlay with the crystal structure of H3.2 (dark blue). (B) Crystal structure of nucleosome with H3.2 exchanged by in silico homology models of H3.X (purple, left) and H3.Y (light blue, right), respectively. (C) IP of <t>mononucleosomes</t> generated from HeLa cells transfected with empty vector, HA-H3.1, -H3.X, and -H3.Y shows incorporation of novel H3 variants into nucleosomes. Bioanalyzer evaluation of purified DNA after IP of <t>MNase-treated</t> chromatin (unbound and bound material) shows digestion of chromatin to mononucleosomes and their successful precipitation (left; see also Fig. S2 A for DNA size and quality). Silver stain of 15% SDS-PAGE with α-HA IPs of mononucleosomes revealed successful binding of HA-tagged H3 variants (asterisks) and pull-down of core histones (top, right). Immunoblot of immunoprecipitates with α-HA (red) and α-H3 C-terminal (green) antibodies visualized by the Odyssey infrared imaging system (bottom, right). Notice that endogenous H3 is coimmunoprecipitated with all H3 variants analyzed. (D) FRAP experiments to evaluate nucleosomal stability of novel H3 variants using spinning disk confocal microscopy. HeLa Kyoto cells were transiently transfected with GFP, GFP-H3.1, -H3.3, -H3.X, and -H3.Y constructs. A small nuclear area was photobleached (box) and the recovery of the fluorescent signal was monitored over 1 min and up to 8 h (see Fig. S2, B–D, for long-term FRAP). Depicted is a short-term FRAP series (selected time points are shown) of GFP-tagged H3 variants compared with GFP alone. Bar, 5 µm. (E) Quantification of short-term FRAP experiment. Mean curves of 10–20 individual cells are shown. Standard deviations were very small (in the range of ± 0.02) and were omitted for clarity (for details see Fig. S2 D). All GFP-H3 variants show almost no recovery within the first 60 s after bleaching, which indicates that all expressed fusion protein was stably incorporated into nucleosomes. In contrast, GFP alone recovers to almost 100% within 5 s.
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Images

1) Product Images from "Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y"

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201002043

Structure and stability of H3.X- and H3.Y-containing nucleosomes. (A) In silico homology model of H3.X (purple, left) and H3.Y (light blue, right) protein structures in overlay with the crystal structure of H3.2 (dark blue). (B) Crystal structure of nucleosome with H3.2 exchanged by in silico homology models of H3.X (purple, left) and H3.Y (light blue, right), respectively. (C) IP of mononucleosomes generated from HeLa cells transfected with empty vector, HA-H3.1, -H3.X, and -H3.Y shows incorporation of novel H3 variants into nucleosomes. Bioanalyzer evaluation of purified DNA after IP of MNase-treated chromatin (unbound and bound material) shows digestion of chromatin to mononucleosomes and their successful precipitation (left; see also Fig. S2 A for DNA size and quality). Silver stain of 15% SDS-PAGE with α-HA IPs of mononucleosomes revealed successful binding of HA-tagged H3 variants (asterisks) and pull-down of core histones (top, right). Immunoblot of immunoprecipitates with α-HA (red) and α-H3 C-terminal (green) antibodies visualized by the Odyssey infrared imaging system (bottom, right). Notice that endogenous H3 is coimmunoprecipitated with all H3 variants analyzed. (D) FRAP experiments to evaluate nucleosomal stability of novel H3 variants using spinning disk confocal microscopy. HeLa Kyoto cells were transiently transfected with GFP, GFP-H3.1, -H3.3, -H3.X, and -H3.Y constructs. A small nuclear area was photobleached (box) and the recovery of the fluorescent signal was monitored over 1 min and up to 8 h (see Fig. S2, B–D, for long-term FRAP). Depicted is a short-term FRAP series (selected time points are shown) of GFP-tagged H3 variants compared with GFP alone. Bar, 5 µm. (E) Quantification of short-term FRAP experiment. Mean curves of 10–20 individual cells are shown. Standard deviations were very small (in the range of ± 0.02) and were omitted for clarity (for details see Fig. S2 D). All GFP-H3 variants show almost no recovery within the first 60 s after bleaching, which indicates that all expressed fusion protein was stably incorporated into nucleosomes. In contrast, GFP alone recovers to almost 100% within 5 s.
Figure Legend Snippet: Structure and stability of H3.X- and H3.Y-containing nucleosomes. (A) In silico homology model of H3.X (purple, left) and H3.Y (light blue, right) protein structures in overlay with the crystal structure of H3.2 (dark blue). (B) Crystal structure of nucleosome with H3.2 exchanged by in silico homology models of H3.X (purple, left) and H3.Y (light blue, right), respectively. (C) IP of mononucleosomes generated from HeLa cells transfected with empty vector, HA-H3.1, -H3.X, and -H3.Y shows incorporation of novel H3 variants into nucleosomes. Bioanalyzer evaluation of purified DNA after IP of MNase-treated chromatin (unbound and bound material) shows digestion of chromatin to mononucleosomes and their successful precipitation (left; see also Fig. S2 A for DNA size and quality). Silver stain of 15% SDS-PAGE with α-HA IPs of mononucleosomes revealed successful binding of HA-tagged H3 variants (asterisks) and pull-down of core histones (top, right). Immunoblot of immunoprecipitates with α-HA (red) and α-H3 C-terminal (green) antibodies visualized by the Odyssey infrared imaging system (bottom, right). Notice that endogenous H3 is coimmunoprecipitated with all H3 variants analyzed. (D) FRAP experiments to evaluate nucleosomal stability of novel H3 variants using spinning disk confocal microscopy. HeLa Kyoto cells were transiently transfected with GFP, GFP-H3.1, -H3.3, -H3.X, and -H3.Y constructs. A small nuclear area was photobleached (box) and the recovery of the fluorescent signal was monitored over 1 min and up to 8 h (see Fig. S2, B–D, for long-term FRAP). Depicted is a short-term FRAP series (selected time points are shown) of GFP-tagged H3 variants compared with GFP alone. Bar, 5 µm. (E) Quantification of short-term FRAP experiment. Mean curves of 10–20 individual cells are shown. Standard deviations were very small (in the range of ± 0.02) and were omitted for clarity (for details see Fig. S2 D). All GFP-H3 variants show almost no recovery within the first 60 s after bleaching, which indicates that all expressed fusion protein was stably incorporated into nucleosomes. In contrast, GFP alone recovers to almost 100% within 5 s.

Techniques Used: In Silico, Generated, Transfection, Plasmid Preparation, Purification, Silver Staining, SDS Page, Binding Assay, Imaging, Confocal Microscopy, Construct, Stable Transfection

2) Product Images from "Neutrophil Extracellular Traps Directly Induce Epithelial and Endothelial Cell Death: A Predominant Role of Histones"

Article Title: Neutrophil Extracellular Traps Directly Induce Epithelial and Endothelial Cell Death: A Predominant Role of Histones

Journal: PLoS ONE

doi: 10.1371/journal.pone.0032366

Neutrophils (PMN) in the BALF of mice produce NET after LPS treatment. (A) PMN, recruited to the BALF of mice following intratracheal LPS administration, were counted at different time intervals. (B) At the same time intervals, neutrophil elastase activity was measured in the supernatant of BALF ( free elastase, open bars ) as well as in the MNase-digested pellet of BALF ( NET-related elastase, filled bars ). (C) PMN were isolated from BALF of mice stimulated intratracheally with LPS for 24 h, and immunofluorescence staining of isolated cells was performed for DNA/histone (red), CD46 (green) and DAPI (blue) ( upper row ). Isolated PMN were further stimulated with PMA for 1.5 h ( lower row ); arrows indicate NET formation which is demonstrated by appearance of the extracellular chromatin, disintegration of the cell membranes as well as chromatin decondensation. Shown are representative data of three independent experiments (mean SD), *** p
Figure Legend Snippet: Neutrophils (PMN) in the BALF of mice produce NET after LPS treatment. (A) PMN, recruited to the BALF of mice following intratracheal LPS administration, were counted at different time intervals. (B) At the same time intervals, neutrophil elastase activity was measured in the supernatant of BALF ( free elastase, open bars ) as well as in the MNase-digested pellet of BALF ( NET-related elastase, filled bars ). (C) PMN were isolated from BALF of mice stimulated intratracheally with LPS for 24 h, and immunofluorescence staining of isolated cells was performed for DNA/histone (red), CD46 (green) and DAPI (blue) ( upper row ). Isolated PMN were further stimulated with PMA for 1.5 h ( lower row ); arrows indicate NET formation which is demonstrated by appearance of the extracellular chromatin, disintegration of the cell membranes as well as chromatin decondensation. Shown are representative data of three independent experiments (mean SD), *** p

Techniques Used: Mouse Assay, Activity Assay, Isolation, Immunofluorescence, Staining

Inhibition of neutrophil elastase does not inhibit NET-induced cytotoxicity. (A) The supernatants of unstimulated (Unstim) or stimulated (Stim) neutrophils (50 nM PMA for 4 h) were collected and analyzed for elastase activity in the absence ( filled bars ) or presence ( open bars ) of neutrophil elastase inhibitor (NEI). Likewise, NET were isolated from stimulated cells and digested with DNase or MNase or kept undigested (−), followed by analysis of elastase activity in the same way. (B) Cytotoxicity of A549 cells was measured after 16 h treatment with NET (DNase-digested) in the absence or presence of NEI. Similar results were seen for MNase- or non-digested NET as well as with different NEI concentrations from 0.125 to 1 mM. Shown are representative data of three independent experiments (mean SD), *** p
Figure Legend Snippet: Inhibition of neutrophil elastase does not inhibit NET-induced cytotoxicity. (A) The supernatants of unstimulated (Unstim) or stimulated (Stim) neutrophils (50 nM PMA for 4 h) were collected and analyzed for elastase activity in the absence ( filled bars ) or presence ( open bars ) of neutrophil elastase inhibitor (NEI). Likewise, NET were isolated from stimulated cells and digested with DNase or MNase or kept undigested (−), followed by analysis of elastase activity in the same way. (B) Cytotoxicity of A549 cells was measured after 16 h treatment with NET (DNase-digested) in the absence or presence of NEI. Similar results were seen for MNase- or non-digested NET as well as with different NEI concentrations from 0.125 to 1 mM. Shown are representative data of three independent experiments (mean SD), *** p

Techniques Used: Inhibition, Activity Assay, Isolation

NET induce cytotoxicity in epithelial and endothelial cells independent of digestion. (A) The extent of cytotoxicity was measured after treatment of A549 cells for 16 h with undigested NET (−), completely (DNase), partially digested (MNase) or boiled forms of NET. The same concentration of DNA alone as DNA-NET (3.4 µg/ml) as well as DNase or MNase alone were used as controls. Shown are representative data of five independent experiments (mean SD), *** p
Figure Legend Snippet: NET induce cytotoxicity in epithelial and endothelial cells independent of digestion. (A) The extent of cytotoxicity was measured after treatment of A549 cells for 16 h with undigested NET (−), completely (DNase), partially digested (MNase) or boiled forms of NET. The same concentration of DNA alone as DNA-NET (3.4 µg/ml) as well as DNase or MNase alone were used as controls. Shown are representative data of five independent experiments (mean SD), *** p

Techniques Used: Concentration Assay

3) Product Images from "Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription"

Article Title: Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription

Journal: Genes & Development

doi: 10.1101/gad.244434.114

TF binding at both category I and category II promoters overlaps with unusually MNase-labile chromatin. ( A ) Chromatin was underdigested or overdigested with MNase and sequenced (see the Materials and Methods). The average relative signal (a proxy for
Figure Legend Snippet: TF binding at both category I and category II promoters overlaps with unusually MNase-labile chromatin. ( A ) Chromatin was underdigested or overdigested with MNase and sequenced (see the Materials and Methods). The average relative signal (a proxy for

Techniques Used: Binding Assay

4) Product Images from "Persistence of an alternate chromatin structure at silenced loci in the absence of silencers"

Article Title: Persistence of an alternate chromatin structure at silenced loci in the absence of silencers

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

doi:

MNase footprinting of chromatin templates. Spheroplasts were made from strains THC27 ( SIR ) and THC28 ( sir3 ), and DNA was digested in situ with MNase (see Materials and Methods ). The indirect end-labeling probe, a LYS2 fragment, hybridizes within the rKWD50N excision cassette adjacent to the Stu I site (these strains lack the LYS2 gene). The positions of HMR E, RS sites, and size markers (in kb) are denoted. (•) identifies a single hypersensitive site outside the excision cassette that appears upon derepression. Digestion of purified chromosomal DNA is also shown (marked Naked). Units of MNase/ml used in each lane: ( Left and Center ) 160, 80, 40, and 20; ( Right ) 10 and 5.
Figure Legend Snippet: MNase footprinting of chromatin templates. Spheroplasts were made from strains THC27 ( SIR ) and THC28 ( sir3 ), and DNA was digested in situ with MNase (see Materials and Methods ). The indirect end-labeling probe, a LYS2 fragment, hybridizes within the rKWD50N excision cassette adjacent to the Stu I site (these strains lack the LYS2 gene). The positions of HMR E, RS sites, and size markers (in kb) are denoted. (•) identifies a single hypersensitive site outside the excision cassette that appears upon derepression. Digestion of purified chromosomal DNA is also shown (marked Naked). Units of MNase/ml used in each lane: ( Left and Center ) 160, 80, 40, and 20; ( Right ) 10 and 5.

Techniques Used: Footprinting, In Situ, End Labeling, Purification

5) Product Images from "Nucleosomes protect DNA from DNA methylation in vivo and in vitro"

Article Title: Nucleosomes protect DNA from DNA methylation in vivo and in vitro

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkr263

Nucleosomal DNA is devoid of CpG methylation in vivo. ( A ) Chromatin from HeLa cells was partially hydrolysed with MNase (lanes 1 and 2) and the purified DNA was analysed by agarose gel electrophoresis. The nucleosome-sized DNA fragments are indicated on the right. Mono-, di- and tri-nucleosomal (1 n, 2 n, 3 n) DNA was isolated and analysed (lanes 3–5). Molecular weight marker (M) and DNA sizes are indicated. ( B ) DNA methylation levels for mononucleosomal (white bars) and trinucleosomal (black bars) DNA using increasing DNA amounts, was determined with a meCpG-sensitive ELISA assay (Sigma). A comparative DNA methylation analysis for 100 ng of different nucleosomal DNAs (1 n, 2 n, 3 n), methylated control DNA (ctrl mDNA), non-methylated and methylated NPS2 DNA (mNPS2) is shown. ( C ) Micellar capillary electrophoresis of nucleosides (∼4 µg/µl) from purified and hydrolysed mononucleosomal DNA (left) and genomic HeLa DNA (right). The absorbance (AU) is plotted against the migration time (t; min). The peaks for 2′-deoxycytidine (C), 5-methyl 2′-deoxycytidine (5-meC) and an impurity (asteriks) are indicated. ( D ) The average ( n = 10) of the 5-meC content of mononucleosomal (light grey) and Hela genomic DNA (dark grey) was calculated as percentage of (Area 5-meC/(Area C + Area 5-meC). Statistical significance of the data was calculated using the paired t -test. The calculated 5-meC content of mononucleosomal DNA (light grey, 147 bp) relative to the 5-meC content of linker DNA (dark grey, 41 bp) is given in the graph on the right.
Figure Legend Snippet: Nucleosomal DNA is devoid of CpG methylation in vivo. ( A ) Chromatin from HeLa cells was partially hydrolysed with MNase (lanes 1 and 2) and the purified DNA was analysed by agarose gel electrophoresis. The nucleosome-sized DNA fragments are indicated on the right. Mono-, di- and tri-nucleosomal (1 n, 2 n, 3 n) DNA was isolated and analysed (lanes 3–5). Molecular weight marker (M) and DNA sizes are indicated. ( B ) DNA methylation levels for mononucleosomal (white bars) and trinucleosomal (black bars) DNA using increasing DNA amounts, was determined with a meCpG-sensitive ELISA assay (Sigma). A comparative DNA methylation analysis for 100 ng of different nucleosomal DNAs (1 n, 2 n, 3 n), methylated control DNA (ctrl mDNA), non-methylated and methylated NPS2 DNA (mNPS2) is shown. ( C ) Micellar capillary electrophoresis of nucleosides (∼4 µg/µl) from purified and hydrolysed mononucleosomal DNA (left) and genomic HeLa DNA (right). The absorbance (AU) is plotted against the migration time (t; min). The peaks for 2′-deoxycytidine (C), 5-methyl 2′-deoxycytidine (5-meC) and an impurity (asteriks) are indicated. ( D ) The average ( n = 10) of the 5-meC content of mononucleosomal (light grey) and Hela genomic DNA (dark grey) was calculated as percentage of (Area 5-meC/(Area C + Area 5-meC). Statistical significance of the data was calculated using the paired t -test. The calculated 5-meC content of mononucleosomal DNA (light grey, 147 bp) relative to the 5-meC content of linker DNA (dark grey, 41 bp) is given in the graph on the right.

Techniques Used: CpG Methylation Assay, In Vivo, Purification, Agarose Gel Electrophoresis, Isolation, Molecular Weight, Marker, DNA Methylation Assay, Enzyme-linked Immunosorbent Assay, Methylation, Electrophoresis, Migration

6) Product Images from "NF-Y Associates with H3-H4 Tetramers and Octamers by Multiple Mechanisms"

Article Title: NF-Y Associates with H3-H4 Tetramers and Octamers by Multiple Mechanisms

Journal: Molecular and Cellular Biology

doi:

MNase accessibility assay of histone–NF-YB–NF-YC combinations. Stoichiometric amounts of the indicated combinations of HFM proteins were reconstituted with fragment 2 (labeled on the top strand [lanes 4 to 9] and bottom strand [lanes 13 to 18]), cut with MNase, and analyzed on sequencing gels. In lane 17, the NF-Y trimer was used to show the NF-Y footprinted area. F refers to free, mock-reconstituted DNA (lanes 4 and 5; uncut and cut with MNase, respectively). Arrows correspond to the major and minor hypersensitive sites. Part of the H3-H4 and H3–H4–NF-YB–NF-YC reconstitutions were cut with DNase I and run in parallel (lanes 1, 2, 10, and 11). Bars correspond to the 10-bp cutting patterns of DNase I. Sequencing reactions (T; lanes 3 and 12) were run in parallel to precisely map the sites of MNase cuts.
Figure Legend Snippet: MNase accessibility assay of histone–NF-YB–NF-YC combinations. Stoichiometric amounts of the indicated combinations of HFM proteins were reconstituted with fragment 2 (labeled on the top strand [lanes 4 to 9] and bottom strand [lanes 13 to 18]), cut with MNase, and analyzed on sequencing gels. In lane 17, the NF-Y trimer was used to show the NF-Y footprinted area. F refers to free, mock-reconstituted DNA (lanes 4 and 5; uncut and cut with MNase, respectively). Arrows correspond to the major and minor hypersensitive sites. Part of the H3-H4 and H3–H4–NF-YB–NF-YC reconstitutions were cut with DNase I and run in parallel (lanes 1, 2, 10, and 11). Bars correspond to the 10-bp cutting patterns of DNase I. Sequencing reactions (T; lanes 3 and 12) were run in parallel to precisely map the sites of MNase cuts.

Techniques Used: Labeling, Sequencing

7) Product Images from "Synergy between histone deacetylase inhibitors and DNA-damaging agents is mediated by histone deacetylase 2 in colorectal cancer"

Article Title: Synergy between histone deacetylase inhibitors and DNA-damaging agents is mediated by histone deacetylase 2 in colorectal cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.9887

HDAC2 controls the chromatin plasticity and its depletion enhances mitotic cell death in drug resistant HT-29 cells upon 5-FU and Oxa treatments A. PARPc measurement in HT-29 treated by 5-FU and Oxa combined with SAHA. After 24 hours, cells were lysed and the proteins separated using SDS-PAGE. B and C. HT-29 cell lines were treated by 5-FU or Oxa alone or combined with SAHA. After 24 hours, Cells were fixed 4% paraformaldehyde, subsequently DNA was stained with DAPI (0.1 μg/ml; Sigma-Aldrich) and the number of apoptotic cells was measured quantitatively by assessing the percentage of cells with fragmented or condensed nuclei. Mitotic cell death (MCD) was quantified by using phosphorylated histone 3 (ser10) as a mitotic cell marker. C) Representative image of mitotic cell death (MCD) in HT-29 upon SAHA + Oxa combined treatment. D. HT-29 or shRNA-HDAC2 HT-29 cells lines were treated with 5-FU or Oxa only or in combination with SAHA. After 24 hours, cells were lysed and the proteins separated using SDS-PAGE. The PARPc and the protein level of HDAC2 were analyzed by WB. Actin was used as a loading control. E. HT-29 cell lines or shRNA-HDAC2 HT-29 cells lines were treated by 5-FU, Oxa or SAHA. After 24 hours, mitotic cell death (MCD) was quantified by using phosphorylated histone 3 (ser10) as a mitotic cell marker. F. HT-29 cell lines were treated by Oxa alone or combined with SAHA and shRNA-HDAC2 HT-29 cells were treated with Oxa. After 24 hours, cells were fixed and HDAC2 protein was detected after immunofluorescence staining. Nucleus was counterstained using DAPI staining. + z-Stack shows nuclear deformation. G. MNase accessibility assay was used to study relaxed chromatin which has higher accessibility to micrococal nuclease enzyme (MNase). Cells HT-29 cells were treated with Oxa or SAHA alone or combined for 24hr and chromatin was extracted and incubated with 0.06U of MNase and fragmented DNA was separated by gel agarose, the arrow represent the undigested DNA. For all the experiments error bars represent ± S.E.M. of three independent experiments ( n =3) and statistical significance is depicted by * for p
Figure Legend Snippet: HDAC2 controls the chromatin plasticity and its depletion enhances mitotic cell death in drug resistant HT-29 cells upon 5-FU and Oxa treatments A. PARPc measurement in HT-29 treated by 5-FU and Oxa combined with SAHA. After 24 hours, cells were lysed and the proteins separated using SDS-PAGE. B and C. HT-29 cell lines were treated by 5-FU or Oxa alone or combined with SAHA. After 24 hours, Cells were fixed 4% paraformaldehyde, subsequently DNA was stained with DAPI (0.1 μg/ml; Sigma-Aldrich) and the number of apoptotic cells was measured quantitatively by assessing the percentage of cells with fragmented or condensed nuclei. Mitotic cell death (MCD) was quantified by using phosphorylated histone 3 (ser10) as a mitotic cell marker. C) Representative image of mitotic cell death (MCD) in HT-29 upon SAHA + Oxa combined treatment. D. HT-29 or shRNA-HDAC2 HT-29 cells lines were treated with 5-FU or Oxa only or in combination with SAHA. After 24 hours, cells were lysed and the proteins separated using SDS-PAGE. The PARPc and the protein level of HDAC2 were analyzed by WB. Actin was used as a loading control. E. HT-29 cell lines or shRNA-HDAC2 HT-29 cells lines were treated by 5-FU, Oxa or SAHA. After 24 hours, mitotic cell death (MCD) was quantified by using phosphorylated histone 3 (ser10) as a mitotic cell marker. F. HT-29 cell lines were treated by Oxa alone or combined with SAHA and shRNA-HDAC2 HT-29 cells were treated with Oxa. After 24 hours, cells were fixed and HDAC2 protein was detected after immunofluorescence staining. Nucleus was counterstained using DAPI staining. + z-Stack shows nuclear deformation. G. MNase accessibility assay was used to study relaxed chromatin which has higher accessibility to micrococal nuclease enzyme (MNase). Cells HT-29 cells were treated with Oxa or SAHA alone or combined for 24hr and chromatin was extracted and incubated with 0.06U of MNase and fragmented DNA was separated by gel agarose, the arrow represent the undigested DNA. For all the experiments error bars represent ± S.E.M. of three independent experiments ( n =3) and statistical significance is depicted by * for p

Techniques Used: SDS Page, Staining, Marker, shRNA, Western Blot, Immunofluorescence, Incubation

8) Product Images from "Histone octamer trans-transfer: a signature mechanism of ATP-dependent chromatin remodelling unravelled in wheat nuclear extract"

Article Title: Histone octamer trans-transfer: a signature mechanism of ATP-dependent chromatin remodelling unravelled in wheat nuclear extract

Journal: Annals of Botany

doi: 10.1093/aob/mcr232

Profile of DNA isolated from the soluble chromatin: DNA was extracted from fractionated nucleosomes obtained from chromatin derived from MNase-treated wheat nuclei and analysed on 1 % agarose gel. The lane numbers (25–39) indicate fraction numbers.
Figure Legend Snippet: Profile of DNA isolated from the soluble chromatin: DNA was extracted from fractionated nucleosomes obtained from chromatin derived from MNase-treated wheat nuclei and analysed on 1 % agarose gel. The lane numbers (25–39) indicate fraction numbers.

Techniques Used: Isolation, Derivative Assay, Agarose Gel Electrophoresis

9) Product Images from "Recruitment of the Nucleolar Remodeling Complex NoRC Establishes Ribosomal DNA Silencing in Chromatin"

Article Title: Recruitment of the Nucleolar Remodeling Complex NoRC Establishes Ribosomal DNA Silencing in Chromatin

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.4.1791-1798.2004

NoRC silences rDNA transcription for chromatin templates. (A) Supercoiling assay. Nucleosomes were assembled on pMrWT-T by salt dialysis and purified in a sucrose gradient. Individual fractions were incubated with topoisomerase I, and the topoisomer distribution of the purified DNA was visualized on agarose gels containing chloroquine. Supercoiled DNA (sc, lane 1), partially relaxed DNA (lane 2), and fractions with decreasing nucleosome density (lanes 3 to 8) are shown. The nucleosomal fraction used for the experiments is indicated by a white triangle. (B) MNase digestion. The indicated chromatin fraction was digested with increasing amounts of MNase. Purified DNA was visualized by agarose gel electrophoresis and ethidium bromide staining. The regular fragment ladder indicative of the nucleosomal array is indicated (1n through 6n). (C) Transcription assay. A minigene (pMrWT-T) containing the rDNA promoter and the termination region was used for in vitro transcription. DNA and chromatin were incubated with the transcription extract in the absence or presence of TTF-I (lanes 1 and 2). Readthrough transcription in the absence and terminated transcription in the presence of TTF is indicated on the left. Increasing amounts of Snf2H (lanes 2 to 4; 25, 50, and 100 fmol, respectively), NoRC (lanes 6 to 8; 25, 50, and 100 fmol, respectively) and ACF (9 to 11; 25, 50, and 100 fmol, respectively), were incubated with TTF-I, resulting in terminated transcripts. Transcription was performed for naked DNA and for chromatin templates as indicated.
Figure Legend Snippet: NoRC silences rDNA transcription for chromatin templates. (A) Supercoiling assay. Nucleosomes were assembled on pMrWT-T by salt dialysis and purified in a sucrose gradient. Individual fractions were incubated with topoisomerase I, and the topoisomer distribution of the purified DNA was visualized on agarose gels containing chloroquine. Supercoiled DNA (sc, lane 1), partially relaxed DNA (lane 2), and fractions with decreasing nucleosome density (lanes 3 to 8) are shown. The nucleosomal fraction used for the experiments is indicated by a white triangle. (B) MNase digestion. The indicated chromatin fraction was digested with increasing amounts of MNase. Purified DNA was visualized by agarose gel electrophoresis and ethidium bromide staining. The regular fragment ladder indicative of the nucleosomal array is indicated (1n through 6n). (C) Transcription assay. A minigene (pMrWT-T) containing the rDNA promoter and the termination region was used for in vitro transcription. DNA and chromatin were incubated with the transcription extract in the absence or presence of TTF-I (lanes 1 and 2). Readthrough transcription in the absence and terminated transcription in the presence of TTF is indicated on the left. Increasing amounts of Snf2H (lanes 2 to 4; 25, 50, and 100 fmol, respectively), NoRC (lanes 6 to 8; 25, 50, and 100 fmol, respectively) and ACF (9 to 11; 25, 50, and 100 fmol, respectively), were incubated with TTF-I, resulting in terminated transcripts. Transcription was performed for naked DNA and for chromatin templates as indicated.

Techniques Used: Purification, Incubation, Agarose Gel Electrophoresis, Staining, In Vitro

10) Product Images from "DNA sequence encoded repression of rRNA gene transcription in chromatin"

Article Title: DNA sequence encoded repression of rRNA gene transcription in chromatin

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq263

rRNA genes are switched off in chromatin. ( A ) Reconstitution of mononucleosomes on a 330 bp (–175 to +155) rDNA fragment. The end-labelled DNA was reconstituted into mononucleosomes by salt dialysis reconstitution (lanes 1 and 2) and analysed by native PAGE. Nucleosomal DNA molecules harbouring a nucleosome on the transcription start site were selected by digestion with the restriction enzyme Rsa I (lane 3). The positions of the nucleosomal DNA, the undigested and digested DNA fragment are indicated. ( B ) Transcription assay with free and nucleosomal rDNA fragments. Increasing amounts of free DNA (lanes 1 and 2), a mixture of free DNA and nucleosomal DNA (lanes 3 and 4) and Rsa I-selected nucleosomal DNA (lanes 5–7) were incubated with the transcription extract. The radioactive labelled transcripts were analysed by native PAGE. The nucleosomal templates used for the transcription reactions are shown above the gel. The positions of the undigested or nucleosomal rDNA fragment, the digested free DNA and the 155-nt-long transcript are indicated on the right. ( C ) Analysis of nucleosome positions on the rDNA promoter. Mononucleosomal templates (–175 to +155) were digested with MNase, and the protected nucleosomal DNA was gel-purified, cloned and sequenced. The graph shows the positions of the nucleosomal dyad axis. The positions of the nucAct and nucRep nucleosomes observed in vivo are indicated with the 5′, 3′ and dyad axis positions relative to the rRNA gene transcription start site. E1 and E2 indicate the dyad axis positions of nucleosomes located at the end of the DNA fragment. ( D ) Prediction of nucleosome positioning by the probability of nucleosome occupancy and the probability of encountering a nucleosomal start site. rRNA sequences from position –5000 to +5000 relative to the transcription start site were used for computational analysis at http://genie.weizmann.ac.il/pubs/nucleosomes06/ ( 13 ). The graph displays a window of the calculated predictions, ranging from position –300 to +300 within the rDNA sequence. Peaks of high p (nucleosomal start) values, indicating a high probability for a nucleosomal start site, are indicated. The two nucleosomal positions identified on the rRNA gene in vivo are indicated [ nucAct –157 to –2 (green); the repressive nucleosome position –132 to +22 (red)].
Figure Legend Snippet: rRNA genes are switched off in chromatin. ( A ) Reconstitution of mononucleosomes on a 330 bp (–175 to +155) rDNA fragment. The end-labelled DNA was reconstituted into mononucleosomes by salt dialysis reconstitution (lanes 1 and 2) and analysed by native PAGE. Nucleosomal DNA molecules harbouring a nucleosome on the transcription start site were selected by digestion with the restriction enzyme Rsa I (lane 3). The positions of the nucleosomal DNA, the undigested and digested DNA fragment are indicated. ( B ) Transcription assay with free and nucleosomal rDNA fragments. Increasing amounts of free DNA (lanes 1 and 2), a mixture of free DNA and nucleosomal DNA (lanes 3 and 4) and Rsa I-selected nucleosomal DNA (lanes 5–7) were incubated with the transcription extract. The radioactive labelled transcripts were analysed by native PAGE. The nucleosomal templates used for the transcription reactions are shown above the gel. The positions of the undigested or nucleosomal rDNA fragment, the digested free DNA and the 155-nt-long transcript are indicated on the right. ( C ) Analysis of nucleosome positions on the rDNA promoter. Mononucleosomal templates (–175 to +155) were digested with MNase, and the protected nucleosomal DNA was gel-purified, cloned and sequenced. The graph shows the positions of the nucleosomal dyad axis. The positions of the nucAct and nucRep nucleosomes observed in vivo are indicated with the 5′, 3′ and dyad axis positions relative to the rRNA gene transcription start site. E1 and E2 indicate the dyad axis positions of nucleosomes located at the end of the DNA fragment. ( D ) Prediction of nucleosome positioning by the probability of nucleosome occupancy and the probability of encountering a nucleosomal start site. rRNA sequences from position –5000 to +5000 relative to the transcription start site were used for computational analysis at http://genie.weizmann.ac.il/pubs/nucleosomes06/ ( 13 ). The graph displays a window of the calculated predictions, ranging from position –300 to +300 within the rDNA sequence. Peaks of high p (nucleosomal start) values, indicating a high probability for a nucleosomal start site, are indicated. The two nucleosomal positions identified on the rRNA gene in vivo are indicated [ nucAct –157 to –2 (green); the repressive nucleosome position –132 to +22 (red)].

Techniques Used: Clear Native PAGE, Incubation, Purification, Clone Assay, In Vivo, Sequencing

PK inhibits nucleosome remodelling at the rDNA promoter. ( A ) MNase digestion of chromatin assembled on the rDNA minigene (pMrWT-T). Chromatin reconstituted with the Drosophila extract was digested with MNase for 0.5–3 min (lanes 1–3) or for 0.5–6 min (lanes 4–8) in the presence of 600 μM PK. The nucleosomal ladder (1n-5n) and the DNA marker (M; 1kb ladder) are indicated. ( B ) Chromatin assembled on pMrWT-T was incubated in the absence or presence of TTF-I and partially digested with MNase. Purified DNA was digested with EcoRI, separated on an agarose gel and transferred onto a nylon membrane. Chromatin configuration around the TTF-I-binding site (T 0 ) was visualized by indirect end-labelling (lanes 1 and 2). Chromatin remodelling was monitored in the presence of K (lanes 4 and 5; 300 and 600 μM) or PK (lanes 6 and 7, 300 and 600 μM). The position of the TTF-I-binding site is indicated by Sal I digestion of the template DNA (lane 3). Open circles mark non-positioned nucleosomes, whereas the gray circles indicate positioned nucleosomes. The position of the TTF-I-binding site (gray box), MNase-protected DNA regions (black triangles) and MNase-sensitive regions (white triangles) are indicated. The strong band in lane 6 (marked with an asterisk) arises due to the relatively lower MNase digestion of this sample.
Figure Legend Snippet: PK inhibits nucleosome remodelling at the rDNA promoter. ( A ) MNase digestion of chromatin assembled on the rDNA minigene (pMrWT-T). Chromatin reconstituted with the Drosophila extract was digested with MNase for 0.5–3 min (lanes 1–3) or for 0.5–6 min (lanes 4–8) in the presence of 600 μM PK. The nucleosomal ladder (1n-5n) and the DNA marker (M; 1kb ladder) are indicated. ( B ) Chromatin assembled on pMrWT-T was incubated in the absence or presence of TTF-I and partially digested with MNase. Purified DNA was digested with EcoRI, separated on an agarose gel and transferred onto a nylon membrane. Chromatin configuration around the TTF-I-binding site (T 0 ) was visualized by indirect end-labelling (lanes 1 and 2). Chromatin remodelling was monitored in the presence of K (lanes 4 and 5; 300 and 600 μM) or PK (lanes 6 and 7, 300 and 600 μM). The position of the TTF-I-binding site is indicated by Sal I digestion of the template DNA (lane 3). Open circles mark non-positioned nucleosomes, whereas the gray circles indicate positioned nucleosomes. The position of the TTF-I-binding site (gray box), MNase-protected DNA regions (black triangles) and MNase-sensitive regions (white triangles) are indicated. The strong band in lane 6 (marked with an asterisk) arises due to the relatively lower MNase digestion of this sample.

Techniques Used: Marker, Incubation, Purification, Agarose Gel Electrophoresis, Binding Assay

TTF-I-dependent chromatin dynamics at the rRNA gene promoter. Reconstituted nucleosomal arrays were incubated for 90 min with the TxE, TTF-I, K (600 μM) or PK (600 μM) as indicated. Nucleosome positions at the rDNA promoter were mapped by partial MNase digestion and primer extension of the purified DNA. DNA fragments were resolved on 8% sequencing gels and quantified with a PhosphorImager. The graph shows the positions (relative to the transcription start site, +1; site is marked by boxes) and relative intensities of the MNase cleavage sites corresponding to the 3′ boundaries of positioned nucleosomes. Boxes highlight the MNase cleavage sites around the transcription start site, correlating with the nucleosome position nucAct . The position of the oligonucleotide used for primer extension and the major MNase-sensitive sites on the rDNA are indicated. The scan of the DNA marker (10-bp ladder) is shown below the graphs.
Figure Legend Snippet: TTF-I-dependent chromatin dynamics at the rRNA gene promoter. Reconstituted nucleosomal arrays were incubated for 90 min with the TxE, TTF-I, K (600 μM) or PK (600 μM) as indicated. Nucleosome positions at the rDNA promoter were mapped by partial MNase digestion and primer extension of the purified DNA. DNA fragments were resolved on 8% sequencing gels and quantified with a PhosphorImager. The graph shows the positions (relative to the transcription start site, +1; site is marked by boxes) and relative intensities of the MNase cleavage sites corresponding to the 3′ boundaries of positioned nucleosomes. Boxes highlight the MNase cleavage sites around the transcription start site, correlating with the nucleosome position nucAct . The position of the oligonucleotide used for primer extension and the major MNase-sensitive sites on the rDNA are indicated. The scan of the DNA marker (10-bp ladder) is shown below the graphs.

Techniques Used: Incubation, Purification, Sequencing, Marker

11) Product Images from "DNA sequence- and conformation-directed positioning of nucleosomes by chromatin-remodeling complexes"

Article Title: DNA sequence- and conformation-directed positioning of nucleosomes by chromatin-remodeling complexes

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

doi: 10.1073/pnas.0702430104

A short DNA element can direct ACF-dependent nucleosome positioning. ( A ) Remodeling reaction with ACF or ISWI with a nucleosome substrate containing a 253-bp-long DNA fragment (K3 DNA) from the pT-K3 plasmid. After nucleosome assembly by salt dialysis on the K3 DNA, a mixed population of a single nucleosome with three main positions (N1, N2, and N4) and one minor position (N3, lane 1) was obtained. This substrate was used in a remodeling reaction with ISWI (lane 2) or ACF (lane 3). ( B ) High-resolution mapping of the remodeler-dependent nucleosome positions on the K3 DNA substrate. MNase protection and subsequent primer extension reactions were conducted. Scans for the primer extension reactions ( Left , forward primer; Right , reverse primer) are shown for the nucleosomal input substrate (green, corresponding to A , lane 1) and the remodeling reaction for ACF (red, corresponding to A , lane 3). The black curve shows a 10-bp DNA marker. The same analysis was conducted with ISWI (data not shown). The peaks reflect nucleosomes positioned adjacent to this site. Considering that 147 bp of DNA are protected by the nucleosome, the major nucleosome positions were identified as 37/45 to 187/195 for N1, 25 to 175 for N2, and 0/7 to 151/157 for N4. ( C ) The ACF- and ISWI-dependent nucleosome positions determined on the 253-bp K3 DNA fragment were plotted together with the predicted DNA curvature. The black arrow refers to the 40-bp DNA sequence encompassing the region of maximal DNA curvature from the rDNA sequence that was cloned into the K3 DNA. ( D ) Same analysis as in C ).
Figure Legend Snippet: A short DNA element can direct ACF-dependent nucleosome positioning. ( A ) Remodeling reaction with ACF or ISWI with a nucleosome substrate containing a 253-bp-long DNA fragment (K3 DNA) from the pT-K3 plasmid. After nucleosome assembly by salt dialysis on the K3 DNA, a mixed population of a single nucleosome with three main positions (N1, N2, and N4) and one minor position (N3, lane 1) was obtained. This substrate was used in a remodeling reaction with ISWI (lane 2) or ACF (lane 3). ( B ) High-resolution mapping of the remodeler-dependent nucleosome positions on the K3 DNA substrate. MNase protection and subsequent primer extension reactions were conducted. Scans for the primer extension reactions ( Left , forward primer; Right , reverse primer) are shown for the nucleosomal input substrate (green, corresponding to A , lane 1) and the remodeling reaction for ACF (red, corresponding to A , lane 3). The black curve shows a 10-bp DNA marker. The same analysis was conducted with ISWI (data not shown). The peaks reflect nucleosomes positioned adjacent to this site. Considering that 147 bp of DNA are protected by the nucleosome, the major nucleosome positions were identified as 37/45 to 187/195 for N1, 25 to 175 for N2, and 0/7 to 151/157 for N4. ( C ) The ACF- and ISWI-dependent nucleosome positions determined on the 253-bp K3 DNA fragment were plotted together with the predicted DNA curvature. The black arrow refers to the 40-bp DNA sequence encompassing the region of maximal DNA curvature from the rDNA sequence that was cloned into the K3 DNA. ( D ) Same analysis as in C ).

Techniques Used: Plasmid Preparation, Marker, Sequencing, Clone Assay

12) Product Images from "Chromatin assembly factor I and Hir proteins contribute to building functional kinetochores in S. cerevisiae"

Article Title: Chromatin assembly factor I and Hir proteins contribute to building functional kinetochores in S. cerevisiae

Journal: Genes & Development

doi: 10.1101/gad.925302

Centromeric chromatin phenotypes in cac1Δ hir1Δ cells. Nuclei were prepared from yeast strains PKY346 (wt), PKY1100 ( cac1Δ ), PKY1154 ( hir1Δ ), and PKY1168 ( cac1Δ hir1Δ ) as indicated and digested with nucleases as follows. ( A – C ) Indirect end-label analysis of CEN3 chromatin. Nuclei were incubated with 0.6 U MNase (Sigma) at 32°C for 0 min (lanes 1,5,9,13 ), 5 min (lanes 2,6,10,14 ), 10 min (lanes 3,7,11,15 ), or 15 min (lanes 4,8,12,16 ) prior to isolation of genomic DNA, restriction enzyme digestion, and DNA blot hybridization with probes as described in the Materials and Methods. ( A ) CDEIII-proximal side of CEN3 in cells grown at 30°C. Samples were digested with Cla I. ( B ) CDEIII-proximal side of CEN3 in cells shifted to 16°C for 36 h prior to isolation of nuclei. Samples were digested with Cla I. ( C ) CDEI-proximal side of CEN3 in nuclei prepared from cells grown at 16°C. Samples were digested with Bam HI. ( D ) Dra I accessibility to CDEII within CEN3 . Nuclei were incubated with Dra I (0, 50, 100, or 150 U/mL) at 37°C for 30 min. DNA was purified, digested with Eco RI, and subjected to Southern blot hybridization. The fold differences in digestion relative to wild-type cells represent the average of three independent experiments with standard deviations indicated by the error bars. ( Lower panel) A region of the same gel used for the Southern blot visualized by ethidium bromide staining, indicating similar extents of Dra I digestion of total chromatin in all four strains.
Figure Legend Snippet: Centromeric chromatin phenotypes in cac1Δ hir1Δ cells. Nuclei were prepared from yeast strains PKY346 (wt), PKY1100 ( cac1Δ ), PKY1154 ( hir1Δ ), and PKY1168 ( cac1Δ hir1Δ ) as indicated and digested with nucleases as follows. ( A – C ) Indirect end-label analysis of CEN3 chromatin. Nuclei were incubated with 0.6 U MNase (Sigma) at 32°C for 0 min (lanes 1,5,9,13 ), 5 min (lanes 2,6,10,14 ), 10 min (lanes 3,7,11,15 ), or 15 min (lanes 4,8,12,16 ) prior to isolation of genomic DNA, restriction enzyme digestion, and DNA blot hybridization with probes as described in the Materials and Methods. ( A ) CDEIII-proximal side of CEN3 in cells grown at 30°C. Samples were digested with Cla I. ( B ) CDEIII-proximal side of CEN3 in cells shifted to 16°C for 36 h prior to isolation of nuclei. Samples were digested with Cla I. ( C ) CDEI-proximal side of CEN3 in nuclei prepared from cells grown at 16°C. Samples were digested with Bam HI. ( D ) Dra I accessibility to CDEII within CEN3 . Nuclei were incubated with Dra I (0, 50, 100, or 150 U/mL) at 37°C for 30 min. DNA was purified, digested with Eco RI, and subjected to Southern blot hybridization. The fold differences in digestion relative to wild-type cells represent the average of three independent experiments with standard deviations indicated by the error bars. ( Lower panel) A region of the same gel used for the Southern blot visualized by ethidium bromide staining, indicating similar extents of Dra I digestion of total chromatin in all four strains.

Techniques Used: Incubation, Isolation, Hybridization, Purification, Southern Blot, Staining

13) Product Images from "Distinct roles for S. cerevisiae H2A copies in recombination and repeat stability, with a role for H2A.1 threonine 126"

Article Title: Distinct roles for S. cerevisiae H2A copies in recombination and repeat stability, with a role for H2A.1 threonine 126

Journal: eLife

doi: 10.7554/eLife.53362

Nucleosome positioning at a (CAG) 85 repeat is not altered in the absence of H2A.1 or H2A.2. A ) Indirect end-labeling of nucleosomal DNA upstream of the CAG repeat. MNase (0, 0.25, 2.5, and 7.5 units) digested DNA was run in 1.5% agarose with ethidium bromide (left) and Southern blotted (right) using a probe ~100 bp proximal to the CAG repeat (red line Figure 1—figure supplement 1A ). Ovals represent nucleosome positions. The experiment was repeated six times; a representative blot is shown. ( B ) Illumina array mapping of nucleosome protection at the CAG repeat. Mononucleosomal DNA from strains containing the (CAG) 85 repeats was hybridized to a custom array of 30-mer probes spanning 425 bp upstream of the repeat to 436 bp downstream of the repeat in YAC CF1. Probes 14–16 contain CAG repeats; probe 15 is composed purely of CAG repeats (probe sequences in Supplementary file 3 ). Error bars represent standard deviation of 2–3 independent experiments.
Figure Legend Snippet: Nucleosome positioning at a (CAG) 85 repeat is not altered in the absence of H2A.1 or H2A.2. A ) Indirect end-labeling of nucleosomal DNA upstream of the CAG repeat. MNase (0, 0.25, 2.5, and 7.5 units) digested DNA was run in 1.5% agarose with ethidium bromide (left) and Southern blotted (right) using a probe ~100 bp proximal to the CAG repeat (red line Figure 1—figure supplement 1A ). Ovals represent nucleosome positions. The experiment was repeated six times; a representative blot is shown. ( B ) Illumina array mapping of nucleosome protection at the CAG repeat. Mononucleosomal DNA from strains containing the (CAG) 85 repeats was hybridized to a custom array of 30-mer probes spanning 425 bp upstream of the repeat to 436 bp downstream of the repeat in YAC CF1. Probes 14–16 contain CAG repeats; probe 15 is composed purely of CAG repeats (probe sequences in Supplementary file 3 ). Error bars represent standard deviation of 2–3 independent experiments.

Techniques Used: End Labeling, Standard Deviation

Nucleosome positioning at a (CAG) 85 repeat is not altered in the absence of H2A.1 or H2A.2. A ) Indirect end-labeling of nucleosomal DNA upstream of the CAG repeat. MNase (0, 0.25, 2.5, and 7.5 units) digested DNA was run in 1.5% agarose with ethidium bromide (left) and Southern blotted (right) using a probe ~100 bp proximal to the CAG repeat (red line Figure 1—figure supplement 1A ). Ovals represent nucleosome positions. The experiment was repeated six times; a representative blot is shown. ( B ) Illumina array mapping of nucleosome protection at the CAG repeat. Mononucleosomal DNA from strains containing the (CAG) 85 repeats was hybridized to a custom array of 30-mer probes spanning 425 bp upstream of the repeat to 436 bp downstream of the repeat in YAC CF1. Probes 14–16 contain CAG repeats; probe 15 is composed purely of CAG repeats (probe sequences in Supplementary file 3 ). Error bars represent standard deviation of 2–3 independent experiments.
Figure Legend Snippet: Nucleosome positioning at a (CAG) 85 repeat is not altered in the absence of H2A.1 or H2A.2. A ) Indirect end-labeling of nucleosomal DNA upstream of the CAG repeat. MNase (0, 0.25, 2.5, and 7.5 units) digested DNA was run in 1.5% agarose with ethidium bromide (left) and Southern blotted (right) using a probe ~100 bp proximal to the CAG repeat (red line Figure 1—figure supplement 1A ). Ovals represent nucleosome positions. The experiment was repeated six times; a representative blot is shown. ( B ) Illumina array mapping of nucleosome protection at the CAG repeat. Mononucleosomal DNA from strains containing the (CAG) 85 repeats was hybridized to a custom array of 30-mer probes spanning 425 bp upstream of the repeat to 436 bp downstream of the repeat in YAC CF1. Probes 14–16 contain CAG repeats; probe 15 is composed purely of CAG repeats (probe sequences in Supplementary file 3 ). Error bars represent standard deviation of 2–3 independent experiments.

Techniques Used: End Labeling, Standard Deviation

14) Product Images from "Distinct roles for S. cerevisiae H2A copies in recombination and repeat stability, with a role for H2A.1 threonine 126"

Article Title: Distinct roles for S. cerevisiae H2A copies in recombination and repeat stability, with a role for H2A.1 threonine 126

Journal: eLife

doi: 10.7554/eLife.53362

Nucleosome positioning at a (CAG) 85 repeat is not altered in the absence of H2A.1 or H2A.2. A ) Indirect end-labeling of nucleosomal DNA upstream of the CAG repeat. MNase (0, 0.25, 2.5, and 7.5 units) digested DNA was run in 1.5% agarose with ethidium bromide (left) and Southern blotted (right) using a probe ~100 bp proximal to the CAG repeat (red line Figure 1—figure supplement 1A ). Ovals represent nucleosome positions. The experiment was repeated six times; a representative blot is shown. ( B ) Illumina array mapping of nucleosome protection at the CAG repeat. Mononucleosomal DNA from strains containing the (CAG) 85 repeats was hybridized to a custom array of 30-mer probes spanning 425 bp upstream of the repeat to 436 bp downstream of the repeat in YAC CF1. Probes 14–16 contain CAG repeats; probe 15 is composed purely of CAG repeats (probe sequences in Supplementary file 3 ). Error bars represent standard deviation of 2–3 independent experiments.
Figure Legend Snippet: Nucleosome positioning at a (CAG) 85 repeat is not altered in the absence of H2A.1 or H2A.2. A ) Indirect end-labeling of nucleosomal DNA upstream of the CAG repeat. MNase (0, 0.25, 2.5, and 7.5 units) digested DNA was run in 1.5% agarose with ethidium bromide (left) and Southern blotted (right) using a probe ~100 bp proximal to the CAG repeat (red line Figure 1—figure supplement 1A ). Ovals represent nucleosome positions. The experiment was repeated six times; a representative blot is shown. ( B ) Illumina array mapping of nucleosome protection at the CAG repeat. Mononucleosomal DNA from strains containing the (CAG) 85 repeats was hybridized to a custom array of 30-mer probes spanning 425 bp upstream of the repeat to 436 bp downstream of the repeat in YAC CF1. Probes 14–16 contain CAG repeats; probe 15 is composed purely of CAG repeats (probe sequences in Supplementary file 3 ). Error bars represent standard deviation of 2–3 independent experiments.

Techniques Used: End Labeling, Standard Deviation

Nucleosome positioning at a (CAG) 85 repeat is not altered in the absence of H2A.1 or H2A.2. A ) Indirect end-labeling of nucleosomal DNA upstream of the CAG repeat. MNase (0, 0.25, 2.5, and 7.5 units) digested DNA was run in 1.5% agarose with ethidium bromide (left) and Southern blotted (right) using a probe ~100 bp proximal to the CAG repeat (red line Figure 1—figure supplement 1A ). Ovals represent nucleosome positions. The experiment was repeated six times; a representative blot is shown. ( B ) Illumina array mapping of nucleosome protection at the CAG repeat. Mononucleosomal DNA from strains containing the (CAG) 85 repeats was hybridized to a custom array of 30-mer probes spanning 425 bp upstream of the repeat to 436 bp downstream of the repeat in YAC CF1. Probes 14–16 contain CAG repeats; probe 15 is composed purely of CAG repeats (probe sequences in Supplementary file 3 ). Error bars represent standard deviation of 2–3 independent experiments.
Figure Legend Snippet: Nucleosome positioning at a (CAG) 85 repeat is not altered in the absence of H2A.1 or H2A.2. A ) Indirect end-labeling of nucleosomal DNA upstream of the CAG repeat. MNase (0, 0.25, 2.5, and 7.5 units) digested DNA was run in 1.5% agarose with ethidium bromide (left) and Southern blotted (right) using a probe ~100 bp proximal to the CAG repeat (red line Figure 1—figure supplement 1A ). Ovals represent nucleosome positions. The experiment was repeated six times; a representative blot is shown. ( B ) Illumina array mapping of nucleosome protection at the CAG repeat. Mononucleosomal DNA from strains containing the (CAG) 85 repeats was hybridized to a custom array of 30-mer probes spanning 425 bp upstream of the repeat to 436 bp downstream of the repeat in YAC CF1. Probes 14–16 contain CAG repeats; probe 15 is composed purely of CAG repeats (probe sequences in Supplementary file 3 ). Error bars represent standard deviation of 2–3 independent experiments.

Techniques Used: End Labeling, Standard Deviation

Related Articles

Centrifugation:

Article Title: Neutrophil Extracellular Traps Directly Induce Epithelial and Endothelial Cell Death: A Predominant Role of Histones
Article Snippet: .. Thereafter, NET samples were digested with DNase or MNase or kept undigested followed by centrifugation at 1000× g for 5 min, and the supernatants were collected for elastase activity in the absence or presence of 0.2 mM N-(methoxysuccinyl)-L-alanyl-L-alanyl-L-prolyl-L-valine chloromethyl ketone (Sigma-Aldrich) as an elastase inhibitor. .. NET-protein quantification For NET-protein quantification the Micro-BCA protein assay reagent kit (Pierce, Germany) or the 2D-Quant kit (GE Healthcare) was used.

Isolation:

Article Title: Nucleosomes protect DNA from DNA methylation in vivo and in vitro
Article Snippet: .. Isolation of endogenous nucleosomal DNA by MNase digest Digestion of chromatin from Hela cells (2 × 107 ) was performed with microccal nuclease (MNase Sigma; 100–1000 U) in 3.0 ml permeabilization buffer (15 mM Tris, pH 7.6, 300 mM sucrose, 60 mM KCl, 15 mM NaCl, 4.0 mM CaCl2 , 0.5 mM EGTA, 0.2% NP-40, 0.5 mM ß-mercaptoethanol) for 3 min at 37°C. ..

Footprinting:

Article Title: Persistence of an alternate chromatin structure at silenced loci in the absence of silencers
Article Snippet: .. Footprinting by MNase was performed in NP-40 (Sigma) permeabilized spheroplasts essentially as described by Kent et al . ( ) with the following exceptions. .. Spheroplasting was accomplished by treatment with 1 ml of spheroplasting solution [20% Sorbitol, 0.3–0.5 mg/ml Zymolyase T100 (Seikagaku, Japan), and 0.5 mM β-mercaptoethanol] for 5 min at 30°C.

Incubation:

Article Title: Histone octamer trans-transfer: a signature mechanism of ATP-dependent chromatin remodelling unravelled in wheat nuclear extract
Article Snippet: .. Approximately 0·03 U MNase (Sigma-Aldrich, India) per milligram of DNA was added to the resuspended nuclei and incubated on ice for 2 min with intermittent mixing at an interval of 30 s. Nuclei were centrifuged at 8500 g for 5 min and the pellet was resuspended in nuclei digestion buffer containing 2 m m CaCl2 and the suspension was incubated at 37 °C in a water bath with continuous stirring for a further 2 min. EDTA was added at 0·5 m to stop MNase activity. .. The pellet obtained after centrifugation at 8500 g at 4 °C was resuspended in 15 m m Tris/HCl, pH 7·5, with 0·2 m m EDTA and incubated for 30 min on ice.

Article Title: Synergy between histone deacetylase inhibitors and DNA-damaging agents is mediated by histone deacetylase 2 in colorectal cancer
Article Snippet: .. A total of 0.06 units of MNase (Sigma-Aldrich, UK) was added to each sample and incubated at 15-20°C for 5 minutes. .. The reaction was stopped by the addition of MNase digestion buffer, MNase stop buffer ((0.5 ml) - 5% SDS; 250 mM EDTA), proteinase K and 20% SDS followed by overnight incubation.

Concentration Assay:

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Article Snippet: .. Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM). .. Centrifugation was performed at 20,000 g for 20 min. Supernatants of four MNase digests were combined, and salt concentration was adjusted to 150 mM KCl.

Generated:

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Article Snippet: .. Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM). .. Centrifugation was performed at 20,000 g for 20 min. Supernatants of four MNase digests were combined, and salt concentration was adjusted to 150 mM KCl.

Activity Assay:

Article Title: Neutrophil Extracellular Traps Directly Induce Epithelial and Endothelial Cell Death: A Predominant Role of Histones
Article Snippet: .. Thereafter, NET samples were digested with DNase or MNase or kept undigested followed by centrifugation at 1000× g for 5 min, and the supernatants were collected for elastase activity in the absence or presence of 0.2 mM N-(methoxysuccinyl)-L-alanyl-L-alanyl-L-prolyl-L-valine chloromethyl ketone (Sigma-Aldrich) as an elastase inhibitor. .. NET-protein quantification For NET-protein quantification the Micro-BCA protein assay reagent kit (Pierce, Germany) or the 2D-Quant kit (GE Healthcare) was used.

Article Title: Histone octamer trans-transfer: a signature mechanism of ATP-dependent chromatin remodelling unravelled in wheat nuclear extract
Article Snippet: .. Approximately 0·03 U MNase (Sigma-Aldrich, India) per milligram of DNA was added to the resuspended nuclei and incubated on ice for 2 min with intermittent mixing at an interval of 30 s. Nuclei were centrifuged at 8500 g for 5 min and the pellet was resuspended in nuclei digestion buffer containing 2 m m CaCl2 and the suspension was incubated at 37 °C in a water bath with continuous stirring for a further 2 min. EDTA was added at 0·5 m to stop MNase activity. .. The pellet obtained after centrifugation at 8500 g at 4 °C was resuspended in 15 m m Tris/HCl, pH 7·5, with 0·2 m m EDTA and incubated for 30 min on ice.

Protease Inhibitor:

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Article Snippet: .. Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM). .. Centrifugation was performed at 20,000 g for 20 min. Supernatants of four MNase digests were combined, and salt concentration was adjusted to 150 mM KCl.

Derivative Assay:

Article Title: Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription
Article Snippet: .. Spheroplasts derived from 100-mL cultures were treated with either 0.5 U (underdigested) or 2 U (overdigested) of MNase (Sigma) for 45 min at 37°C. .. Purified and precipitated DNA was sequenced using the paired-end TruSeq protocol (Illumina).

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    85
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    Scheme of the EBV genome and experimental design for the analyses of pre-RC and SNS zones as well as mapping of MR profiles. (A) Scheme of the circular EBV genome. In addition to the latent origins oriP (blue box) and the 14-kbp “Raji origin” (blue line), the lytic origin (oriLyt) is shown. The latent EBV nuclear antigens 1, 2, 3A–C, EBNA-LP genes (turquoise), and LMP1 and -2 (purple) are depicted, including their transcripts and promoters. The EBER1 and -2 and the miRNAs regions (BART and BARF) are indicated (green lines). The Raji genome harbors two deletions (red Δ, nt 86,000–89,000 and 163,978–166,635). These regions do not produce array signals in comparison to the reference strain type I used for the design of the EBV microarray. (B) Chart of the experimental set up to map pre-RC zones (left), <t>MNase</t> profiles (central), and SNS <t>DNA</t> (right). (C) Cell cycle phases of logarithmically growing Raji cells were separated by centrifugal elutriation. The DNA content of the different fractions was determined by FACS analysis (top, I–VI). The FACS profiles of one out of three experiments are shown. The quality of coprecipitated DNA was determined by quantitative PCR. The histograms show the mean values of three independent Orc2 (bottom left) and Mcm3 (bottom right) immunoprecipitations. The enrichments of Orc2 (red bars) and Mcm3 enrichments (blue bars) at the DS region are shown. The black bars indicate the enrichments of Orc2 and Mcm3 to a reference site. Error bars indicate mean ± SEM.
    Mnase Digested Chip Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Millipore mnase
    Scheme of the EBV genome and experimental design for the analyses of pre-RC and SNS zones as well as mapping of MR profiles. (A) Scheme of the circular EBV genome. In addition to the latent origins oriP (blue box) and the 14-kbp “Raji origin” (blue line), the lytic origin (oriLyt) is shown. The latent EBV nuclear antigens 1, 2, 3A–C, EBNA-LP genes (turquoise), and LMP1 and -2 (purple) are depicted, including their transcripts and promoters. The EBER1 and -2 and the miRNAs regions (BART and BARF) are indicated (green lines). The Raji genome harbors two deletions (red Δ, nt 86,000–89,000 and 163,978–166,635). These regions do not produce array signals in comparison to the reference strain type I used for the design of the EBV microarray. (B) Chart of the experimental set up to map pre-RC zones (left), <t>MNase</t> profiles (central), and SNS <t>DNA</t> (right). (C) Cell cycle phases of logarithmically growing Raji cells were separated by centrifugal elutriation. The DNA content of the different fractions was determined by FACS analysis (top, I–VI). The FACS profiles of one out of three experiments are shown. The quality of coprecipitated DNA was determined by quantitative PCR. The histograms show the mean values of three independent Orc2 (bottom left) and Mcm3 (bottom right) immunoprecipitations. The enrichments of Orc2 (red bars) and Mcm3 enrichments (blue bars) at the DS region are shown. The black bars indicate the enrichments of Orc2 and Mcm3 to a reference site. Error bars indicate mean ± SEM.
    Mnase, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mnase/product/Millipore
    Average 98 stars, based on 93 article reviews
    Price from $9.99 to $1999.99
    mnase - by Bioz Stars, 2020-05
    98/100 stars
      Buy from Supplier

    Image Search Results


    Scheme of the EBV genome and experimental design for the analyses of pre-RC and SNS zones as well as mapping of MR profiles. (A) Scheme of the circular EBV genome. In addition to the latent origins oriP (blue box) and the 14-kbp “Raji origin” (blue line), the lytic origin (oriLyt) is shown. The latent EBV nuclear antigens 1, 2, 3A–C, EBNA-LP genes (turquoise), and LMP1 and -2 (purple) are depicted, including their transcripts and promoters. The EBER1 and -2 and the miRNAs regions (BART and BARF) are indicated (green lines). The Raji genome harbors two deletions (red Δ, nt 86,000–89,000 and 163,978–166,635). These regions do not produce array signals in comparison to the reference strain type I used for the design of the EBV microarray. (B) Chart of the experimental set up to map pre-RC zones (left), MNase profiles (central), and SNS DNA (right). (C) Cell cycle phases of logarithmically growing Raji cells were separated by centrifugal elutriation. The DNA content of the different fractions was determined by FACS analysis (top, I–VI). The FACS profiles of one out of three experiments are shown. The quality of coprecipitated DNA was determined by quantitative PCR. The histograms show the mean values of three independent Orc2 (bottom left) and Mcm3 (bottom right) immunoprecipitations. The enrichments of Orc2 (red bars) and Mcm3 enrichments (blue bars) at the DS region are shown. The black bars indicate the enrichments of Orc2 and Mcm3 to a reference site. Error bars indicate mean ± SEM.

    Journal: The Journal of Cell Biology

    Article Title: Open chromatin structures regulate the efficiencies of pre-RC formation and replication initiation in Epstein-Barr virus

    doi: 10.1083/jcb.201109105

    Figure Lengend Snippet: Scheme of the EBV genome and experimental design for the analyses of pre-RC and SNS zones as well as mapping of MR profiles. (A) Scheme of the circular EBV genome. In addition to the latent origins oriP (blue box) and the 14-kbp “Raji origin” (blue line), the lytic origin (oriLyt) is shown. The latent EBV nuclear antigens 1, 2, 3A–C, EBNA-LP genes (turquoise), and LMP1 and -2 (purple) are depicted, including their transcripts and promoters. The EBER1 and -2 and the miRNAs regions (BART and BARF) are indicated (green lines). The Raji genome harbors two deletions (red Δ, nt 86,000–89,000 and 163,978–166,635). These regions do not produce array signals in comparison to the reference strain type I used for the design of the EBV microarray. (B) Chart of the experimental set up to map pre-RC zones (left), MNase profiles (central), and SNS DNA (right). (C) Cell cycle phases of logarithmically growing Raji cells were separated by centrifugal elutriation. The DNA content of the different fractions was determined by FACS analysis (top, I–VI). The FACS profiles of one out of three experiments are shown. The quality of coprecipitated DNA was determined by quantitative PCR. The histograms show the mean values of three independent Orc2 (bottom left) and Mcm3 (bottom right) immunoprecipitations. The enrichments of Orc2 (red bars) and Mcm3 enrichments (blue bars) at the DS region are shown. The black bars indicate the enrichments of Orc2 and Mcm3 to a reference site. Error bars indicate mean ± SEM.

    Article Snippet: Southern blotting 500 ng of sonicated and MNase-digested ChIP DNA or nascent strand DNA were separated on a 1.0% TAE gel and transferred to membrane (Immobilon Ny+; EMD Millipore).

    Techniques: Microarray, FACS, Real-time Polymerase Chain Reaction