nucleosome  (New England Biolabs)


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    Structured Review

    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 .
    Nucleosome, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    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- 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

    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

    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, Polyacrylamide Gel Electrophoresis, Staining

    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

    Stability and dynamics of H2B K34ub nucleosomes
    Figure Legend Snippet: Stability and dynamics of H2B K34ub nucleosomes

    Techniques Used:

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    New England Biolabs di nucleosome dna bands
    Arrangement of nucleosomes around neocentromere (A) Top, ideogram depicting HSA3 and Neo3 chromosomes with enlargement of 3 Mb region around the neocentromere (marked by CENP-C). Bottom, genomic location of the capture baits used to enrich for 1.5 Mb of neocentromeric region. (B) Agarose gel electrophoresis of DFF digested nuclei isolated from HSA3 and Neo3 containing cells. Mono and <t>di-nucleosome</t> fragments were excised, and the <t>DNA</t> extracted by β-agarase treatment. (C) Agarose gel electrophoresis of purified mono- and di-nucleosomes fragments used for nucleosome mapping. (D) Size distribution of mono and di nucleosomes (bp) isolated from HSA3 and Neo3 cells for 1.5 Mb around the region corresponding to the neocentromere. (E) Mono nucleosome size distribution in 10 kb windows across the 1.5 Mb captured domain (neocentromere marked in blue). ( F) Left, mono nucleosome size (median) in 1 kb windows across the 1.5 Mb captured domain and focussed region covering the neocentromere (marked in blue). Right, variance (interquartile range) in mono nucleosome size in 1 kb windows across the 1.5 Mb captured domain and focussed region covering the neocentromere (marked in blue). (G) Nucleosome dyad coverage on the HSA3 and Neo3 chromosome at the neocentromere domain (blue) with enlarged region (below) (H) Autocorrelation of nucleosome dyad coverage at left flank, centromere core and right flank for different lag (bp).
    Di Nucleosome Dna Bands, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Arrangement of nucleosomes around neocentromere (A) Top, ideogram depicting HSA3 and Neo3 chromosomes with enlargement of 3 Mb region around the neocentromere (marked by CENP-C). Bottom, genomic location of the capture baits used to enrich for 1.5 Mb of neocentromeric region. (B) Agarose gel electrophoresis of DFF digested nuclei isolated from HSA3 and Neo3 containing cells. Mono and di-nucleosome fragments were excised, and the DNA extracted by β-agarase treatment. (C) Agarose gel electrophoresis of purified mono- and di-nucleosomes fragments used for nucleosome mapping. (D) Size distribution of mono and di nucleosomes (bp) isolated from HSA3 and Neo3 cells for 1.5 Mb around the region corresponding to the neocentromere. (E) Mono nucleosome size distribution in 10 kb windows across the 1.5 Mb captured domain (neocentromere marked in blue). ( F) Left, mono nucleosome size (median) in 1 kb windows across the 1.5 Mb captured domain and focussed region covering the neocentromere (marked in blue). Right, variance (interquartile range) in mono nucleosome size in 1 kb windows across the 1.5 Mb captured domain and focussed region covering the neocentromere (marked in blue). (G) Nucleosome dyad coverage on the HSA3 and Neo3 chromosome at the neocentromere domain (blue) with enlarged region (below) (H) Autocorrelation of nucleosome dyad coverage at left flank, centromere core and right flank for different lag (bp).

    Journal: bioRxiv

    Article Title: Centromere formation remodels chromatin fibre structure

    doi: 10.1101/2021.08.01.454615

    Figure Lengend Snippet: Arrangement of nucleosomes around neocentromere (A) Top, ideogram depicting HSA3 and Neo3 chromosomes with enlargement of 3 Mb region around the neocentromere (marked by CENP-C). Bottom, genomic location of the capture baits used to enrich for 1.5 Mb of neocentromeric region. (B) Agarose gel electrophoresis of DFF digested nuclei isolated from HSA3 and Neo3 containing cells. Mono and di-nucleosome fragments were excised, and the DNA extracted by β-agarase treatment. (C) Agarose gel electrophoresis of purified mono- and di-nucleosomes fragments used for nucleosome mapping. (D) Size distribution of mono and di nucleosomes (bp) isolated from HSA3 and Neo3 cells for 1.5 Mb around the region corresponding to the neocentromere. (E) Mono nucleosome size distribution in 10 kb windows across the 1.5 Mb captured domain (neocentromere marked in blue). ( F) Left, mono nucleosome size (median) in 1 kb windows across the 1.5 Mb captured domain and focussed region covering the neocentromere (marked in blue). Right, variance (interquartile range) in mono nucleosome size in 1 kb windows across the 1.5 Mb captured domain and focussed region covering the neocentromere (marked in blue). (G) Nucleosome dyad coverage on the HSA3 and Neo3 chromosome at the neocentromere domain (blue) with enlarged region (below) (H) Autocorrelation of nucleosome dyad coverage at left flank, centromere core and right flank for different lag (bp).

    Article Snippet: Mono and di-nucleosome DNA bands were excised from the gel and purified by β-agarase (NEB) digestion followed by phenol/chloroform extraction and ethanol precipitation.

    Techniques: Agarose Gel Electrophoresis, Isolation, Purification

    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- 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

    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

    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, Polyacrylamide Gel Electrophoresis, Staining

    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

    Stability and dynamics of H2B K34ub nucleosomes

    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 and dynamics of H2B K34ub nucleosomes

    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:

    Single-molecule analysis shows that Cse4-nucleosomes have weakened histone–DNA interactions in the peripheral regions. Nucleosome disruption signatures under a constant loading rate (8 pN s −1 ). Representative traces are shown for canonical Sc-nucleosomes (left panel) and Cse4-nucleosomes (right panel), where each colour represents data obtained from a single-nucleosomal DNA molecule. Interaction regions are defined according to the crystal structure 27 as super-helical location (SHL) −2.5 to +2.5 (H3/H4 tetramer interaction region) and SHL ±3.5 to ±6.5 (H2A/H2B dimer interaction region). Within the SHL −2.5 to +2.5 region the maximum disruption forces observed are similar between Sc- and Cse4-nucleosomes. Cse4-nucleosomes have significantly weaker interactions throughout the peripheral regions (SHL ±3.5 to ±6.5). A quantitative comparison of the peak forces measured within each interaction region is shown (centre panel). SHL ±3.5 to ±6.5 is shown in red bar and SHL −2.5 to +2.5 shown in blue. Error bars represent s.d. See Supplementary Figures S4–6 for additional data.

    Journal: Nature Communications

    Article Title: Structure and Scm3-mediated assembly of budding yeast centromeric nucleosomes

    doi: 10.1038/ncomms1320

    Figure Lengend Snippet: Single-molecule analysis shows that Cse4-nucleosomes have weakened histone–DNA interactions in the peripheral regions. Nucleosome disruption signatures under a constant loading rate (8 pN s −1 ). Representative traces are shown for canonical Sc-nucleosomes (left panel) and Cse4-nucleosomes (right panel), where each colour represents data obtained from a single-nucleosomal DNA molecule. Interaction regions are defined according to the crystal structure 27 as super-helical location (SHL) −2.5 to +2.5 (H3/H4 tetramer interaction region) and SHL ±3.5 to ±6.5 (H2A/H2B dimer interaction region). Within the SHL −2.5 to +2.5 region the maximum disruption forces observed are similar between Sc- and Cse4-nucleosomes. Cse4-nucleosomes have significantly weaker interactions throughout the peripheral regions (SHL ±3.5 to ±6.5). A quantitative comparison of the peak forces measured within each interaction region is shown (centre panel). SHL ±3.5 to ±6.5 is shown in red bar and SHL −2.5 to +2.5 shown in blue. Error bars represent s.d. See Supplementary Figures S4–6 for additional data.

    Article Snippet: The nucleosome samples (reconstituted onto ∼5 μg DNA) were digested with MNase (4,000 gel units; NEB) for the indicated times (in ) and quenched by adding EDTA to final concentration of 50 mM.

    Techniques:

    Cse4-nucleosomes exhibit an extended morphology. ( a ) Normalized SAXS curves of nucleosomes. Cse4 Δ102 -nucleosomes (red) produce a clearly different scattering curve compared with Sc-nucleosomes (magenta) or Xl-nucleosomes (blue). ( b ) Distance distribution functions ( P (r)) of the three nucleosomes shown in a . Xl-nucleosome is shown in blue, Sc-nucleosome in green and Cse4 Δ102 -nucleosomes in red. Differences in the maximum dimensions of the three nucleosomes (see also Supplementary Table S2 ) are obvious. Ab initio envelopes, calculated from the intensity data, are shown in Supplementary Figure S2 . ( c – e ) Comparison of experimental scattering profiles (black) with a scattering profile calculated using FoXS 51 from the Xl-nucleosome crystal structure (1kx5; red). Experimental profiles from Xl-, Sc- and Cse4 Δ102 -nucleosomes are in c – e , respectively. The χ 2 values of the fit between the two curves are shown.

    Journal: Nature Communications

    Article Title: Structure and Scm3-mediated assembly of budding yeast centromeric nucleosomes

    doi: 10.1038/ncomms1320

    Figure Lengend Snippet: Cse4-nucleosomes exhibit an extended morphology. ( a ) Normalized SAXS curves of nucleosomes. Cse4 Δ102 -nucleosomes (red) produce a clearly different scattering curve compared with Sc-nucleosomes (magenta) or Xl-nucleosomes (blue). ( b ) Distance distribution functions ( P (r)) of the three nucleosomes shown in a . Xl-nucleosome is shown in blue, Sc-nucleosome in green and Cse4 Δ102 -nucleosomes in red. Differences in the maximum dimensions of the three nucleosomes (see also Supplementary Table S2 ) are obvious. Ab initio envelopes, calculated from the intensity data, are shown in Supplementary Figure S2 . ( c – e ) Comparison of experimental scattering profiles (black) with a scattering profile calculated using FoXS 51 from the Xl-nucleosome crystal structure (1kx5; red). Experimental profiles from Xl-, Sc- and Cse4 Δ102 -nucleosomes are in c – e , respectively. The χ 2 values of the fit between the two curves are shown.

    Article Snippet: The nucleosome samples (reconstituted onto ∼5 μg DNA) were digested with MNase (4,000 gel units; NEB) for the indicated times (in ) and quenched by adding EDTA to final concentration of 50 mM.

    Techniques:

    Scm3 is not stably incorporated into nucleosome-like particles. ( a ) Two different concentrations of Cse4 Δ127 -octamer (lanes 2 and 3), (Cse4 Δ127 /H4) 2 tetramer (lane 4) and Cse4 Δ127 /H4/Scm3 (lane 5) were reconstituted on 601 DNA (207 bp) by salt dilution and analysed by gel-shift on native 5% PAGE. In lane 5, 'a' and 'b' refer to the lower discrete band and upper multiple bands, respectively. ( b ) Individual bands from gel shown in a were excised and analysed by 16% SDS–PAGE. Lanes 1: control Cse4 Δ127 -nucleosome; 2: Cse4 Δ127 -nucleosome band (from lane 3 in a ); 3: control (Cse4 Δ127 /H4) 2 tetrasome; 4: (Cse4 Δ127 /H4) 2 tetrasome band (lane 4 in a ); 5: control Cse4 Δ127 /H4/Scm3 complex; 6 and 7 correspond to the bands labelled as 'a' and 'b', respectively from lane 5 in a . ( c ) Sucrose density gradient fractionation of Cse4 Δ127 /H4/Scm3 reconstituted on 601, 207 bp DNA. Samples were fractionated in a 5–25% sucrose density gradient. Upper panel: the fractions were analysed on 5% native PAGE and stained with ethidium bromide. Lanes: DNA: 207-bp 601; IN, input sample; 1–13: fractions from low- to high-density sucrose. Lower panel: samples 1–12 shown in the upper panel were analysed by SDS–PAGE stained with SYPRO Ruby (Invitrogen).

    Journal: Nature Communications

    Article Title: Structure and Scm3-mediated assembly of budding yeast centromeric nucleosomes

    doi: 10.1038/ncomms1320

    Figure Lengend Snippet: Scm3 is not stably incorporated into nucleosome-like particles. ( a ) Two different concentrations of Cse4 Δ127 -octamer (lanes 2 and 3), (Cse4 Δ127 /H4) 2 tetramer (lane 4) and Cse4 Δ127 /H4/Scm3 (lane 5) were reconstituted on 601 DNA (207 bp) by salt dilution and analysed by gel-shift on native 5% PAGE. In lane 5, 'a' and 'b' refer to the lower discrete band and upper multiple bands, respectively. ( b ) Individual bands from gel shown in a were excised and analysed by 16% SDS–PAGE. Lanes 1: control Cse4 Δ127 -nucleosome; 2: Cse4 Δ127 -nucleosome band (from lane 3 in a ); 3: control (Cse4 Δ127 /H4) 2 tetrasome; 4: (Cse4 Δ127 /H4) 2 tetrasome band (lane 4 in a ); 5: control Cse4 Δ127 /H4/Scm3 complex; 6 and 7 correspond to the bands labelled as 'a' and 'b', respectively from lane 5 in a . ( c ) Sucrose density gradient fractionation of Cse4 Δ127 /H4/Scm3 reconstituted on 601, 207 bp DNA. Samples were fractionated in a 5–25% sucrose density gradient. Upper panel: the fractions were analysed on 5% native PAGE and stained with ethidium bromide. Lanes: DNA: 207-bp 601; IN, input sample; 1–13: fractions from low- to high-density sucrose. Lower panel: samples 1–12 shown in the upper panel were analysed by SDS–PAGE stained with SYPRO Ruby (Invitrogen).

    Article Snippet: The nucleosome samples (reconstituted onto ∼5 μg DNA) were digested with MNase (4,000 gel units; NEB) for the indicated times (in ) and quenched by adding EDTA to final concentration of 50 mM.

    Techniques: Stable Transfection, Electrophoretic Mobility Shift Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Fractionation, Clear Native PAGE, Staining

    Cse4-nucleosomes do not preferentially assemble on CEN3 DNA. ( a ) Experimental scheme of Cse4- and Sc-nucleosomes reconstitution on 5S, 601 and CEN3 DNA. Nucleosomes were reconstituted under limiting octamer concentration by salt dilution on either a 207-bp atto647-labelled CEN3 or cy3-labelled 601 DNA probe in the presence of an excess of 147-bp 5S DNA and analysed by 5% native PAGE. ( b ) The reconstitution on total DNA (≈5S DNA) was visualized by ethidium bromide staining. Nucleosomes assembled on 601 DNA and CEN3 DNA were detected by scanning for cy3 601 ( c ) and atto647 ( d ), respectively. The ratio of octamer to DNA and the types of histone octamer (Cse4, Sc) used are indicated above the lanes. ( e ) Quantification of nucleosome assembly. The fraction of DNA incorporated into nucleosomes in b – d was calculated by dividing the signal of the shifted band(s) by the sum of free DNA and shifted band signal, and multiplying by 100. We excluded the material in the wells, but added all nucleosome bands. The graph of nucleosomes reconstituted using octamer–DNA ratio of 0.32:1 (Cse4-Nuc, shown in white) and 0.28:1 (Sc-nuc, shown in black) is shown. The mean and standard deviation values from 4 to 5 different experiments were used to plot the graph.

    Journal: Nature Communications

    Article Title: Structure and Scm3-mediated assembly of budding yeast centromeric nucleosomes

    doi: 10.1038/ncomms1320

    Figure Lengend Snippet: Cse4-nucleosomes do not preferentially assemble on CEN3 DNA. ( a ) Experimental scheme of Cse4- and Sc-nucleosomes reconstitution on 5S, 601 and CEN3 DNA. Nucleosomes were reconstituted under limiting octamer concentration by salt dilution on either a 207-bp atto647-labelled CEN3 or cy3-labelled 601 DNA probe in the presence of an excess of 147-bp 5S DNA and analysed by 5% native PAGE. ( b ) The reconstitution on total DNA (≈5S DNA) was visualized by ethidium bromide staining. Nucleosomes assembled on 601 DNA and CEN3 DNA were detected by scanning for cy3 601 ( c ) and atto647 ( d ), respectively. The ratio of octamer to DNA and the types of histone octamer (Cse4, Sc) used are indicated above the lanes. ( e ) Quantification of nucleosome assembly. The fraction of DNA incorporated into nucleosomes in b – d was calculated by dividing the signal of the shifted band(s) by the sum of free DNA and shifted band signal, and multiplying by 100. We excluded the material in the wells, but added all nucleosome bands. The graph of nucleosomes reconstituted using octamer–DNA ratio of 0.32:1 (Cse4-Nuc, shown in white) and 0.28:1 (Sc-nuc, shown in black) is shown. The mean and standard deviation values from 4 to 5 different experiments were used to plot the graph.

    Article Snippet: The nucleosome samples (reconstituted onto ∼5 μg DNA) were digested with MNase (4,000 gel units; NEB) for the indicated times (in ) and quenched by adding EDTA to final concentration of 50 mM.

    Techniques: Concentration Assay, Clear Native PAGE, Staining, Standard Deviation

    Scm3 is a Cse4-specific chaperone that assembles nucleosomes with a negative supercoil. ( a ) As illustrated in the schematic, Cse4- or Sc-nucleosomes were assembled on relaxed circular plasmid DNA using full-length Scm3 or truncated Scm3 63–189 as a histone chaperone; nucleosome assembly was analysed by plasmid supercoiling assay. ( b , c ) Deproteinized DNA samples were analysed on 1% agarose gel in the absence and presence of chloroquine (as indicated), and visualized by SYBR Gold staining. ( b ) Full-length Scm3 and Scm3 63–189 assemble nucleosomes equally well. Lanes 1 and 2: negatively supercoiled or relaxed pCR2.1-(CEN3+CEN6) 4 ; lane 3: Cse4-octamer (Oct) without histone chaperone; lanes 4 and 5: Cse4-octamer with 25 pmol Scm3 63–189 and full-length Scm3, respectively. ( c ) Scm3 is specific for the Cse4-octamer: Cse4- or Sc-nucleosomes were assembled and analysed as described in a . Lanes 1 and 2: negatively supercoiled and relaxed plasmid, respectively; lanes 3 and 4: Cse4-nucleosome assembled in the presence of 25 and 50 pmol of Scm3 63–189 , respectively; lanes 5 and 6: Sc-nucleosomes assembled in the presence of 25 and 50 pmol Scm3 63–189 , respectively; lane 7: 50 pmol Scm3 63–189 , no histone octamer. ( d ) Cse4-nucleosomes assembled by Scm3 contain all four histones but not Scm3: As diagramed in e , Cse4-nucleosomes were reconstituted on a 561-bp DNA containing three 147-bp 601 nucleosome positioning sequences using Scm3 63–189 as the chromatin assembly factor. The assembled chromatin was purified by sucrose gradient sedimentation. Incremental fractions 13–34 (top to bottom of the sucrose gradient) were analysed by 4% native PAGE and visualized by ethidium bromide (upper panel). Input samples and fractions 2, 4, and incremental fractions13–34 were analysed by SDS–PAGE stained with SYPRO Ruby (Invitrogen; lower panel). Control experiments with Nap1 instead of Scm3 63–189 as the assembly factor and assembly by full-length Scm3, and assembly on 207-bp CEN3 and 5S DNA by Scm3 63–189 are shown in Supplementary Figure S9 . ( e ) Experimental scheme of nucleosome reconstitution for data shown in d .

    Journal: Nature Communications

    Article Title: Structure and Scm3-mediated assembly of budding yeast centromeric nucleosomes

    doi: 10.1038/ncomms1320

    Figure Lengend Snippet: Scm3 is a Cse4-specific chaperone that assembles nucleosomes with a negative supercoil. ( a ) As illustrated in the schematic, Cse4- or Sc-nucleosomes were assembled on relaxed circular plasmid DNA using full-length Scm3 or truncated Scm3 63–189 as a histone chaperone; nucleosome assembly was analysed by plasmid supercoiling assay. ( b , c ) Deproteinized DNA samples were analysed on 1% agarose gel in the absence and presence of chloroquine (as indicated), and visualized by SYBR Gold staining. ( b ) Full-length Scm3 and Scm3 63–189 assemble nucleosomes equally well. Lanes 1 and 2: negatively supercoiled or relaxed pCR2.1-(CEN3+CEN6) 4 ; lane 3: Cse4-octamer (Oct) without histone chaperone; lanes 4 and 5: Cse4-octamer with 25 pmol Scm3 63–189 and full-length Scm3, respectively. ( c ) Scm3 is specific for the Cse4-octamer: Cse4- or Sc-nucleosomes were assembled and analysed as described in a . Lanes 1 and 2: negatively supercoiled and relaxed plasmid, respectively; lanes 3 and 4: Cse4-nucleosome assembled in the presence of 25 and 50 pmol of Scm3 63–189 , respectively; lanes 5 and 6: Sc-nucleosomes assembled in the presence of 25 and 50 pmol Scm3 63–189 , respectively; lane 7: 50 pmol Scm3 63–189 , no histone octamer. ( d ) Cse4-nucleosomes assembled by Scm3 contain all four histones but not Scm3: As diagramed in e , Cse4-nucleosomes were reconstituted on a 561-bp DNA containing three 147-bp 601 nucleosome positioning sequences using Scm3 63–189 as the chromatin assembly factor. The assembled chromatin was purified by sucrose gradient sedimentation. Incremental fractions 13–34 (top to bottom of the sucrose gradient) were analysed by 4% native PAGE and visualized by ethidium bromide (upper panel). Input samples and fractions 2, 4, and incremental fractions13–34 were analysed by SDS–PAGE stained with SYPRO Ruby (Invitrogen; lower panel). Control experiments with Nap1 instead of Scm3 63–189 as the assembly factor and assembly by full-length Scm3, and assembly on 207-bp CEN3 and 5S DNA by Scm3 63–189 are shown in Supplementary Figure S9 . ( e ) Experimental scheme of nucleosome reconstitution for data shown in d .

    Article Snippet: The nucleosome samples (reconstituted onto ∼5 μg DNA) were digested with MNase (4,000 gel units; NEB) for the indicated times (in ) and quenched by adding EDTA to final concentration of 50 mM.

    Techniques: Plasmid Preparation, Agarose Gel Electrophoresis, Staining, Purification, Sedimentation, Clear Native PAGE, SDS Page

    In vitro assembled Cse4-containing nucleosomes exhibit retarded gel mobility and organize DNA in left-handed superhelix. ( a ) Cse4-nucleosomes were reconstituted onto 147-bp DNA segments by salt dilution and analysed by 5% native PAGE followed by ethidium bromide staining. Lane 1: 147-bp α-satellite DNA; lanes 2, 4 and 5: nucleosomes with full-length Cse4, Cse4 Δ102 and Cse4 Δ127 , respectively, assembled on α-sat DNA; lanes 3 and 6: S. cerevisiae (Sc) and Xenopus laevis (Xl) nucleosomes on α-sat DNA, respectively. Lanes 7 and 8: Cse4- and Sc-nucleosomes reconstituted on yeast centromeric DNA (CEN3); lane 9: 147-bp 601 DNA; lanes 10 and 11: Cse4- and Xl-nucleosomes reconstituted on 147-bp 601 DNA. ( b ) Nucleosome bands were eluted from the gel shown in a , lanes 2 and 6, and analysed by SDS–PAGE and Coomassie Blue staining. Lanes 1 and 3: Cse4- and Xl-nucleosome bands; 2 and 4: Cse4- and Xl-octamers; M: protein molecular weight standards. Bands indicated as 'a': yeast H2A; 'b': yeast H2B. ( c – i ) Cse4-nucleosomes induce negative supercoiling: ( c ) Nucleosomes were assembled on a circular pBR322 plasmid using yNap1 in the presence of Topoisomerase I. Deproteinized DNA was analysed on 1.2% agarose gel in the absence (left panel) and presence of 2 μg ml −1 chloroquine (right panel). Lane 1: negatively supercoiled plasmid; lane 2: relaxed plasmid; lanes 3 and 4: Cse4- and Sc-nucleosomes, respectively; lane 5: Cse4 Δ127 /H4/Scm3; lane 6: positively supercoiled pBR322 plasmid (purchased from Inspiralis). ( d – i ) Two-dimensional gel analysis of plasmid supercoiling. ( d ) and ( e ): negatively and positively supercoiled plasmid DNA, respectively, ( f ): samples in d , e combined; plasmid supercoiling induced by Cse4-nucleosomes ( g ), Cse4 Δ127 /H4/Scm3/DNA complex ( h ) and Sc-nucleosomes ( i ). −S and +S refer to negatively and positively supercoiled plasmid DNA. The arrow in g – i indicates wherein the most supercoiled topoisomers run if they were positively supercoiled.

    Journal: Nature Communications

    Article Title: Structure and Scm3-mediated assembly of budding yeast centromeric nucleosomes

    doi: 10.1038/ncomms1320

    Figure Lengend Snippet: In vitro assembled Cse4-containing nucleosomes exhibit retarded gel mobility and organize DNA in left-handed superhelix. ( a ) Cse4-nucleosomes were reconstituted onto 147-bp DNA segments by salt dilution and analysed by 5% native PAGE followed by ethidium bromide staining. Lane 1: 147-bp α-satellite DNA; lanes 2, 4 and 5: nucleosomes with full-length Cse4, Cse4 Δ102 and Cse4 Δ127 , respectively, assembled on α-sat DNA; lanes 3 and 6: S. cerevisiae (Sc) and Xenopus laevis (Xl) nucleosomes on α-sat DNA, respectively. Lanes 7 and 8: Cse4- and Sc-nucleosomes reconstituted on yeast centromeric DNA (CEN3); lane 9: 147-bp 601 DNA; lanes 10 and 11: Cse4- and Xl-nucleosomes reconstituted on 147-bp 601 DNA. ( b ) Nucleosome bands were eluted from the gel shown in a , lanes 2 and 6, and analysed by SDS–PAGE and Coomassie Blue staining. Lanes 1 and 3: Cse4- and Xl-nucleosome bands; 2 and 4: Cse4- and Xl-octamers; M: protein molecular weight standards. Bands indicated as 'a': yeast H2A; 'b': yeast H2B. ( c – i ) Cse4-nucleosomes induce negative supercoiling: ( c ) Nucleosomes were assembled on a circular pBR322 plasmid using yNap1 in the presence of Topoisomerase I. Deproteinized DNA was analysed on 1.2% agarose gel in the absence (left panel) and presence of 2 μg ml −1 chloroquine (right panel). Lane 1: negatively supercoiled plasmid; lane 2: relaxed plasmid; lanes 3 and 4: Cse4- and Sc-nucleosomes, respectively; lane 5: Cse4 Δ127 /H4/Scm3; lane 6: positively supercoiled pBR322 plasmid (purchased from Inspiralis). ( d – i ) Two-dimensional gel analysis of plasmid supercoiling. ( d ) and ( e ): negatively and positively supercoiled plasmid DNA, respectively, ( f ): samples in d , e combined; plasmid supercoiling induced by Cse4-nucleosomes ( g ), Cse4 Δ127 /H4/Scm3/DNA complex ( h ) and Sc-nucleosomes ( i ). −S and +S refer to negatively and positively supercoiled plasmid DNA. The arrow in g – i indicates wherein the most supercoiled topoisomers run if they were positively supercoiled.

    Article Snippet: The nucleosome samples (reconstituted onto ∼5 μg DNA) were digested with MNase (4,000 gel units; NEB) for the indicated times (in ) and quenched by adding EDTA to final concentration of 50 mM.

    Techniques: In Vitro, Clear Native PAGE, Staining, SDS Page, Molecular Weight, Plasmid Preparation, Agarose Gel Electrophoresis, Two-Dimensional Gel Electrophoresis

    Mhrt inhibits chromatin targeting and gene regulation by Brg1 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: Mhrt inhibits chromatin targeting and gene regulation by Brg1 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, Chromatin Immunoprecipitation, Fluorescence, Luciferase, Activity Assay, Plasmid Preparation, Polymerase Chain Reaction, Expressing