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Extranucleosomal DNA enhances the activity of the LSD1/CoREST histone demethylase complex.

Kim SA, Chatterjee N, Jennings MJ, Bartholomew B, Tan S - Nucleic Acids Res. (2015)

Bottom Line: Our studies of LSD1/CoREST's enzyme activity and nucleosome binding show that extranucleosomal DNA dramatically enhances the activity of LSD1/CoREST, and that LSD1/CoREST binds to the nucleosome as a 1:1 complex.Our photocrosslinking experiments further indicate both LSD1 and CoREST subunits are in close contact with DNA around the nucleosome dyad as well as extranucleosomal DNA.Our results suggest that the LSD1/CoREST interacts with extranucleosomal DNA when it productively engages its nucleosome substrate.

View Article: PubMed Central - PubMed

Affiliation: Center for Eukaryotic Gene Regulation, Department of Biochemistry & Molecular Biology, 108 Althouse Laboratory, The Pennsylvania State University, University Park, PA 16802-1014, USA.

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Fluorescence positions on the nucleosome affected by LSD1/CoREST binding. Oregon Green 488 was conjugated site-specifically to histones engineered with unique cysteines, reconstituted with appropriate histones and Widom 601 147 bp DNA and used in fluorescence quenching nucleosome binding experiments. The results are mapped on the space filling representation of the 1.9 Å crystal structure of the nucleosome core particle (PDB code 1KX5). Nucleosomes labeled on H2B K20, H3 A21 and H3 K27 showed >15% fluorescence quenching upon LSD1/CoREST binding (side chains shown in red), nucleosome labeled on H2A T10, H2A D72 and H2B S120 produced 10–15% fluorescence quenching (purple residues) while nucleosomes labeled on H2A E91, H2A S113 and H4 Q27 produced less than 10% fluorescence quenching (blue residues). The four H2A residues in the nucleosome acidic patch, E61, E64, D90 and E92, are shown in yellow. Figure prepared using PyMOL molecular graphics software (37).
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Figure 2: Fluorescence positions on the nucleosome affected by LSD1/CoREST binding. Oregon Green 488 was conjugated site-specifically to histones engineered with unique cysteines, reconstituted with appropriate histones and Widom 601 147 bp DNA and used in fluorescence quenching nucleosome binding experiments. The results are mapped on the space filling representation of the 1.9 Å crystal structure of the nucleosome core particle (PDB code 1KX5). Nucleosomes labeled on H2B K20, H3 A21 and H3 K27 showed >15% fluorescence quenching upon LSD1/CoREST binding (side chains shown in red), nucleosome labeled on H2A T10, H2A D72 and H2B S120 produced 10–15% fluorescence quenching (purple residues) while nucleosomes labeled on H2A E91, H2A S113 and H4 Q27 produced less than 10% fluorescence quenching (blue residues). The four H2A residues in the nucleosome acidic patch, E61, E64, D90 and E92, are shown in yellow. Figure prepared using PyMOL molecular graphics software (37).

Mentions: To scout for suitable nucleosome positions, we prepared histones containing Cys substitution at nine histone positions across the nucleosome surface: on H2A Thr10, Asp72, Glu91, Ser113; on H2B Lys20, Ser120, H3 Ala21, Lys27; and on H4 Gln27 (Figure 2). The Oregon Green 488 maleimide was conjugated to the individual Cys mutant histone protein, and each fluorescently labeled histone was then reconstituted into recombinant nucleosomes. We tested these fluorescently labeled nucleosomes in binding experiments with LSD1/CoREST complex at 50 mM NaCl and found little or no (<10%) change in fluorescence for nucleosomes labeled on H2A E91C, H2A S113C and H4 Q27C (data not shown), suggesting that these positions are either located far away from LSD1/CoREST or alternatively, that fluorescent labeling at these positions inhibits binding of LSD1/CoREST to the nucleosome. Nucleosomes labeled on H2A T10C, H2A D72C and H2B S120C produced measurable fluorescence changes (between 10 and 15%) when mixed with LSD1/CoREST complex. However, incubation of LSD1/CoREST with nucleosomes labeled at H2B K20C, H3 A21C and H3 K27C resulted in large changes (>15%) in fluorescence. The fluorescent changes for nucleosomes labeled at H3 A21C and H3 K27C were particularly dramatic, with changes of 30–60% detected (data not shown), more than the minimum of 10% recommended for quantitative studies (25).


Extranucleosomal DNA enhances the activity of the LSD1/CoREST histone demethylase complex.

Kim SA, Chatterjee N, Jennings MJ, Bartholomew B, Tan S - Nucleic Acids Res. (2015)

Fluorescence positions on the nucleosome affected by LSD1/CoREST binding. Oregon Green 488 was conjugated site-specifically to histones engineered with unique cysteines, reconstituted with appropriate histones and Widom 601 147 bp DNA and used in fluorescence quenching nucleosome binding experiments. The results are mapped on the space filling representation of the 1.9 Å crystal structure of the nucleosome core particle (PDB code 1KX5). Nucleosomes labeled on H2B K20, H3 A21 and H3 K27 showed >15% fluorescence quenching upon LSD1/CoREST binding (side chains shown in red), nucleosome labeled on H2A T10, H2A D72 and H2B S120 produced 10–15% fluorescence quenching (purple residues) while nucleosomes labeled on H2A E91, H2A S113 and H4 Q27 produced less than 10% fluorescence quenching (blue residues). The four H2A residues in the nucleosome acidic patch, E61, E64, D90 and E92, are shown in yellow. Figure prepared using PyMOL molecular graphics software (37).
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Figure 2: Fluorescence positions on the nucleosome affected by LSD1/CoREST binding. Oregon Green 488 was conjugated site-specifically to histones engineered with unique cysteines, reconstituted with appropriate histones and Widom 601 147 bp DNA and used in fluorescence quenching nucleosome binding experiments. The results are mapped on the space filling representation of the 1.9 Å crystal structure of the nucleosome core particle (PDB code 1KX5). Nucleosomes labeled on H2B K20, H3 A21 and H3 K27 showed >15% fluorescence quenching upon LSD1/CoREST binding (side chains shown in red), nucleosome labeled on H2A T10, H2A D72 and H2B S120 produced 10–15% fluorescence quenching (purple residues) while nucleosomes labeled on H2A E91, H2A S113 and H4 Q27 produced less than 10% fluorescence quenching (blue residues). The four H2A residues in the nucleosome acidic patch, E61, E64, D90 and E92, are shown in yellow. Figure prepared using PyMOL molecular graphics software (37).
Mentions: To scout for suitable nucleosome positions, we prepared histones containing Cys substitution at nine histone positions across the nucleosome surface: on H2A Thr10, Asp72, Glu91, Ser113; on H2B Lys20, Ser120, H3 Ala21, Lys27; and on H4 Gln27 (Figure 2). The Oregon Green 488 maleimide was conjugated to the individual Cys mutant histone protein, and each fluorescently labeled histone was then reconstituted into recombinant nucleosomes. We tested these fluorescently labeled nucleosomes in binding experiments with LSD1/CoREST complex at 50 mM NaCl and found little or no (<10%) change in fluorescence for nucleosomes labeled on H2A E91C, H2A S113C and H4 Q27C (data not shown), suggesting that these positions are either located far away from LSD1/CoREST or alternatively, that fluorescent labeling at these positions inhibits binding of LSD1/CoREST to the nucleosome. Nucleosomes labeled on H2A T10C, H2A D72C and H2B S120C produced measurable fluorescence changes (between 10 and 15%) when mixed with LSD1/CoREST complex. However, incubation of LSD1/CoREST with nucleosomes labeled at H2B K20C, H3 A21C and H3 K27C resulted in large changes (>15%) in fluorescence. The fluorescent changes for nucleosomes labeled at H3 A21C and H3 K27C were particularly dramatic, with changes of 30–60% detected (data not shown), more than the minimum of 10% recommended for quantitative studies (25).

Bottom Line: Our studies of LSD1/CoREST's enzyme activity and nucleosome binding show that extranucleosomal DNA dramatically enhances the activity of LSD1/CoREST, and that LSD1/CoREST binds to the nucleosome as a 1:1 complex.Our photocrosslinking experiments further indicate both LSD1 and CoREST subunits are in close contact with DNA around the nucleosome dyad as well as extranucleosomal DNA.Our results suggest that the LSD1/CoREST interacts with extranucleosomal DNA when it productively engages its nucleosome substrate.

View Article: PubMed Central - PubMed

Affiliation: Center for Eukaryotic Gene Regulation, Department of Biochemistry & Molecular Biology, 108 Althouse Laboratory, The Pennsylvania State University, University Park, PA 16802-1014, USA.

Show MeSH