Extranucleosomal DNA enhances the activity of the LSD1/CoREST histone demethylase complex.
Bottom Line: We examine here the role of extranucleosomal DNA beyond the nucleosome core particle for LSD1/CoREST function.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.
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
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).
Affiliation: Center for Eukaryotic Gene Regulation, Department of Biochemistry & Molecular Biology, 108 Althouse Laboratory, The Pennsylvania State University, University Park, PA 16802-1014, USA.