Extended Data Fig. 6: Analysis of Nucleosome Deacetylase Activity for Fluorescein-labeled WT LHC and Y391K LHC.
From: Uncoupling histone modification crosstalk by engineering lysine demethylase LSD1
(a) Assessment of WT LHC deacetylase activity against H3K9ac nucleosomes. WT LHC concentrations of 90 nM (top) and 120 nM (bottom) were subjected to a 120-minute incubation with 100 nM of 185 bp H3K9ac nucleosomes, and variations in H3K9ac levels were monitored. (b) Evaluation of Y391K LHC deacetylase activity targeting H3K9ac-marked nucleosomes. Similar to (a), 90 nM (top) and 120 nM (bottom) Y391K LHC was incubated with 100 nM of 185 bp H3K9ac nucleosomes, and changes in H3K9ac levels were tracked over 120 minutes. Panels (c) and (d) illustrate the relative intensities obtained from (a) and (b), respectively, subjected to fitting into an exponential decay equation that includes constraints of Y0 at 1 and plateau at 0. (e) V/[E] (min−1) values from (a) and (b) were extrapolated (mean ± SEM). Anti-H3 blot at each time point from every other replicate was shown as a representative loading control.
