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. 2018 Jun 5;7:e37231. doi: 10.7554/eLife.37231

Figure 2. LHC purity and its demethylase activity with H3 tail peptide substrate.

(A) Coomassie-stained SDS-PAGE of the purified ternary CoREST complex LHC. (B) Demethylation of 100 μM H3K4me2 H3 tail peptide (aa1-21) peptide by LHC (130 nM) exhibits a bi-phasic activity. In the presence of 100 μM InsP6, the first phase becomes longer. In contrast, the demethylation by GST-LSD1 (88 nM) exhibits a linear activity that best matches the second phase of LHC as shown in panel C. The y-axis is shown as [Product]/[Enzyme]. (C) Summary of the steady-state kinetic parameters of peptide demethylation by LHC and GST-LSD1. (p<0.05 for Km and p<0.005 for kcat of the first phase vs. the second phase); (n = 3 for all measurements); kinetic parameters shown are ± S.E.M. (D) DNA accelerates the peptide demethylation by LHC (50 nM) in a dose dependent manner. (E) BHC80 modulates the peptide demethylation by LHC (100 nM), altering the bi-phasic activity (>100 nM).

Figure 2.

Figure 2—figure supplement 1. Non-linear curve fitting to the Michaelis–Menten equation was used to calculate the observed Km and kcat values of LHC and GST-LSD1.

Figure 2—figure supplement 1.

(n = 3 for all measurements).
Figure 2—figure supplement 2. Size exclusion chromatography analysis of LHC after the demethylase reaction.

Figure 2—figure supplement 2.

(A) Size exclusion chromatography analysis of the demethylation mixture after 20 min reaction at 25°C with LHC (200 nM) and H3 peptide substrate (60 μM). (B) SDS-PAGE analysis of the fractions from size exclusion chromatography. It demonstrated that the complex was still intact after the 20 min demethylation reaction.
Figure 2—figure supplement 3. LHC demethylase activity with H3 tail peptide substrate in the presence of DNA or BHC80.

Figure 2—figure supplement 3.

(A) dsDNA (146 bp, 50 ng/μL) addition did not alter the rate of the demethylation of H3K4me2 21aa peptide (60 μM) by GST-LSD1 (300 nM) (left). dsDNA (146 bp or 4.9 kb, 50 ng/μL) effects on H3K4me2 21aa peptide demethylation by LHC (50 nM) (middle). 60 bp dsDNA (100 ng/μL) did not alter the rate of the demethylation (right). (B) The dose-response relationship of the rate of demethylation of H3K4me2 21aa peptide (60 µM) by LHC (50 nM) vs DNA concentration. The estimated EC50 is shown. (C) Effects of BHC80 (aa331-430) on LHC (100 nM) catalyzed demethylation of H3K4me2 21aa peptide (60 µM); (n = 2 for all measurements).