Supplementary Figure 1: LSD1n removes H4K20 methylation in vitro
From: LSD1n is an H4K20 demethylase regulating memory formation via transcriptional elongation control
a) Diagram of LSD1 locus, revealing two alternatively-spliced exons: E2a and E8a;
b) RT-PCR detecting alternatively spliced isoforms of LSD1 in mouse embryonic stem cells and Neuro2A cell lines. LSD1 E8a is induced upon RA-induced differentiation of mouse embryonic stem cells;
c) Amino acid sequence showing the alternative splicing events of LSD1 identified in mammals, turtles and fishes;
d) Coomassie blue staining of different isoforms of LSD1 recombinant proteins obtained by two-step affinity purification;
e) In vitro histone demethylase assay on core histones substrates using control (without enzyme), LSD1 WT, E8A, E2A or E2A&8A recombinant proteins in the presence of recombinant CoREST protein. Western blot analysis of histone H3 and H4 methylations revealing the removal of H4K20 methylation specifically by E8A or E2&8A. The amount of specific modification is quantified using ImageJ software; the 2x control lane has an arbitrarily assigned value of 100;
f) In vitro histone demethylase assay on core histones substrates using LSD1n or LSD1m (K685A) mutant recombinant proteins. Western blot analysis of histone H3 and H4 methylations revealing the removal of H4K20 methylation specifically by LSD1n but not by LSD1m mutant. The amount of specific modification is quantified using ImageJ software; the first control lane has an arbitrarily assigned value of 100;
g) In vitro histone demethylase assay on core histones substrates using LSD1n or LSD1m (K685A) mutant recombinant proteins. Western blot analysis of histone H3 and H4 methylations revealing the removal of H4K20 methylation specifically by LSD1n but not by LSD1m mutant. The amount of specific modification is quantified using ImageJ software; the first control lane has an arbitrarily assigned value of 100.
