Supplementary Figure 4: Both LSD1n and LSD1c occupy gene promoters and enhancers

a) Schematic diagram of the generation of FLAG-LSD1n and FLAG-LSD1c transgenic mice;

b) Western blot analysis of the expression of FLAG-LSD1 upon Cre-mediated recombination, assessed by Western blot using anti-LSD1 or anti-FLAG antibody;

c) LSD1 genome occupancy in cortical neurons is changed after KCl treatment. Heatmaps display of LSD1 (total), FLAG-LSD1c and FLAG-LSD1n genome occupancies before and after KCl-mediated depolarization, centered on LSD1-peaks in resting cortical neurons. Additional 3kb from the center of the peaks is shown;

d) Box-and-whisker plots of LSD1n/LSD1c tag-density ratio of LSD1 binding on promoters and enhancers revealing similar binding intensity for both isoforms;

e) Western blot analysis revealing decreased H4K20 methylation levels in primary cortical neurons derived from FLAG-LSD1n but not FLAG-LSD1c transgenic mice. The amount of protein is quantified using ImageJ software; the first control lane has an arbitrarily assigned value of 100;

f) Top five enriched transcription factor binding motifs by de novo motif analysis of lost LSD1-peaks on enhancers after KCl treatment, including CTCF motif;

g) Top five enriched transcription factor binding motifs by de novo motif analysis of gained LSD1-peaks on enhancers after KCl treatment, including MEF2c motif;

h) Top five enriched transcription factor binding motifs by de novo motif analysis of gained LSD1-peaks on promoters after KCl treatment, including CREB motif;

i) Immuno-precipitation of brain extracts using LSD1 antibody. Western blot analysis of LSD1-interacting proteins: CoREST, MEF2 and CREB;

j) Table showing the number of overlapping peaks in MEF2 and LSD1 ChIP-seqs;

k) Venn diagram showing combined (resting and depolarized neurons) peaks identified in LSD1 and MEF2 ChIP-seqs;

l) Heatmaps display of LSD1 and MEF2 genome occupancies by ChIP-seqs, centered on LSD1-peaks. Additional 3kb from the centers of the peaks is shown;

m) UCSC genome browser image of Arc locus revealing overlapping peaks from MEF2 and LSD1 ChIP-seqs;

n) UCSC genome browser image of Egr1 locus revealing overlapping peaks from MEF2 and LSD1 ChIP-seqs.