Supplementary Figure 6: LSD1n removes H4K20 methylation in vivo
From: LSD1n is an H4K20 demethylase regulating memory formation via transcriptional elongation control
a) LSD1n does not remove H3K4me1 in vivo. Histogram plots of normalized ChIP-seq tag intensities of H3K4me1 on LSD1n-binding sites at promoters and enhancers of neuronal activity-regulated genes in WT and LSD1n KO cortical neurons after KCl treatment (1 hour);
b) LSD1n does not remove H3K9me2 in vivo. Histogram plots of normalized ChIP-seq tag intensities of H3K9me2 on LSD1n-binding sites at promoters and enhancers of neuronal activity-regulated genes in WT and LSD1n KO cortical after upon KCl treatment (1 hour);
c) LSD1n does not remove H3K36me3 in vivo. Histogram plots of normalized ChIP-seq tag intensities of H3K36me3 on LSD1n-binding sites at promoters and enhancers of neuronal activity-regulated genes in WT and LSD1n KO cortical neurons after KCl treatment (1 hour);
d) LSD1n removes H4K20me1 in vivo. Histogram plots of normalized ChIP-seq tag intensities of H4K20me1 on LSD1n-binding sites at promoters and enhancers of neuronal activity-regulated genes in WT and LSD1n KO cortical neurons after KCl treatment (1 hour);
e) ChIP-qPCR analysis of H3K4me2 level on Npas4 and Arc promoters in WT and LSD1n KO cortical neurons after KCl treatment (1 hour). Data are shown as mean ± SD; N.S. = non statistically significant (p=0.904 Npas4, p=0.494 Arc, n=4 technical replicates from pool of 8-12 embryos; unpaired t-test);
f) ChIP-qPCR analysis of H3K9me2 level on Npas4 and Arc promoters in WT and LSD1n KO cortical neurons after KCl treatment (1 hour). Data are shown as mean ± SD; N.S. = non statistically significant (p=0.247 Npas4, p=0.019 Arc, n=4 technical from pool of 8-12 embryos, unpaired t-test);
g) ChIP-qPCR analysis of H3K79me2 level on Npas4 and Arc promoters in WT and LSD1n KO cortical neurons after KCl treatment (1 hour). Data are shown as mean ± SD; N.S. = non statistically significant (p=0.363 Npas4, p=0.014 Arc, n=4 technical from pool of 8-12 embryos, unpaired t-test);
h) Increased H4K20 methylation in LSD1n KO cortical neurons measured by Western blot. The amount of protein is quantified using ImageJ software; the 1x control lane has an arbitrarily assigned value of 100;
i) Analysis of gene expression of LSD1, Phf8 and Svil in mouse cortical neurons by RT-qPCR (normalized by Actb) and by RNA-seqs. Data are shown as mean ± SD or FPKM value (Fragments Per Kilobase Of Exon Per Million Fragments Mapped);
j) Phf8 knockdown efficiency in cortical neurons using shRNA is assessed by RT-qPCR. Data are shown as mean ± SEM; * = P-value<0.01 (p=0.0074 on minus KCl condition; p=0.0052 on plus KCl condition; n=3 biological replicates/condition; unpaired t-test);
k) Neuronal activity-regulated gene expression is not significantly affected upon Phf8 knockdown. Gene expression of Arc, Btg2, Cyr61, Egr3, Npas4 and Pcsk1 in control or Phf8 deficient cortical neurons is measured by RT-qPCR. Values are normalized against Actb. Data are shown as mean ± SEM; N.S. = non statistically significant (p=0.4562 Arc; p=0.3708 Btg2; p=0.1894 Cyr61; p=0.3875 Egr3; p=0.2828 Npas4; p=0.3663 Pcsk1; n=3 biological replicates/condition; unpaired t-test).
