Extended Data Fig. 8: Kacme is affected by acetylation and methylation pathways, and in vitro deacetylation of H4 peptides.
From: Acetyl-methyllysine marks chromatin at active transcription start sites
a, Representative western blots of two biological replicates of HEK293T histones treated with A-485 and NU9056 (10 μM for 24 h). b, Bar plot of normalized western blot signal for Kacme in two biological replicates of HEK293T histones treated with A-485 (10 μM for 24 h). Bar plot shows the mean of the individual normalized values. c, Kacme (top) and H3K27ac (bottom) ChIP-qPCR in HEK293T cells after A-485 treatment (10 μM for 24 h). Bar plots show the mean of two biological replicates. GD = gene desert. d, qPCR results showing relative expression levels of indicated transcripts in HEK293T cells after A-485 treatment (10 μM for 24 h). Bar plots show the mean of two biological replicates. e, Replicate blots for Kacme in wild type and set5 knockout (KO) S. pombe. Blots were repeated independently three times with similar results. f, qPCR results showing relative expression levels of D. melanogaster Smyd3 and Smyd5 after RNAi knockdown. g, Western blots of histones extracted from D. melanogaster cells after RNAi knockdown, with * indicating a lane from an unsuccessful knockdown. Blots were repeated independently twice with similar results. h, Distribution of H4K5acme peptides from HEK293T histones with and without trichostatin A (TSA) treatment (400 nM for 18 h). K5acmeKac1, 2, and 3 represent H4 4-17 peptides with K5acme and 1, 2, or 3 acetyl modifications at remaining lysine residues (K8, K12, K16). i, Extracted ion chromatograms of H4Kac (left) and H4K (right) peptides after in vitro deacetylation by HDAC1. j, Extracted ion chromatograms of H4Kacme (left) and H4Kme1 (right) peptides after in vitro deacetylation by HDAC1. k, Extracted ion chromatograms of H4Kac (left) and H4K (right) peptides after in vitro deacetylation by HDAC3. l, Extracted ion chromatograms of H4Kacme (left) and H4Kme1 (right) peptides after in vitro deacetylation by HDAC3. m, Relative transcriptional activity after TSA treatment (400 nM for 18 h) for genes in the 25th or 75th percentiles of H4Kacme levels as binned by H4Kac ChIP-seq signal. n, Relative transcriptional activity after TSA treatment (400 nM for 18 h) for genes in the 25th or 75th percentiles of H4Kac levels as binned by H4Kacme ChIP-seq signal. m-n, Distribution means compared with two-tailed unpaired Wilcoxon test for two biological replicates. The box plots indicate the median (centre line), the third and first quartiles (box limits), 1.5x interquartile range (IQR) above and below the box (whiskers), and the 95% confidence interval (notches). Outlier points are not plotted. * p = 0.019; *** p < 0.001; NS = not significant. n = 389 (m-n).
