Supplementary Figure 2: Chromatin marking at stable, regulated and silent genes during fly development.

We performed a number of controls to rule out the possibility that our observations arose from undetected confounding factors. (a) Normalized levels of H3K4me3, H3K9ac, H3K4me1, H3K27ac, H3K27me3 and H3K9me3 at the time point of maximum expression during development. Because of the differences in heights between modENCODE ChIP-seq tracks, we identified the highest peak of each mark in the genome by checking all expressed genes and used this value to normalize the corresponding profiles. The distributions correspond to the maximum height of the ChIP-seq peak within the gene body for H3K4me3, H3K9ac, H3K4me1 and H3K27ac and the average height of the ChIP-seq signal over the gene body for H3K27me3 and H3K9me3. Patterns are the same or even stronger than those in Figure 1b. The bottom and top of the boxes are the first and third quartiles, and the line within is the median. The whiskers denote the interval within 1.5 times the IQR from the median. Outliers are plotted as dots. (b) Distribution of expression of the top 1,000 stable, regulated and silent genes and of the set of the top 1,000 regulated genes divided into 3 groups according to expression (low, medium, high) at the time point of maximum expression for each gene. Gene expression was computed as FPKM by the modENCODE consortium. (c) Levels of H3K4me3 at the time point of maximum expression for the gene sets defined in b. Values represent the maximum height of the ChIP-seq peak within the gene body. The three sets of regulated genes show similar levels of H3K4me3, comparable to silent genes. (d) Levels of H3K4me3 at the time point of maximum gene expression computed as the average signal over the gene body instead of as the maximum peak. The pattern is the same as that in Figure 1b. (e) Lengths of stable and regulated genes. Regulated genes have a lower number of exons than stable genes (2.6 versus 5.7 on average) and shorter introns (600 bp versus 1,000 bp); as a consequence, regulated genes are shorter than stable genes (1,136 bp versus 2,864 bp). To rule out the possibility that gene size is a confounding factor, we selected the 520 shortest stable genes. These have an average length (1,188 bp) and number of exons (2.6) very similar to those of variable genes. (f) The H3K4me3 maximum peak at short stable genes is comparable to the peak at the previous set of stable genes, and it is much higher than the H3K4me3 peak at regulated genes. Therefore, there is no effect of gene length and number of exons in our observations.