Drosophila Chromosome 4 Chromatin Structure Figure 2. The relationship between marks of classical heterochromatin and gene expression are altered on chromosome 4. The strength of correlation between marks is illustrated in this diagram by the color intensity (red - positive correlation; blue - negative correlation). In pericentric heterochromatin, the black outline demarcates the strong correlation structure observed between H3K9me2, H3K9me3, and HP1a (right). This strong correlation is not present on chromosome 4; HP1a and H3K9me3 instead are positively correlated with H3K36me3, a mark of elongation, and the chromosome 4-specific protein POF (left). doi:10.1371/journal.pgen.1002954.g002 S2 cells). GRO-seq data confirmed this assessment, indicating that 47.6% of euchromatic genes were being actively transcribed in S2 cells, compared to 40.4% of those in heterochromatin. On chromosome 4, 54.3% of the genes were associated with GRO-seq signal, a fraction slightly higher but not significantly different from that of euchromatin (p = 0.147; Figure 4A). However, the GRO- seq signal on chromosome 4 and within euchromatin was higher than that in pericentric heterochromatin (p,0.01; Figure 4A). Next, we assessed polymerase pausing using a pausing index (PI) that measures the level of polymerase at the 59 end of the gene compared to that over the gene body [27]. Specifically, we used the ratio of GRO-seq read density in the first 500 bp of the gene and the read density of the first 25% of the remaining length of the gene (for details, see [26]). With a stringent threshold for pausing, the results indicate that in pericentric heterochromatin and in euchromatin, the fraction of genes associated with a paused polymerase is similar, 12.5% and 15.0%, respectively (Figure 4A). In contrast, only 1.6% of the RNA polymerase-associated chromosome 4 genes exhibit such pausing, a significantly smaller percentage than what was observed in either euchromatin or pericentric heterochromatin (p,0.0005; Figure S7). While the absolute number of paused genes varies depending on the threshold, the difference in pausing frequency between chromo- some 4 and the other genome domains using this analytical definition was observed over a wide range of PI thresholds (Figure 4B). This finding is consistent with recent results from Johannsen and colleagues using the same GRO-seq dataset [19]. We also validated this result using ChIP-chip data from S2 and BG3 cells with an alternative definition of pausing (see Materials and Methods; Table S3). The overlap in the genes identified as exhibiting pausing by these two methods is ,50%, significantly more than the random expectation (p,1610216, Figure S8). These data demonstrate that paused polymerase (no matter how defined) is not uniformly distributed across genomic domains, and further establish the unique properties of chromosome 4. Whether the observations on chromosome 4 reflect a difference in the protein machinery identified with ‘‘classical’’ RNA pol II pausing (i.e. dependent on NELF and DSIF) or with some other aspect of RNA pol II regulation (e.g. elongation) remains to be explored. Given that our observations are based on experimental approach- es widely used to study pausing, and for convenience, we will refer to this particular distribution of RNA pol II as ‘pausing’ in the text below. Sequence characteristics of chromosome 4 are congruent with its low level of paused polymerase Previous work has shown that chromosome 4 is distinct from other domains in a number of sequence-associated features [4,28]. As various gene features (e.g. sequence composition [29], gene expression levels [27], and gene ontology [30,31]) have also been associated with the use of polymerase pausing, we considered whether these features of chromosome 4 might be correlated with PLOS Genetics | www.plosgenetics.org 5 September 2012 | Volume 8 | Issue 9 | e1002954