Drosophila Chromosome 4 Chromatin Structure Figure 4. Chromosome 4 has a very low incidence of polymerase pausing identified by GRO-seq data. A. The bar graph shows the percentage of transcripts associated with RNA polymerase (grey) and the percentage of those RNA polymerase-associated genes exhibiting significant pausing (black) for euchromatin, pericentric heterochromatin, and chromosome 4. A pausing index (PI) threshold value of 10 was used. B. The low frequency of polymerase pausing observed for chromosome 4 is independent of the PI selected. C. The average Tm of 9-mers downstream of the TSS for chromosome 4 genes tends to be lower than that of other genes. Tm (Y-axis) of 9-mers in the 100 bp downstream of the TSS (bp, X-axis) is compared for genes on chromosome 4 (red) to genes classified as either paused (black) or not-paused (grey) by GRO-seq analysis. Data from S2 cells [23]. doi:10.1371/journal.pgen.1002954.g004 the observed lack of polymerase pausing. ‘‘Developmental control’’ genes (gene ontology), which preferentially exhibit pausing [30,31], occur on chromosome 4 at the same frequency as in other genomic domains (Table S4), and the average copy number for chromosome 4 segments in S2 cells is similar to that of the other autosomes [32]. The low fraction of genes displaying pausing is also not due to proximity to the centromere: the percentage of genes displaying pausing within the first 1.2 Mb of contiguously assembled sequence on chromosome arms 2L, 2R, and 3L is 13.6% in S2 cells. Genes on chromosome 4 are larger than those in euchromatin and heterochromatin (median: 8,001 bp vs. 1,907 bp vs. 1,844 bp; Wilcoxon test: p,0.0001, Figure S9A and S9D), and the average number of exons for genes on chromosome 4 is higher than for genes in euchromatin and pericentric heterochromatin (median: 6 vs. 3 vs. 2; p,0.0001; Figure S9B and S9E). However, larger, more exon-rich genes tend to have higher PI values (Figure S6E), indicating that this feature does not contribute to the lack of pausing on chromosome 4. Genes on chromosome 4 can be biased towards higher expression levels (e.g. in third instar larvae: p,0.05; Figure S9C; no higher expression in S2 cells), but this bias is cell type-specific, while the lack of pausing is observed in all cell types examined. Thus, these sequence features alone cannot account for the low incidence of polymerase pausing observed on chromosome 4. Previous reports have identified several sequences that are preferentially associated with paused polymerase, including GAGA factor (TRL) binding motifs, the Inr motif, as well as the so-called ‘‘pause button’’ (PB) sequence KCGRWCG [29]. We find that the fraction of promoters with a PB or Inr motif is similar in euchromatin and on chromosome 4 (Table S5), and thus unlikely to contribute to the differences we observe in pausing incidence. In contrast, the fraction of promoters containing TRL motifs (or their inversions) differs significantly between euchroma- tin and chromosome 4. While TRL binding sites are observed in 24% of euchromatic promoters, we find TRL binding sites in only 11% of the promoters on chromosome 4 (Table S5; p,2.69e-3; inverted TRL binding site: 25% vs. 13%, p,4.88e-3). The underrepresentation of TRL binding sites on chromosome 4 is reflected also in the significantly lower number of TRL-bound sites detected in TRL ChIP-chip data. In euchromatin, 33% of promoters are bound by TRL, while on chromosome 4 only 17% show TRL binding in S2 cells (p,1.52e-3). Thus, the paucity of TRL binding sites on chromosome 4 might contribute to the low occurrence of polymerase pausing, but cannot explain it entirely. Recently, it has been reported that genes exhibiting polymerase pausing have a distinct Tm (melting temperature) peak for 9-mers approximately 25–30 bp downstream of the TSS [33]. Therefore, we examined Tm values of 9-mers in the TSS-proximal 100 bp of each D. melanogaster mRNA by a sliding window analysis. Interestingly, the Tm values for TSS-associated sequences on chromosome 4 are lower than those on the other chromosomes for both pausing and non-pausing genes (11.50 degrees vs. 15.03– 15.84 degrees; see Table S6) over the entire 100 bp interval (see Figure 4C). Thus, chromosome 4 genes show a different sequence organization at their 59 end, which may contribute to the low incidence of pausing. Lack of HP1a alters RNA pol II distribution and decreases gene expression levels on chromosome 4 Given the low frequency of paused polymerase on chromosome 4, but not in pericentric regions of heterochromatin, we tested the hypothesis that chromosome 4’s distinct chromatin composition is responsible for this difference. First, we disrupted the typical chromatin structure, using third instar larvae lacking HP1a, trans- heterozygous for Su(var)20504 and Su(var)20505. These trans- heterozygotes do not produce zygotic HP1a and survive to the third larval instar by utilizing maternally loaded HP1a protein and/or mRNA. By the third instar, little detectable HP1a protein remains, and in ChIP-chip experiments, .95% of peaks observed in wildtype are absent in the mutants (Figure S10A). RNA pol II enrichment on chromosome 4 in Su(var)205 mutants is reduced in the gene bodies, leading to an increase in PI, as RNA pol II now is relatively more concentrated at the TSS compared to the wildtype distribution (Figure 5A, compare red [HP1a 2/2] to grey [+/+]). For RNA pol II ChIP-chip data, the PI is defined as the ratio between the maximum enrichment value around the TSS (+/ 2300 bp) and the medium enrichment values over the gene body PLOS Genetics | www.plosgenetics.org 7 September 2012 | Volume 8 | Issue 9 | e1002954