Rrd1 Regulates Elongation of RNA Polymerase II Figure 1. Rrd1 affects RNAPII occupancy on rapamycin responsive genes. (A) ChIP of Rpb1 (RNAPII) from WT cells followed by Q-PCR on the indicated genes. Dashed bars indicate rapamycin treatment (100 ng/ml for 30 min). The data is represented as a ratio of the % IP over Input on the ORF compared to % IP over Input in a non transcribed region (noORF). (B) ChIP of RNAPII from rrd1D mutant cells performed as in panel A. (C) ChIP of Rrd1-MYC (Myc antibodies) performed as in panel A. Results for all panels are shown as an average of at least three independent experiments, error bars indicate the standard deviation and the asterix (*) indicates if the P-value is below 0.05 between the untreated and treated condition using the Student T test. doi:10.1371/journal.pone.0023159.g001 To further confirm that Rrd1 changes in occupancy correlate with RNAPII in response to rapamycin, we plotted the difference in RNAPII occupancy between untreated and rapamycin-treated cells versus the difference in Rrd1 occupancy between these two conditions (Fig. 2E). This analysis revealed that both, Rrd1 and RNAPII are downregulated on a large group of genes and recruited to another group of genes in response to rapamycin (R2 = 0.34). Taken together, this data suggest that Rrd1 and RNAPII co-localize on the coding region of most of the actively transcribed genes, even after massive transcriptional changes such as the ones caused by rapamycin treatment. RRD1 is required for proper RNAPII relocalization in response to rapamycin Since Rrd1 co-localizes with RNAPII on actively transcribed genes and Rrd1 was previously shown to be required to modulate the expression of a few rapamycin-responsive genes [20], we analyzed the genome-wide localization of RNAPII in the rrd1D mutant. To compare RNAPII levels from the wild-type and rrd1D cells, we plotted RNAPII occupancy in wild-type and rrd1D cells, both in absence and presence of rapamycin (Fig. 3A). Whereas RNAPII levels were similar (R2 = 0.87) in untreated cells, they were less correlated in response to rapamycin (R2 = 0.75), suggesting that Rrd1 is required to regulate RNAPII occupancy in response to rapamycin as observed at individual genes in figure 1. To further confirm this, we mapped RNAPII occupancy from wild-type and rrd1D mutant on the six clusters from Figure 2A (Fig. 3B). Interestingly, at down regulated genes, RNAPII in the rrd1D mutant failed to reach levels as low as in wild type cells (cluster W1 and W6). On the other hand, on upregulated genes (cluster W3 and W4) RNAPII also failed to reach wild-type levels in the rrd1D mutant. We note that RNAPII occupancy was similar for the untreated RNAPII levels (full line) as well as for clusters that were not affected by rapamycin (cluster W2 and W5). Taken PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e23159