[6,20]. However, we found that the Gln3 target gene MEP2 was activated independently of Rrd1, suggesting that this latter factor has an additional role in the response to rapamycin [20]. Consistent with this, we found that Rrd1 exerts an effect at the transcriptional level: genes known to be upregulated (the diauxic shift genes CPA2 and PYC1) and down-regulated (the ribosomal protein genes including RPS26A, RPL30, and RPL9) following rapamycin exposure showed an altered transcription pattern in rrd1D mutants [20]. Since ribosomal biogenesis results from the concerted action of all three RNA polymerases, which are controlled by a tight regulatory network, we expected that Rrd1 plays a broader role in transcription of these genes [21]. Indeed, we subsequently found that Rrd1 is associated with the chromatin and that it interacts with the major subunit of RNAPII [22]. Further, biochemical analysis revealed that Rrd1 is able to release RNAPII from the chromatin in vivo and in vitro, which we ascribed to the peptidyl prolyl isomerase activity acting on the C-terminal domain (CTD) of RNAPII [22]. This mechanism of RNAPII regulation resembles that of the peptidyl prolyl isomerase, Pin1, and its yeast homologue Ess1 which are also known to regulate transcription [23,24,25,26,27]. Both Pin1 and Ess1 are thought to isomerize the CTD of RNAPII and regulate elongation [26,27]. In yeast, the CTD consists of 26 repeats of the YS2PTS5PS7 heptad sequence which are differentially phosphorylated on Ser2, Ser5 and Ser7 [28,29,30,31]. These different phosphorylation patterns act as a recruitment platform for multiple factors involved in chromatin remodelling, mRNA processing and transcription termination [28,29,30,31]. For example, Ess1 has been shown to stimulate the dephosphorylation of Ser5 to efficiently terminate transcription of a subset of genes [24]. In this study, we analyzed how Rrd1 regulates transcription by RNAPII. We mapped Rrd1 and RNAPII occupancy using ChIP- chip analysis in the presence and the absence of rapamycin. We found that Rrd1 colocalized with RNAPII on actively transcribed genes under both conditions. Furthermore, rrd1D deletion affected RNAPII occupancy on a large set of rapamycin responsive genes. This was independent of TATA binding protein (TBP) recruit- ment to the promoter, suggesting that Rrd1 acts downstream of PIC formation during transcriptional initiation and elongation. The observation that Rrd1 modulated Ser5 and Ser2 phosphor- ylation of the RNAPII CTD further supported a role for Rrd1 in elongation. Finally, we demonstrate that Rrd1 is required to regulate gene expression in response to a variety of environmental stresses, thus establishing Rrd1 as a new elongation factor required for effective transcriptional responses to environmental challenges. Results Rrd1 affects RNAPII occupancy on rapamycin responsive genes Recently, we have shown that Rrd1 interacts with and isomerizes RNA polymerase II (RNAPII) in response to rapamycin [22]. Also it was demonstrated that Rrd1 is required to regulate the expression of some rapamycin responsive genes [20]. To further investigate this, we used ChIP analysis to measure the association of Rpb1, the major subunit of RNAPII (henceforth referred to as RNAPII), within the ORFs of four known rapamycin-responsive genes in wild-type cells and rrd1D mutant cells [32]. The rapamycin-upregulated genes, such as HSP104 and PUT4, were significantly enriched for RNAPII in the wild-type strain, but this association was reduced in the rrd1D mutant (Fig. 1A and B). Examination of RPL32 and RPS2, which are downregulated by rapamycin, revealed that RNAPII dissociated from both genes upon rapamycin treatment of wild-type yeast but Rrd1 Regulates Elongation of RNA Polymerase II remained bound in the rrd1D (Fig. 1A and B). Localization of RNAPII to ACT1, a gene unaffected by rapamycin treatment, was not altered upon addition of rapamycin in wild-type cells and or by RRD1 deletion (Fig. 1A and B). These data indicate that Rrd1 is required to modulate expression of a larger set of genes than previously discovered [20]. To better understand by what mechanism Rrd1 affects transcription, we tagged Rrd1 with a Myc epitope and asked if Rrd1 also localizes to the set of genes assayed above. Similar to RNAPII, Rrd1 occupancy was increased on the ORFs of HSP104 and PUT4, depleted on those of RPL32 and RPS2, and remained constant on ACT1, in response to rapamycin (Fig. 1C). These data suggest that Rrd1 directly regulates these rapamycin-responsive genes. Rrd1 localization correlates with RNAPII along actively transcribed genes To extend our initial analysis from a small set of genes to the entire genome, we used the ChIP-chip assay to examine the genome-wide distribution of both RNAPII and Rrd1. We first examined RNAPII occupancy under exponential growth condi- tions and after a 30 minutes treatment with rapamycin, the time point at which the rapamycin transcriptional response is most prominent based on mRNA expression analysis [32]. ChIP samples were hybridized on tiling arrays covering the whole yeast genome with an average resolution of four probes per kilobase. We first calculated the median RNAPII occupancy over the coding region of approximately 5000 ORFs and clustered these enrichment values using self organizing maps (SOM) with Cluster 3.0 and used Java TreeView to visualize the data [33]. Figure 2A shows the average RNAPII signal over each ORF in the absence (column 1), or the presence (column 2) of rapamycin. In order to better visualize the genes that were affected by rapamycin, we also show the difference in RNAPII occupancy between the two conditions (rapamycin treated data was subtracted from the untreated; column 3). We identified 6 clusters of genes (W1–W6) with different behaviors upon rapamycin treatment. These clusters were then analyzed for gene ontology (GO) category using funcassociate 2.0 (Fig. 2B and Suppl. File S1) [34]. This analysis revealed that upon rapamycin treatment, RNAPII occupancy was sharply reduced on metabolic genes including those involved in ribosome biogenesis (see cluster W1, W6 and Fig. 2B). In contrast, RNAPII was strongly recruited to genes involved in nitrogen discrimination, the Krebs cycle, stress response and catabolic processes after rapamycin treatment (W3, W4 and Fig. 2B). These data are consistent with the transcriptional changes reported for rapamycin treatment as well as for environmental stress responses [32,35]. We next performed ChIP-chip analysis on Myc-tagged Rrd1 and compared its distribution on the ORFs with that of RNAPII. As shown in Figure 2C, RNAPII occupancy correlates with that of Rrd1, indicating that Rrd1 is recruited to transcribed genes. Interestingly, when cells were treated with rapamycin, Rrd1 and RNAPII displayed a similar correlation (Suppl. Fig S1A), suggesting that under normal growth conditions, as well as after drastic transcriptional changes, Rrd1 remains associated to actively transcribed genes. To further investigate this relationship, we mapped RNAPII occupancy along a group of genes with the lowest levels of RNAPII as well as a group with the highest levels of RNAPII and then mapped Rrd1 on the same groups (Fig. 2D). Indeed, when RNAPII levels were low, the Rrd1 levels were also low, and consistently, when RNAPII levels were high Rrd1 levels were also increased (Fig. 2D). This was also the case for rapamycin treated cells (Suppl. Fig. S1B). PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e23159