Rrd1 Regulates Elongation of RNA Polymerase II Figure 4. Rrd1 is required for RNAPII association independently of TBP recruitment. (A) Mapping of TBP-MYC occupancy on genes from clusters W1–W6, from WT (blue) and from rrd1D mutant (orange) under normal growth condition (full line) and rapamycin treatment (dotted line). (B) SOM clustering of cluster W1 and W6 of RNAPII under normal growth conditions (2RAP), the difference of RNAPII from treated and untreated conditions (diff) and the difference of TBP from treated and untreated conditions (diff). The red colour indicates enriched (bound) regions and blue colour represents depleted regions. (C) Mapping analysis of the clusters S1–S4 generated from (A) of TBP (left panel) and RNAPII (right panel). Legend is the same as (A) for TBP and the same as Fig. 3B for RNAPII. doi:10.1371/journal.pone.0023159.g004 of RRD1. This shows that the upregulation of RNAPII is controlled by Rrd1 independently of TBP binding and suggest that Rrd1 may regulate transcriptional elongation as will be addressed below. We next checked if the regulation of the rapamycin-downreg- ulated genes by Rrd1 is TBP-independant. Preliminary analysis of the downregulated genes (clusters W1 and W6) suggested that these are heterogeneous with respect to TBP binding (Fig. 4A). We therefore generated new clusters from clusters W1 and W6 using both RNAPII and TBP occupancy values from wild type cells (Fig. 4B). This clustering revealed that although RNAPII is downregulated for all genes TBP occupancy is only reduced in cluster S2 and S3 in wild-type cells treated with rapamycin. This suggests that, in wild-type cells, rapamycin leads to gene expression downregulation by at least two distinct mechanisms. The first involving the regulation of TBP recruitment and the latter involving the regulation of a downstream step. To look at the function of Rrd1 in that regulation, we looked at TBP and RNAPII occupancies in rrd1D cells on these clusters (Figure 4C). As predicted from Figure 4A, deletion of RRD1 affected RNAPII occupancy of all these genes upon rapamycin treatment. Interestingly, however, TBP levels were not affected by the deletion of RRD1 for the genes included in clusters S1 and S4, while they were significantly crippled at genes from clusters S2 and S3. Taken together these data indicate that RNAPII downregu- lation upon rapamycin treatment is regulated by two mechanisms, one which does not depend on TBP depletion (cluster S1 and S4). Interestingly, Rrd1 is required for both mechanisms by optimizing TBP depletion (S2 and S3) and RNAPII levels (S1 and S4). This suggests that Rrd1 has an influence on TBP binding, probably by affecting the signaling cascade upstream of PIC assembly [19] but also functions downstream of TBP binding, likely during transcription initiation/elongation. Rrd1 functions during transcription elongation The above data suggest that Rrd1 might regulate RNAPII during transcription initiation and/or elongation. To test this possibility we used a well known transcription elongation assay that is based on an artificial arrest site (ARTAR) within the E.coli b- galactosidase ORF and is thought to cause RNAPII stalling/ pausing downstream of the promoter during elongation [25,36]. This ARTAR is composed of three arrest sites which promote backtracking and arresting of RNAPII and elongation factors such as Dst1 are required to restart RNAPII so that it can resume transcription [25,36]. We introduced this reporter gene expression plasmid in the wild-type, rrd1D mutant and dst1D mutant strains and measured LacZ expression (Fig. 5A). Similar to wild-type, rrd1D mutants were able to overcome the ARTAR and express LacZ as opposed to the dst1D mutants, suggesting that Rrd1 is not essential to overcome RNAPII arrests in this system. We next tested whether increased dosage of Rrd1 would rescue the elongation defect of dst1D mutants (Fig. 5A). Strikingly, ectopic overexpression of Rrd1 partially restored the lacZ expression in the dst1D mutant. This clearly indicates that although Rrd1 is not essential for transcription elongation it is likely to perform a backup function during elongation or alternatively that Rrd1 promotes elongation of RNAPII in such a way that it overcomes the ARTAR. Many mutants defective in transcription elongation, such as dst1D and spt4D, are known to be sensitive to 6-azauracil (6-AU), an inhibitor of the IMP dehydrogenase, which decreases GTP pools and thereby causes transcriptional arrest [36]. We found that rrd1D mutants were sensitive to 6-AU, although not as sensitive as dst1D cells (Fig. 5B), further supporting a role for Rrd1 in transcription elongation. Finally, we examined whether Rrd1 might influence elongation by altering the phosphorylation status of the RNAPII C-terminal domain (CTD). The CTD is highly phosphorylated on Ser5 at the promoter and in the early elongation phase, and is progressively dephosphorylated as transcription progresses. In contrast, Ser2 phosphorylation gradually increases throughout the ORF, reach- ing maximum levels at the 39 end of the gene [28,29,30,31]. These differential phosphorylation states can be used to monitor transcription elongation efficiency [24,27]. We used phosphospe- cific antibodies to perform ChIP-chip analysis for Ser5 phosphor- ylation (Ser5-P) and Ser2 phosphorylation (Ser2-P) in wild-type and rrd1D strains with and without rapamycin. Figure 5C shows the correlation between wild-type and rrd1D for Ser5-P and Ser2-P under untreated and rapamycin treated conditions. The correla- tion of Ser2-P shifted from a R2 = 0.89 to an R2 = 0.68 upon rapamycin treatment and the correlation of Ser5-P was even more affected upon treatment (R2 = 0.84 and R2 = 0.53). This suggests that Rrd1 influences the phosphorylation status of RNAPII in response to rapamycin. We next mapped Ser5-P and Ser2-P from wild-type and rrd1D mutant on clusters W1 to W6 (Fig. 5D and Suppl. Fig. S2A). We found that in general the pattern of Ser5-P and Ser2-P is similar between wild-type and rrd1D mutant under normal growth condition (full line). However, when treated with rapamycin (dashed line) the patterns of both phosphorylations were altered in the rrd1D mutant in each cluster, leading to an increase in the 39 region of the genes. On down regulated genes, Ser5-P largely remained at the 39end in the mutant as compared to wild-type for cluster W1 and increased for cluster W6 (Fig. 5D). In the case of the upregulated genes (W3 and W4), Ser5-P increased in the wild-type at promoters and within the ORF but not in the mutant where it remained low except for being elevated at the 39 end of these genes. Similarly to Ser5-P, Ser2-P remained high at the 39 end of genes in the mutant on down regulated genes (W1 and W6). Also for the upregulated genes, Ser2-P was lower in the mutant throughout the ORFs but then peaked only at the 39 end as compared to wild-type (W3 and W4). We next analyzed Ser5-P and Ser2-P on the clusters S1 to S4 and here also, both phosphorylations were similarly altered in response to rapamycin (Suppl. Fig S2B). Taken together, this indicates that Rrd1 affects the phosphorylation states of RNAPII in response to a transcrip- tional stress (see discussion). Rrd1 plays a role in environmental stress responses The above data indicate that Rrd1 is required for an optimal transcriptional response following rapamycin exposure. Since rapamycin mimics nutrient starvation conditions, we next asked whether Rrd1 is required for optimal response to other PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e23159