Rrd1 Regulates Elongation of RNA Polymerase II Figure 2. Rrd1 localization correlates with RNAPII along actively transcribed genes. (A) Self-organizing map (SOM) clustering analysis of the median enrichment of RNAPII on all ORFs from the WT strain. The red colour indicates enriched (bound) regions and blue colour represents depleted regions. The difference (diff (+/2)) (right column) was calculated by subtracting the complete untreated (2RAP) RNAPII data set (left column) from the treated (+RAP) RNAPII data set (middle row) (RAP = rapamycin 100 ng/ml for 30 min). The brackets (W1–W6) indicate clusters that were analyzed by gene ontology (GO). (B) Functional annotation of cluster W1–W6 with representative GO groups is shown. Funcassociate 2.0 was used to generate the full GO analysis, available in supplemental file S1. The X- axis represents the P-value. (C) Linear correlation of RNAPII and Rrd1- Myc enriched genes under normal growth condition. Each dot represents a single gene containing the median enrichment value from RNAPII on the y-axis and the Rrd1-Myc median enrichments on the x-axis. For the rapamycin treated data sets refer to supplemental figure S1A. (D) Mapping of RNAPII and Rrd1 on groups of genes with different median enrichment of RNAPII under normal growth conditions. The red line represents RNAPII along the 10% of genes with the highest amount of RNAPII and the blue line represents Rrd1 on this same group of genes. The orange line represents RNAPII on the lowest 10% RNAPII enriched genes and the green line represents Rrd1 on this same group of genes. (E) Linear correlation of RNAPII and Rrd1 gene occupancy differences between normal growth and rapamycin conditions. This is the same analysis as (C) only that the difference of the untreated and treated data set for the median enrichment of RNAPII and Rrd1 was plotted. doi:10.1371/journal.pone.0023159.g002 together, this data clearly indicates that Rrd1 is required for the optimal up and down regulation of RNAPII levels during rapamycin response. Rrd1 regulates RNAPII occupancy independently of TBP binding To find out at what step Rrd1 influences RNAPII transcription, we examined the genome wide association of the yeast Myc-tagged TATA box binding protein (TBP) encoded by SPT15 using ChIP- chip. If, upon rapamycin treatment, Rrd1 affects steps upstream of PIC formation, TBP occupancy would be affected in rrd1D cells. In the other scenario, whereby Rrd1 was to affect a step downstream from PIC formation (such as elongtion for example), TBP levels (contrarily to RNAPII levels, Figure 3B) would not necessarily be affected. We therefore mapped TBP occupancy in WT (blue) and rrd1D cells (orange) on genes from clusters W1–W6 (Fig. 4A). Interestingly, at upregulated genes (cluster W3 and W4) TBP binding (unlike RNAPII occupancy) is not affected by the deletion PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e23159