Locus-specific proteome decoding reveals Fpt1 as a chromatin-associated negative regulator of RNA Polymerase III assembly
Figure 3 is aimed to study the characteristics of Fpt1. To determine whether increased Fpt1 binding to tDNAs during repressive conditions was caused by increased Fpt1 protein expression, we measured global cellular protein levels of Fpt1-TAP in response to changing nutrient conditions. Immunoblotting showed that increased Fpt1-TAP occupancy at tRNA genes was accompanied by an increase in Fpt1-TAP protein levels upon a switch to repressive conditions. To further explore whether recruitment of Fpt1 is regulated by expression levels, we generated strains in which Fpt1-TAP is overexpressed by a strong TDH3 promoter at the native FPT1 locus. This caused a 20-fold increase in Fpt1 protein levels. We also determined the cellular localization of Fpt1 in nutrient-rich and repressive conditions. GFP-tagged Fpt1 localized to the nucleus in all tested conditions and showed only a modest increase in nuclear enrichment in repressive conditions. In figure 6 we aimed to determine how Fpt1 occupancy depends on other members of the RNAPIII transcription machinery, and to get insights into Fpt1’s mechanism. We treated cells for 30 minutes with 1,10-phenanthroline (PH). Rpo31-TAP and Brf1-TAP occupancy and protein levels decreased upon treatment with PH. In agreement with the competitive model between RNAPIII and TFIIIC, TFIIIC (Tfc3-TAP) binding increased upon treatment with PH, albeit protein levels were decreased. Similar to TFIIIC, occupancy of Fpt1 increased in PH treated cells. Fpt1-TAP protein levels also increased, corroborating the observed increase of Fpt1 protein levels in repressive conditions. To explore the dependency of Fpt1 on the RNAPIII transcription machinery in more detail, we used the anchor away system to conditionally deplete proteins from the nucleus and check whether Fpt1 binding to tRNA genes is perturbed. The anchor away system was validated using microscopy. Immunoblotting showed that effects on Fpt1 binding were not caused by altered Fpt1-TAP protein levels.