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.图3旨在探究Fpt1的相关特性。为明确抑制条件下Fpt1与转运RNA基因（tDNAs）的结合增强是否由Fpt1蛋白表达上调所导致，我们检测了不同营养条件下细胞内Fpt1-TAP的整体蛋白水平。免疫印迹实验结果显示，当细胞切换至抑制性培养条件时，Fpt1-TAP在tRNA基因上的结合富集水平升高，同时伴随Fpt1-TAP蛋白水平的上调。为进一步探究Fpt1的募集是否受其蛋白表达水平调控，我们构建了在天然FPT1基因座处通过强启动子TDH3过表达Fpt1-TAP的菌株。该操作使Fpt1的蛋白水平提升了20倍。我们还检测了富营养及抑制性培养条件下Fpt1的细胞定位情况。经绿色荧光蛋白（GFP，green fluorescent protein）标签标记的Fpt1在所有检测条件下均定位于细胞核，且仅在抑制性培养条件下表现出小幅的核富集增强。 图6旨在探究Fpt1的结合富集水平如何依赖于RNA聚合酶III（RNAPIII，RNA polymerase III）转录机器的其他组分，并深入解析Fpt1的作用机制。我们用1,10-邻菲啰啉（1,10-phenanthroline, PH）处理细胞30分钟。经PH处理后，Rpo31-TAP与Brf1-TAP的结合富集水平及其蛋白水平均出现下降。这与RNA聚合酶III与转录因子IIIC（TFIIIC，transcription factor IIIC）之间的竞争模型相符：经PH处理后，转录因子IIIC（Tfc3-TAP）的结合水平出现上升，尽管其蛋白水平有所下降。与转录因子IIIC类似，经PH处理的细胞中Fpt1的结合富集水平也出现升高。Fpt1-TAP的蛋白水平同样出现上调，这与抑制性培养条件下观察到的Fpt1蛋白水平上调结果相印证。为更细致地探究Fpt1对RNA聚合酶III转录机器的依赖关系，我们采用锚定撤离（anchor away）系统条件性地将细胞核内的蛋白耗竭，并检测Fpt1与转运RNA基因的结合是否受到干扰。我们通过显微镜成像验证了该锚定撤离系统的有效性。免疫印迹实验结果表明，对Fpt1结合水平的影响并非由Fpt1-TAP蛋白水平的变化所导致。