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旨在研究Fpt1的特性。为明确阻遏条件下Fpt1与tRNA基因的结合增强是否由Fpt1蛋白表达上调所致，我们检测了不同营养条件下Fpt1-TAP的整体细胞蛋白水平。免疫印迹实验结果显示，当切换至阻遏条件时，tRNA基因处的Fpt1-TAP结合占有率升高，同时伴随Fpt1-TAP蛋白水平的提升。为进一步探究Fpt1的招募是否受蛋白表达水平调控，我们构建了工程菌株：在内源FPT1基因座处，通过强TDH3启动子过表达Fpt1-TAP，该操作使Fpt1蛋白水平提升了20倍。我们还检测了富营养与阻遏条件下Fpt1的细胞定位情况，经绿色荧光蛋白（GFP）标记的Fpt1在所有测试条件下均定位于细胞核，且仅在阻遏条件下出现小幅的核富集增强。在图6中，我们旨在明确Fpt1的结合占有率如何依赖RNA聚合酶III（RNAPIII）转录机器的其他组分，并解析Fpt1的作用机制。我们用1,10-菲咯啉（1,10-phenanthroline, PH）处理细胞30分钟，结果显示Rpo31-TAP与Brf1-TAP的结合占有率及蛋白水平均有所下降。与RNA聚合酶III与转录因子IIIC（TFIIIC）之间的竞争模型相符，经PH处理后，TFIIIC（Tfc3-TAP）的结合占有率升高，尽管其蛋白水平有所降低。与TFIIIC类似，经PH处理的细胞中Fpt1的结合占有率也有所升高，且Fpt1-TAP的蛋白水平同样上调，这验证了阻遏条件下Fpt1蛋白水平升高的观察结果。为更深入地探究Fpt1对RNA聚合酶III转录机器的依赖关系，我们采用锚定远离系统从细胞核中条件性耗竭靶蛋白，以检测Fpt1与tRNA基因的结合是否受到扰动。该系统通过显微镜实验完成验证，免疫印迹实验证实，Fpt1结合的变化并非由Fpt1-TAP蛋白水平的改变所导致。