Supplementary MaterialsSupplementary information biolopen-7-037911-s1. common regulator of Pol I and Pol III, PWP1 might contribute to coordinated control of ribosomal gene GOAT-IN-1 expression in response to nutrition. This article has an associated First Person interview with the first author of the paper. mutant larvae lacking PWP1 show a prominent growth retardation. In addition to the role in Pol I regulation, we observed earlier that mutant larvae display reduced expression of RNA polymerase III targets, including 5S rRNA (Liu et al., 2017). However, it remained to be tested whether PWP1 controls Pol III-mediated transcription directly and in a nutrient-responsive manner, and whether its role in this setting is conserved in animals. Here we provide GOAT-IN-1 evidence that PWP1 critically contributes to nutrient-responsive expression of 5S rRNA in larvae and it regulates 5S rRNA expression in a conserved manner, likely through a direct mechanism at the 5S rDNA chromatin. Our data suggest that PWP1 is a common regulator of Pol I and III and therefore has the potential to act as a coordinator of their activities. RESULTS Our previous study showed that PWP1 acts downstream of nutrient-responsive mTOR signaling in and that PWP1 is essential for the induced expression of the Pol I-dependent rRNAs in response to protein-rich diet (Liu et al., 2017). As the Pol III-dependent gene expression is also dependent on nutrition (Marshall et al., 2012), we wanted to test if PWP1 (dPWP1, encoded by the gene) controls the expression of Pol III-dependent 5S rRNA in this setting. This was indeed the case, as control larvae displayed strongly elevated 5S rRNA expression upon refeeding on protein-rich yeast food following protein starvation, but this effect was blunted in polytene chromosomes. A control without primary antibody was used to assess the specificity of the staining (Fig.?S1). As previously shown (Casper et al., 2011), dPWP1 displayed a specific staining pattern, with strongest signal in weakly DAPI stained interband regions (Fig.?S1). We focused our attention on the division 56E, which contains Pol III targets, including the cluster of 5S rDNA as well GOAT-IN-1 as several tRNA-encoding loci (Fig.?2A). Previous work has shown localization of BRF, a Pol III initiation factor subunit, into this specific region (Takada et al., 2000). Strong PWP1 staining was observed in 56E, in particular in subdivisions 56E1 and 56E2, which harbor the cluster of Pol III targets (Fig.?2B,C). In conclusion, our data implies that PWP1 associates with chromatin in the vicinity of Pol III targets. Open in a separate window Fig. 2. In polytene chromosomes PWP1 binds to the region 56E1-2 that contains the 5S rDNA cluster. (A) The upper panel shows division 57 of chromosome 2R of the Bridges map (Bridges and GOAT-IN-1 Bridges, 1939). The lower figure reveals the subdivisions 57CCF in a wpolytene chromosome. The red arrow indicates the localization of 5S locus and different tRNAs at the polytene band 56E1C2. (B) Representative images of the subdivisions 56CCF. Immunofluorescence staining with PWP1 antibody and Hoechst revealed the localization of PWP1 in the polytene band 56E1C2. Scale bar: 1?m. (C) Scheme of the tRNA genes and tandemly repeated 5S rDNA genes as they are represented in Flybase genome browser (http://flybase.org/). The specific tRNA and 5S rDNA clusters are represented in the green and blue boxes, respectively. The blue arrow refers to the neighboring Rabbit Polyclonal to SPTA2 (Cleaved-Asp1185) gene OR56a. To achieve a better spatial resolution, we used chromatin immunoprecipitation (ChIP) in human cell lines. A significant enrichment of PWP1 was observed in the 5S rDNA gene region (Fig.?3). PWP1 was earlier shown to regulate.