Fig 2: Pol III depletion alters the Pol II interactome and significantly impairs recruitment of the FACT complex. A Experimental setup for mass spectrometry of anti-Pol II immunoprecipitates obtained from crosslinking chromatin fractions of Pol III_degron cells that were untreated or treated with IAA for 1 h (upper). The protein factors listed here showed a similar trend of differential interactions with Pol II in both the canonical DDA and DIA-MS experiments (see the detailed procedure in the Supplementary Methods section). The color bar indicates proteins differentially interacting with Pol II, characterized by the log2FC and -log10(P value) with DIA-MS data (bottom). B Western blot analysis of anti-Pol II immunoprecipitates obtained from crosslinking chromatin fractions of Pol III_degron cells that were untreated or treated with IAA for 1 h revealed decreased interaction of FACT (SSRP1 and SPT16) and integrator (INTS3 and INTS11) complex components with Pol II after Pol III depletion. RPB3 is a subunit of Pol II with no change in its interaction with Pol II after the depletion of Pol III. The quantitative data shown under each panel were measured by ImageJ and normalized to the input under untreated conditions based on the representative figure shown. Three independent western blotting analyses were performed. Each showed a similar trend regarding a decreased interaction between FACT and Pol II upon Pol III depletion. However, the technical limitations of western blotting, such as large molecular weight or poor antibody sensitivity, may cause experimental variations. C MA plots showing differential enrichment of SSRP1, INTS3, and TBP ChIP-Seq signals in Pol III_degron cells under 1 h IAA treatment versus untreated conditions at Pol II peaks called in wild-type mESCs. Each dot represents one peak. Red indicates a significant change that meets both criteria of a false discovery rate (FDR) < 0.05 and fold change > 2 (upper). Venn diagrams comparing the Pol II-bound regions that had significantly altered binding affinities for SSRP1 (N = 5520, left), INTS3 (N = 332, middle), or TBP (N = 190, right) after Pol III depletion, as shown in the top panel, and the differential Pol II peaks upon Pol III depletion (N = 2526), as shown in Fig. 2D (bottom). D Venn diagrams showing the overlap of active promoters with significantly reduced SSRP1 (N = 4147, upper) or INTS3 (N = 95, lower) binding affinities with genebody-up mRNA genes upon Pol III depletion (N = 773). E Under the same representations described in Fig. 4A, downregulated mRNA genes upon Pol III depletion in SSRP1, INTS3, and TBP ChIP-Seq as identified in Fig. 6C were clustered based on their distance from the nearest Pol III-bound peaks and expressed tRNAs in 50 bp bins. Equal numbers of unchanged active genes and silent genes were selected as the control group. The y-axis in Fig. 6E represents the normalized counts of Pol III peaks or tRNAs by calculating their average density within 10 kb of the Pol II gene TSS for each category. F Bar graphs showing relative SSRP1, Pol II, and Pol III ChIP enrichment normalized to input (5%) at the presented example genes under Pol III untreated, + IAA 10 min, 20 min, 30 min, and 1 h conditions. Each sample was analyzed with two technical replicates per biological replicate and two biological replicates in total. Statistical significance was evaluated by Student’s t test (**: <0.01, *: <0.05). G Bar graphs showing relative SSRP1 ChIP enrichment normalized to input (5%) at the presented example genes under Pol III untreated, + IAA 1 h and +IAA 1 h followed by 6 h IAA withdrawal conditions. Each sample was analyzed with two technical replicates per biological replicate and two biological replicates in total. Statistical significance was evaluated by Student’s t test (**: <0.01, *: <0.05). H Under the same representations described in Fig. 4F, NET-seq data in mES cells were obtained from the public dataset (GSE90906). The average signal levels at the gene bodies using the gene groups from Fig. 4A were calculated and presented as metaplots and violin plots. The p value was calculated using the Mann–Whitney test