Fig 2: GATA4 regulates the pol III transcription machinery assembly at the promoters of pol III target genes by affecting Brf1 and Gtf3c2 gene expression.A, ChIP–qPCR results showing the effect of GATA4 knockdown on the occupancy of the pol III transcription machinery at the 5S rRNA promoter. ChIP assays were performed using the 293T cell lines expressing GATA4 shRNA or control shRNA and the antibodies against the factor as indicated in the graph. qPCR was performed using 1 μl of the DNA samples (40 μl in total) recovered from the ChIP assay for individual factors. Relative occupancy for each factor at the promoter was obtained by comparing the relative quantity of 5S rRNA gene promoter from 1 μl of the ChIP DNA sample to that from 0.1 ng genomic DNA, which is equivalent to 0.01% of input DNA (left panel). Fold change was obtained by comparing the relative quantity of the 5S rRNA gene promoter from the control shRNA-expressing cells to that from the GATA4 shRNA-expressing cells (right panel). B–D, ChIP–qPCR data showing the effect of GATA4 knockdown on the occupancies of the pol III transcription machinery at the promoters of U6 RNA (B), 7SL RNA (C), and tRNA-Met (D) genes. ChIP–qPCR was performed using the ChIP samples obtained in A and the primers used for the detection of their respective promoters as indicated. The data from ChIP assays were obtained as described for the 5S rRNA promoter and presented as for A. E–G, Western blot data of the effect of GATA4 knockdown on the expression of TBP, BRF1, and GTF3C2 proteins in 293T, HeLa, and HT29 cells. The 293T (or HeLa and HT29) cell lines expressing GATA4 shRNA or control shRNA were cultured in 6-well plates. At 90% confluence, cells were harvested; TBP, BRF1, and GTFC3C2 expression were analyzed by Western blot. H and I, GATA4 knockdown inhibited the activities of BRF1P4 and GTF3C2P2. The promoter-driving reporter vectors were transfected into the 293T cell lines expressing GATA4 shRNA or control shRNA cultured in 12-well plates, and luciferase activity was detected using the Dual-Light Luciferase Detection kit (Promega). J and K, GATA4 overexpression enhanced the activities of BRF1P4 and GTF3C2P2. The promoter-driving reporter vectors were transfected into the 293T cell lines expressing HA-GATA4 and its control cell line, and luciferase activity was detected as described in H and I. L, ChIP–qPCR data showing the GATA4 occupancy on the promoters of BRF1P4 and GTF3C2P2. Each column in A–D (left panels) and H–L represents the mean ± SD of three independent experiments. ∗p ≤ 0.05; ∗∗p ≤ 0.01. p Values were obtained by one-way ANOVA. BRF1P4, the Brf1 gene promoter 4; ChIP, chromatin immunoprecipitation assay; GATA4, GATA-binding protein 4; GTF3C2, general transcription factor 3C subunit 2; GTF3C2P2, the Gtf3c2 gene promoter 2; pol III, RNA polymerase III; qPCR, quantitative PCR.

Fig 3: Knockdown of TFIIIC components leads to reduced EZH2 occupancy on TFIIIC targets. (A, C and E) SUM159 cells were transfected with specific siRNAs for TFIIIC63, TFIIIC102 or TFIIIC110. Western blots with indicated antibodies showed the knockdown efficiency and effects of siRNAs on EZH2 expression. (B, D and F) ChIP assays were performed with an EZH2 antibody in these TFIIIC knockdown cells, followed with qPCR using indicated primers. Results were presented by mean ± S.D. from representative of three independent experiments. **P < 0.01, ***P < 0.001 by Student's t-test.

Fig 4: PRC2 component SUZ12 also interacts with TFIIIC complex and affects the function of EZH2 on Pol III transcription by maintaining EZH2 stability. (A) Co-IP assays were performed using SUM159 cell lysate with IgG and SUZ12 antibodies and detected by western blot with indicated antibodies. (B and C) Reverse Co-IP assays were performed with TFIIIC102 and TFIIIC110 antibodies and detected by western blot with indicated antibodies. (D) GST pull-down assays were carried out with GST-SUZ12 protein and HeLa cell lysates, detected by western blot with indicated antibodies. (E–H) SUM159 cells were transfected with SUZ12 siRNA 2 or control siRNA for 48 h; ChIP assays were performed with indicated antibodies to detect EZH2 and H3K27me3 occupancy. (e) Occupancy of EZH2 and H3K27me3 at promoters of tRNATyr, 5S rRNA and 7SL RNA genes were detected by qPCR with indicated primers. (F) As a positive control, the EZH2 and H3K27me3 occupancy was examined at MYT-1 promoter by qPCR after ChIP assay. (g and h) Quantitative PCR and western blot assays were conducted to examine the knockdown efficiency of SUZ12 and its effect on EZH2 expression. Results were presented by mean ± S.D. from three independent experiments. *P < 0.05, *** P < 0.001 by Student's t-test.

Fig 5: TFAP2A is required for the activation of Pol III–directed transcription induced by FLNA silencing.A and B, generation of SaOS2 cell lines expressing both FLNA shRNA and TFAP2A shRNA. TFAP2A expression in the cell lines as indicated was analyzed by RT-qPCR (A) and Western blot (B). C, TFAP2A depletion inhibited the activation of Pol III–directed transcription induced by FLNA silencing in SaOS2 cells. Pol III products were analyzed by RT-qPCR. D and E, TFAP2A depletion inhibited the enhancement of SaOS2 cell proliferation induced by FLNA silencing in SaOS2 cells. Cell counting (D) and MTT (E) assays were performed using cell lines established in A and B and the corresponding control cell line. F, BRF1 and GTF3C2 expression in SaOS2 cell lines was detected by RT-qPCR. G, BRF1 and GTF3C2 expression in SaOS2 cell lines was detected by Western blot. H, a proposed model showing the regulatory mechanisms by which TFAP2A modulates Pol III–directed transcription. Each column in histograms represents the mean ± SD of three biological replicates. ∗p < 0.05; ∗∗p < 0.01. p Values were obtained by Student’s t test. RT-qPCR, reverse transcription–quantitative polymerase chain reaction.