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. 2021 Jun 4;10:e58342. doi: 10.7554/eLife.58342

Figure 4. Apoptosis causes reduced RNAPII transcriptional output and promoter occupancy in an mRNA decay-dependent manner.

(A) Western blots performed with lysates from HCT116 cells depleted of the indicated decay factors with and without 2 hr TRAIL treatment. Blot representative of three biological replicates. Apoptosis induction was confirmed by disappearance of the full-length CASP3 band, quantified in the graph below by measuring band intensity normalized to an α-tubulin (TUBA1A) loading control (n = 3). (B, C) Changes in total (B) and nascent 4sU-labeled (C) RNA upon 2 hr TRAIL treatment in cells nucleofected with the indicated siRNAs were quantified by RT-qPCR (n = 4). Fold changes were calculated from Ct values normalized to 18S rRNA. Also see Figure 4—figure supplement 1A–B. (D, E) Chromatin immunoprecipitation (ChIP)-qPCR was used to measure occupancy of the indicated promoters by hypophosphorylated RNAPII (D) or TBP (E) under cellular conditions described in (A). Rabbit and mouse IgG antibodies were included in parallel immunoprecipitation reactions with chromatin from scramble siRNA-treated non-apoptotic cells in lieu of TBP and RNAPII antibodies, respectively, as a control. Also see Figure 4—figure supplement 1D–E. (F) Relative band intensity ratios from four replicates of the representative western blots depicted in Figure 4—figure supplement 1D, using primary antibodies specific to the indicated RPB1 CTD phosphorylation state under cellular conditions described in (A). Band intensity values were first normalized to a vinculin (VCL) loading control in each blot. All bar graphs display mean ± SEM with individual biological replicates represented as dots. Statistically significant deviation from a null hypothesis of 1 was determined using one sample t test and indicated with asterisks directly above bars, while student’s t tests with the Holm-Sidak correction for multiple comparisons were performed to compare mean values between groups indicated with brackets. *p<0.05, **p<0.00.1, ***p<0.001.

Figure 4.

Figure 4—figure supplement 1. Apoptosis causes reduced RNAPII transcriptional output and promoter occupancy in an mRNA decay-dependent manner.

Figure 4—figure supplement 1.

(A, B) RT-qPCR quantification of the change in total (A) and 4sU-labeled (B) RNA levels in HCT116 cells treated with the indicated siRNAs upon 2 hr TRAIL treatment (n = 4). Fold changes were calculated from Ct values normalized to 18S rRNA. (C) Chromatin immunoprecipitation (ChIP)-qPCR was used to measure the change in hypophosorylated RNAPII occupancy at the indicated promoters in cells nucleofected with the indicated siRNAs (n = 4). Mouse IgG antibody was included in parallel immunoprecipitation reactions with chromatin from scramble siRNA-treated non-apoptotic cells in lieu of RNAPII antibody as a control. (D) Representative western blots on lysates of HCT116 cells depleted of either 3’ DFs (DIS3L2, EXOSC4, and PNPT1) or XRN1 before and after 2 hr TRAIL treatment, probing for the indicated protein or RPB1 C-terminal domain (CTD) phosphorylation state. Apoptosis induction was confirmed by observing CASP3 cleavage. A vinculin (VCL) loading control was imaged for the blots of each phosphorylation state, but a single representative panel is shown. Quantification of RPB1 phosphorylation states from four replicates displayed in Figure 4F. (E) Band intensity fold changes of hypophosphorylated RPB1 and TBP derived from four biological replicates of the experiment described in (D). Bar graph displays mean ± SEM with individual biological replicates represented as dots. Statistically significant deviation from a null hypothesis of 1 was determined using one sample t test and indicated with asterisks directly above bars, while student’s t tests with the Holm-Sidak correction for multiple comparisons were performed to compare mean values between groups indicated with brackets. *p<0.05, **p<0.00.1, ***p<0.001.