Figure 2. RNAPII transcription is globally repressed during early apoptosis.

(A, B) RT-qPCR measurements of total (A) and nascent 4sU-labeled (B) RNA fold changes after 2 hr TRAIL treatment of HCT116 cells, including a 1 hr pre-treatment with either 40 μM zVAD or an equal volume of DMSO (‘mock’). Also see Figure 2—figure supplement 1A. RNA fold change values were calculated in reference to 18S rRNA. Bar graphs display mean ± SEM with individual biological replicates (n = 4) 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 were performed to compare mean fold change values for mock inhibitor or scramble treated cells to those treated with inhibitor or a targeting siRNA and indicated with brackets. *p<0.05, **p<0.00.1, ***p<0.001. (C, D) rRNA-depleted cDNA sequencing libraries were reverse transcribed from 4sU-labeled RNA isolated from cells under the conditions described in (A, B). Transcripts that aligned to genes in the human genome are graphed with differential log₂ fold change expression values (log₂FC) on the y axis and fragments per kilobase per million reads (FKPM) expression values (normalized to ERCC spike-in controls) on the x axis. All values were averaged from two biological replicates. Data points for transcripts upregulated or downregulated by twofold or greater are colored green and purple, respectively. Percentages of transcripts in each expression class are indicated with an arrow and in their corresponding colors. Also see Figure 2—figure supplement 1C–D and Figure 2—source data 1A–B. (E) Top statistically significant hits from gene ontology analysis performed for the list of transcripts that were upregulated upon TRAIL treatment with DMSO in a statistically significant manner across biological duplicates. Also see Figure 2—source data 1C. (F) Top hits from transcription factor (TF) enrichment analysis for the same list of genes as above. The lower the MeanRank value, the more statistically significant enrichment for genes regulated by the indicated TF. Also see Figure 2—source data 1D.

Figure 2—figure supplement 1. RNAPII transcription is globally repressed during early apoptosis.

(A) Western blot of HCT116 lysates showing cleavage (or lack thereof) of the caspase 8 (CASP8) targets BID and caspase 3 (CASP3), as well as the CASP3 target PARP1 upon 2 hr 100 ng/μL TRAIL treatment in the presence or absence of 40 μM zVAD. Blot representative of that from three biological replicates. (B) Log₂ fold change (log₂FC) in abundance of the ACTB transcript upon 2 hr TRAIL treatment in the presence of either zVAD or DMSO vehicle (‘mock’). Fold changes from the same 4sU-labeled RNA samples were quantified by next-generation sequencing and differential expression analysis, as well as RT-qPCR normalized to 18S rRNA using both intronic and exonic gene-specific primers. Graph displays mean ± SEM with individual biological replicates (n = 2) represented as dots. Student’s t tests were performed to compare the log₂FC values upon TRAIL treatment in the presence of DMSO vehicle or zVAD. **p<0.00.1 (C, D) Volcano plot depicting the –log₁₀p values across biological duplicates for the fragment per kilobase per million read fold changes in the sequencing libraries depicted in Figure 2C,D. Each point represents a transcript mapped to the human genome. Upregulated and downregulated transcripts with |FC| > 2 and p<0.05 are colored in green and purple, respectively. Number of transcripts in each expression class are indicated with an arrow and in their corresponding colors.