Transcriptomic impacts of TFIIB D207G editing in Jurkats during 2 h TRAIL treatment

Cellular stressors often cause widespread repression of RNA polymerase II (RNAP II) activity, which is thought to facilitate a focused transcriptional output towards stress resolution. In many cases, however, the underlying regulatory mechanisms remain unknown. Here, we demonstrate that stress-induced downregulation of the general transcription factor TFIIB tempers expression of specific stimulus response genes. Following a variety of stressors, TFIIB is proteolytically cleaved between its cyclin folds at conserved aspartic acid residue D207 by caspases- 3 and 7. Cleavage in this portion of the protein significantly reduces the ability of TFIIB to form a TBP-TFIIB-DNA promoter complex in vitro. Using both overexpression and endogenous base-editing, we find that B and T cells that are unable to cleave TFIIB upregulate expression of a select gene set during apoptosis. These TFIIB-sensitive genes are primarily short, stimulus-responsive and proto-oncogenic loci, and cleavage of TFIIB temporally restricts their expression. Failure to cleave TFIIB during stress leads to aberrant lymphocyte proliferation during chemical perturbation. Hence, caspase targeting of TFIIB destabilizes transcription to tune gene expression, allowing for proper stress resolution. Overall design: Using Jurkat T cells, which are sensitive to apoptotic stimuli, two different sgRNAs targeting the TFIIB D207 locus were validated (D207G-#1 and D207G-#2; also referred to as sgRNA1 and sgRNA2 in the sequencing below). A control dinucleotide base edit was made in the AAVS1 locus (Control/sgRNA3). Pooled populations with = 85% editing at each target locus were validated by amplicon sequencing. Pooled populations were expanded for = 10 days after delivery of A•G base editor RNA and the sgRNA, and then submitted to SLAM-sequencing (edit stability was monitored for all experiments and protein-level impacts were confirmed in parallel by western blotting). Cells were treated with SUPERKILLER TRAIL (Enzo) and 4sU (to label nascent RNA) for 2 h prior to harvesting . This was followed by RNA extraction, alkylation, library preparation, RNA-sequencing, and differential gene expression analysis.