Promoter-associated RNA polymerase III shapes RNA polymerase II-dependent inflammatory gene expression during viral infection

Key results

1. ShownMHV68 infection of NIH3T3 fibroblasts redistributes Polr3A from Pol II protein-coding promoters to B1/B2 SINE retrotransposons, with total Polr3A signal largely unchanged and four core subunits stable at the protein level.

   7,429 Polr3A peaks lost and 4,293 gained (FDR < 0.05). Lost peaks: 85% at Pol II promoters, 95% of those SINE-free. Gained peaks: 83% at B1/B2 SINEs, 92.3% of the promoter ones overlapping a SINE. · RNA polymerase III is redistributed from…; Figure legends

2. ShownIn uninfected primary BMDMs, Brf1 depletion lowers expression of innate immune and inflammatory genes, which carry higher promoter Polr3A than the genes that rise.

   255 genes down (log2FC ≤ -1.5) vs 175 up among 14,416 protein-coding genes (p < 0.05). The down set was enriched for TNFα signaling via NF-κB, inflammatory response, and interferon alpha and gamma response (Hallmark). · RNA polymerase III supports basal innate immune…; Figure legends

3. ShownIn uninfected NIH3T3 cells, Brf1 depletion reduces promoter accessibility in proportion to Polr3A occupancy. This dose-dependence is substantially attenuated during MHV68 infection.

   Top Polr3A quintile (Q5; mean spike-in-normalized signal = 95) showed a 6% absolute reduction vs the lowest quintile. 377 mapped Brf1-dependent inflammatory promoters lost more signal than 19,635 unchanged genes (ATAC-seq, n=3 per condition). · RNA polymerase III maintains chromatin…; Visualization; Figure legends

4. ShownDuring MHV68 infection of BMDMs, Brf1 depletion raises inflammatory gene expression without changing viral gene expression or DNA replication. HSV-1 and influenza A infection of human A549 cells show the same TNFα/NF-κB derepression.

   373 genes up vs 381 down at 48 hpi in BMDMs. After removing cell-cycle gene sets, TNFα/NF-κB led in HSV-1 (31 genes) and IAV (22 genes, adjusted p = 0.042). Mock-infected A549 cells showed no TNFα/NF-κB signal. · RNA polymerase III activity restrains…; RNA polymerase III also regulates inflammatory…; Figure legends

5. SuggestedPromoters restrained by Pol III are enriched for TATA boxes, TBP dependency and baseline TBP occupancy. This supports, but does not directly test, a model in which Pol III competes with Pol II for TBP.

   Human inflammatory subset (n=48): 23% with a TATA-box vs 5% genome-wide; 34.9% TBP-dependent vs 4.2% in background (OR = 12.1). TAF1 dependency was not enriched. TBP ChIP-seq signal was higher than at expression-matched Brf1-dependent genes in human and mouse. · TBP dependency underlies RNA Polymerase III…; Analysis of TBP, TAF1, and TFIIB dependency from…; Figure legends