Fig. 5: VCP presence, hexamerization, and ATPase activity impact AT3-related transcription.
From: ASPSCR1-TFE3 reprograms transcription by organizing enhancer loops around hexameric VCP/p97
a Correlation of fold-change (FC; to control) FU-UR-1 RNA-seq transcription comparing siRNAs that deplete VCP or AT3 among target genes (highly enriched AT3 region genes with >2FC by AT3 depletion. (n = 3 × 2 siRNAs each for VCP and AT3, pooled.) b Similar targets correlation for ASPS-1 (also n = 3 × 2 pooling). c RT-qPCR for target genes (controls HPRT, VCP) for HEK293T expressing TFE3, type 1 or 2 ASPSCR1::TFE3 (AT3.1, AT3.2), each with or without exogenous VCP (VCPex) overexpression. (n = 3, pooled; mean ± standard deviation; homoscedastic two-tailed t-tests comparing added VCPex to each alone.) d Genes plotted had at least 8-fold upregulated expression by RNA-seq for TFE3, AT3.1, and AT3.2 relative to control GFP in the absence of CΔ in HEK293T cells. The plot indicates log transformed p-values and FC comparing each transcription factor with co-transfected CΔ to that without CΔ. Co-transfected CΔ to HEK293T cells changes TFE3 targets in both directions, mostly insignificantly. CΔ significantly downregulates most AT3.1/AT3.2 targets (n = 3 each group, homoscedastic two-tailed t-tests). e Less pronounced correlation between the differential expression by RNA-seq in ASPS-1 from CB-5083 relative to vehicle at 48 h (n = 3 for each) to siRNAs depleting VCP relative to control at 48 h (n = 3 × 2 pooled for each). f The same for FU-UR-1 cells (for CB-5083 experiment, n = 2 for each). g Plot of log-transformed p-values and FCs of CB-5083 or DMSO treated HEK293T cells transfected with TFE3. The genes included were 8-fold upregulated by TFE3, AT3.1 and AT3.2 over GFP control transfection. Most significant changes showed further upregulation by CB-5083. (n = 3, pooled, homoscedastic two-tailed t-tests). h Genes upregulated by CB-5083 added to TFE3 are mostly downregulated by CB-5083 added to AT3.1 or AT3.2. (n = 3, pooled, homoscedastic two-tailed t-tests). i Schematic working model where VCP hexamers interact with chromatin via AT3 to activate transcription, abrogated by VCP depletion, hexamer disassembly, or enzymatic inhibition, the last with mixed impacts. j RT-qPCR for target genes after TFE3, AT3.1, or AT3.2 expression in HEK293T with co-transfection of control, VCP, or VCPE305Q (hexamer-assembling, enzymatically inactive mutant), which increases some targets’ expression (n = 3, pooled; mean ± st.dev.; homoscedastic two-tailed t-tests).
