Figure 5
CUL1 represses splicing-associated genes. (A) HeLa cells stably transduced with shRNA against CUL1 show a stable reduction in CUL1 expression based on immunoblot against endogenous CUL1. Knockdown construct KD 1 represents TRCN0000010781; CUL1 protein levels are reduced to 11.82% relative to GAPDH control. Knockdown construct KD 2 is TRCN0000003391; CUL1 protein levels are reduced to 26.17% relative to GAPDH control. The left lane contains lysate from cells transduced with control vector TRCN0000241922. Protein lysates were normalized by Bradford assay. Densitometry was performed with ImageJ. Uncropped immunoblots are shown in Suppl. Figure 2. (B) Analysis of transcript expression changes upon CUL1 knockdown for genes that show bona fide peaks for c-MYC and both CUL1 replicates in their promoter regions. Splicing-associated genes show a significant upregulation upon CUL1 depletion (p = 2.46E−2, Wilcoxon rank-sum test). (C) CUL1 knockdown cells show a significant increase in transcripts of genes encoding splicing factors by RT-qPCR compared to cells expressing the TRC control vector (data are expressed as mean ± standard deviation, all significant p values < 1.21E−2, two-sided homoscedastic t test). RPS14 was used as reference transcript for ΔΔCt quantification. (D) HeLa cells transiently overexpressing 3xFLAG-CUL1 show a significant reduction in splicing-associated gene transcripts compared to the cells expressing the 3xFLAG vector alone (data are expressed as mean ± standard deviation, all significant p values < 2.42E−8, two-sided homoscedastic t test). RPS14 was used as reference transcript for ΔΔCt quantification. (E) Genome browser tracks of CUL1, H3K27ac, degradative ubiquitin, and c-MYC at select splicing-associated CUL1 and c-MYC target genes. Tracks from 3xFLAG-Ubiquitin-expressing HeLa cells treated with proteasome inhibitor represent degradative ubiquitination sites8, “Deg. Ubiq.”. Red boxes indicate promoter regions. Asterisks denote statistical significance.
