WSB1/2 target chromatin-bound lysine-methylated RelA for proteasomal degradation and NF-?B termination

Proteasome-mediated degradation of chromatin-bound NF-?B is critical in terminating the transcription of pro-inflammatory genes and can be triggered by Set9-mediated lysine methylation of RelA subunit. However, the E3 ligase targeting methylated RelA remains unknown. Here, we identified two structurally similar WD-40 repeat (WDR) proteins, WSB1 and WSB2, as the E3s that recognize chromatin-bound methylated RelA for polyubiquitination and proteasomal degradation. WSB1/2 specifically recognized methylated lysines (K) 314 and 315 of RelA via their WDR domains. Deletion of WRD in WSB1/2 or mutation of K314/315 of RelA to arginines abolished the interaction between WSB1/2 and RelA. RNA-sequencing of TNF-?-stimulated WSB1/2 knockdown cells revealed that WSB1/2 negatively regulated a subset of NF-?B target genes with reduced polyubiquitination of chromatin-bound RelA. TNF-? stimulated the methylation of RelA and the subsequent recruitment of WSB1/2 to the promoters of these NF-?B target genes to terminate the transcription. Computational modeling demonstrated that a highly conserved Asp within repeat 3 of WDR domains of WSB1/2 coordinated its interaction with K314/K315 of RelA, with a higher pKa when either of the lysines is methylated. Together, these findings identify novel E3 ligases that target chromatin-bound methylated RelA for proteolysis to prevent sustained NF-?B activation, providing new targets for therapeutic intervention of NF-?B-mediated inflammatory diseases. Overall design: Comparative gene expression profiling analysis of RNA-seq data for U2OS cells and its KD derivatives (shWSB2) under normal conditions and TNF-a stimulation (two replicates per sample) Please note that each processed data file contains processed data for two samples and is linked to the corresponding '1' sample records.