ABSTRACT
During eukaryotic transcription elongation, RNA polymerase II (RNAP2) is regulated by a chorus of factors. Here, we identified a common binary interaction module consisting of TFIIS N-terminal domains (TNDs) and natively unstructured TND-interacting motifs (TIMs). This module was conserved among the elongation machinery and linked complexes including transcription factor TFIIS, Mediator, super elongation complex, elongin, IWS1, SPT6, PP1-PNUTS phosphatase, H3K36me3 readers, and other factors. Using nuclear magnetic resonance, live-cell microscopy, and mass spectrometry, we revealed the structural basis for these interactions and found that TND-TIM sequences were necessary and sufficient to induce strong and specific colocalization in the crowded nuclear environment. Disruption of a single TIM in IWS1 induced robust changes in gene expression and RNAP2 elongation dynamics, which underscores the functional importance of TND-TIM surfaces for transcription elongation.
Subject(s)
Intrinsically Disordered Proteins/chemistry , RNA Polymerase II/metabolism , RNA-Binding Proteins/chemistry , Transcription Elongation, Genetic , Transcription Factors/chemistry , Transcriptional Elongation Factors/chemistry , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Cell Line, Tumor , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Gene Expression , Humans , Intrinsically Disordered Proteins/metabolism , Models, Molecular , Mutation , Protein Binding , Protein Domains , Protein Interaction Domains and Motifs/genetics , Protein Interaction Maps , RNA Polymerase II/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Elongation Factors/metabolismABSTRACT
Dimerization of many eukaryotic transcription regulatory factors is critical for their function. Regulatory role of an epigenetic reader lens epithelium-derived growth factor/p75 (LEDGF/p75) requires at least two copies of this protein to overcome the nucleosome-induced barrier to transcription elongation. Moreover, various LEDGF/p75 binding partners are enriched for dimeric features, further underscoring the functional regulatory role of LEDGF/p75 dimerization. Here, we dissected the minimal dimerization region in the C-terminal part of LEDGF/p75 and, using paramagnetic NMR spectroscopy, identified the key molecular contacts that helped to refine the solution structure of the dimer. The LEDGF/p75 dimeric assembly is stabilized by domain swapping within the integrase binding domain and additional electrostatic "stapling" of the negatively charged α helix formed in the intrinsically disordered C-terminal region. We validated the dimerization mechanism using structure-inspired dimerization defective LEDGF/p75 variants and chemical crosslinking coupled to mass spectrometry. We also show how dimerization might affect the LEDGF/p75 interactome.
