Single-molecule dynamics and genome-wide transcriptomics reveal that NF-kB (p65)-DNA binding times can be decoupled from transcriptional activation

• Published in: PLoS genetics 2019-01, Vol.15 (1), p.e1007891-e1007891
• Main Authors: Callegari, Andrea, Sieben, Christian, Benke, Alexander, Suter, David M, Fierz, Beat, Mazza, Davide, Manley, Suliana
• Format: Article
• Language: English
• Subjects: Binding sites; Biochemistry; Bioengineering; Bioinformatics; Biology and life sciences; Clonal deletion; Deoxyribonucleic acid; DNA; DNA binding; DNA binding proteins; DNA-Binding Proteins - genetics; Gene deletion; Gene expression; Gene Expression - genetics; Genome, Human - genetics; Genome-wide association studies; Genomes; Genomics; HeLa Cells; Humans; Kinases; Kinetics; Life sciences; Mutant Proteins - chemistry; Mutant Proteins - genetics; NF-kappa B - chemistry; NF-kappa B - genetics; NF-κB protein; Nucleotide sequence; Observations; Physics; Physiological aspects; Prokaryotes; Promoters (Genetics); Protein interaction; Protein Interaction Domains and Motifs - genetics; Protein-protein interactions; Proteins; Research and Analysis Methods; RNA polymerase; Single Molecule Imaging; Stem cells; Supervision; Transcription (Genetics); Transcription activation; Transcription Factor RelA - chemistry; Transcription Factor RelA - genetics; Transcription factors; Transcription Factors - chemistry; Transcription Factors - genetics; Transcriptional Activation - genetics; Tumor necrosis factor-TNF
• ISSN: 1553-7404; 1553-7390; 1553-7404
• DOI: 10.1371/journal.pgen.1007891

Transcription factors (TFs) regulate gene expression in both prokaryotes and eukaryotes by recognizing and binding to specific DNA promoter sequences. In higher eukaryotes, it remains unclear how the duration of TF binding to DNA relates to downstream transcriptional output. Here, we address this question for the transcriptional activator NF-κB (p65), by live-cell single molecule imaging of TF-DNA binding kinetics and genome-wide quantification of p65-mediated transcription. We used mutants of p65, perturbing either the DNA binding domain (DBD) or the protein-protein transactivation domain (TAD). We found that p65-DNA binding time was predominantly determined by its DBD and directly correlated with its transcriptional output as long as the TAD is intact. Surprisingly, mutation or deletion of the TAD did not modify p65-DNA binding stability, suggesting that the p65 TAD generally contributes neither to the assembly of an "enhanceosome," nor to the active removal of p65 from putative specific binding sites. However, TAD removal did reduce p65-mediated transcriptional activation, indicating that protein-protein interactions act to translate the long-lived p65-DNA binding into productive transcription.