Abstract
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.
Highlights
Transcription factors (TFs) are fundamental regulatory components of transcription in both prokaryotes and eukaryotes, which can activate or repress the expression of specific genes
To control the rate of transcription of genes, both eukaryotes and prokaryotes express specialized proteins, transcription factors (TF), that bind promoter sequences to mark them for the transcriptional machinery including DNA polymerase II
While p65 DNA binding times are dominated by the DNA-binding domain (DBD), transcriptional output relies upon functional protein-protein interaction subunit
Summary
Transcription factors (TFs) are fundamental regulatory components of transcription in both prokaryotes and eukaryotes, which can activate or repress the expression of specific genes. The NF-κB family of TFs, universal among most animal cell types, is involved in many signaling pathways and when dysregulated can contribute to several pathologies, including cancer and inflammatory diseases [1]. This is exemplified by the RELA (v-rel reticuloendotheliosis viral oncogene homolog A), or p65 TF, which is implicated in regulating the activation of ~150 genes involved in wide-ranging functions from immune response to metabolism [1]. Co-regulators of transcription are recruited at the promoters of target genes via protein-protein interactions mediated by TAD1 and TAD2, eventually leading to the recruitment of RNA polymerase II (RNA pol-II) and subsequent activation of gene expression [8]. Deletion of one or both TADs has been shown to heavily impair p65-dependent transcriptional activation, suggesting a dominantnegative effect of such truncation mutants [7]
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