UC San Diego

The NF-κB family of dimeric transcription factors function as master regulators of inflammation and the innate immune response. Upon pathway activation, cytoplasmic NF-κB dimers translocate to the nucleus and bind DNA response elements, known as κB DNAs or κB sites, at the promoters or enhancers of pro-inflammatory genes to activate transcription. Like other transcription factors, NF-κB binding affinity to κB DNA is sequence-dependent, however in vitro binding affinity is not a determinant of transcriptional output in the cell. Further, recent reports suggest that nuclear cofactors influence NF-κB DNA binding to κB DNA. This thesis explores what factors contribute to DNA binding by the NF-κB subunit RelA. Chapter 1 introduces gene regulation by NF-κB and outlines current gaps in our understanding of DNA binding by NF-κB. Chapter 2 explores how the central nucleotide of κB DNA modulates RelA DNA binding affinity. Our results show that the central nucleotide of κB DNA dramatically influences RelA:κB DNA complex stability through transient and dynamic interactions not observed in crystal structures. Chapter 3 investigates how locally distributed low affinity κB sites contribute to DNA binding by RelA. Our results show that low affinity binding sites impact RelA DNA binding kinetics and overall affinity, and tandemly organized low affinity κB sites can synergistically activate RelA-dependent transcription. Additionally, DNA-dependent cofactors can associate with RelA on κB DNA to collectively increase promoter occupancy and activate transcription. Chapter 4 explores how nuclear cofactors contribute to RelA DNA binding affinity. Our results show that nuclear cofactors enhance RelA DNA binding in vitro and we identify NME1 as a κB site-specific nuclear cofactor for RelA.