UC San Diego

NF-[kappa]B is a family of dimeric transcription factors made up of five family members, p50 (NF-[kappa]B1), RelA (p65), p52 (NF-[kappa]B2), c-Rel and RelB, that regulate the expression of a large number of genes involved in innate and adaptive immunity. The NF-[kappa]B dimers regulate gene expression by binding to a class of decameric DNA duplexes known as [kappa]B sites. RelB is the most poorly understood and unique member of the NF- [kappa]B family. Unlike other members, RelB does not form a stable detectable homodimer in vivo. In uninduced cells, RelB preferentially associates with p100 and to a lesser extent with p50. Gene knockout studies demonstrated that RelB and p52 are involved in common biological functions such as secondary lymphoid organ development and B-cell maturation. It was subsequently found that these two proteins act together as a heterodimeric transcription factor that activates a set of genes in the developmental programs. The promoters of several of these genes have shown to contain [kappa]B sites that somewhat deviate from the classical [kappa]B sites. There are two important questions that remain to be answered. These questions are 1) how is the RelB/p52 heterodimer generated and 2) how does this heterodimer bind to [kappa]B sites? The purpose of this dissertation was to provide answers to these questions. The RelB/p52 heterodimer is the product of NF- [kappa]B signaling pathways, referred to as the non- canonical pathways, which involve processing of the precursor protein p100 into p52. However, whether the RelB /p52 heterodimer is generated from the p100/RelB complex observed in uninduced cells was unclear. Results shown in this dissertation demonstrate that NF-[kappa]B RelB is an unstable protein that is stabilized by forming complexes with both p100 and p52. The p100/RelB complex involves all functional domains of each protein and is not processing competent. A large body of experimental results shown here clearly suggests that p52 is primarily generated from newly synthesized p100 and not from the pre-existing p100/ RelB complex. Processing of the newly synthesized p100 is facilitated when bound to a newly synthesized NF-[kappa]B subunit, including RelB. However, the binding of newly synthesized p100 with RelB can also lead to the formation of an inactive p100/RelB complex. Therefore a dynamic binding mode between p100 and RelB, where one is processing competent and the other is not, is proposed. To understand the [kappa]B DNA recognition by the RelB/p52 heterodimer, the complex between the heterodimer and [kappa]B DNA has been crystallized and the 3.05 Å crystal structure is reported in this dissertation. The structure has revealed a new mode of DNA interaction by an NF- [kappa]B dimer that has never been observed previously. In addition to the conserved DNA contacts made by all NF- [kappa]B family members, a conserved arginine of RelB contacts the outermost G:C base pair of the RelB DNA half site. At the same time RelB loses one contact with the innermost A:T base pair of the half site normally mediated by a conserved tyrosine residue. Binding affinity measurements demonstrate that the RelB/p52 heterodimer binds to the [kappa]B DNA tested, including the newly identified ones indicated above, with similar affinities. We propose that the RelB/p52 heterodimer binds to different [kappa]B sites with high affinity by altering the DNA contact. The RelB subunit allows for different binding modes by binding to some [kappa]B sequences using the tyrosine contacts and to other [kappa]B sequences by utilizing the arginine contact. Specific DNA sequences at the center and flanking region dictate the RelB binding modes