DESCRIPTION (provided by applicant): Dimeric NF-κB transcription factors regulate gene expression by binding to specific DNA sequences, known as the κB site DNA, located in the promoter/enhancer of target genes. These dimers can be classified into two groups, one containing the transcription activation domain (AD) and one without. p50 and p52 homodimers do not possess AD but in association with cofactors they can participate in transcription. We found that the p52 homodimer complexed to Bcl3 (p52:Bcl3 complex) is involved in transcription by binding to two types of kB sites. Recent experiments have shown the both RelA dimers and p52:Bcl3 complex can both activate and repress transcription by binding to distinct kB DNA sequences. So far we found no correlation that links NF-κB: κB DNA complex binding affinity and transcriptional output. The focus of this proposal is to understand the relationship between κB site sequence, its binding mechanism to NF-κB dimers, and transcriptional output. We hypothesize that the kinetics of NF-κB dimer binding to a κB site determines whether it acts as a repressor or activator of transcription. We propose that the binding kinetics of NF-κB to DNA is coupled to NF-κB's interactions with corepressors and coactivators. We will test our hypothesis through the following experiments: Under Aim 1, we will investigate how Bcl3 is activated by lipo-polysaccharide (LPS) stimulation and how it assembles with p52 homodimer to interact with kB sites. We will further test using genome-wide ChIP-se analysis to strengthen out claim that two distinct classes of kB sites acre acted differently by this NF-kB complex. We will further use structural, kinetic, and mutational studies to determine the correlation between κB site sequence, binding kinetics, and transcriptional potential. Under Aim 2, we will investigate the correlation between kB sequence pattern, RelA dimer binding and transcriptional output using in vitro chromatin templates. Aim 3 will investigate how NF-kB dimers find κB sites embedded in mononucleosome templates by directly measuring binding kinetics of NF-kB dimer to nucleosome bound coactivator or corepressor complexes. We will also test if in vitro binding kinetics correlates with in vivo binding kinetics for the same pair of NF-kB dimer and kB site.

PUBLIC HEALTH RELEVANCE: Transcription of protein coding genes in eukaryotes is a highly complex process that requires communication between the promoter DNA, sequence-specific DNA binding transcription factors such as NF-kappaB, mediator complex and basal transcription factors including RNA polymerase. Our proposed research plans to elucidate if and how kinetics of promoter DNA binding by NF-kappaB is able to establish the proper communication leading to transcriptional activation or establishes a different communication leading to transcriptional repression.