The dimeric NF-KB transcription factors are formed from five family members, p50 (NFKBI), ReI A (p65), p52 (NF-KB2), c-Rel, and RelB. p50 and p52 are the processed products of precursor proteins, p105 and p100, respectively. RelA homo-and heterodimer are activated through classical NF-KB signaling pathways that are linked to IKKj3 activation. These dimers are responsible for rapid activation of genes that are important for inflammation and immune response. RelB/p52 are generated from the inactive p100/RelB complex through non-classical NF-KB signaling pathways, which involve IKKa activation. The RelB/p52 heterodimer is known to be important for organogenesis and cell survival. Aberrant processing of p100 into p52 is seen in a large number of cancers.

I have been working on two related projects; first, determination of NF-KB dimer specificity for target DNA sequences, and second, investigation of the role of RelB in p100 processing/degradation. The first project has two components: To determine affinities of different NFKB dimers for KB DNA sequences and to determine the X-ray crystal structure of RelB/p52 heterodimer. The affinity measurement experiments may indicate if there is a correlation between an NF-KB dimer and its target DNA sequences. The structure of the RelB/p52/DNA complex will be the last NF-KB/DNA complex to be determined. If I find dimer specificity for target DNA sequences, three-dimensional structures might provide an explanation for the basis of the biochemical specificity. I have determined the binding affinities of most physiological NF-KB dimers for a large number of known target DNA sequences. I have found that whereas RelA dimers can discriminate target DNAs, the RelB/p52 heterodimer binds to all KB DNAs with similar affinities tested so far. I have grown crystals of RelB/p52/DNA complex that diffract to 3.0A resolution, and I am in the process of completing the structure. We hope that the structure might explain as to why RelB/p52 is more promiscuous in DNA binding than the RelA dimers. In my second project, I have shown that RelB protects p100 from degradation and processing in resting cells. I have further shown that dimerization of p100 with RelB is absolutely essential for this protective function. Moreover, I found that the strength of the RelB/p100 complex will determine if p100 can be processed into p52 or be completely degraded. This work clearly suggests that RelB/p52 is not generated from the p100/RelB complex. I have completed the in vitro biochemistry work using reconstituted knock out cell lines, and a manuscript will be submitted shortly either to EMBO or Molecular cell.

PUBLICATIONS (resulting from this training)

Huang DB, Phelps CB, Fusco AJ, Ghosh G. (2004) Crystal structure of a free kappaB DNA: insights into DNA recognition by transcription factor NF-kappaB. Mol. Biol. 346: 147-160.

Fusco A, Vu D, Huang DB, Savinova O, Talwar R, Kerns J, Hoffmann A, Ghosh G. (2007) RelB forms a unique complex with p100/p52 leading to its stabilization and inhibition of p100 processing/degradation. Nature Struc. & Mol. Biol., In Review (revised manuscript).

Fusco A, Vu D, Huang DB, Ghosh G. (2007) The stability of RelB dimerization domaini is a critical regulator of the RelB/p52 formation. Manuscript to be submitted.