Role of NF-kappaB Interactions with its Binding Partners in NF-kappaB driven Transcription

Abstract

The NF- family of inducible transcription factors (TF) plays a major role in immune response by regulating the expression of hundreds of target genes. These target genes in turn are vital for normal cell physiology through their actions in cellular immune and inflammatory response, survival, proliferation, growth and development. The NF- family is comprised of five proteins all of which form dimers with self or another family member in various combinations. The NF- family members, namely, RelA (also known as p65), RelB, c-Rel (also known as rel), p50 and p52 share a ~300 amino acid Rel homology region (RHR) which binds to B DNA as a dimer. The family can be classified into two classes based on the presence of a highly acidic transcription activation domain (TAD). RelA, RelB and c-rel belong to Class I that contains the TAD, and p50 and p52 to Class II where the TAD is absent. The RHR is solely responsible for B DNA binding and dimerization of the NF-B members whereas the TAD interacts with various cofactor proteins and is required for the assembly of the basic transcription machinery on the transcription start site. Hence, homodimers of p50 or p52 cannot activate transcription despite its ability to bind to B DNA on the promoter/enhancer region of NF-B target genes. According to the canonical NF-B pathway, in the resting state, NF-B (p50-RelA heterodimer) is arrested in its inactive form in the cytoplasm bound to Inhibitor B family (IB) of proteins. The NF-B pathway is activated by a number of stimuli ranging from cytokines, growth factors, UV-irradiation, oxidative stress and more. Post stimulation, a cascade of events leads to the proteazomal degradation of IBα thereby releasing the NF-B dimer into the nucleus to activate transcription of its target genes. Unlike RelA dimers, p50-p50 (homodimer) is also present in the nucleus in the cell resting state. The nuclear p50-p50 is found to bind with B DNA on various promoter/enhancer regions but is unable to activate transcription of those genes due to lack of TAD. Thus, p50-p50 acts as repressor of such genes. How these NF-B repressed genes are activated remains unclear. Here I have used NMR spectroscopy to understand the mechanism activation of NF-B repressed genes. As a first step, the resonance assignments of the RHR of NF-B p50 subunit was obtained in both free (73.1 kDa) as well as DNA bound (>80 kDa) forms. To achieve this, the strategy of transfer of resonance assignments from constituent domains of p50RHR was used. The resonance assignments revealed the independent nature of the two constituent domains, namely, the DNA-binding N-terminal domain (NTD) and the dimerization domains (DD) of the p50RHR. A comparison of p50NTD was made with RelA NTD which revealed more flexible regions present in the p50NTD. The resonance assignments were then used to understand the differential binding of two different B DNA sites by p50RHR. To activate transcription of its target genes, apart from NF-:DNA interaction, NF-B also interacts with various other co-factor proteins like the CREB-binding protein (CBP) and the gigantic Mediator complex. Mediator is a variable multi-subunit protein complex, ~1.2 MDa with 26 core protein subunits. The wellstudied RelA subunit of NF-B is known to physically interact with the MED17 subunit of the Mediator complex. In my thesis, the interaction of RelATAD with various fragments of MED17 protein was studied.