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Regulation of gene expression by NF-kB RelA

H. Thien Nguyen

Appointment Period: 2015-2016, Grant Year: [29]


How the transcriptional activator proteins such as NF-kappaB (NF-kB), AP1, STAT, NFAT, etc, regulate transcription remains an open question. It is believed that multiple factors bound to tandem DNA sites interact cooperatively to form a stable ‘enhanceosome’ which recruits basal transcription machinery including the polymerase at the site of transcription initiation. But these activators can also repress transcription. Findings in our laboratory challenge the model. We have recently reported that single bp variations in NF-kB binding sites can have profound effect in transcriptional output. We showed using genome wide and in vitro biochemistry that NF-kB p52 represses transcription by binding the A/Tcentric (GGGAAATTCC) site by recruiting corepressors but activates transcription by binding to a G/Ccentric site (GGGAAGTTCC) by recruiting coactivators. We further noted that NF-kB binds to A/Tcentric sites more tightly than the G/C-centric site.

We have now found that RelA also behaves similarly, consistent with data reported by the Baltimore laboratory. We found that stronger equilibrium binding affinity is linked to transcriptional repression by RelA dimers. The major objective of my thesis project is to investigate the mechanism by which the same transcription factor induces recruitment of coactivator or corepressor by binding to only ‘slightly’ different DNA target sequences. Our model is that the half-life of RelA on a DNA site dictates if it couples to co-repressor or co-activator. My experiments will test the model in vitro using chromatinized template where single NF-kB binding sites are incorporated behind the transcription start site and monitor transcription using pure RelA and HeLa nuclear extracts. I have recently prepared chromatin templates and started transcription assays recently, and we are working closely with the Kadonaga group in this part of the project.

In the an extension of the project described above, I will also be investigating how a single methylation event alters RelA’s DNA binding activity using x-ray crystallography, in vitro binding and transcription assays. Other work has shown that the residue Arg30 in RelA is methylated by PRMT5 (proline arginine methyltransferase 5) resulting in an altered gene expression profile. I will utilize a semisynthetic approach to generate RelA-meR30 to prepare the protein. This work is in collaboration with Tao Lu at the Indiana University (expert on NF-kB methylation-dependent gene expression) and Eileen Kennedy at University of Georgia (expert on synthetic peptide preparation).

Misregulation of NF-kB activation are linked to many human diseases, particularly ones associated with chronic inflammation, immunodeficiency and cancer. Several pathway-affecting mutations that cause gene amplification, single amino acid change or deletions have been reported. Many other cancers such as breast, lung and skin are also affected by NF-kB misregulation. No mutation of RelA has been reported to date, perhaps due to its essential functions, but increased nuclear RelA activity is found in many cancers. RelA and all other NF-kB regulators are known for their cell survival and proliferation activities. We believe that our mechanistic studies will be helpful to understand how misregulation of NF-kB altered gene expression of several proteins that are directly affect cell growth and proliferation resulting in oncogenesis.

PUBLICATIONS (resulting from this training)

Trainee recently appointed to grant: publications are still in progress.