The transcription factor NF-kB is a central regulator in the body's response to infection and injury. NF-kB is a family of homo- and hetero-dimers that together regulate genes involved in the immune and inflammatory responses, such as pro-inflammatory cytokines, chemokines, adhesion molecules and inducible enzymes. A key mechanism that has emerged in the regulation of certain NF-kB target genes is the cooperative binding of NF-kB dimers with the high mobility group proteins HGMA1a, and HMGA1b. Stimulus-dependent, cooperative binding of NF-kB dimers and HMGA1 proteins to DNA has been shown necessary for the full inducible expression of genes such as IFN-b, IL-2, GM-CSF, E-selectin, CXCL1/MGSA, COX-2 and iNOS. Importantly, not all NF-kB binding sites can bind NF-kB:HMGA complexes, while others sites can bind only complexes containing specific NF-kB dimers. It remains unclear the extent to which DNA binding site sequence alone defines the role HMGA1 cofactors play in the NF-kB response. The goal of this proposal is to comprehensively characterize the DNA binding specificity of different NF-kB:HMGA1 complexes, and to relate this specificity to HMGA1-dependence of NF-kB target gene expression.
Aim 1 is to use protein binding microarrays (PBMs) to characterize the DNA binding of NF-kB:HMGA1 complexes using purified protein.
Aim 2 is to characterize any potential differences in NF-kB:HMGA1 complexes that might arise due to post-translational modifications or unknown cofactors. To do this PBM experiments will be performed using nuclear lysates from stimulated cells, these results will then be compared to results obtained using purified protein.
Aim 3 is to related the binding specificity of NF-kB:HMGA1 complexes to NF-kB target gene expression using bioinformatics approaches and cell-based reporter assays. Successful completion of this work will provide a clearer picture of how co-factors, such as HMGA1, provide specificity to NF-kB-dependent gene regulation. Inhibition of the NF-kB transcriptional response has been a focus of therapeutic efforts to treat many inflammatory disorders;insights gained from the proposed work may suggest new ways to selectively modulate the NF-kB response for therapeutic purposes by targeting HMGA1 or other co-factors.
STATEMENT: This research on genome sequences that allow the proteins HMGA1 and NF-kB to regulate key genes of the immune and inflammatory responses will provide insight into both the body's normal response to invading microbes and situations where these proteins are mis-regulated as is found in many cancers and inflammatory diseases. It may also allow us to better understand and predict how the sequence differences in each of our genomes, either hereditary differences or mutations, affects our individual immune and inflammatory responses and susceptibility to disease.
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