The role of HMGA1 proteins in modulating NF-kB-dependent gene regulation

描述（由申请人提供）：转录因子NF-kB是机体对感染和损伤反应的中枢调节因子。NF-kB是一个同源和异源二聚体家族，它们共同调节参与免疫和炎症反应的基因，如促炎细胞因子、趋化因子、粘附分子和诱导酶。NF-kB靶基因调控的一个关键机制是NF-kB二聚体与高迁移率基团蛋白HGMA1a和HMGA1b的协同结合。刺激依赖的NF-kB二聚体和HMGA1蛋白与DNA的协同结合已被证明是IFN-b、IL-2、GM-CSF、E-selectin、CXCL1/MGSA、COX-2和iNOS等基因完全诱导表达的必要条件。重要的是，并非所有NF-kB结合位点都能结合NF-kB:HMGA复合物，而其他位点只能结合含有特定NF-kB二聚体的复合物。目前尚不清楚DNA结合位点序列在多大程度上单独定义HMGA1辅助因子在NF-kB应答中的作用。

DESCRIPTION (provided by applicant): 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.