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

描述(由申请人提供)：转录因子核因子-kB是人体对感染和损伤反应的中央调节因子。核因子-kB是一个同源和异源二聚体家族，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、黏附分子和诱导酶。在某些核因子-kB靶基因的调控中出现的一个关键机制是核因子-kB二聚体与高迁移率基团蛋白HGMA1a和HMGA1b的协同结合。依赖于刺激的核因子-kB二聚体和HMGA1蛋白与DNA的协同结合是干扰素-b、白介素2、粒-巨噬细胞集落刺激因子、E-选择素、CXCL1/MGSA、COX-2和iNOS等基因完全诱导表达所必需的。重要的是，并不是所有的核因子-kB结合位点都能结合核因子-kB：HMGA复合体，而其他结合位点只能结合含有特定核因子-kB二聚体的复合体。目前尚不清楚DNA结合位点序列本身在多大程度上定义了HMGA1辅助因子在NF-kB反应中所起的作用。 该建议的目的是综合表征不同的核因子-kB：HMGA1复合体的DNA结合特异性，并将这种特异性与核因子-kB靶基因表达的HMGA1依赖性联系起来。目的1利用蛋白质结合微阵列(PBMS)研究纯化蛋白与核因子-kB：HMGA1复合体的DNA结合。目标2是表征由于翻译后修饰或未知辅因子可能产生的任何潜在差异。为了做到这一点，将使用来自刺激细胞的核裂解物进行PBM实验，然后将这些结果与使用纯化蛋白获得的结果进行比较。目的3利用生物信息学方法和基于细胞的报告分析，研究核因子-kB：HMGA1复合体与核因子-kB靶基因表达的结合特异性。 这项工作的成功完成将为辅助因子，如HMGA1，如何为依赖于核因子-kB的基因调控提供特异性提供更清晰的图景。抑制核因子-kB的转录反应一直是治疗许多炎症性疾病的努力的焦点；从拟议的工作中获得的见解可能会提出新的方法，通过靶向HMGA1或其他辅助因子来选择性地调节核因子-kB的反应以达到治疗目的。

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.