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-κ B是机体对感染和损伤反应的中心调节因子。NF-kB是同源和异源二聚体的家族，其共同调节参与免疫和炎症反应的基因，例如促炎细胞因子、趋化因子、粘附分子和诱导酶。在某些NF-κ B靶基因的调节中出现的关键机制是NF-κ B二聚体与高迁移率族蛋白HGMA 1a和HMGA 1b的协同结合。已经证明，NF-κ B二聚体和HMGA 1蛋白与DNA的刺激依赖性协同结合对于基因如IFN-β、IL-2、GM-CSF、E-选择素、CXCL 1/MGSA、考克斯-2和iNOS的完全诱导表达是必需的。重要的是，并非所有的NF-kB结合位点都可以结合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结合位点序列单独定义HMGA 1辅因子在NF-κ B应答中的作用程度。 该提案的目的是全面表征不同NF-kB：HMGA 1复合物的DNA结合特异性，并将这种特异性与NF-kB靶基因表达的HMGA 1依赖性相关。目的1：利用蛋白结合微阵列（PBMs）研究NF-κ B：HMGA 1复合物与DNA的结合。目的2是表征可能由于翻译后修饰或未知辅因子而引起的NF-κ B：HMGA 1复合物中的任何潜在差异。为此，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细胞报告基因检测技术，研究NF-κ B：HMGA 1复合物与NF-κ B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HMGA 1）如何为NF-κ B依赖性基因调控提供特异性提供更清晰的图像。抑制NF-kB转录反应一直是治疗许多炎症性疾病的治疗努力的焦点;从所提出的工作中获得的见解可能提出通过靶向HMGA 1或其他辅因子选择性调节NF-kB反应以用于治疗目的的新方法。

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.