DNA-Binding Activity of Human Transcription Factors

描述（由申请人提供）：人类转录因子（HTF）和修饰它们的调节因子是协调人类基因组表达所必需的，它们的功能障碍与引起人类癌症和其他疾病有关。这项研究的长期目标是了解控制全球转录调控网络的分子机制，这是开发治疗方案以治愈HTF相关癌症和疾病的先决条件。我们假设HTFs（单体或晚餐）和DNA基序之间的许多相互作用可以是收敛的或发散的，并且可能受到磷酸化的调节。作为实现这一目标的第一步，模体预测算法和蛋白质芯片技术的结合力量将被应用于发现和验证HTFs的新模体。HTFs的DNA结合活性的调节将通过集中于人类中的选定激酶来检查。数据将被整合以启动基于生化活性的转录电路的组装。这些目标将通过以下四个具体目标来实现：（1）制备HTF蛋白芯片并确定最佳检测条件。1003个纯化的HTF和96个异源二聚体将用于构建400个HTF蛋白芯片。(2)通过使用HTF蛋白芯片上的预测基序来分析基序结合活性。将在HTF芯片上确定DNA基序与其结合因子之间的关联。(3)确定MAPKs对HTF的基序结合活性的影响。将在基于芯片的测定中鉴定可被MAPK和相关激酶磷酸化的TF，并确定这种修饰对HTF的DNA结合活性的影响。(4)对上述实验结果进行统计分析，构建转录调控网络图。为了验证结果，我们计划首先应用数据集成步骤来为每种类型的高通量数据集生成鲁棒的命中列表，然后使用常规方法（例如，用于DNA基序验证的染色质-IP测定）。利用人类细胞系，我们将在体内验证激酶底物：我们期望该项目将完全建立一个研究HTF活性和调控的平台，并为人类转录组调控机制提供重要见解，从而帮助我们更好地了解相关人类疾病和癌症发展的分子机制，并为改善公共健康提供线索。

DESCRIPTION (provided by applicant): Human Transcription factors (HTFs) and regulators that modify them are required to coordinate expression of the human genome/and their malfunction is implicated in causing cancers and other diseases in humans. The long-term goal of the proposed research here is to understand the molecular mechanisms governing the global transcription regulatory networks, as a prerequisite to developing therapeutic protocols to cure HTF- related cancers and diseases. We hypothesize that the many interactions between HTFs (monomers or dinners) and DNA motifs can be either convergent or divergent, and may be regulated by phosphorylation. As the first step towards this goal, the combined power of motif prediction algorithms and protein chip technologies will be applied to discover and validate novel motifs of HTFs. Regulation of the DNA-binding activity of HTFs will be examined by focusing on selected kinases in humans. Data will be integrated to initiate assembly of biochemical activity-based transcription circuitry. These goals will be achieved through the following four specific aims: (1) Fabricate HTF protein chips and determine the optimal assay conditions. 1003 purified HTFs and 96 heterodimers will be used to construct 400 HTF protein chips. (2) Profile motif- binding activities by using predicted motifs on the HTF protein chips. The association between a DNA motif and its binding factor will be determined on the HTF chips. (3) Determine the effects of MAPKs on motif- binding activity of HTFs. TFs that can be phosphorylated by MAPKs and related kinases will be identified in chip-based assays, and the effects of this modification on the DNA-binding activity of HTFs will be determined. (4) Validate the results from the above assays and build a map of transcription regulatory network. To validate the results, we plan to first apply a data integration step to generate robust hit lists for each type of high-throughput data set, followed by careful validation using conventional approaches (e.g., "gel shift" and chromatin-IP assays for DNA motif validation). Using human cell lines, we will validate the kinase substrates in vivo: We expect that this project will fully establish a platform to inverstigate HTF activities and regulation, and to provide significant insight into the mechanisms of regulation of the human transcriptome and thus, help us to better understand the molecular mechanisms of related human diseases and cancer development and provide clues to improve public health.