DNA-Binding Activity of Human Proteins

人类蛋白质的 DNA 结合活性

• 批准号: 8248692
• 负责人: Heng Zhu
• 金额: $ 40.66万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-04 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8248692
• 关键词: Amino Acids; Area; Basic Science; Bioinformatics; Biological; Calculi; Communities; Complex; DNA; DNA Binding; DNA-Binding Proteins; DNA-Protein Interaction; Data; Data Set; Defect; Development; Disease; Epigenetic Process; Functional RNA; Gene Expression; Gene Targeting; Generations; Genetic Transcription; Goals; Human; Human Activities; Interferon Type II; Knowledge; Laboratories; Lead; MAPK1 gene; Malignant Neoplasms; Metabolism; Microarray Analysis; Mission; Mitogen-Activated Protein Kinases; Molecular; Open Reading Frames; Outcome; Participant; Pathway interactions; Physiological; Play; Protein Microchips; Proteins; Proteome; Public Health; Regulation; Regulatory Element; Research; Role; Series; Signal Pathway; Signal Transduction; Specific qualifier value; Specificity; Structure; Surveys; Testing; Therapeutic; Transcription Repressor/Corepressor; Transcriptional Regulation; Work; base; cofactor; human disease; innovation; novel; preference; public health relevance; tool; transcription factor

DESCRIPTION (provided by applicant): It has long been puzzling how a relatively small number of transcription factors (TFs) can precisely control expression of ~21,000 ORFs and probably 10-fold more non-coding RNAs in humans. Another major gap in the transcription field is a lack of a simple set of rules that explain the specificity of protein-DNA interactions. These gaps represent a major problem because, until they are filled, understanding of the transcription circuitry and its underlining principles will remain highly incomplete. The long-term goals are to characterize the human protein-DNA interaction (PDI) network and elucidate the underlining molecular mechanisms of transcriptional regulation using the combined force of protein microarray technologies and bioinformatics. The objectives of this particular application are to identify a comprehensive list of sequence-specific unconventional DNA-binding proteins (uDBPs) and to better define rules that specify TF-DNA interactions. The central hypothesis is that unbiased, high-throughput profiling of PDIs will reveal rules and organization of transcriptional regulatory networks and pathways. This hypothesis has been formulated on the basis of preliminary data produced in the applicants< laboratories. The rationale for the proposed research is that, once a comprehensive list of sequence-specific uDBPs is generated and once a comprehensive analysis of PDIs and crystal structures of TFs is completed, we will be able to predict and test novel physiological roles of uDBPs and generate better rules that define DNA-binding specificity for many TF subfamilies in humans. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Comprehensively identify DNA-binding proteins using human proteome microarrays; and 2) Identify and characterize TF recognition domains that define DNA-binding preference. Under the first aim, 500 predicted and known DNA motifs will be probed to a human proteome microarray composed of ~17,000 individually purified proteins, a new tool fabricated at the applicants' laboratory, to generate a comprehensive list of uDBPs. On the basis of a series of bioinformatics analysis and prediction, 1-2 uDBPs will be characterized in-depth to elucidate the physiological roles in transcription regulation in the applicants' laboratories. Under the second aim, the existing crystal structures of TF-DNA complexes of various species will be analyzed to identify non-contacting amino acid residues (AAs) of TFs that dictate PDIs. A selected set of the identified residues will be further tested experimentally. The approach is innovative, because it utilizes activity-based screens for uDBPs in the human proteome and an unbiased survey for contribution by non-contacting AAs to dictate DNA-binding specificity. This proposed research is significant, because it will be the first systematic profiling of DNA-binding activities in humans that will offer a comprehensive list of participants in transcriptional control and regulation, and because a set of better-defined rules will ultimately provide the scientific community with a Rosetta Stone for decoding human transcriptional regulatory circuitry. Ultimately, such knowledge has the potential to inform the development of better therapeutics for TF-related diseases. PUBLIC HEALTH RELEVANCE: The propose research is relevant to public health because generation of a comprehensive list of participants in human transcriptional control and regulation and characterization of the rules that define DNA-binding specificity is ultimately expected to increase understanding of diseases that result from defects in transcription factor function. The proposed research is thus relevant to part of the NIH's mission as this advance in fundamental biological knowledge will help identify better therapeutics for a broad range of human diseases.

描述（由申请人提供）： 长期以来，它一直困扰着相对较少的转录因子（TFS）如何精确地控制约21,000个ORF的表达，并且在人类中可能多10倍的非编码RNA。转录字段中的另一个主要差距是缺乏一组简单的规则来解释蛋白质-DNA相互作用的特异性。这些差距代表了一个主要问题，因为在填补之前，对转录电路的理解及其强调原则将仍然很不完整。长期目标是表征人类蛋白-DNA相互作用（PDI）网络，并使用蛋白质微阵列技术和生物信息学的联合力阐明转录调节的分子机制。该特定应用程序的目标是确定序列特异性非常规DNA结合蛋白（UDBPS）的综合列表，并更好地定义指定TF-DNA相互作用的规则。中心假设是，PDI的公正，高通量分析将揭示转录监管网络和途径的规则和组织。该假设是根据申请人<实验室产生的初步数据提出的。 The rationale for the proposed research is that, once a comprehensive list of sequence-specific uDBPs is generated and once a comprehensive analysis of PDIs and crystal structures of TFs is completed, we will be able to predict and test novel physiological roles of uDBPs and generate better rules that define DNA-binding specificity for many TF subfamilies in humans.在强有力的初步数据的指导下，将通过追求两个具体目的来检验该假设：1）使用人蛋白质组微阵列全面鉴定DNA结合蛋白； 2）确定并表征定义DNA结合偏好的TF识别域。在第一个目标下，将探测500个预测和已知的DNA基序，以探测约17,000个单独纯化蛋白质的人类蛋白质组微阵列，这是一种在申请人实验室制造的新工具，以生成UDBPS的全面列表。根据一系列生物信息学分析和预测，将对1-2个UDBPS进行深入的特征，以阐明申请人实验室中转录调控中的生理作用。在第二个目标下，将分析各种物种的TF-DNA复合物的现有晶体结构，以鉴定决定PDI的TFS的非接触式氨基酸残基（AAS）。选定的一组确定的残基将通过实验进行进一步测试。该方法具有创新性，因为它利用了人类蛋白质组中的UDBP的基于活动的筛选，以及对非触发AAS进行贡献的无偏见调查来决定DNA结合特异性。这项拟议的研究非常重要，因为它将是人类中DNA结合活动的首次系统分析，这些研究将为转录控制和调节提供全面的参与者列表，并且因为一系列更好定义的规则最终将为科学界提供一个罗塞塔石，用于解码人类转录调节循环。最终，这种知识有可能为与TF相关疾病的更好的治疗剂提供信息。 公共卫生相关性： 提出的研究与公共卫生有关，因为最终期望定义DNA结合特异性的人类转录控制和调节的参与者的全面名单，以及定义DNA结合特异性的规则，将增加对转录因子功能缺陷的疾病的理解。因此，拟议的研究与NIH任务的一部分有关，因为基本生物学知识的这一进步将有助于确定各种人类疾病的更好的治疗疗法。