Epigenetics-mediated transcription regulation in mammals

表观遗传学介导的哺乳动物转录调控

基本信息

批准号：
9115212
负责人：
Jiang Qian
金额：
$ 34.43万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-01 至 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9115212
关键词：
Address Algorithms Base Pairing Binding Binding Proteins Binding Sites Biological Biological Assay Biological Models Biological Process Cell Line Cells Coupled Cytosine DNA DNA Binding DNA Methylation DNA Probes DNA Sequence DNA Transposable Elements DNA-Protein Interaction Data Deoxyribonuclease I Developmental Biology EMSA Enhancers Epigenetic Process Eukaryota Gel Gene Expression Gene Expression Regulation Gene Silencing Gene Targeting Genetic Transcription Genomic DNA Genomic Imprinting Goals Health Histones Human In Vitro Knowledge Luciferases Mammals Maps Mass Spectrum Analysis Mediating Methylation Mission Modeling Modification Molecular Monitor Neuronal Injury Nucleic Acid Regulatory Sequences Physiological Play Population Positioning Attribute Promoter Regions Protein Binding Domain Protein Microchips Proteins Public Health Publications Qualifying Reader Reagent Recruitment Activity Regenerative Medicine Regulator Genes Regulatory Element Research Resolution Role Sequence Analysis Side Site Site-Directed Mutagenesis Specificity Surveys Techniques Tertiary Protein Structure Testing Therapeutic Transcriptional Regulation X Inactivation Zinc Fingers axon regeneration base carcinogenesis deep sequencing factor A human DNA in vivo Model insight methyl group methylome mutant novel novel strategies promoter success transcription factor

Address Algorithms Base Pairing Binding Binding Proteins Binding Sites Biological Biological Assay Biological Models Biological Process Cell Line Cells Coupled Cytosine DNA DNA Binding DNA Methylation DNA Probes DNA Sequence DNA Transposable Elements DNA-Protein Interaction Data Deoxyribonuclease I Developmental Biology EMSA Enhancers Epigenetic Process Eukaryota Gel Gene Expression Gene Expression Regulation Gene Silencing Gene Targeting Genetic Transcription Genomic DNA Genomic Imprinting Goals Health Histones Human In Vitro Knowledge Luciferases Mammals Maps Mass Spectrum Analysis Mediating Methylation Mission Modeling Modification Molecular Monitor Neuronal Injury Nucleic Acid Regulatory Sequences Physiological Play Population Positioning Attribute Promoter Regions Protein Binding Domain Protein Microchips Proteins Public Health Publications Qualifying Reader Reagent Recruitment Activity Regenerative Medicine Regulator Genes Regulatory Element Research Resolution Role Sequence Analysis Side Site Site-Directed Mutagenesis Specificity Surveys Techniques Tertiary Protein Structure Testing Therapeutic Transcriptional Regulation X Inactivation Zinc Fingers axon regeneration base carcinogenesis deep sequencing factor A human DNA in vivo Model insight methyl group methylome mutant novel novel strategies promoter success transcription factor

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项目摘要

DESCRIPTION (provided by applicant): DNA methylation is an important epigenetic modification that plays crucial roles in multiple biological processes. Technical advance, especially a variety of deep sequencing-based techniques, have made it possible to monitor DNA methylome changes at a single-base resolution. However, how to interpret the epigenetic information encoded by the fast accumulating methylome data is still challenging. DNA methylation is traditionally considered to disrupt the interactions between transcription factors (TFs) and cis-regulatory regions and thus, silence the expression of downstream target genes. However, recent studies, especially our recent discoveries, suggest that many TFs and co-factors preferentially bind to methylated DNA motifs and, in some cases, transactivate downstream gene expression, challenging the current paradigm of DNA methylation in transcription regulation. Identification of comprehensive sets of functional methylation sites and their interacting partners will greatly expand the protein-DNA interaction landscape in a new direction and promise significant advances in the understanding of the biological roles of DNA methylation. To achieve these goals, we propose four specific aims in this R01 application. First, we will survey all possible 8-base DNA sequence combinations to identify methylated sequences that can be recognized by human TFs. A pool of methylated DNA motifs will be probed on the human TF protein microarrays and the DNA fragments that are captured by the proteins on the microarrays will be recovered and their sequences determined with deep-sequencing. Second, we will predict which of these methylated motifs are likely to play a role in gene regulation and interact with proteins. Those 8-mer sequences that are statistically enriched in the recovered population will be mapped to the available methylomes and examine whether they overlap with the known regulatory regions. The qualified motifs will be synthesized and individually probed on the protein microarrays to identify their binding partners. Third, we will predict the protein domains that are responsible fo methylated DNA binding. The sequences from the same TF subfamilies will be compared and the positions that can best separated the proteins with and without methylated binding activities will be the candidates for methylation binding. The prediction will be tested by site-directed mutagenesis coupled with gel shift and cell-based luciferase assays. Finally, we will use both in vitro and in vivo models of mammalian axon regeneration to investigate the physiological roles of newly identified mCpG-dependent TF-DNA interactions. The positive results provided by this Aim will not only reveal novel epigenetic mechanisms of mammalian axon regeneration, but also provide proof-of-concept evidence that mCpG-dependent TF-DNA interactions are physiological regulators of gene expression.

描述（由申请人提供）： DNA甲基化是一种重要的表观遗传修饰，在多种生物过程中起着至关重要的作用。技术进步，尤其是各种基于测序的技术，已使以单基碱分辨率监测DNA甲基化体的变化成为可能。但是，如何解释由快速积累甲基化的数据编码的表观遗传信息仍然具有挑战性。传统上，DNA甲基化被认为破坏了转录因子（TFS）和顺式调节区域之间的相互作用，从而使下游靶基因的表达保持沉默。然而，最近的研究，尤其是我们最近的发现，表明许多TF和辅助因子优先与甲基化的DNA基序结合，在某些情况下，在某些情况下是反式激活下游基因表达，在转录调节中挑战了当前DNA甲基化的范例。鉴定全面的功能性甲基化位点及其相互作用伙伴将在新方向上大大扩展蛋白质-DNA相互作用景观，并有望在理解DNA甲基化生物学作用方面取得重大进步。为了实现这些目标，我们在此R01应用程序中提出了四个具体目标。首先，我们将调查所有可能的8-基DNA序列组合，以鉴定可以被人类TF识别的甲基化序列。将在人类TF蛋白微阵列上探测甲基化的DNA基序，并将恢复由微阵列上的蛋白捕获的DNA片段，并通过深层序列确定其序列。其次，我们将预测这些甲基化基序中的哪些可能在基因调节中起作用并与蛋白质相互作用。在统计上富集在回收人群中的8-Mer序列将映射到可用的甲基瘤，并检查它们是否与已知的调节区域重叠。合格的基序将在蛋白质微阵列上合成并单独探测，以识别其结合伴侣。第三，我们将预测负责FO甲基化DNA结合的蛋白质结构域。将比较来自相同TF亚家族的序列，并且可以最好地分离有和没有甲基化结合活性的蛋白质的位置将是甲基化结合的候选者。该预测将通过定点诱变，凝胶移位和基于细胞的荧光素酶测定进行测试。最后，我们将同时使用哺乳动物轴突再生的体外和体内模型来研究新鉴定的MCPG依赖性TF-DNA相互作用的生理作用。该目标提供的积极结果不仅会揭示哺乳动物轴突再生的新型表观遗传机制，而且还提供了概念验证证据，表明MCPG依赖性TF-DNA相互作用是基因表达的生理调节剂。