Building a unified framework for understanding bacterial gene regulation and chromosomal architecture

建立理解细菌基因调控和染色体结构的统一框架

• 批准号: 9892610
• 负责人: Lydia Freddolino
• 金额: $ 6.78万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9892610
• 关键词: ATAC-seq; Architecture; Area; Bacteria; Bacterial Antibiotic Resistance; Bacterial Chromosomes; Bacterial Genes; Bacterial Genome; Behavior; Binding; Binding Sites; Bioinformatics; Biotechnology; DNA-Binding Proteins; DNA-Protein Interaction; Data Set; Escherichia coli; Eukaryota; Food; Gene Expression Regulation; Genetic Transcription; Growth; Heterochromatin; Impairment; Infection; Investigation; Logic; Maps; Molecular; Molecular Biology; Organism; Orphan; Physiological; Play; Process; Proteins; Role; Signal Transduction; Site; Source; Stress; Study models; Technology; Therapeutic; Time; Transcriptional Regulation; Virulence; combat; experimental study; follow-up; global health; improved; innovation; interest; prevent; protein profiling; tool; transcription factor

Transcriptional regulation via protein-DNA interactions plays an important role in the regulatory networks of all known organisms. Bacterial regulatory networks are now an especially fruitful target for detailed investigation: as antibiotic-resistant bacteria continue to emerge as a global health threat, new and innovative approaches to either preventing virulence or impairing bacterial growth are required. As our ability to predict and exploit bacterial behavior for therapeutic purposes hinges on our understanding of the logic behind their regulatory networks, it is of great utility to fully map those networks and the molecular mechanisms underlying them. Several challenges, both old and newly recognized, stand in the way of a comprehensive understanding of regulatory logic, even in well-studied models such as Escherichia coli. Progress in mapping bacterial regulatory networks has in general been slow, requiring a steady march of mapping binding sites of one transcription factor (TF) at a time. Even when such experiments are done, they can typically be performed only under a handful of physiological conditions, and thus may miss key contributions of a transcription factor in responding to specific environmental triggers. In addition, contrary to prevailing dogma over the last several decades, we and others have recently gathered substantial evidence that bacterial chromosomes are in fact not universally accessible to transcription, but rather, that they are packaged by densely protein occupied heterochromatin-like regions that we refer to as EPODs, which influence both overall chromosomal architecture and transcriptional regulation in particular. Progress in the area of fully charting bacterial regulation of transcription via DNA binding proteins thus simultaneously requires more efficient coverage of transcription factor space and an improved understanding of the role of larger-scale protein occupancy in gene regulation. We have optimized a technology referred to as IPODHR for overall profiling of protein occupancy on bacterial genomes, similar to the signal provided by ATAC-seq in eukaryotes. Building on IPODHR data sets as a cornerstone, we are pursuing several highly innovative and efficient approaches to expand our understanding of bacterial regulatory networks: Massively parallel profiling of TF occupancy. Tracking IPODHR signal across known TF binding sites, in tandem with appropriate bioinformatic analysis, provides occupancy information on dozens of known TFs in a single experiment. We will utilize this technology to profile TF binding under a broad range of conditions. Identification of orphan TFs. IPODHR profiles enable us to identify active regulatory sites under conditions of interest, and identify the responsible TFs through follow-up experiments and bioinformatics. Regulatory roles and molecular biology of EPODs. IPODHR has revealed the presence of EPODs across a wide range of bacterial taxa, and we will determine the full impact of EPODs on condition-dependent gene regulation, and the molecular mechanisms through which these regions are established.

通过蛋白质-DNA相互作用进行的转录调控在所有的调控网络中起着重要的作用。 已知的生物。细菌监管网络现在是详细调查的一个特别富有成果的目标： 随着抗药性细菌继续成为全球健康威胁， 需要防止毒性或削弱细菌生长。我们预测和利用 用于治疗目的的细菌行为取决于我们对其调控背后的逻辑的理解。 网络，它是非常有用的，以充分映射这些网络和它们背后的分子机制。 一些新老挑战阻碍了全面实现千年发展目标， 对调控逻辑的理解，即使是在经过充分研究的模型中，如大肠杆菌。制图进展 细菌的调控网络通常是缓慢的，需要一个稳定的行进图的结合位点， 一次一个转录因子。即使这样的实验已经完成， 仅在少数生理条件下，因此可能错过转录因子的关键贡献 对特定环境触发的反应。此外，与过去几年流行的教条相反， 几十年来，我们和其他人最近收集了大量证据，证明细菌染色体实际上是 不是普遍可用于转录，而是，它们被密集的蛋白质占据包装， 异染色质样区域，我们称之为EPOD，它影响整个染色体 特别是结构和转录调控。细菌调控研究进展 因此，通过DNA结合蛋白进行转录的同时需要更有效的转录覆盖率。 因子空间和更好地理解大规模蛋白质占据在基因调控中的作用。 我们已经优化了一种称为IPODHR的技术，用于对蛋白质占据率进行整体分析。 细菌基因组，类似于真核生物中ATAC-seq提供的信号。以国际人口与发展研究所的数据集为基础， 作为基石，我们正在寻求几种高度创新和有效的方法来扩大我们的 了解细菌调控网络： TF占用的大规模平行分析。在已知TF结合位点上跟踪IPODHR信号， 与适当的生物信息学分析相结合，提供了几十个已知TF的占用信息， 单一实验我们将利用这项技术在广泛的条件下分析TF结合。 孤儿TF的鉴定。IPODHR配置文件使我们能够在以下条件下识别活跃的监管网站： 通过后续实验和生物信息学鉴定出负责的转录因子。 EPODs的调节作用和分子生物学。IPODHR揭示了EPOD在整个 广泛的细菌分类群，我们将确定EPODs对条件依赖性基因的全面影响， 调节，以及这些区域建立的分子机制。