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

摘要 通过蛋白质-DNA相互作用的转录调控在所有已知的转录调控网络中起着重要作用。 有机体细菌调控网络现在是详细研究的一个特别富有成效的目标：作为抗生素- 耐药细菌继续成为全球健康威胁，新的和创新的方法， 需要毒性或削弱细菌生长。我们预测和利用细菌行为的能力 治疗目的取决于我们对其调控网络背后逻辑的理解， 完全绘制出这些网络及其背后的分子机制。 一些新老挑战阻碍了全面了解 甚至在研究得很好的模型中，如大肠杆菌。除了经典的顺式调控逻辑外， 转录因子和sigma因子，我们实验室和其他人最近的工作揭示了由于 染色体背景，大的异染色质样区域的抑制性占用的核相关蛋白， 整体三维染色体结构，以及DNA结合蛋白和DNA结合蛋白的表观遗传修饰。 DNA本身进一步调节转录调节。此外，对于非模式细菌，即使是基本的 经典转录调控的逻辑通常特征不佳。因此，背后的基本监管逻辑 诸如代谢开关、运动性和毒力诱导的细胞决定通常特征不足。 我们已经开发了几种创新技术，以帮助快速表征细菌转录 调节逻辑，包括：IPOD-HR，其允许细菌上的蛋白质占据的总体概况;基于转座子的 用于快速分析遗传背景对转录的全基因组影响的方法;以及基于所述方法的方法。 转座噬菌体Mu用于基因组3D结构的无交联测量。使用这些方法 除了细菌遗传学和ChIP-seq等经典方法外，我们还在寻求几种研究途径， 研究细菌转录调控网络。主要关注领域包括： 快速阐明新的转录调控网络：利用IPOD-HR技术，我们 已经表明可以很容易地应用于新的细菌物种，我们正在绘制一组顺式调节相互作用， 几种临床相关细菌物种的重要环境反应。 扩展蛋白质占据结构域（EPODs）的动态和组成：我们已经表明， 被占据的异染色质样EPOD存在于广泛的细菌物种中，并在调节EPOD的表达中发挥关键作用。 原噬菌体、毒力基因和代谢基因。我们将继续研究EPOD的监管作用， 包括它们的蛋白质，以及决定它们在一系列细菌分类群中形成/解离的因素。 细菌表观遗传标记与TF结合和EPOD的相互作用：通过连接我们的IPOD-HR和ChIP-seq 通过追踪共价DNA和DNA结合蛋白修饰的实验，我们将阐明这些 修饰调节细菌中的局部和大规模蛋白质占据和转录。

ABSTRACT 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. In additional to classical cis-regulatory logic by transcription factors and sigma factors, recent work by our laboratory and others has revealed contributions due to chromosomal context, large heterochromatin-like regions of repressive occupancy of nucleoid-associated proteins, overall three-dimensional chromosomal structure, and epigenetic modifications of both DNA-binding proteins and the DNA itself that further modulate transcriptional regulation. In addition, for non-model bacteria even the fundamental logic of classical transcriptional regulation is often poorly characterized. Thus, the fundamental regulatory logic behind cellular decisions such as metabolic switches, motility, and induction of virulence is often under-characterized. We have developed several innovative technologies to assist in rapid characterization of bacterial transcriptional regulatory logic, including: IPOD-HR, which allows overall profiling of protein occupancy on bacterial; transposon based methods for rapidly profiling genome-wide effects of genetic context on transcription; and a method based on the transposable phage Mu for crosslinking-free measurement of the 3D structure of the genome. Using these methods alongside classical approaches such as bacterial genetics and ChIP-seq, we are pursuing several avenues of research to investigate bacterial transcriptional regulatory networks. Key areas of interest include: Rapid elucidation of new transcriptional regulatory networks: Leveraging the IPOD-HR technology, which we have shown can be readily applied to new bacterial species, we are mapping the set of cis regulatory interactions driving important environmental responses in several clinically relevant bacterial species. Dynamics and composition of extended protein occupancy domains (EPODs): We have shown that highly protein occupied, heterochromatin like EPODs are present in a broad range of bacterial species, and play key roles in regulating prophages, virulence genes, and metabolic genes. We will continue to investigate the regulatory roles of EPODs, the proteins that comprise them, and the factors dictating their formation/dissociation across a range of bacterial taxa. Interplay of bacterial epigenetic marks with TF binding and EPODs: By interfacing our IPOD-HR and ChIP-seq experiments with tracking of covalent DNA and DNA-binding protein modifications, we will elucidate how these modifications modulate both local and large-scale protein occupancy and transcription in bacteria.