Methods for Multiscale and Integrative Characterization of Bacterial Epigenomes

 描述(申请人提供)：在细菌世界中，甲基化腺嘌呤和胞嘧啶残基最常与限制酶修饰系统联系在一起，该系统提供了一种防御外来基因组入侵的机制。此外，它们中的某些形式还在细胞周期、基因表达、毒力和抗生素耐药性的调节中发挥重要作用。直到最近单分子实时测序(SMRTseq)技术的出现，才有可能对细菌甲基化事件进行高效和高分辨率的分析。单分子实时测序技术可以检测N6-甲基腺嘌呤(6 MA)和4-甲基胞嘧啶(4mC)残基，这是细菌世界中两种主要的甲基化类型，此外还可以检测5-甲基胞嘧啶(5mC)。这项技术使我们能够在单核苷酸分辨率下表征第一批全基因组细菌甲基组之一，即整个甲基化。迅速增长的细菌数量正在 特征，揭示了细菌甲基组出人意料的复杂程度和多样性。然而，主要在种群水平上进行的使用SMRTseq数据的现有方法无法解决通常存在于单个种群中的表观遗传异质性，并使细菌能够更好地适应不断变化的条件。此外，SMRTseq也有其自身的局限性，需要与其他现有的补充技术相结合。最后但并非最不重要的一点是，以前的研究大多集中在甲基化介导的基因表达调控的基因特异性机制上，这些机制只占表观遗传扰动引起的基因表达全局变化的很小一部分。由于SMRTseq的独特优势和这些新出现的挑战，我们建议开发新的方法来进行细菌DNA甲基化的多尺度检测和综合功能表征。这些新方法将结合多个学科的创新发展：混合测序设计、多维分子图谱、统计学和系统生物学。它们将使人们更好地了解不同类型细菌的表观遗传异质性的机制和动力学，并推动将表观遗传变异整合到细菌的系统生物学框架中。我们将应用这些方法研究具有不同甲基组复杂性、甲基化相关表型和临床意义的不同细菌集合。我们还将在为更广泛的研究社区制定的综合计划中实施在项目期间开发的所有方法。其影响将不仅限于目前对细菌甲基组的研究，而且还将影响到假定DNA甲基化独立于感兴趣的生物过程的细菌研究，部分原因是缺乏技术和工具。其中包括一些具有重要临床相关性的因素：毒力、抗生素耐药性和持久性，这些因素正在造成严重的健康危机。

 DESCRIPTION (provided by applicant): In the bacterial world, methylated adenine and cytosine residues are most commonly associated with restriction-modification systems that provide a defense mechanism against invading foreign genomes. In addition, some forms of them also play important roles in the regulation of cell cycle, gene expression, virulence, and antibiotic resistance. Efficient and high resolution profiling of bacterial methylation events has not been possible until the recent advent of Single Molecule Real-Time sequencing (SMRTseq) technique that can detect N6-methyladenine (6mA) and 4-methylcytosine (4mC) residues, the two major types of methylation in the bacterial world, in addition to 5-methylcytosine (5mC). This technique enabled us to characterize one of the first whole-genome bacterial methylomes, the entire set of methylations, at single- nucleotide resolution. A fast growing number of bacteria are being characterized, revealing unexpected degrees of complexity and diversity in bacterial methylomes. However, existing methods using SMRTseq data mainly carried out at population levels cannot resolve epigenetic heterogeneity that often exists in a single population and empowers bacteria to better adapt to changing conditions. Also, SMRTseq has its own limitations that call for combinations with other existing complementary techniques. Last but not least, previous studies have mostly focused on gene-specific mechanisms of methylation-mediated regulation of gene expression, which only account for a very small fraction of global changes of gene expression induced by epigenetic perturbations. Motivated by both the unique advantages of SMRTseq and these emerging challenges, we propose to develop novel methods for multiscale detection and integrative functional characterization of bacterial DNA methylation. The novel methods will combine innovative developments across multiple disciplines: hybrid sequencing design, multi-dimensional molecular profiling, statistics and systems biology. They will enable the better understanding of the mechanisms and dynamics of epigenetic heterogeneity in different types of bacteria, and advance the integration of epigenetic variations into a systems biology framework for bacteria. We will apply these methods to study a diverse collection of bacteria with different methylome complexity, methylation-related phenotypes and clinical significance. We will also implement all the methods developed over the project period in an integrated program for the broader research community. The impact will not be restricted to current research on bacterial methylomes, but also to bacterial research in which DNA methylations are assumed to be independent of the biological processes of interest, partly due to the lack of techniques and tools. These include some that have important clinical relevance: virulence, antibiotic resistance and persistence that are causing critical health crisis