Methods for Multiscale and Integrative Characterization of Bacterial Epigenomes

 描述（由申请人提供）：在细菌世界中，甲基化腺嘌呤和胞嘧啶残基最常与限制修饰系统相关，该系统提供了针对入侵的外源基因组的防御机制。此外，它们的某些形式还在细胞周期、基因表达、毒力和抗生素抗性的调节中发挥重要作用。细菌甲基化事件的有效和高分辨率谱分析一直是不可能的，直到最近出现的单分子实时测序（SMRTseq）技术，该技术可以检测N6-甲基腺嘌呤（6 mA）和4-甲基胞嘧啶（4 mC）残基，这是细菌世界中除了5-甲基胞嘧啶（5 mC）之外的两种主要类型的甲基化。该技术使我们能够以单核苷酸分辨率表征第一个全基因组细菌甲基化组之一，即整个甲基化组。快速增长的细菌数量 被表征，揭示了细菌甲基化中意想不到的复杂性和多样性程度。然而，主要在群体水平上使用SMRTseq数据的现有方法无法解决通常存在于单个群体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有其自身的局限性，需要与其他现有的互补技术相结合。最后但并非最不重要的是，以前的研究主要集中在甲基化介导的基因表达调控的基因特异性机制，这只占表观遗传扰动诱导的基因表达的全球变化的一小部分。受SMRTseq的独特优势和这些新出现的挑战的启发，我们提出开发新的方法用于细菌DNA甲基化的多尺度检测和综合功能表征。这些新方法将联合收割机结合多个学科的创新发展：杂交测序设计、多维分子分析、统计学和系统生物学。它们将使我们能够更好地理解不同类型细菌中表观遗传异质性的机制和动力学，并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来研究具有不同甲基化组复杂性、甲基化相关表型和临床意义的各种细菌。我们还将在更广泛的研究社区的综合计划中实施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化的研究，而且还将影响到细菌研究，其中DNA甲基化被认为与感兴趣的生物过程无关，部分原因是缺乏技术和工具。其中包括一些具有重要临床意义的因素：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