Extent and Significance of Bacterial DNA Integrations in the Human Cancer Genome

 描述(由申请人提供)：将外源DNA整合到人类基因组中可导致与肿瘤发生相关的体细胞突变。例如，将HPV DNA插入人类染色体是导致宫颈癌发生的最重要的事件。现在也可以通过接种HPV疫苗来预防。与病毒DNA整合相比，细菌DNA整合到体细胞人类基因组中的实例和影响不太清楚。然而，被发现与肿瘤发生有关的细菌DNA整合也可以通过限制接触细菌的疫苗等疗法来防止。这项提议有三个目标，旨在解决我们在细菌DNA整合方面的知识差距。首先，将为我们的生物信息学工具LGTSeek和LGTview开发虚拟机，我们以前曾使用这些工具来检测人类基因组测序项目中的细菌DNA整合。这些虚拟机将使这些工具能够由各种各样的用户运行，从了解生物信息的人到天真的人。这为社区提供了一种资源，使更多种类的科学家能够检测到这种整合。此外，LGTSeek和LGTView将被用来进一步询问可公开获得的癌症基因组数据，在这些数据中，由于组织暴露在微生物组(例如结肠)中，可能会发生这种整合。其次，将对新的胃腺癌样本和急性髓系白血病样本进行基因组和转录组测序。这个目标的目的是重现以前的结果，这些结果表明细菌DNA整合的存在。这些测序工作将包括在样本中加入外源细菌核酸的对照样本，以便在现代测序技术中量化嵌合体的形成。第三，以前检测到的细菌DNA整合对转录的影响将使用荧光素酶报告结构进行询问。导致基因上调的整合将通过使用CRISPR/Cas9系统重建细胞中的整合来进一步研究。总的来说，这项研究有望提高我们对细菌DNA在体细胞人类基因组中整合的程度和意义的理解。

 DESCRIPTION (provided by applicant): The integration of exogenous DNA into the human genome can cause somatic mutations associated with oncogenesis. For example, the insertion of HPV DNA into human chromosomes is the single most important event leading to tumorigenesis in cervical cancer. It is also now preventable with vaccines against HPV. In contrast to viral DNA integrations, the instances and repercussions of bacterial DNA integration into the somatic human genome are less clear. Yet bacterial DNA integrations found to be associated with tumorigenesis could also be prevented using therapeutics like vaccines that limit exposure to the bacteria. This proposal has three objectives aimed at addressing our gap in knowledge about bacterial DNA integrations. First, virtual machines will be developed for LGTSeek and LGTview, our bioinformatics tools that we have used previously to detect bacterial DNA integrations in human genome sequencing projects. These virtual machines would enable these tools to be run by a wide variety of users, from the bioinformatically savvy to the naïve. This provides a resource to the community to enable detection of such integrations by a wider variety of scientists. In addition, LGTSeek and LGTView will be used to further interrogate publicly available cancer genome data where such integrations are likely to occur because the tissues are exposed to the microbiome (e.g. colon). Second, genome and transcriptome sequencing will be undertaken of new stomach adenocarcinoma samples and acute myeloid leukemia samples. This objective is aimed at reproducing previous results that suggest the presence of bacterial DNA integrations. These sequencing efforts would include control samples with exogenous bacterial nucleic acids added to the sample in order to quantify the formation of chimeras in modern sequencing techniques. Third, the effect that previously detected bacterial DNA integrations have on transcription will be interrogated using luciferase reporter constructs. Integrations that lead to up-regulation of the gene will be further interrogated by reconstructing the integrations in cells using the CRISPR/Cas9 system. Collectively, this research is expected to improve our understanding of the extent and significance of bacterial DNA integrations in the somatic human genome.