Bacterial Rogue Methyltransferases Inducing Human Epimutations

项目摘要：DNA甲基化在任何形式的癌症中都是不受调控的。虽然有大量的 关于跨肿瘤类型的异常DNA甲基化的位置和功能的知识，我们严重 缺乏了解这种异常的DNA甲基化是如何在癌症形成过程中建立的。结直肠 癌症(CRC)是世界上第三种最常见的癌症类型，与 然而，DNA甲基化/去甲基化途径基因的突变是罕见的，而且不能 解释了DNA过度甲基化的原因。大约1.5公斤的细菌存在于我们的肠道中，并已被 经证明，它在儿童权利公约的发展中发挥了重要作用。最近的证据表明，这些细菌可以 通过诱导异常的DNA超甲基化改变结肠细胞表观基因组，尽管其机制促进 这一点仍然没有完全被理解。一种这样的微生物，核梭杆菌，能够入侵到 结肠上皮细胞，与结直肠癌的发生以及结直肠癌的高甲基化密切相关。 肿瘤抑制基因。大约90%的细菌，包括核梭杆菌，都含有DNA 甲基转移酶作为其限制修饰系统的组成部分；一种防御机制 旨在通过甲基化他们DNA中的特定基序来保护他们的基因组免受入侵病毒的侵袭 区分它们自己的和入侵的病毒DNA。到目前为止，已经确定了3500个不同的R-M基序， 因此，我们的肠道微生物群充满了微生物基因组修改酶，但它们可能会修改 人类的DNA还没有被评估。这项建议的长期目标是评估核盘藻是否 DNA甲基转移酶能够访问并异常甲基化结肠细胞DNA，潜在地给出 上升到在《儿童权利公约》发展过程中观察到的变化。这将以三个具体目标进行测试。第一个目标 将通过异位表达F. 核型DNA甲基转移酶在大肠癌细胞系中的表达及其进入细胞核的能力 甲基化DNA，以及确定DNA甲基化的位置。第二个目标将利用 勒纳研究所的微生物组核心设施用于预制细菌与F. 经基因改造含有标记DNA甲基转移酶的有核细胞，以检测 核盘藻DNA甲基转移酶是否能访问和甲基化结肠细胞DNA。第三个目标是 通过在CRC高甲基化位置寻找核镰刀菌R-M基序的富集区，检测体内核镰刀菌的活性 Loci，使用我们的实验室公布的MBD-SEQ数据和TCGA CRC队列。结果可能揭示一种新的范式 在宿主-微生物组相互作用中，由于微生物和宿主细胞之间的直接表观遗传串扰尚未被 充分探索。然而，如果这是真的，这一新的范式将改变对细菌相关癌症和 为治疗和风险管理开辟新的治疗途径。

PROJECT SUMMARY: DNA methylation is dysregulated in every form of cancer. While there is substantial knowledge regarding the location and function of abnormal DNA methylation across tumor types, we are severely lacking in understanding how such abnormal DNA methylation is established during cancer formation. Colorectal cancer (CRC), the third most common cancer type worldwide, has been heavily associated with alterations in DNA methylation, however, mutations in DNA methylation/demethylation pathway genes are rare and cannot account for the exaggerated DNA hypermethylation. Roughly 1.5 kg of bacteria reside in our gut and have been shown to play a substantial role in the development of CRC. Recent evidence suggests that these bacteria can alter the colon cell epigenome by inducing aberrant DNA hypermethylation, although the mechanism promoting this is still not completely understood. One such microorganism, Fusobacterium nucleatum, is able to invade into colon epithelial cells and has been heavily associated with CRC development, as well as hypermethylation of tumor suppressor genes. Roughly 90% of bacteria, including Fusobacterium nucleatum, contain DNA methyltransferase enzymes as a component of their restriction-modification system; a defense mechanism aimed at protecting their genome against invading viruses by methylating specific motifs within their DNA as to distinguish between their own and invading viral DNA. To date, >3500 different R-M motifs have been identified, thus our gut microbiome is filled with microbial genome-modifying enzymes, however their potential to modify human DNA has not been evaluated. The long-term objective of this proposal is to evaluate whether F. nucleatum DNA methyltransferase enzymes are able to access and aberrantly methylate colon cell DNA, potentially giving rise to the epimutations observed in CRC development. This will be tested with three specific aims. The first aim will experimentally evaluate F. nucleatum methyltransferase activity in vitro, by ectopically expressing F. nucleatum DNA methyltransferase enzymes in CRC cell lines and evaluating their ability to enter the nucleus, methylate DNA, as well as determine the locations of DNA methylation. The second aim will take advantage of the microbiome core facilities at Lerner Research Institute to preform bacterial co-culture experiments using F. nucleatum that have been genetically modified to contain tagged-DNA methyltransferase enzyme, to test whether F. nucleatum DNA methyltransferase can access and methylate colon cell DNA. The third aim is to detect F. nucleatum activity in vivo, by searching for F. nucleatum R-M motif enrichment at CRC hypermethylated loci, using our labs published MBD-seq data and the TCGA CRC cohort. Results could reveal a novel paradigm in host-microbiome interactions, as direct epigenetic crosstalk between microbial and host cells has not been fully explored. However, if true, this new paradigm will transform studies of bacterial-associated cancers and open new therapeutic avenues for treatment and risk-management.