Bacterial Rogue Methyltransferases Inducing Human Epimutations

项目概述：DNA甲基化在每种癌症中都是失调的。虽然有大量的 关于肿瘤类型中异常DNA甲基化的位置和功能的知识，我们严重怀疑 缺乏对这种异常DNA甲基化在癌症形成过程中是如何建立的理解。结直肠 癌症（CRC）是世界上第三大常见癌症类型，其与以下改变密切相关： 然而，DNA甲基化/去甲基化途径基因的突变是罕见的， 导致了DNA过度甲基化大约有1.5公斤的细菌存在于我们的肠道中， 在CRC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， 这一点还没有完全弄清楚。一种这样的微生物，具核梭杆菌，能够侵入 结肠上皮细胞，并且与CRC的发生以及结肠上皮细胞的超甲基化密切相关 肿瘤抑制基因大约90%的细菌，包括具核梭杆菌，含有DNA 甲基转移酶作为其限制修饰系统的组成部分;防御机制 目的是通过甲基化DNA中的特定基序来保护它们的基因组免受入侵病毒的侵害， 区分自己的和入侵的病毒DNA到目前为止，已经鉴定了超过3500种不同的R-M基序， 因此，我们的肠道微生物组充满了微生物基因组修饰酶，但它们的潜力， 人类的DNA还没有被评估过。这项建议的长期目标是评估F。具核 DNA甲基转移酶能够进入结肠细胞DNA并使其异常甲基化， 在CRC发展中观察到的表突变。这将通过三个具体目标进行测试。第一个目标 将通过实验评估F。体外表达F. 在CRC细胞系中的核质DNA甲基转移酶并评价它们进入细胞核的能力， 甲基化DNA，以及确定DNA甲基化的位置。第二个目标将利用 Lerner研究所的微生物组核心设施使用F. 已被遗传修饰为含有标记DNA甲基转移酶的核质，以测试 F. nucleatum DNA甲基转移酶可以进入结肠细胞DNA并使其甲基化。第三个目标是 检测F. nucleatum活性，通过寻找F. CRC高甲基化的核仁R-M基序富集 基因座，使用我们的实验室发表的MBD-seq数据和TCGA CRC队列。结果可能揭示了一种新的范式 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，这种新的范式将改变细菌相关癌症的研究， 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.