Histone demethylases and regulation of chromatin and transcription in eukaryotes

摘要：细胞的生存需要保持基因组的完整性，而基因组的完整性一直受到威胁。 由细胞内和外部来源的DNA损伤。DNA损伤的一个普遍来源是紫外线 光，产生环丁烷嘧啶二聚体(CPDS)和6-4嘧啶-嘧啶酮(6-4 PP) 加合物。核苷酸切除修复(NER)途径主要负责去除和 修复这些损伤。NER途径的两个不同分支参与修复：转录 偶联修复(TCR)识别转录活性位点上的CPD，而全局基因组修复 (GGR)标记基因组其他部分的损伤。跨病变合成(TLS)并行运行 绕过所有S期未修复的病变。其中许多成分的突变会导致 光敏性疾病，特别是着色性干皮病和Cockayne综合征。 最近，我们发现腺苷(6 MA)6位的RNA甲基化是一种令人兴奋的 DNA修复的新贡献者。6 mA RNA在下列损伤部位迅速而瞬时地诱导 紫外线照射，我们已经确定RNA甲基转移酶METTL3负责 这一修改。我们的初步结果表明，METTL3可能参与了TCR的分支 在METTL3基因敲除中，NER途径，即转录区域的修复被特异性地破坏 (Ko)细胞。因此，METTL3 KO失去6 mA RNA会降低紫外线照射后细胞的存活率， 它被野生型，但不是无催化活性的METTL3所拯救。这项工作的目标是 确定6 mA在DNA修复中的作用和机制。我们将确定6 mA是否 RNA修复全基因组转录和/或非转录区域的功能，并识别 它参与了NER的哪些路径/步骤。我们还将探讨它在TLS中的作用以及修复其他 DNA损伤类型以确定6 mA RNA在DNA损伤反应中的作用范围 (解甲返乡)。其次，我们将调查假设，6毫安标志着来自受损的转录 DNA模板用于降解，和/或6 mA影响选择性剪接或翻译 修饰的转录本，尤指那些编码对DNA修复或细胞生存重要的蛋白质的转录本。 最后，我们将鉴定和鉴定能够识别6 mA RNA的“阅读器”蛋白，以便 调查这样一种假设，即这些读取器在将6 mA RNA与DDR联系起来方面起到了机械性作用。 我们发现了一种介导DNA修复的RNA修饰，这揭示了一个新的令人兴奋的方面 对DDR和我们对细胞如何维持基因组完整性的了解。这项研究建议在 这一应用将为6 mA RNA如何调节DDR提供重要的新见解，以及我们的 这一发现将对理解和治疗光敏性疾病具有重要意义。

ABSTRACT: Cell viability requires maintenance of genome integrity, which is continuously threatened by intracellular and external sources of DNA damage. One prevalent source of DNA damage is UV light, which generates cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone (6-4 PP) adducts. The nucleotide excision repair (NER) pathway is mainly responsible for the removal and repair of these lesions. Two different branches of the NER pathway contribute to repair: transcription coupled repair (TCR) recognizes CPDs at transcriptionally active loci, while global genomic repair (GGR) marks lesions in other parts of the genome. Trans-lesion synthesis (TLS) operates in parallel to bypass any unrepaired lesions in S phase. Mutations in many of these components cause photosensitivity diseases, notably Xeroderma Pigmentosa and Cockayne Syndrome. Recently, we have identified methylation of RNA at the 6 position of adenosines (6mA) as an exciting new contributor to DNA repair. 6mA RNA is rapidly and transiently induced at damage sites following UV exposure, and we have identified the RNA methyltransferase METTL3 as being responsible for this modification. Our preliminary results suggest that METTL3 may participate in the TCR branch of the NER pathway, as repair of transcribed regions is specifically compromised in METTL3 knock-out (KO) cells. Accordingly, loss of 6mA RNA by METTL3 KO decreased cell survival after UV exposure, which was rescued by wild-type, but not catalytically-inactive, METTL3. The goals of this work are to determine the role and mechanism of action of 6mA in DNA repair. We will determine whether 6mA RNA functions to repair transcribed and/ or non-transcribed regions genome-wide, and identify in which pathways/ steps of NER it participates. We will also explore its role in TLS and repair of other types of DNA damage to determine how broadly 6mA RNA operates in the DNA damage response (DDR). Secondly, we will investigate the hypotheses that 6mA marks transcripts arising from damaged DNA templates for degradation, and/ or that 6mA influences alternative splicing or translation of modified transcripts, particularly of those encoding proteins important for DNA repair or cell survival. Finally, we will identify and characterize "reader" proteins that recognize 6mA RNA, in order to investigate the hypothesis that these readers play a mechanistic role in linking 6mA RNA to the DDR. Our discovery of an RNA modification mediating DNA repair has uncovered a new and exciting facet to the DDR and to our knowledge of how cells maintain genome integrity. The research proposed in this application will provide important new insight into how 6mA RNA regulates the DDR, and our findings will be important for understanding and treating photosensitivity diseases.