Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America.
PLoS Pathog. 2024 Aug 23;20(8):e1012454. doi: 10.1371/journal.ppat.1012454. eCollection 2024 Aug.
R-loops are trimeric nucleic acid structures that form when an RNA molecule hybridizes with its complementary DNA strand, displacing the opposite strand. These structures regulate transcription as well as replication, but aberrant R-loops can form, leading to DNA breaks and genomic instability if unresolved. R-loop levels are elevated in many cancers as well as cells that maintain high-risk human papillomaviruses. We investigated how the distribution as well as function of R-loops changed between normal keratinocytes and HPV positive cells derived from a precancerous lesion of the cervix (CIN I). The levels of R-loops associated with cellular genes were found to be up to 10-fold higher in HPV positive cells than in normal keratinocytes while increases at ALU1 elements increased by up to 500-fold. The presence of enhanced R-loops resulted in altered levels of gene transcription, with equal numbers increased as decreased. While no uniform global effects on transcription due to the enhanced levels of R-loops were detected, genes in several pathways were coordinately increased or decreased in expression only in the HPV positive cells. This included the downregulation of genes in the innate immune pathway, such as DDX58, IL-6, STAT1, IFN-β, and NLRP3. All differentially expressed innate immune genes dependent on R-loops were also associated with H3K36me3 modified histones. Genes that were upregulated by the presence of R-loops in HPV positive cells included those in the DNA damage repair such as ATM, ATRX, and members of the Fanconi Anemia pathway. These genes exhibited a linkage between R-loops and H3K36me3 as well as γH2AX histone marks only in HPV positive cells. These studies identify a potential link in HPV positive cells between DNA damage repair as well as innate immune regulatory pathways with R-loops and γH2AX/H3K36me3 histone marks that may contribute to regulating important functions for HPV pathogenesis.
R 环是由 RNA 分子与其互补 DNA 链杂交形成的三聚体核酸结构，取代了相反的链。这些结构调节转录和复制，但如果未解决，异常的 R 环会形成，导致 DNA 断裂和基因组不稳定。许多癌症以及维持高危人乳头瘤病毒的细胞中 R 环水平升高。我们研究了正常角质形成细胞和源自宫颈癌前病变（CIN I）的 HPV 阳性细胞之间 R 环的分布和功能如何变化。与细胞基因相关的 R 环水平在 HPV 阳性细胞中比在正常角质形成细胞中高 10 倍，而 ALU1 元件的增加高达 500 倍。增强的 R 环的存在导致基因转录水平发生改变，相等数量的基因增加和减少。虽然由于 R 环水平的增强而没有检测到转录的统一全局影响，但在 HPV 阳性细胞中，只有几个途径中的基因表达协同增加或减少。这包括下调先天免疫途径中的基因，如 DDX58、IL-6、STAT1、IFN-β 和 NLRP3。依赖 R 环的差异表达的先天免疫基因也与 H3K36me3 修饰的组蛋白相关。在 HPV 阳性细胞中，由于 R 环的存在而上调的基因包括 ATM、ATR 和范可尼贫血途径的成员等 DNA 损伤修复基因。这些基因在 HPV 阳性细胞中表现出 R 环与 H3K36me3 以及 γH2AX 组蛋白标记之间的联系。这些研究在 HPV 阳性细胞中确定了 DNA 损伤修复以及先天免疫调节途径与 R 环和 γH2AX/H3K36me3 组蛋白标记之间的潜在联系，这些联系可能有助于调节 HPV 发病机制的重要功能。
