HPV induced R-loop formation represses innate immune gene expression while activating DNA damage repair pathways

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-b, 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 gH2AX 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 gH2AX/H3K36me3 histone marks that may contribute to regulating important functions for HPV pathogenesis Overall design: 1 × 107 cells were harvested and collected in Southern lysis buffer before being treated with RNase A (5 ng/mL) and Proteinase K (7.5 ng/mL) at 37 °C overnight. DNA was purified from these samples using phenol-chloroform extractions, and 25 to 50 mg of DNA was used for each sample. DNA was sheared using a Bioruptor (Diagenode) on high power, 30 s on/90 s off cycles for 20 min or digested using 1U of mung bean nuclease for 1 h at 37 °C. Input DNA was removed before loading the samples into preblocked magnetic beads in IP buffer containing 2 mg of the RNA:DNA hybrid antibody. Immunoprecipitations were allowed to incubate overnight at 4 °C while rotating. The next day, samples were washed 8 times with RIPA buffer for 5 min while rotating. One wash in TE buffer was performed before samples were eluted for 10 min at 65 °C in 10% sodium dodecyl sulfate (SDS), 10 mM Tris pH 7.4, 50 mM ethylenediaminetetraacetic acid (EDTA). DNA was purified from these elutions using a PCR purification kit (Qiagen) and stored at -20 °C. Samples were stored at -80? C until being shipped to Admera Biosciences (NJ), who performed the sequencing experiments. Briefly, the library was prepared using a KAPA HyperPrep Kit (Kapa Biosystems) following the manufacturer's recommendation. Input DNA was end-repaired and 3'-dA tailed. Adapter was then ligated to the DNA, and the ligated product was PCR amplified and cleaned up using the SPRIselect Reagent (Beckman Coulter). Quality control was then performed for the final library, followed by sequencing.

R环（R-loops）是当RNA分子与其互补DNA链杂交、置换出另一条DNA单链时形成的三聚体核酸结构。这类结构可调控转录与复制过程，但异常形成的R环若未被及时清除，会引发DNA断裂与基因组不稳定。诸多癌症以及携带高危型人乳头瘤病毒（HPV）的细胞中，R环水平均会升高。本研究探究了正常角质形成细胞与源自宫颈上皮内瘤变I级（CIN I）癌前病变的HPV阳性细胞之间，R环的分布与功能变化。

研究发现，与细胞基因相关的R环水平在HPV阳性细胞中较正常角质形成细胞最高上调10倍，而ALU1元件区域的R环水平增幅更是高达500倍。R环水平升高会导致基因转录水平发生改变，上调与下调的基因数量相当。尽管未检测到R环水平升高对转录产生全局性的统一影响，但在HPV阳性细胞中，多条通路的基因表达呈现协同上调或下调。其中包括先天免疫通路基因的下调，例如DDX58、IL-6、STAT1、IFN-β及NLRP3。所有依赖R环的差异表达先天免疫基因，均与H3K36me3修饰的组蛋白相关联。

在HPV阳性细胞中，受R环调控而上调的基因包括DNA损伤修复相关基因（如ATM、ATRX）及范可尼贫血通路成员。这类基因仅在HPV阳性细胞中，同时与R环、H3K36me3以及γH2AX（gH2AX）组蛋白修饰标记存在关联。本研究揭示了HPV阳性细胞中，DNA损伤修复与先天免疫调控通路、R环及γH2AX/H3K36me3组蛋白修饰标记之间的潜在关联，这可能在HPV致病过程中发挥重要调控功能。

总体实验设计：
收集1×10⁷个细胞，用Southern裂解缓冲液重悬后，于37℃下用核糖核酸酶A（RNase A，5 ng/mL）与蛋白酶K（Proteinase K，7.5 ng/mL）孵育过夜。随后通过酚-氯仿抽提纯化DNA，每份样本使用25~50 μg DNA。将DNA以高功率超声破碎（使用Diagenode品牌Bioruptor超声破碎仪），循环参数为30秒开启/90秒关闭，总时长20分钟；或使用1 U绿豆核酸酶于37℃下消化1小时。加载样本至预先封闭的磁珠前，先去除输入DNA。磁珠需悬浮于含有2 mg RNA:DNA杂交抗体的免疫沉淀（IP）缓冲液中。免疫沉淀反应于4℃下旋转孵育过夜。次日，用RIPA缓冲液旋转洗涤样本8次，每次5分钟；再用TE缓冲液洗涤1次。随后于65℃下用洗脱液（10%十二烷基硫酸钠[SDS]、10 mM Tris-HCl pH 7.4、50 mM乙二胺四乙酸[EDTA]）洗脱样本10分钟。从洗脱液中使用Qiagen品牌PCR纯化试剂盒纯化DNA，于-20℃保存。所有样本于-80℃保存，直至送往新泽西州的Admera Biosciences公司进行测序实验。

测序流程简述如下：依照Kapa Biosystems品牌KAPA HyperPrep建库试剂盒的制造商指南制备测序文库。首先对输入DNA进行末端修复并添加3'端dA尾，随后连接测序接头，通过PCR扩增连接产物并使用Beckman Coulter品牌SPRIselect试剂纯化。对最终文库进行质量控制后，进行高通量测序。