HPV induced R-loop formation represses innate immune gene expression while activating DNA damage repair pathways [H3K9me3 ChIP seq]

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 Formaldehyde was added to 1 × 107 cells to a final concentration of 1% for 10 min at room temperature. Excess formaldehyde was quenched upon adding 0.125M glycine before then washing samples with PBS. Cells were then incubated in collection buffer (0.1M TrisHCl pH 9.4 and 10mM DTT containing Roche Protease Inhibitor Cocktail) for 10min on ice. Cells were then collected and spun down before being sequentially washed and incubated with NCP1 (10mM EDTA, 0.5mM EGTA, 10mM HEPES pH 6.5, 0.25% Triton X100) and NCP2 (1mM EDTA, 0.5mM EGTA, 10mM HEPES, and 200mM NaCl) before being lysed in 0.5% Empigen BB, 1% SDS, 10mM EDTA, 50mM Tris HCl pH 8.0 containing Roche Protease Inhibitor Cocktail for 30 min on ice. Samples were then sonicated using a Bioruptor (Diagenode) on high power, 30 s on/90 s off cycles for 20 min. After sonication, Input DNA was removed before loading the samples into preblocked magnetic beads in IP buffer containing 2 ug of the H3K36me3 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.R环（R-loops）是一类三聚体核酸结构，当RNA分子与其互补DNA链杂交并置换出另一条DNA链时即可形成。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。在多种癌症以及携带高危型人乳头瘤病毒（high-risk human papillomaviruses）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癌前病变（CIN I）的人乳头瘤病毒（human papillomavirus, HPV）阳性细胞之间，R环的分布与功能变化情况。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升高10倍，而ALU1元件区域的R环水平增幅最高可达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，但仅在HPV阳性细胞中，多条通路的基因表达出现了协同上调或下调的情况。其中包括先天免疫通路相关基因的下调，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（H3K36me3 modified histones）相关。在HPV阳性细胞中，因R环水平升高而上调的基因包括DNA损伤修复相关基因（如ATM、ATRX）以及范可尼贫血通路（Fanconi Anemia pathway）的相关成员。这些基因仅在HPV阳性细胞中，呈现出R环与H3K36me3及gH2AX组蛋白修饰标记之间的关联。本研究明确了HPV阳性细胞中，DNA损伤修复与先天免疫调控通路、R环以及gH2AX/H3K36me3组蛋白修饰标记之间的潜在关联，这一关联可能参与调控HPV致病过程中的关键功能。向1×10^7个细胞中加入甲醛，使其终浓度达到1%，于室温下孵育10分钟。加入0.125M甘氨酸以淬灭过量甲醛，随后用磷酸盐缓冲液（PBS）洗涤样本。随后将细胞置于含Roche蛋白酶抑制剂混合物的收集缓冲液（0.1M Tris-HCl pH 9.4、10mM DTT）中，于冰上孵育10分钟。收集细胞并离心沉淀，依次用NCP1缓冲液（10mM EDTA、0.5mM EGTA、10mM HEPES pH 6.5、0.25% Triton X-100）与NCP2缓冲液（1mM EDTA、0.5mM EGTA、10mM HEPES、200mM NaCl）洗涤并孵育细胞，之后将细胞置于含Roche蛋白酶抑制剂混合物的裂解液（0.5% Empigen BB、1%十二烷基硫酸钠（sodium dodecyl sulfate, SDS）、10mM EDTA、50mM Tris-HCl pH 8.0）中，于冰上裂解30分钟。随后使用Bioruptor（Diagenode）以高功率对样本进行超声破碎，参数为30秒开启/90秒关闭，循环20分钟。超声破碎完成后，取部分输入对照DNA（Input DNA），将剩余样本加入预封闭的磁珠与免疫沉淀（immunoprecipitation, IP）缓冲液中，其中包含2μg H3K36me3抗体。将免疫沉淀反应体系于4℃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。先用TE缓冲液（Tris-EDTA缓冲液）洗涤一次样本，随后于65℃下在洗脱液（10% SDS、10mM Tris pH7.4、50mM乙二胺四乙酸（ethylenediaminetetraacetic acid, EDTA））中洗脱样本10分钟。使用PCR纯化试剂盒（Qiagen）从洗脱产物中纯化DNA，并将其保存于-20℃环境中。