PO.IM02.06 · 免疫学
卵巢癌中转座元件的调控与表达
Regulation and expression of transposable elements in ovarian cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：转座元件（TEs）是高度重复的DNA序列，当转录为RNA时可结合免疫原性双链RNA传感器。通过这一机制，TEs的表达可诱导I型干扰素（IFN）信号，该信号在改善肿瘤对免疫调节疗法的应答方面已显示出效力。我们此前已发表，与野生型p53肿瘤细胞相比，R175H突变型p53卵巢癌细胞系激活了慢性TE表达。尽管如此，在R175H p53肿瘤中并未观察到持久免疫应答的激活，这提示存在未知的耐受机制，抑制了TE诱导的IFN。为此，我们试图阐明p53状态如何改变TEs的表达并影响下游IFN信号。
方法：为确定p53直接结合对TE转录的影响，我们将p53靶向染色质免疫沉淀测序（chIP-seq）与bulk RNA测序表达进行关联。接下来，我们利用MAVS/MDA-5信号介导因子IRF3/7的磷酸化状态，来确定dsRNA感知下游激酶活性的破坏情况。
结果：我们发现，大多数被转录的TEs并未表现出直接的p53结合，而是作为下游p53靶基因内插入的副产物被转录。此外，R175H突变型或缺失型p53的bulk RNA测序结果表明，许多TE抑制因子发生下调，包括KRAB锌指蛋白（ZNF43、ZNF93和ZNF561）和DNA甲基转移酶1（DNMT1）。这提示在突变型R175H或缺失型p53背景下，TEs丧失了DNA甲基化和/或KRAB锌指沉默。对IRF3/7的评估表明，与野生型p53细胞相比，R175H p53细胞的磷酸化水平降低。我们假设，突变型R175H p53蛋白对TBK1的失活作用阻止了IRF3的磷酸化，从而在TE表达较高的情况下仍阻止了IFN的刺激。
讨论：总体而言，这些数据表明，KRAB锌指蛋白的DNA甲基化/表达是p53状态失调TE转录的间接机制。我们计划利用全基因组亚硫酸氢盐测序，表征野生型、R175H或缺失型p53卵巢癌细胞中TEs的DNA甲基化状态。此外，我们希望利用磷酸化蛋白质印迹分析来剖析抗病毒应答介导因子的激活情况，以确定这些相同细胞系中效应信号的破坏。
查看英文原文 English abstract
Background: Transposable Elements (TEs) are highly repetitive DNA sequences that, when transcribed into RNA, bind immunogenic double-stranded RNA sensors. Through this, expression of TEs can induce type-I interferon (IFN) signaling which has shown potency in improving the response of tumors to immune-modulating therapies. We have published the R175H mutant p53 ovarian cancer cell lines activate chronic TE expression compared to wildtype p53 tumor cells. Despite this, activation of durable immune responses are not seen in R175H p53 tumors, indicating unknown tolerance mechanisms that dampen TE-induced IFN. To this end, we sought to characterize how p53 status alters the expression of TEs and influences downstream IFN signaling.
Methods: To determine the effect that direct binding of p53 has on TE transcription, we correlated p53-targeted chromatin immunoprecipitation sequencing (chIP-seq) with bulk RNA-sequencing expression. Next, we used phosphorylation status of MAVS/MDA-5 signaling mediators, IRF3/7, to determine disruptions in kinase activity downstream of dsRNA sensing.
Results: We found the majority of transcribed TEs did not display direct p53 binding, but rather were transcribed as a biproduct of intragenic insertion within downstream p53 target genes. Additionally, R175H mutant or null p53 bulk RNA sequencing results indicated the downregulation of many TE repressors including KRAB zinc finger proteins (ZNF43, ZNF93, and ZNF561) and DNA Methyltransferase 1 (DNMT1). This suggests TEs lose either DNA methylation and/or KRAB zinc finger silencing in mutant R175H or null p53 backgrounds. The assessment of IRF3/7 indicate R175H p53 cells have reduced phosphorylation levels as compared to wildtype p53 cells. We hypothesize that inactivation of TBK1 by the mutant R175H p53 protein prevents phosphorylation of IRF3 and thus prevents IFN stimulation despite higher TE expression.
Discussion: Overall, these data suggest DNA methylation/expression of KRAB zinc finger proteins are indirect mechanisms by which p53 status dysregulates TE transcription. We aim to characterize DNA methylation status of TEs in wildtype, R175H, or null p53 ovarian cancer cells using whole genome bisulfite sequencing. Additionally, we hope to profile the activation of antiviral response mediators using phosphorylated western blot analyses to determine the disruption of effector signaling in these same cell lines.
利益披露 Disclosure
R. R. Walker, None..
K. Nestler, None..
M. Hadley, None..
K. B. Chiappinelli, None.