mrRNA-directed DNA methylation enables selective mutant oncogene silencing therapy without exogenous proteins

The outcome of recent demethylation therapy is limited by the unspecific hypomethylation of oncogenes. The mechanism underlying differential DNA methylation distribution in the same topological domain remains unclear. Here, we established an efficient framework based on heterogeneity across pan-cancers to systemically characterize DNA methylation regulatory boxes (mrBoxes). Through plasmid-based functional motif interactome analysis assay, the role of mrBoxes on DNA methylation was validated. Moreover, the U1 small nuclear ribonucleoprotein A (SNPRA) was found to regulate the chromatin localization of DNMT1 to mrBox. In this process, long noncoding RNAs were identified to act as a bridge, connecting the targeted mrBox element and SNRPA/DNMT1 complex. Based on this regulatory model, we synthesized exogenous methylation-regulating RNA (mrRNA), which contains only the mrBox-recognizing and SNRPA/DNMT1 complex-binding motif. Furthermore, we developed a methylation-regulating therapy called “OncoMutation Silence”, which regulates the DNA methylation of mutated oncogenes through mrRNA. The efficacy and specificity of this therapy have been verified in tumors with KrasG12D mutation. Collectively, this study reveals mrBox-related regulatory model of DNA methylation, and provides mrRNA-based tumor therapy for silencing specific mutated oncogenes. Overall design: ChIP-seq of DNMT1 dynamics in HEK293T cell lines treated with siZFPM2AS1 and siSNRPA

现有去甲基化疗法的疗效因癌基因发生非特异性低甲基化而受到显著限制。目前，同一拓扑结构域内DNA甲基化分布差异的潜在调控机制仍未阐明。本研究基于泛癌层面的异质性构建了一套高效分析框架，以系统性表征DNA甲基化调控盒（methylation regulatory boxes, mrBoxes）。通过基于质粒的功能基序相互作用组分析实验，验证了mrBox对DNA甲基化的调控功能。此外，研究发现U1小核核糖核蛋白A（SNRPA）可调控DNA甲基转移酶1（DNMT1）在mrBox位点的染色质定位。在此调控过程中，长链非编码RNA（long noncoding RNAs, lncRNAs）被证实可作为桥梁分子，连接靶向mrBox元件与SNRPA/DNMT1复合物。基于该调控模型，本研究合成了外源性甲基化调控RNA（methylation-regulating RNA, mrRNA），其仅包含mrBox识别基序与SNRPA/DNMT1复合物结合基序。进一步，本研究开发了名为“癌突变沉默（OncoMutation Silence）”的甲基化调控疗法，该疗法通过mrRNA靶向调控突变癌基因的DNA甲基化水平。该疗法的疗效与特异性已在携带KrasG12D突变的肿瘤模型中得到验证。综上，本研究揭示了mrBox相关的DNA甲基化调控模型，并提供了基于mrRNA的肿瘤治疗策略，以特异性沉默突变癌基因。整体实验设计：在转染siZFPM2AS1与siSNRPA的HEK293T细胞系中开展DNMT1动态变化的染色质免疫沉淀测序（ChIP-seq）实验。