R-loop homeostasis and cancer mutagenesis promoted by the DNA cytosine deaminase APOBEC3B

The single-stranded DNA cytosine-to-uracil deaminase APOBEC3B is an antiviral protein implicated in cancer. However, its substrates in cells are not fully delineated. Here, APOBEC3B proteomics reveal interactions with a surprising number of R-loop factors. Biochemical experiments show APOBEC3B binding to R-loops in human cells and in vitro. Genetic experiments demonstrate R-loop increases in cells lacking APOBEC3B and decreases in cells overexpressing APOBEC3B. Genome-wide analyses show major changes in the overall landscape of physiological and stimulus-induced R-loops with thousands of differentially altered regions as well as binding of APOBEC3B to many of these sites. APOBEC3 mutagenesis impacts overexpressed genes and splice factor mutant tumors preferentially, and APOBEC3-attributed kataegis are enriched in RTCW consistent with APOBEC3B deamination. Taken together with the fact that APOBEC3B binds single-stranded DNA and RNA and preferentially deaminates DNA, these results support a mechanism in which APOBEC3B mediates R-loop homeostasis and contributes to R-loop mutagenesis in cancer. Overall design: In MCF10A cells: ChIP-seq of eGFP-tagged APOBEC3B (A3B-eGFP) in non-induced (DMSO), induced (DMSO + Doxycycline), and induced + PMA 2h (Doxycyline + PMA) conditions. DRIP-seq in WT or A3B-KO cells treated with DMSO, PMA 2h, or PMA 6h.

单链DNA胞嘧啶向尿嘧啶脱氨酶APOBEC3B（APOBEC3B）是一类兼具抗病毒活性的功能蛋白，且与癌症发生发展密切相关，但其在细胞内的底物尚未得到完全阐明。本研究通过蛋白质组学分析发现，APOBEC3B可与大量R环（R-loop）相关因子产生相互作用，该结果颇为出人意料。生化实验证实，APOBEC3B可在人体细胞及体外体系中与R环特异性结合。遗传学实验表明，敲除APOBEC3B的细胞内R环水平显著升高，而过表达APOBEC3B的细胞内R环水平则明显降低。全基因组分析显示，生理状态及刺激诱导下的R环整体景观发生显著改变，存在数千个差异调控区域；同时APOBEC3B可结合其中多数位点。APOBEC3介导的诱变作用优先影响过表达基因及剪接因子突变型肿瘤，且由APOBEC3介导的kataegis突变簇（kataegis）在RTCW基序中富集，这与APOBEC3B的脱氨酶活性特征相符。结合APOBEC3B可结合单链DNA与RNA、且优先对DNA进行脱氨的固有特性，上述结果共同支持如下作用机制：APOBEC3B可介导R环稳态维持，并参与癌症中R环依赖的诱变过程。整体实验设计：在MCF10A细胞中，设置三组处理条件：未诱导组（二甲基亚砜（DMSO））、诱导组（DMSO + 多西环素（Doxycycline））以及诱导+PMA处理2小时组（多西环素 + PMA），对eGFP标记的APOBEC3B（A3B-eGFP）开展染色质免疫沉淀测序（ChIP-seq）检测。同时在野生型（WT）或APOBEC3B敲除（A3B-KO）细胞中，分别经DMSO、PMA处理2小时或6小时后，进行DNA-RNA免疫沉淀测序（DRIP-seq）检测。