Mapping the genomic occupancy by flag-tagged APOBEC3B protein. Mapping the genomic occupancy by flag-tagged APOBEC3B protein

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3B) is a key molecular driver inducing mutations in multiple human cancers. APOBEC3B belongs to the APOBEC3 enzyme family, which consists seven closely related DNA deaminases that catalyse cytosine-to-uracil (C>U) editing of single-stranded DNA (ssDNA). In order to investigate the genomic binding sites of APOBEC3B, high through-put sequencing experiments were conducted using DNA samples acquired by standard chromatin immunoprecipitation (ChIP) and ssDNA protein immunoprecipitation (SPI) samples. Substrate preference by APOBEC3B were characterised by comparing these results. Overall design: T-47D breast cancer cells transduced with a lentiviral vector that allows inducible expression of flag-tagged APOBEC3B protein. After induction of the lentiviral APOBEC3B cassette by doxycycline, cells were transfected with plasmid that allow transient expression of human RNase H, or treated with transfection agent as control. Treated cells were subjected to ChIP-seq and SPI-seq with anti-flag antibody (M2, Sigma, F1804), which detect APOBEC3B-bound dsDNA or ssDNA respectively.

载脂蛋白B mRNA编辑酶催化多肽样3（APOBEC3B）是诱导多种人类癌症发生突变的关键分子驱动因子。APOBEC3B隶属于APOBEC3酶家族，该家族包含7个紧密相关的DNA脱氨酶，可催化单链DNA（ssDNA）的胞嘧啶向尿嘧啶（C→U）编辑反应。为探究APOBEC3B的基因组结合位点，研究人员采用标准染色质免疫共沉淀（ChIP）与单链DNA蛋白质免疫共沉淀（SPI）获取的DNA样本开展了高通量测序实验，并通过对比两组实验结果表征了APOBEC3B的底物偏好性。实验整体设计：将可诱导表达Flag标签融合APOBEC3B蛋白的慢病毒载体转导至T-47D乳腺癌细胞中。以多西环素诱导慢病毒载体携带的APOBEC3B表达盒后，将细胞分为两组：一组转染可瞬时表达人类RNase H的质粒，另一组仅用转染试剂处理作为空白对照。随后分别采用抗Flag抗体（"M2, Sigma, F1804"）对两组处理后的细胞进行ChIP-seq与SPI-seq实验，分别检测结合APOBEC3B的双链DNA（dsDNA）与单链DNA（ssDNA）。