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Mapping and quantification of single-strand DNA bound by flag-tagged APOBEC3B

ABSTRACT: Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3B, A3B) is a key molecular driver inducing mutations in multiple human cancer. A3B 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). Here, we adopted single-strand DNA protein-immunoprecipitation sequencing (SPI-seq) to capture the ssDNA bound by flag-tagged A3B protein. Quantification on sequence reads was carried out to investigate the effect of 17-beta estradiol (E2) on A3B binding to DNA.

ORGANISM(S): Homo sapiens

PROVIDER: GSE193228 | GEO | 2025/01/06

REPOSITORIES: GEO

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Mapping the genomic occupancy by flag-tagged APOBEC3B protein

Project description: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.

2025-01-06 | GSE193227 | GEO

RNA-seq of T-47D cells treated with 17-beta-estradiol and/or APOBEC3B-targeting siRNA

Project description:Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3B, A3B) is a key molecular driver inducing mutations in multiple human cancer. A3B 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). Here, we used RNA-seq technology to study the effect of A3B knockdown on the effect of estradiol induction in T-47D cells.

2025-01-06 | GSE193231 | GEO

Whole genome sequencing of T-47D cell colonies overexpressed with APOBEC3B and hUGI

Project description:Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3B, A3B) is a key molecular driver inducing mutations in multiple human cancer. A3B 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). Here, we used a lentiviral inducible system to overexpress A3B and hUGI in T-47D cells. This system will allow augmented genomic editing by A3B, while preserving the C>U editing sites from DNA repair by inhibiting base excision repair mechanism.

2025-01-06 | GSE193226 | GEO

ChIP-seq of TDRD3, CARM1 and H3R17me2a in T-47D cells

Project description:Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3B, A3B) is a key molecular driver inducing mutations in multiple human cancer. A3B 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). By using mass-spec proteomics, we identified TDRD3, a ‘reader’ of asymmetrically methylated arginine marks, as the binding partner of A3B.

2025-01-06 | GSE193232 | GEO

Detection of DNA double-strand breaks in T-47D cells using DSBCapture-seq

Project description:Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3B, A3B) is a key molecular driver inducing mutations in multiple human cancer. A3B 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). Here, we used DSBCapture-seq to study the formation of DNA double-strand breaks (DSB) by estrogen receptor activation upon 17-estradiol (E2) induction. The effect of A3B-depletion on DSB formation was investigated using RNA interference.

2025-01-06 | GSE193230 | GEO

Strand-specific DNA:RNA immunoprecipitation sequencing on T-47D breast cancer cells.

Project description:Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3B) is a key molecular driver inducing mutations in multiple human cancer. A3B is a member of the APOBEC3 enzyme family, which consists seven closely related DNA deaminases that catalyse cytosine-to-uracil (C>U) editing of single-stranded DNA (ssDNA). Using a cell model that lacks base excision repair function, we sampled A3B editing sites in bulk. Analyses conducted on these A3B editing sites point to a function of A3B in editing DNA:RNA hybrid structure known as R-loops. In order to verify this result, we conducted strand-specific DNA:RNA immunoprecipitation sequencing (ssDRIP-seq; S9.6, KeraFAST, ENH001) on T-47D breast cancer cells.

2025-01-06 | GSE193225 | GEO

Quantified strand-specific DNA:RNA immunoprecipitation sequencing on T-47D breast cancer cells.

Project description:Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3B) is a key molecular driver inducing mutations in multiple human cancer. A3B 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). Using a cell model that lacks base excision repair function, we sampled A3B editing sites in bulk. Analyses conducted on these A3B editing sites point to a function of A3B in editing DNA:RNA hybrid structure known as R-loops. In order to verify this result, we conducted strand-specific DNA:RNA immunoprecipitation sequencing (ssDRIP-seq; S9.6, KeraFAST, ENH001) on T-47D breast cancer cells.

2025-01-06 | GSE193229 | GEO

Detection of APOBEC3B-mediated RNA editing sites in T-47D cells [RNA-seq]

Project description:RNA editing is a crucial post-transcriptional process that diversifies proteomic outcomes and influences gene expression. Particularly, the APOBEC3 family has emerged as a significant player in this mechanism, with APOBEC3A (A3A) showing notable roles in immune response and stress conditions. APOBEC3B (A3B), another family member, has garnered attention for its potential role in breast cancer genomic mutations. In this study, we employ an inducible expression cell model and a novel methodology for identifying differential variants in RNA (DVRs) to map A3B-mediated RNA editing sites in a breast cancer cell model. Our findings indicate that A3B engages in selective RNA editing and targets NEAT1 and MALAT1 long non-coding RNAs. Notably, the binding of these RNAs sequesters A3B’s catalytic activity, and thereby affects A3A’s activity through a feedback loop.

2024-08-14 | GSE245700 | GEO

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

Project description: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.

2023-06-10 | GSE148581 | GEO

SNP microarray of T-REx-293-based clones overexpressing the DNA cytosine deaminase APOBEC3B

Project description:A conditional and isogenic system for APOBEC3B (A3B) induction in T-REx-293 cells. Cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs and subjected to 10 rounds of A3B-eGFP exposure causing 80-90% cell death. Control pools (eGFP) were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. DNA was extracted and subjected to SNP analyses using the Human OmniExpress-24v1-0 BeadChip (Illumina, San Diego, CA). Genotyping was performed using the humanomniexpress_24v1-0_a cluster file. SNP data is avaiable for a total of 16 samples, all derived from single cells mother lines A and C. For each series, each consisting of 8 samples, A3B-eGFP and eGFP daugther clones were processed on the SNP microarray, as well as 3 A3B-eGFP and 3 eGFP grand-daugther clones.

2016-06-30 | E-GEOD-78710 | biostudies-arrayexpress

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