R-loop homeostasis and cancer mutagenesis promoted by the DNA cytosine deaminase APOBEC3B
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PROVIDER: PRJNA625079 | ENA |
REPOSITORIES: ENA
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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
Project description:Mutations in cancer are due in part to DNA cytosine deamination by APOBEC3B (A3B). While low in healthy tissues, A3B expression and activity are elevated in tumors and further increase in metastases. However, molecular mechanisms responsible for A3B transcriptional regulation are poorly understood. Here, we address whether the RB/E2F pathway, which is often dysregulated in breast cancer, is a molecular trigger of A3B overexpression. First, an A3B promoter-driven luciferase reporter was used to demonstrate reporter activation by disruption of only one out of five predicted E2F binding sites. Second, A3B-luciferase reporter activation was also triggered by expressing the BK polyomavirus T-antigen, which inactivates RB and thereby alleviates repression of E2F regulated genes. Importantly, A3B-luciferase reporter induction by BK polyomavirus T-antigen could not be further increased by mutating the functional E2F binding site. Third, both CRISPR disruption and targeted base substitutions in the endogenous E2F binding site caused strong A3B upregulation and confirmed importance of this regulatory element. Fourth, proteomics experiments showed that members of the DREAM and PRC1.6-complexes including multiple E2F family members are able to bind to wild-type but not E2F mutant promoter sequences. Finally, a combination of genetic and biochemical studies implicated E2F4 and E2F6 in endogenous A3B gene repression. Altogether, our studies demonstrate that A3B expression is suppressed in normal cells by the RB/E2F axis and that viral or mutational disruption of this pathway causes overexpression of this DNA deaminase in cancer and contributes mutational fuel to tumor evolution.
2020-10-19 | PXD020473 | Pride
Project description:Oestrogen receptor-α (ER) is the principal transcription factor in the majority of breast cancers, driving expression of genes that control cell growth and endocrine response. Understanding the mechanisms of ER action is crucial for improving response to endocrine treatments. Recent studies show that cytosine deaminase (CD) activity is an important source of cancer mutations. In particular, APOBEC3B (A3B) promotes mutagenesis in breast cancer cells1. Our analysis of breast cancer expression datasets showed that A3B expression predicts for poor survival in ER-positive, but not in ER-negative patients, indicative of a link with ER activity. Chromatin immunoprecipitation coupled to deep sequencing (ChIP-seq) used to map global A3B binding sites showed a remarkable oestrogen-stimulated recruitment of A3B to ER binding sites. Functionally, A3B was critical for ER transcriptional activity and regulated breast cancer cell proliferation. We show that A3B regulates ER transcription by promoting cytosine deamination and activation of DNA strand break repair at ER binding regions. We propose that cytosine deamination and DNA strand break generation by A3B facilitates gene expression by aiding chromatin remodeling at ER target genes. Our findings also suggest a mechanism by which subversion of transcription factor mediated recruitment of cytosine deaminases promotes cancer mutations. Hormone-depleted MCF-7 breast cancer cell line was treated with estrogen (100 nM), H2O2 (10 mM) or vehicle for 45 minutes. H2AX ChIP-seq was performed using Illumina methodology.
2015-09-07 | E-GEOD-57426 | biostudies-arrayexpress
Project description:<p><b>Reprinted from Roberts et al. "An APOBEC cytidine deaminase mutagenesis pattern is widespread in human cancers", Nature Genetics, 45:970-976, 2013, with permission of Nature Publishing Group:</b></p> <p>Recent studies indicate that a subclass of APOBEC cytidine deaminases, which convert cytosine to uracil during RNA editing and retrovirus or retrotransposon restriction, may induce mutation clusters in human tumors. We show here that throughout cancer genomes APOBEC-mediated mutagenesis is pervasive and correlates with APOBEC mRNA levels. Mutation clusters in whole-genome and exome data sets conformed to the stringent criteria indicative of an APOBEC mutation pattern. Applying these criteria to 954,247 mutations in 2,680 exomes from 14 cancer types, mostly from The Cancer Genome Atlas (TCGA), showed a significant presence of the APOBEC mutation pattern in bladder, cervical, breast, head and neck, and lung cancers, reaching 68% of all mutations in some samples. Within breast cancer, the HER2-enriched subtype was clearly enriched for tumors with the APOBEC mutation pattern, suggesting that this type of mutagenesis is functionally linked with cancer development. The APOBEC mutation pattern also extended to cancer-associated genes, implying that ubiquitous APOBEC-mediated mutagenesis is carcinogenic.</p>
| phs000677 | dbGaP
Project description:The APOBEC3 cytosine deaminases are implicated as the cause of a prevalent somatic mutation pattern found in cancer genomes. The APOBEC3 enzymes act as viral restriction factors by mutating viral genomes. Mutation of the cellular genome is presumed to be an off-target activity of the enzymes, although the regulatory measures for APOBEC3 expression and activity remain undefined. It is therefore difficult to predict the circumstances that enable APOBEC3 interaction with cellular DNA that leads to mutagenesis. The APOBEC3A (A3A) enzyme is the most potent deaminase of the family. Using proteomics, we evaluated protein interactors of A3A to identify potential regulators. We found that A3A interacts with the Chaperonin Containing TCP-1 (CCT) complex, a cellular machine that assists in protein folding and function. Importantly, depletion of CCT resulted in increased A3A-induced cytotoxicity. Evaluation of cancer genomes demonstrated an enrichment of A3A mutational signatures in cancers with silencing mutations in CCT subunit genes. Together, these data suggest that the CCT complex interacts with A3A, and that disruption of CCT function results in increased A3A mutational activity.
2021-07-02 | PXD024191 | Pride
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.
2016-06-30 | GSE78710 | GEO
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
Project description:Oestrogen receptor-α (ER) is the principal transcription factor in the majority of breast cancers, driving expression of genes that control cell growth and endocrine response. Understanding the mechanisms of ER action is crucial for improving response to endocrine treatments. Recent studies show that cytosine deaminase (CD) activity is an important source of cancer mutations. In particular, APOBEC3B (A3B) promotes mutagenesis in breast cancer cells1. Our analysis of breast cancer expression datasets showed that A3B expression predicts for poor survival in ER-positive, but not in ER-negative patients, indicative of a link with ER activity. Chromatin immunoprecipitation coupled to deep sequencing (ChIP-seq) used to map global A3B binding sites showed a remarkable oestrogen-stimulated recruitment of A3B to ER binding sites. Functionally, A3B was critical for ER transcriptional activity and regulated breast cancer cell proliferation. We show that A3B regulates ER transcription by promoting cytosine deamination and activation of DNA strand break repair at ER binding regions. We propose that cytosine deamination and DNA strand break generation by A3B facilitates gene expression by aiding chromatin remodeling at ER target genes. Our findings also suggest a mechanism by which subversion of transcription factor mediated recruitment of cytosine deaminases promotes cancer mutations.
2015-09-30 | GSE56979 | GEO
Project description:Oestrogen receptor-α (ER) is the principal transcription factor in the majority of breast cancers, driving expression of genes that control cell growth and endocrine response. Understanding the mechanisms of ER action is crucial for improving response to endocrine treatments. Recent studies show that cytosine deaminase (CD) activity is an important source of cancer mutations. In particular, APOBEC3B (A3B) promotes mutagenesis in breast cancer cells1. Our analysis of breast cancer expression datasets showed that A3B expression predicts for poor survival in ER-positive, but not in ER-negative patients, indicative of a link with ER activity. Chromatin immunoprecipitation coupled to deep sequencing (ChIP-seq) used to map global A3B binding sites showed a remarkable oestrogen-stimulated recruitment of A3B to ER binding sites. Functionally, A3B was critical for ER transcriptional activity and regulated breast cancer cell proliferation. We show that A3B regulates ER transcription by promoting cytosine deamination and activation of DNA strand break repair at ER binding regions. We propose that cytosine deamination and DNA strand break generation by A3B facilitates gene expression by aiding chromatin remodeling at ER target genes. Our findings also suggest a mechanism by which subversion of transcription factor mediated recruitment of cytosine deaminases promotes cancer mutations.
2015-09-07 | GSE57426 | GEO
Project description:APOBEC Cytidine Deaminase Mutagenesis Pattern is Widespread in Human Cancers
| PRJNA226256 | ENA