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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE148581 | GEO | 2023/06/10

REPOSITORIES: GEO

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Protein interaction map of APOBEC3 enzyme family

Project description:Human APOBEC3 enzymes are a family of single-stranded (ss)DNA and RNA cytidine deaminases that act as part of the intrinsic immunity against viruses and retroelements. APOBEC3s deaminate cytosine to form uracil which can functionally inactivate or cause degradation of viral or retroelement genomes. In addition, APOBEC3 proteins have deamination independent anti-viral activity and aberrant regulation of APOBEC3 expression can result in the deamination and mutagenesis of the genome contributing to cancer initiation and evolution. To further understand their cellular roles, we used affinity purification mass spectrometry (AP-MS) to determine the protein-protein interaction (PPI) network for the human APOBEC3 enzymes and uncovered a diverse number of protein-protein and protein-RNA mediated interactions. PPIs with the Prefoldin family of protein folding chaperones were identified for APOBEC3B, APOBEC3D, and APOBEC3F. The APOBEC3B and prefoldin 5 (PFD5) interaction disrupted the ability of PFD5 to induce degradation of the oncogene cMyc, implicating a deamination independent contribution of APOBEC3B to cancer. Altogether, the results uncover novel functions and interactions of the APOBEC3 family and suggest that they may have fundamental roles in cellular RNA biology, their roles are not redundant, and there is a deamination independent influence on cancer.

2024-04-16 | PXD044275 | Pride

Genome-wide profiling of γH2AX in Arabidopsis

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2019-04-17 | GSE124943 | GEO

ALBA protein complex reads genic R-loops to maintain genome stability in Arabidopsis

Project description:The R-loop, composed of a DNA-RNA hybrid and the displaced single-stranded DNA, regulates diverse cellular processes. However, how cellular R-loops are sensed remain poorly understood. Here we report the discovery of the evolutionally conserved ALBA proteins (AtALBA1&2) functioning as the R-loops sensor of genic regions in higher plant Arabidopsis thaliana. AtALBA1 and 2 form a heterodimer protein complex and localize in the nucleus. While AtALBA1 binds to the DNA-RNA hybrid strand, AtALBA2 associates with single-stranded DNA in the R-loops. AtALBA1&2 preferentially binds to the genic R-loop regions that are associated with active epigenetic marks. Depletion of AtALBA1 or AtALBA2 results in hypersensitivity of plants to DNA damaging agents. Our results demonstrate the AtALBA1&2 protein complex is the Arabidopsis genic R-loops sensor for maintaining genome stability in Arabidopsis.

2019-04-17 | GSE121683 | GEO

DDX17 helicase promotes resolution of R-loop-mediated transcription-replication conflicts in human cells

Project description:R-loops are three-stranded nucleic acid structures composed of an RNA:DNA hybrid and displaced DNA strand. These structures can halt DNA replication when formed co- transcriptionally in the opposite orientation to replication fork progression. Recent studies have shown that replication forks stalled by co-transcription R-loops can be restarted by a mechanism involving fork cleavage by MUS81 endonuclease, followed by reactivation of transcription, and fork religation by the DNA ligase IV (LIG4)/XRCC4 complex. However, how R-loops are eliminated to allow the sequential restart of transcription and replication in this pathway remains elusive. Here, we identified the human DDX17 helicase as a factor that associates with R-loops and counteracts R-loop-mediated replication stress to preserve genome stability. We show that DDX17 unwinds RNA:DNA hybrids in vitro and promotes MUS81-dependent restart of R-loop-stalled forks in human cells in a manner dependent on its helicase activity. Loss of DDX17 helicase induces accumulation of R-loops and the formation of R-loop-dependent anaphase bridges and micronuclei. These findings establish DDX17 as a component of the MUS81-LIG4 pathway for resolution of R-loop-mediated transcription- replication conflicts, which may be involved in R-loop unwinding.

2022-11-02 | PXD034331 | Pride

ChIP-Seq in mouse embryonic stem cells of endogenous HA-tagged Tet1

Project description:R-loops are prevalent three-stranded nucleic acid structures, composed of a DNA:RNA hybrid and a displaced single-stranded DNA loop. Although aberrant R-loop formation is a threat to genome stability, R-loops are enriched at CpG islands (CGI) and are implicated in maintaining CGI promoters unmethylated. Mapping of genomic regions where Tet1 binds in an R-loop dependent manner we here identify almost exclusively CGIs. The results suggest that R-loops are genomic address labels for TET1.

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Project description:Intratumor mutational heterogeneity has been documented in primary non-small cell lung cancer. Here, we elucidate mechanisms of tumor evolution and heterogeneity in metastatic thoracic tumors (lung adenocarcinoma and thymic carcinoma) using whole-exome and transcriptome sequencing, SNP array for copy number alterations (CNA) and mass spectrometry-based quantitative proteomics of metastases obtained by rapid autopsy. APOBEC-mutagenesis, promoted by increased expression of APOBEC3 region transcripts and associated with a high-risk germline APOBEC3 variant, strongly correlated with mutational tumor heterogeneity. TP53 mutation status was associated with APOBEC hypermutator status. Interferon pathways were enriched in tumors with high APOBEC mutagenesis and IFN- induced expression of APOBEC3B in lung adenocarcinoma cells in culture suggesting a role for the immune microenvironment in the generation of mutational heterogeneity. CNA occurring late in tumor evolution correlated with downstream transcriptomic and proteomic heterogeneity, although global proteomic heterogeneity was significantly greater than transcriptomic and CNA heterogeneity. These results illustrate key mechanisms underlying multi-dimensional heterogeneity in metastatic thoracic tumors.

2019-05-07 | PXD012845 | Pride

Rapid Autopsy in Thoracic Malignancy Cancer Patients to Investigate Tumor Heterogeneity

Project description:<p>Integrated proteo-genomics studies to characterize tumor heterogeneity of metastatic lung adenocarcinoma and thymic carcinoma are lacking. We carried out whole exome sequencing, RNA sequencing, copy number estimation and mass spectrometry-based proteomics of 40 tumors from five rapid/warm autopsy patients. We found highly variable mutational heterogeneity that was largely driven by APOBEC-mutagenesis with the expression of APOBEC3B (<a href="https://www.ncbi.nlm.nih.gov/gene/?term=APOBEC3B">Gene ID: 9582</a>) and APOBEC3A_B (<a href="https://www.ncbi.nlm.nih.gov/gene/100913187">Gene ID: 100913187</a>) due to underlying APOBEC3 region germline variants as the likely mediating mechanism. APOBEC3A_B expression was associated with increased immune tumor microenvironment and CD274 (<a href="https://www.ncbi.nlm.nih.gov/gene/?term=CD274">Gene ID: 29126</a>) expression while smoking was associated with increased APOBEC-mutagenesis in TP53 (<a href="https://www.ncbi.nlm.nih.gov/gene/?term=TP53">Gene ID: 7157</a>) mutant tumors with high APOBEC3B expression. Heterogeneity at the level of the transcriptome and proteome occurred in association with copy number heterogeneity, not APOBEC-induced mutational heterogeneity. Arm and focal copy number alterations (CNAs) occurred as an evolutionarily late event in a subset of patients, with corresponding changes in the transcriptome and proteome, implicating CNAs as a driving force of heterogeneity in the evolution of metastatic disease.</p>

| phs001432 | dbGaP

R-loop proximity proteomics identifies a role of DDX41 in transcription-associated genomic instabilityR-loop proximity proteomics identifies a role of DDX41 in transcription-associated genomic instability

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2021-11-25 | PXD024517 | Pride

The extruded non-template strand determines the architecture of R-loops

Project description:We show that these R-loop objects impose specific physical constraints on the DNA, as revealed by the presence of stereotypical angles in the surrounding DNA. Biochemical probing and mutagenesis experiments revealed that the formation of R-loop objects at Airn is dictated by the extruded non-template strand, suggesting that R-loops possess intrinsic sequence-driven properties. Consistent with this, we show that R-loops formed at the fission yeast gene sum3 do not form detectable R-loop objects. Our results reveal that R-loops differ by their architectures and that the organization of the non-template strand is a fundamental characteristic of R-loops, which could explain that only a subset of R-loops is associated with replication-dependent DNA breaks.

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2021-06-03 | MSV000087568 | MassIVE

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