Lela Lackey

APOBEC3B is an enzymatic source of mutation in breast cancer

Several mutations are required for cancer development, and genome sequencing has revealed that many cancers, including breast cancer, have somatic mutation spectra dominated by C-to-T transitions. Most of these mutations occur at hydrolytically disfavoured non-methylated cytosines throughout the genome, and are sometimes clustered. Here we show that the DNA cytosine deaminase APOBEC3B is a probable source of these mutations. APOBEC3B messenger RNA is upregulated in most primary breast tumours and breast cancer cell lines. Tumours that express high levels of APOBEC3B have twice as many mutations as those that express low levels and are more likely to have mutations in TP53. Endogenous APOBEC3B protein is predominantly nuclear and the only detectable source of DNA C-to-U editing activity in breast cancer cell-line extracts. Knockdown experiments show that endogenous APOBEC3B correlates with increased levels of genomic uracil, increased mutation frequencies, and C-to-T transitions. Furthermore, induced APOBEC3B overexpression causes cell cycle deviations, cell death, DNA fragmentation, γ-H2AX accumulation and C-to-T mutations. Our data suggest a model in which APOBEC3B-catalysed deamination provides a chronic source of DNA damage in breast cancers that could select TP53 inactivation and explain how some tumours evolve rapidly and manifest heterogeneity.

Human and Rhesus APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H Demonstrate a Conserved Capacity to Restrict Vif-deficient HIV-1

ABSTRACT Successful intracellular pathogens must evade or neutralize the innate immune defenses of their host cells and render the cellular environment permissive for replication. For example, to replicate efficiently in CD4+ T lymphocytes, human immunodeficiency virus type 1 (HIV-1) encodes a protein called viral infectivity factor (Vif) that promotes pathogenesis by triggering the degradation of the retrovirus restriction factor APOBEC3G. Other APOBEC3 proteins have been implicated in HIV-1 restriction, but the relevant repertoire remains ambiguous. Here we present the first comprehensive analysis of the complete, seven-member human and rhesus APOBEC3 families in HIV-1 restriction. In addition to APOBEC3G, we find that three other human APOBEC3 proteins, APOBEC3D, APOBEC3F, and APOBEC3H, are all potent HIV-1 restriction factors. These four proteins are expressed in CD4+ T lymphocytes, are packaged into and restrict Vif-deficient HIV-1 when stably expressed in T cells, mutate proviral DNA, and are counteracted by HIV-1 Vif. Furthermore, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H of the rhesus macaque also are packaged into and restrict Vif-deficient HIV-1 when stably expressed in T cells, and they are all neutralized by the simian immunodeficiency virus Vif protein. On the other hand, neither human nor rhesus APOBEC3A, APOBEC3B, nor APOBEC3C had a significant impact on HIV-1 replication. These data strongly implicate a combination of four APOBEC3 proteins—APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H—in HIV-1 restriction.

Methylcytosine and Normal Cytosine Deamination by the Foreign DNA Restriction Enzyme APOBEC3A

Background: APOBEC3A is a myeloid-specific interferon-inducible DNA C to U deaminase implicated in innate immunity. Results: APOBEC3A also elicits MeC to T editing activity in vitro with deoxy-oligonucleotides and in vivo with transfected plasmids. Conclusion: APOBEC3A accommodates both normal and larger DNA cytosine substrates. Significance: The developmental specialization and broader substrate range of APOBEC3A may be an evolutionary adaptation for physiological function in foreign DNA restriction. Multiple studies have indicated that the TET oxidases and, more controversially, the activation-induced cytidine deaminase/APOBEC deaminases have the capacity to convert genomic DNA 5-methylcytosine (MeC) into altered nucleobases that provoke excision repair and culminate in the replacement of the original MeC with a normal cytosine (C). We show that human APOBEC3A (A3A) efficiently deaminates both MeC to thymine (T) and normal C to uracil (U) in single-stranded DNA substrates. In comparison, the related enzyme APOBEC3G (A3G) has undetectable MeC to T activity and 10-fold less C to U activity. Upon 100-fold induction of endogenous A3A by interferon, the MeC status of bulk chromosomal DNA is unaltered, whereas both MeC and C nucleobases in transfected plasmid DNA substrates are highly susceptible to editing. Knockdown experiments show that endogenous A3A is the source of both of these cellular DNA deaminase activities. This is the first evidence for nonchromosomal DNA MeC to T editing in human cells. These biochemical and cellular data combine to suggest a model in which the expanded substrate versatility of A3A may be an evolutionary adaptation that occurred to fortify its innate immune function in foreign DNA clearance by myeloid lineage cell types.

Atomic force microscopy studies provide direct evidence for dimerization of the HIV restriction factor APOBEC3G

APOBEC3G (A3G) is an antiviral protein that binds RNA and single-stranded DNA (ssDNA). The oligomerization state of A3G is likely to be influenced by these nucleic acid interactions. We applied the power of nanoimaging atomic force microscopy technology to characterize the role of ssDNA in A3G oligomerization. We used recombinant human A3G prepared from HEK-293 cells and specially designed DNA substrates that enable free A3G to be distinguished unambiguously from DNA-bound protein complexes. This DNA substrate can be likened to a molecular ruler because it consists of a 235-bp double-stranded DNA visual tag spliced to a 69-nucleotide ssDNA substrate. This hybrid substrate enabled us to use volume measurements to determine A3G stoichiometry in both free and ssDNA-bound states. We observed that free A3G is primarily monomeric, whereas ssDNA-complexed A3G is mostly dimeric. A3G stoichiometry increased slightly with the addition of Mg2+, but dimers still predominated when Mg2+ was depleted. A His-248/His-250 Zn2+-mediated intermolecular bridge was observed in a catalytic domain crystal structure (Protein Data Bank code 3IR2); however, atomic force microscopy analyses showed that the stoichiometry of the A3G-ssDNA complexes changed insignificantly when these residues were mutated to Ala. We conclude that A3G exchanges between oligomeric forms in solution with monomers predominating and that this equilibrium shifts toward dimerization upon binding ssDNA.

Subcellular localization of the APOBEC3 proteins during mitosis and implications for genomic DNA deamination

Humans have seven APOBEC3 DNA cytosine deaminases. The activity of these enzymes allows them to restrict a variety of retroviruses and retrotransposons, but may also cause pro-mutagenic genomic uracil lesions. During interphase the APOBEC3 proteins have different subcellular localizations: cell-wide, cytoplasmic or nuclear. This implies that only a subset of APOBEC3s have contact with nuclear DNA. However, during mitosis, the nuclear envelope breaks down and cytoplasmic proteins may enter what was formerly a privileged zone. To address the hypothesis that all APOBEC3 proteins have access to genomic DNA, we analyzed the localization of the APOBEC3 proteins during mitosis. We show that APOBEC3A, APOBEC3C and APOBEC3H are excluded from condensed chromosomes, but become cell-wide during telophase. However, APOBEC3B, APOBEC3D, APOBEC3F and APOBEC3G are excluded from chromatin throughout mitosis. After mitosis, APOBEC3B becomes nuclear, and APOBEC3D, APOBEC3F and APOBEC3G become cytoplasmic. Both structural motifs as well as size may be factors in regulating chromatin exclusion. Deaminase activity was not dependent on cell cycle phase. We also analyzed APOBEC3-induced cell cycle perturbations as a measure of each enzyme’s capacity to inflict genomic DNA damage. AID, APOBEC3A and APOBEC3B altered the cell cycle profile, and, unexpectedly, APOBEC3D also caused changes. We conclude that several APOBEC3 family members have access to the nuclear compartment and can impede the cell cycle, most likely through DNA deamination and the ensuing DNA damage response. Such genomic damage may contribute to carcinogenesis, as demonstrated by AID in B cell cancers and, recently, APOBEC3B in breast cancers.

APOBEC3B and AID Have Similar Nuclear Import Mechanisms

Members of the APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) protein family catalyze DNA cytosine deamination and underpin a variety of immune defenses. For instance, several family members, including APOBEC3B (A3B), elicit strong retrotransposon and retrovirus restriction activities. However, unlike the other proteins, A3B is the only family member with steady-state nuclear localization. Here, we show that A3B nuclear import is an active process requiring at least one amino acid (Val54) within an N-terminal motif analogous to the nuclear localization determinant of the antibody gene diversification enzyme AID (activation-induced cytosine deaminase). Mechanistic conservation with AID is further suggested by A3Bs capacity to interact with the same subset of importin proteins. Despite these mechanistic similarities, enforced A3B expression cannot substitute for AID-dependent antibody gene diversification by class switch recombination. Regulatory differences between A3B and AID are also visible during cell cycle progression. Our studies suggest that the present-day A3B enzyme retained the nuclear import mechanism of an ancestral AID protein during the expansion of the APOBEC3 locus in primates. Our studies also highlight the likelihood that, after nuclear import, specialized mechanisms exist to guide these enzymes to their respective physiological substrates and prevent gratuitous chromosomal DNA damage.

Endogenous APOBEC3A DNA Cytosine Deaminase Is Cytoplasmic and Nongenotoxic

Background: APOBEC3A is a potentially genotoxic DNA cytosine deaminase expressed in myeloid lineage cells. Results: Exogenous APOBEC3A genotoxicity correlates with expression level and nuclear localization. Endogenous APOBEC3A is nontoxic and cytoplasmic, despite its capacity to be highly induced by interferon. Conclusion: Cytoplasmic localization prevents endogenous APOBEC3A from accessing nuclear DNA. Significance: Endogenous APOBEC3A is not genotoxic. APOBEC3A (A3A) is a myeloid lineage-specific DNA cytosine deaminase with a role in innate immunity to foreign DNA. Previous studies have shown that heterologously expressed A3A is genotoxic, suggesting that monocytes may have a mechanism to regulate this enzyme. Indeed, we observed no significant cytotoxicity when interferon was used to induce the expression of endogenous A3A in CD14+-enriched primary cells or the monocytic cell line THP-1. In contrast, doxycycline-induced A3A in HEK293 cells caused major cytotoxicity at protein levels lower than those observed when CD14+ cells were stimulated with interferon. Immunofluorescent microscopy of interferon-stimulated CD14+ and THP-1 cells revealed that endogenous A3A is cytoplasmic, in stark contrast to stably or transiently transfected A3A, which has a cell-wide localization. A3A constructs engineered to be cytoplasmic are also nontoxic in HEK293 cells. These data combine to suggest that monocytic cells use a cytoplasmic retention mechanism to control A3A and avert genotoxicity during innate immune responses.

Abstract 505: Somatic and inherited riboSnitches inTPT1andLCP1mRNA secondary structures

RiboSnitches are single nucleotide variants that change the secondary structures of RNA. These structural changes can have a regulatory or functional impact on the RNA. Many inherited single nucleotide variants function as riboSNitches. We wanted to investigate the potential of somatic mutations to act as riboSNitches. We determined the secondary structure of two cancer-associated mRNAs, TPT1 and LCP1, using the chemical probing SHAPE-Map technique. Both of these mRNAs are highly structured and have similar folds in vivo and in vitro. We analyzed the structural impact of all the synonymous somatic mutations identified in the COSMIC cancer sequencing database for both TPT1 and LCP1 mRNAs. In TPT1 we also tested several inherited variants. We found that both somatic and inherited variants can be riboSNitches, at approximately the same ratio. Our results are in accord with previous work indicating that ~15% of SNVs are capable of acting as riboSNitches. Although the impact of non-coding and synonymous somatic mutations are difficult to measure, it is becoming clear that these mutations can contribute to cancer progression and phenotype. The majority of our identified riboSNitches are detrimental according to FATHMM-MKL functional impact prediction. Our experiments indicate that a substantial fraction of somatic mutations can cause a measurable change to RNA structure, potentially changing the regulatory or functional behavior of that mRNA. Citation Format: Lela L. Lackey, Aaztli Coria, Chanin Tolson, Evonne McArthur, Alain Laederach. Somatic and inherited riboSnitches in TPT1 and LCP1 mRNA secondary structures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 505. doi:10.1158/1538-7445.AM2017-505

Corrigendum: APOBEC3B is an enzymatic source of mutation in breast cancer

This corrects the article DOI: 10.1038/nature11881

116 Deamination of both methyl- and normal-cytosine by the foreign DNA restriction enzyme APOBEC3A

Multiple studies have indicated that the TET oxidases and, more controversially, the AID/APOBEC deaminases have the capacity to convert genomic DNA 5-methyl-cytosine (MeC) into altered nucleobases that provoke excision repair and culminate in the replacement of the original MeC with a normal cytosine (C). We show that human APOBEC3A (A3A) efficiently deaminates both MeC to thymine (T) and normal C to uracil (U) in single-stranded DNA substrates. In comparison, the related enzyme APOBEC3G (A3G) has undetectable MeC-to-T activity and 10-fold less C-to-U activity. Upon 100-fold induction of endogenous A3A by interferon, the MeC status of bulk chromosomal DNA is unaltered whereas both MeC and C nucleobases in transfected plasmid DNA substrates are highly susceptible to editing. Knockdown experiments show that endogenous A3A is the source of both of these cellular DNA deaminase activities. This is the first evidence for non-chromosomal DNA MeC-to-T editing in human cells. These biochemical and cellular data combine to suggest a model in which the expanded substrate versatility of A3A may be an evolutionary adaptation that occurred to fortify its innate immune function in foreign DNA clearance by myeloid lineage cell types.