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

Abstract

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.

Keywords: APOBEC3; DNA cytosine deamination; cancer; cell cycle; mitosis; subcellular localization; uracil.

Figure 1. A3A-E72A, A3C and A3H are excluded from DNA as the chromosomes condense but become cell-wide during telophase. (A) Images of HeLa cells in prophase expressing the indicated APOBEC3-eGFP constructs (top) merged with Hoechst stain to visualize the nuclei (merge, middle). Boxed regions (bottom) are magnified below each image with APOBEC3-eGFP exclusion from DNA indicated (white arrows). (B) Images of HeLa cells in telophase expressing indicated APOBEC3-eGFP constructs (top), merged with nuclear stain (middle) and magnified (bottom). All images are representative of at least three mitotic cells. See Figure S2 for APOBEC3-eGFP localization during metaphase and anaphase and Videos S1 and S2 for time-lapse images of A3A-E72A-mCherry localization during mitosis.

Figure 2. A3B, A3D, A3F and A3G are excluded from DNA during cell division. (A) Images of HeLa cells in prophase expressing the indicated APOBEC3-eGFP constructs (top). Cells were stained with Hoechst dye to identify the nuclei (merge, middle). Boxed regions (bottom) are blown up below each image with APOBEC3 exclusion indicated (white arrows). (B) Images of HeLa cells in telophase expressing indicated APOBEC3-eGFP constructs (top), merged with nuclear stain (middle) and magnified (bottom). All images are representative of at least three mitotic cells. See Figure S4 for APOBEC3-eGFP localization during metaphase and anaphase and Videos S3 and S4 for time-lapse images of A3F-mCherry localization during mitosis.

Figure 3. Untagged A3B and A3G are excluded from genomic DNA in the same manner as HA- and eGFP-tagged derivatives. (A) Images of HeLa cells transfected with untagged A3B labeled with anti-A3B and anti-rabbit FITC (top) then stained with Hoechst dye to illuminate the DNA (bottom). The indicated progression through telophase (early and late) was based on chromatin condensation. (B) Images of HeLa cells transfected with untagged A3G labeled with anti-A3G and anti-rabbit FITC (top) and DNA stain as before (bottom). Representative images are based on several telophase cells.

Figure 4. Localization of single domain variants of A3B, A3D, A3F and A3G. (A) Images of HeLa cells in telophase expressing N-terminal or (B) C-terminal halves of the indicated APOBEC3-eGFP constructs. DAPI and merged images below. Wild-type A3D-CTD was toxic, so a catalytic mutant was used (A3D-CTD-E264A; indicated by *). All images are representative of several telophase cells. (C) Representative images of HeLa cells in interphase expressing the N-terminal halves of the indicated APOBEC3-eGFP constructs.

Figure 5. Similar A3B deaminase activity in untreated, S-phase and mitotic cell lysates. (A) Cell cycle profiles of asynchronous cells (red), S-phase cells (blue) and mitotic cells (green). See methods for synchronization details. (B) Deaminase assay using lysates from cell populations shown in (A). (C) Immunoblot of protein lysates from (A) probed with anti-A3B or anti-tubulin antibodies.

Figure 6. APOBEC3 effects on cell cycle progression in HEK293T cells. (A) DNA flow cytometry profiles of HEK293T cells transfected with APOBEC3-eGFP constructs. Representative profiles of triplicate independent PI-staining experiments are shown at 48 h. APOBEC3-eGFP expressing cell profiles (red) are overlaid on untransfected cells in the same population (blue). The shift toward S-phase in A3A-eGFP expressing cells is indicated (black arrow). (B) Expression of the indicated APOBEC3-eGFP proteins in HEK293T cells as shown by flow cytometry (top) and immunoblotting (bottom).