The Cytidine Deaminase APOBEC3 Family Is Subject to Transcriptional Regulation by P53 Daniel Menendez, Thuy-Ai Nguyen, Joyce Snipe, and Michael A

Published OnlineFirst February 23, 2017; DOI: 10.1158/1541-7786.MCR-17-0019

Oncogenes and Tumor Suppressors Molecular Cancer Research The Cytidine Deaminase APOBEC3 Family Is Subject to Transcriptional Regulation by p53 Daniel Menendez, Thuy-Ai Nguyen, Joyce Snipe, and Michael A. Resnick

Abstract

The APOBEC3 (A3) family of proteins are DNA cytidine dea- Interestingly, overexpression of a group of tumor-associated p53 minases that act as sentinels in the innate immune response against mutants in TP53-null cancer cells promoted A3B expression. These retroviral infections and are responsive to IFN. Recently, a few A3 findings establish that the "guardian of the genome" role ascribed genes were identified as potent enzymatic sources of mutations in to p53 also extends to a unique component of the immune system, several human cancers. Using human cancer cells and lympho- the A3 genes, thereby integrating human immune and chromo- cytes, we show that under stress conditions and immune chal- somal stress responses into an A3/p53 immune axis. lenges, all A3 genes are direct transcriptional targets of the tumor suppressor p53. Although the expression of most A3 genes (includ- Implications: Activated p53 can integrate chromosomal stresses ing A3C and A3H) was stimulated by the activation of p53, and immune responses through its influence on expression of treatment with the DNA-damaging agent doxorubicin or the APOBEC3 genes, which are key components of the innate p53 stabilizer Nutlin led to repression of the A3B gene. Further- immune system that also influence genomic stability. Mol Cancer more, p53 could enhance IFN type-I induction of A3 genes. Res; 1–9. 2017 AACR.

Introduction new p53-regulated target genes. From our p53 cistrome studies in human cancer (3) and human primary lymphocytes (unpub- The well-known tumor suppressor p53 is a transcriptional lished data), we have identified new p53 targets involved in master regulator that regulates the expression of genes associated immune response signaling. Included are several members of the with a wide range of functions, including cell-cycle arrest, apo- innate immune gene family APOBEC3 (A3; apolipoprotein B ptosis, and senescence in response to genotoxic and nongenotoxic mRNA editing enzyme, catalytic polypeptide-like 3). stresses that challenge cellular genomic integrity (1). Following The A3 genes, which consist of seven highly related DNA various stresses, the activated p53 protein is accumulated in the cytidine deaminases that are tandemly distributed on human nucleus, where it binds to DNA as a tetramer at p53 response chromosome 22, catalyze the deamination of cytidine to uracil elements (p53RE; ref. 1). (12). The A3 genes are a key component of the innate immune The commonly held view of p53 as a "guardian of the system in vertebrates that inhibit replication of a variety of retro- genome" has expanded greatly during the past few years to viruses, endogenous retroelements, and DNA viruses (13, 14). cover many biological processes (1–3), including the role of Recently, the C-to-T hypermutagenesis genomic changes in mul- p53 in modulating the human immune system and antiviral tiple cancers have been attributed to A3A and A3B (15–17). defense (4–7). Previously, we found that activation of p53 by The A3 genes are generally viewed as constitutively expressed in common antitumor agents in human primary and cancer cell immune-related cells as well as in immune-associated cancer cells. lines directly alters the expression of several members of the In the context of innate immune responses to infection, the innate immune Toll-like receptor (TLR) gene family, which is expression of several A3 genes is modulated by IFNs that may involved in host defense against invading pathogens (8, 9). be cell type and IFN-type dependent (18, 19). However, little is This results in modulation of TLR downstream responses to known about the transcription factors involved in the expression cognate ligands (10, 11). of the A3 genes in response to immune challenges and other p53 chromatin immunoprecipitation (ChIP-seq) approaches environmental stressors. in combination with transcriptome analysis have revealed many Here, we describe a new role for p53: regulation of the A3 gene family in response to common anticancer drugs and direct activation of p53. Using both a panel of human cancer cell lines as Genome Integrity and Structural Biology Laboratory, National Institute of well as primary immune cells obtained directly from human Environmental Health Sciences, NIH, Research Triangle Park, North Carolina. subjects, we discovered that most A3 genes are transcriptionally Note: Supplementary data for this article are available at Molecular Cancer responsive to p53. Furthermore, cancer-associated p53 mutants Research Online (http://mcr.aacrjournals.org/). can dramatically impact the pattern of expression of the A3 genes, Corresponding Author: Daniel Menendez, National Institute of Environmental including novel responses of the cancer hypermutator A3B gene. Health Sciences, 111 TW Alexander Drive, MD-D3-01, Building 101, Research In addition, p53 can participate and influence the IFN-induced Triangle Park, NC 27709. Phone: 919-541-0972; Fax: 919-541-7593; E-mail: transcriptional responses of several A3 family members. Overall, [email protected] we provide the first evidence that p53 is a key node linking DNA doi: 10.1158/1541-7786.MCR-17-0019 damage, immune-induced responses, and A3 gene expression in 2017 American Association for Cancer Research. human cells.

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Materials and Methods The A3B gene differed in that both drug treatments repressed it. In addition to A3A and A3D, we confirmed upregulation of A3C and Cell lines and treatments A3H as well as the p53 target gene CDKN1A (p21) used as a Human cancer cell lines were cultured in RPMI1640, McCoy's positive control. We also observed that doxorubicin, but not 5A, or DMEM supplemented with 10% of FBS and 100 U/mL Nutlin, increased A3G mRNA. Except for repression of A3B, a penicillin/streptomycin as described elsewhere. More informa- similar pattern of expression of A3 genes was observed in A549 tion about the cancer and primary human cells is provided in the lung cancer cells (Supplementary Fig. S1A). The expression of A3A Supplementary Material. was not detected in A549 cells. Overall, we establish that the expression of A3 genes can be regulated in response to genotoxic RNA isolation and qPCR stress and p53 activation. fi Total RNA was isolated with RNeasy Kits (Qiagen). Quanti - Given that the induction of innate immune genes is relevant cation and purity of the samples were determined using Nano- in disease and cancer treatments and to determine whether the fi m Drop spectrophotometer (Thermo Fisher Scienti c), and 1 g total impact of Nutlin and doxorubicin on A3 genes was general RNA was reverse transcribed using Transcriptor reverse transcrip- rather than cell type specific, we expanded the analysis for A3 tase with random hexameric primers (Roche) following the man- responses to DNA-damaging agents and p53 activation in a ufacturer's recommendations. qPCR was performed following panel of cell lines harboring wild-type (WT), null, and mutated established procedures, primers, and Universal Primary Library p53 (Supplementary Table S2). Presented as heatmaps in Fig. System probes as described previously (19) using 7000 ABI 1B (Nutlin) and C (doxorubicin) are the mRNA fold changes sequence Detection System (Applied Biosystems). All reactions for each A3 gene and cell line (see Supplementary Table S3 for fi were done in triplicate, and relative quanti cation values were raw data). Expression of A3A was mainly observed in cell lines DDCt calculated on the basis of the 2 method using expression of immune origin (LCL35, GM12878, THP1, Jurkat, and RAJI). TBP from the housekeeping gene Tata-Binding Protein ( ). The expression of the A3 genes was clearly induced by drug Primers and probes are described in Supplementary Table treatments only in WT p53 cells, where there was consistent S6. Additional material and methods can be found in the induction of A3C and A3H as well as p21 among the cell lines. Supplementary Material. Cell type and agent-specific effects in mRNA changes were observed for A3A, A3D, A3F,andA3G. As observed in U2OS Statistical analysis and A549, A3B was repressed in all WT p53 cell lines exam- Analysis was performed using GraphPad Prism statistical soft- ined in response to Nutlin and doxorubicin. We also found ware. Data are represented as mean SDs from at least three that other chromosomal stressors, such as etoposide and separate experiments. Two-tailed Student t test was applied ionizing radiation, altered the A3 genes expression profile in for comparisons of two groups. P values <0.05 were considered various cancer cell lines in a p53-dependent manner (Sup- significant. plementary Fig. S1B and S1C). Although there are dramatic differences in the expression spectra for some A3 genes between cell lines, we establish that many genes of the A3 Results family are responsive to genotoxic stress and p53 activation in Chromosomal stress and p53 activation alters A3 gene family human cancer cells. expression As most of the A3 genes are expressed in immune cells, we Recently, in our p53 ChIP-seq study that included associated examined their response to p53 induction in human primary gene expression analysis in osteosarcoma U2OS cells following lymphocytes. Relative levels of mRNA expression were de- p53 activation by Nutlin-3 (Nutlin) or doxorubicin (DXR), we termined in phytohemagglutinin-stimulated lymphocytes identified several new p53 target genes related to immune obtained from peripheral blood mononuclear cells freshly iso- responses (3). Binding of p53 to the regulatory regions of the lated from 11 healthy human volunteers. Despite variability A3C and A3H members of the A3 gene family was associated with among subjects, the overall A3 gene family expression profiles increased transcription based on TaqMan analysis. Given that induced by Nutlin and doxorubicin were like those in WT p53 homology is high among the A3 family sequences (20), we cancer cell lines, as described in Supplementary Fig. S2A and S2B. hypothesized that p53 responsiveness may have been conserved The expression of the internal controls p21 and Mdm2 were also among other members of the A3 family. induced in all donors after Nutlin or doxorubicin treatments. In Because of the redundancy in the regulatory and coding regions agreement with previous reports (18, 19), A3A expression was for A3 genes, there have been inconsistent conclusions about the undetectable in T lymphocytes. A3B mRNA was not detected in transcriptional regulation of the A3 genes as well as the quanti- all donors, but in those that it was observed (8/11), both fication of A3 expression by common approaches (microarray and treatments induced repression of this gene. Pretreatment of qPCR; ref. 19). To validate our previous findings and explore the lymphocytes from 4 volunteers with the p53 inhibitor pifi- extent to which p53 regulates the expression of other A3 members, thrin-a (21) prior to Nutlin or doxorubicin strongly altered we measured expression of all A3 genes in U2OS cells following expression, identifying a direct role for p53 in regulating Nutlin- exposure to Nutlin and doxorubicin using the Universal Primary and doxorubicin-induced A3 gene expression (Supplementary Library System technology [this technology, which is more robust Fig. S2C and S2D). than the general SYBR Green primers or Applied Biosystems TaqMan primers/probes assays for evaluating A3 mRNA changes p53 directly modulates A3 gene family expression (19), allows highly related sequences to be distinguished]. Acti- in human cells vation of p53 by these agents led to time-dependent upregulation A direct comparison between the A3 expression profiles þ of several A3 genes (from 2.5- to 13-fold), as shown in Fig. 1A. for HCT116 p53 and its isogenic version lacking p53

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Figure 1. Induced expression of the A3 gene family by DNA stress and activation of the p53 pathway in human cancer cell lines. A, Expression of A3 genes in U2OS cells treated for 24 hours with DMSO (vehicle 0.1%), the p53-activating drug Nutlin (10 mmol/L) and doxorubicin (DXR, 1.5 mmol/L). Changes in gene expression presented as fold change compared with untreated cells (value of 1) were analyzed by real-time qPCR. p21 expression was a positive control for a p53 transcriptional target. , P < 0.05 compared with untreated cells. B and C, Nutlin (B) or doxorubicin (C) heatmaps for expression of A3 genes after 24-hour treatment in human cancer cell lines that are p53 proﬁcientordeﬁcient. The heatmap fold change values compared with untreated cells and statistical analysis are available in Supplementary Table S3.

(HCT116 p53 ) revealed that most of the changes in A3 genes reduced in all p53-depleted cell lines regardless of tissue origin, were dependent on p53 (Fig. 1B and C). Few significant changes in confirming that these are p53 targets. The A3B-induced repres- expression were observed for the A3 genes after Nutlin or doxo- sion was prevented by silencing of p53 expression. rubicin in the null and mutant p53 cell lines. When changes were To investigate further a direct connection between p53 and detected in the p53-mutant cell lines, they were typically opposite expression of A3 genes, WT p53 was overexpressed in the p53-null to the changes observed in WT p53 cell lines. An example is cancer cell lines SaOS2, HCT116 p53 , and H1299. Although cell A3C, which was repressed in p53-mutant cells but significantly type differences were observed, the functional restoration of WT induced in WT p53 cells. However, the most dramatic example p53 expression resulted in approximately 2.5- to 14-fold upre- was found for the A3B response to doxorubicin in that it was gulation of several A3 genes along with p21 mRNA used as a upregulated in several mutant cell lines but repressed in WT positive control (Fig. 2D). The A3F gene was the only family p53 cells. The p53-dependent gene expression changes in A3 member that was not modified in any of the cell lines transfected. genes were addressed by expressing RNAi p53 from a cassette There was a lack of basal A3A or A3H expression in SaOS2 cells that was integrated in MCF7 (22) or in U2OS (3), or using a that appeared to be due to the absence of p53 as transfection of lentiviral vector harboring short hairpin RNAs that target TP53 WT p53 resulted in expression of both genes (Fig. 1B and C). mRNA (termed p53sh-3755 and p53sh-3756) in A549 and Consistent with our previous observations, the restoration of LCL35 cells (Fig. 2; Supplementary Fig. S3). As shown in Fig. functional p53 resulted in a significant inhibition of A3B gene 2A–C and Supplementary Fig. S3, the upregulation observed expression in both transfected cell lines. Overall, these results for A3 genes following doxorubicin or Nutlin exposure in confirm the direct participation of p53 in the regulation of A3 gene control cells (parental or scrambled) was completely lost or family members.

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Figure 2. p53 regulates expression of A3 genes in human cancer cells. A–C, Changes in A3 mRNA levels were evaluated after 24-hour treatment with Nutlin (10 mmol/L) or doxorubicin (DXR; 1.5 mmol/L) in U2OS (A), A549 (B), and MCF7 cells (C) with stably expressing scrambled shRNA, vector, or p53 shRNAi (p53sh-3756 or p53i). Values are displayed as fold changes (log2 scale) relative to their respective parental cell lines (value set to 1). D, p53-null SaOS2, HCT116 p53-, and H1299 cells were transfected either with empty vector or WT p53. A3 mRNA expression levels following transfection were examined by qPCR; p21 expression was a positive control. , P < 0.05 relative to untransfected cells (i.e., value of 1). ND, not detected. Calculations in SAOS2 cells for A3A and A3H after WT p53 transfection are approximations. See Supplementary Material for explanation.

Activated p53 binds transcriptional regulatory regions of Overall, these results support a broad role for p53 in the A3 A3 genes gene family regulation and suggest that the p53-binding The p53-induced transcription of several A3 genes led us regions containing the putative p53REs may regulate the to investigate public p53 ChIP-seq datasets (summarized in expression of A3 genes. The top predicted functional p53REs Supplementary Table S4) for the impact of p53 across a broad are shown in Supplementary Table S1. A complete list of range of cell types and exposures. Included are the responses of p53REs associated with A3 genes is presented in Supplementary normal diploid fibroblasts, embryonic stem cells, lymphoblasts Table S5. We note that for some of the putative p53REs, the as well as breast, colon and bone cancer cells. As shown in Fig. sequences were 95% to 100% conserved between several A3 3A, p53 can bind to the transcriptional regulatory regions of genes, as found for p53REs of the A3D, A3F,andA3G genes, most of the A3 genes, A3A, A3B, A3C, and A3H genes, in which is consistent with the evolutionary duplication of the A3 response to the following stress conditions that activate p53: genes (20). Although potential p53REs in A3B, A3D, A3F,and doxorubicin, cisplatin, 5-FU, actinomycin-D, ionizing radia- A3G were identified around their TSS, none exhibited binding tion, Nutlin, and RITA. To determine whether A3 genes are in reported p53 ChIP-seq studies. However, for the case of A3B, direct transcriptional targets of p53, we screened for potential an intronic region containing a p53RE was bound by p53. The p53REs in the promoter and regulatory regions of each A3 gene, p53RE associated with A3F was previously reported as a p53- from 5toþ1.7 kb flanking the transcription start site (TSS) as bound region in a ChIP-PET assay (25). As a lack of binding in wellastheregionswhereChIP-seq p53-binding peaks were previous reports might be due to relatedness of the A3 genes, observed. Using established guidelines for transcriptional we investigated p53 binding to specific sites using a ChIP-PCR functionality of potential p53REs (23) and the p53Scan soft- analysis that targets each of the putative p53REs (due to high ware (24), we identified several potential transactivation- sequence conservation, no reliable primers were identified responsive p53REs located in the transcriptional regulatory to analyze some of the top predicted functional p53REs for regions of all A3 genes (Fig. 3A). A3D, A3F,andA3G). With this approach, we confirmed that

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Potential p53REs

Figure 3. Activated p53 binds transcriptional regulatory region of A3 genes. A, Binding peak analysis of public p53 ChIP-seq datasets for human primary and cancer cells treated with DNA- damaging agents and p53-activating drugs. Depicted is the region of human chromosome 22 containing the seven A3 genes. Red bars, relative position of potential p53REs identiﬁed by p53Scan software (24); blue bars, p53-binding regions identiﬁed in the B public p53 ChIP-seq studies (see 1.6 Supplementary Table S4). Validation p53 U2OS 1.2 of p53 binding to transcriptional IgG 0.8 regulatory regions of A3 genes containing putative p5REs in Nutlin (10 mmol/L) or doxorubicin (DXR; 1.5 0.4 0.3 mmol/L) is described in U2OS (B) and % Input 0.2 A549 cells (C). p53 occupancy, 0.1 determined by ChIP-PCR, is presented 0.0 as the percentage of total input DNA. Binding to the p21 promoter is NT NT NT NT NT NT NT DXR DXR DXR DXR DXR DXR DXR used as a positive control. The DMSONutlin DMSONutlin DMSONutlin DMSONutlin DMSONutlin DMSONutlin DMSONutlin A3 superscript in genes corresponds to a b b c d a p53RE sequences described in A3A A3A A3B A3C A3F A3G A3H Supplementary Table S1. IgG serves as a negative control. Presented are means and SDs from three C P < 1.0 independent experiments. , 0.05 p53 A549 when compared with no treatment 0.8 IgG ("NT") samples. 0.6 0.4

% Input 0.2 0.0

NT NT NT NT NT DXR DXR DXR DXR DXR DMSONutlin DMSONutlin DMSONutlin DMSONutlin DMSONutlin c d a A3Bb A3C A3F A3G A3H activated p53 can directly bind to the p53RE sequences iden- Tumor-associated p53 mutants can promote A3B expression tiﬁed in silico and/or previously found to bind in vivo for several As described above, all the A3 genes were upregulated in WT A3 genes after Nutlin or doxorubicin treatment in U2OS and p53 cancer cell lines in response to p53 activation except A3B, A459 cells, as described in Fig. 3B and C and Supplementary which was repressed. However, in cell lines with mutant p53 Fig. S4. Binding of p53 to the promoter region of its target gene alleles, A3B was generally upregulated. As shown in Fig. 4A, p21 was used as positive control, while a random region of the transient expression in SaOS2 p53-null cells of the cancer-asso- GAPDH promoter was used as negative control (Supplementary ciated, hotspot p53 mutants R175H, G245S, R248Q, R273H, and Fig. S4). Collectively, these results demonstrate that most R280K resulted in upregulation of the A3B gene with little effect p53RE sites considered as associated with p53-induced expres- on expression of A3C, A3H, or the internal control p21. The results sion of the A3 family members can be directly bound by p53 were similar when the same p53 mutants were transiently over- and strongly indicate that in response to chromosomal stresses, expressed in HCT116 p53 cells, conﬁrming that mutant p53 p53 is a major transcription factor regulating the expression of induces the expression of A3B in human cancer cells (Fig. 4B). A3 genes. Interestingly, change-of-spectrum mutants A138Y, H178Y, and

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Figure 4. Expression of mutant p53 in cancer cells leads to A3B upregulation. A and B, p53-null SaOS2 (A)and HCT116 cells (B) were transiently transfected with empty vector or p53 expression plasmids for WT or tumor- associated mutants. After 24 hours, mRNA levels for A3B were quantiﬁed by qRT-PCR. Expression of A3C, A3H, and p21 were used as internal controls for genes upregulated by WT but not mutant p53. Presented are mRNA fold changes relative to untransfected cells. For presentation purposes, the y-axis corresponding to mRNA fold changes is presented in a log2 scale format. , P < 0.05 compared with expression induced by WT p53 transfection. *, P < 0.05 compared with cells transfected with empty vector.

R337H (10, 26) that retain different levels of WT p53 transactiva- sion and p53 occupancy of several A3 genes was also observed in tion change the expression profiles of A3 genes, including A3B. A549 cells (Supplementary Fig. S5B and S5C). Unlike for U2OS Altogether, these results imply that tumor-associated p53 mutants cells, the expression of the A3B gene in the A549 cells was initially can influence the expression of several A3 genes, especially upregulated after IFN-I at early time points (3 and 6 hours) when increasing the A3B mRNA levels. p53 was not bound in the A3B transcriptional regulatory region. Notably, A3B mRNA levels were returned to basal levels after 24 p53 modulates IFN-induced expression of A3 genes hours, while p53 occupancy increased overall, consistent with a in cancer cells p53-repressive activity on this gene. Consistent with its antiviral roles, several A3 genes are upre- Theroleofp53inmodulatingIFN-I–induced expression of gulated by various IFNs (18, 19). We therefore assessed whether A3 genes was further confirmed in SaOS2 p53 null cells tran- p53 can influence the expression of A3 genes in response to IFN siently transfected with WT p53. Restoration of WT p53 type-I (IFN-I) in various cancer cell lines that differed in TP53 enhanced the IFN-I–inducedgeneexpressionofmostofthe status. A3 genes except for A3C (Supplementary Fig. S5D). There is a As shown in the heatmap of Fig. 5A, all A3 genes were modestly difference with overexpressed p53 compared with nontrans- upregulated after 24 hours of IFN-I treatment in most cell lines fected cells, in that IFN-I treatment in parental cells failed to regardless of p53 functional status, except for the substantial induce the A3A and A3H genes over the undetectable basal increase in A3G. To address whether p53 directly affects IFN-I– levels, while in p53 transfected cells, the mRNA levels of both mediated gene expression of A3 genes, U2OS cells with genes along with other A3 genes were synergistically increased, p53shRNAi or scramble shRNAi were treated with IFN-I. The except for A3B. Consistent with our other observations, A3B IFN-I treatment stimulated the expression of all A3 genes (Fig. was repressed after p53 expression. Although IFN-I had no 5B), except A3B and A3C (Supplementary Fig. S5A) in a time- effect on its own, the IFN-I abolished the p53 induced repres- dependent manner in U2OS scramble cells. Consistent with sion of A3B, returning its expression to basal levels. Collective- previous findings (27), there was a modest approximately 3-fold ly, our results demonstrate a role for p53 in IFN-mediated increase in TP53 mRNA levels after 24 hours of IFN-I treatment expression of A3 gene family members. (Fig. 5) in the scramble cells. Although the absence of p53 due to p53shRNAi does not seem to have any impact on the early upregulation of the A3 genes by IFN-I challenge (1 to 6 hours), Discussion the presence of p53 was clearly required to maintain the upre- The A3 cytosine deaminases are key innate immune effector gulation of the A3A, -D, -F, -H, and -G genes at 24 hours (Fig. 5B). proteins providing defense against a range of viruses and retro- Correspondingly, the protein levels of total p53 and phosphor- elements through their ability to mutagenize viral DNA, restrict ylated Ser15 p53, a marker for activation, were increased (Fig. 5C) viral replication, and silence retrotransposition (28, 29). As the A3 at 24 hours. proteins can deaminate cellular DNA and contribute to genomic Similarly, p53 occupancy at the p53REs for A3A and A3G was instability, a fine-tuned control of expression of the A3 genes and greatly increased only at 18 hours of IFN-I treatment (Fig. 5D), enzymatic activities is needed to avoid collateral damage to the suggesting that activated p53 is needed for IFN-I regulation of A3 host genome. However, little was known about the transcriptional genes at late times. The impact of p53 on IFN-I–induced expres- control of the A3 genes at basal or stress conditions beyond

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Figure 5. p53 modulates IFN-induced expression of A3 genes. A, IFN-I–induced expression of A3 genes in human cancer cell lines with different p53 functional status. The heatmap shows changes in A3 mRNA levels determined by qPCR after IFN-I treatment (500 U, 24 hour). Fold change values compared with untreated cells (value of 1) and statistical analyses are available in Supplementary Table S3. B, TP53 depletion inﬂuences IFN-induced responses of the A3 gene family expression. Expression of A3 genes, presented as fold change relative to untreated parental cells, was evaluated in parental, scramble, and TP53 shRNA U2OS cells after being challenged with 500 U IFN-I during the indicated times. C, Immunoblot of total p53 and phosphorylated Ser15 p53, used as an activation maker in U2OS cells treated with IFN-I (500 U) for 6 or 24 hours. The lysate from doxorubicin (DXR)-treated U2OS was used as positive control. Actin was used as loading control. D, p53 occupancy for A3A and A3G transcriptional regulatory regions in U2OS cells treated for 6 or 18 hours with 500 U of IFN-I. The A3 gene superscripts correspond to p53RE sequences described in Supplementary Table S1. p53 binding is expressed as the percentage of total input DNA. IgG was a negative control. , P < 0.05 when compared with parental no treatment ("NT") samples.

regulation by type-I IFN stimulation, estrogen, and recently by We also found that p53 can influence expression of the A3 gene replication stress (18, 19, 30, 31). family in response to IFN related immune challenges, suggesting Here, we show that p53 can be a major transcriptional regulator p53 may act as a central mediator of global innate immune of all A3 genes in response to chromosomal stress. Consistent with responses. IFNs are the major cytokines produced by the innate these observations, depletion of p53, inhibition by pifithrin-a, immune system in response to viral infections, and several viral and expression of loss-of-function tumor-associated p53 mutants infections trigger p53 (32). Included in the IFN-stimulated group alters A3 expression. Also, all A3 genes had p53 binding near TSSs, of genes (33) are TP53 (27) and A3 genes. (18, 19, 27). p53 was many of which were bound by activated p53. We observed that found to only influence late IFN-mediated transcriptional although p53 binding to these novel p53REs was relatively lower responses of the A3 genes. We suggest that p53 can enhance the when compared with the binding of p53 to the p21 target gene, the antiviral arm of the innate immune response via the upregulation binding of p53 in the A3 regulatory regions was consistent in two of A3 genes as well in consort with the antiviral response itself. cell lines observed, not only following activation with the classical On the basis of our observations, we propose that activated p53 activator drugs doxorubicin and Nutlin but also after IFN-I p53 can serve as an integrator of DNA damage and immune treatment. Furthermore, we found that numerous CHIP-seq data- responses. This is consistent with the recent report in which sets across cell lines, exposures, and tissue types provide further drug-induced replication stress can promote A3 activation (31). evidence for direct regulation of the A3 family by p53. Although The role of p53 was not assessed in this work; however, we attempted to relate mRNA to protein changes with commer- replication stress typically involves p53 activation (31). We cially available antibodies, results for A3 proteins were inconsis- liken the action of p53 to a two-edged sword that has the tent, presumably due to the polyclonal nature of the antibodies potential to both increase genome stability through actions on used and/or multiple nonspecific signals. However, a strong viruses and retrotransposons or to decrease genome stability correlation has been reported for A3F and A3G mRNA and protein through mutagenesis of chromosomal DNA by the A3 deam- expression using noncommercial antibodies (19). inase activity. Through increased expression of A3 genes, p53

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might guard against retrotransposition of large repeats that are sion (particularly A3A and A3B) and subsequent appearance of active in the genome (34–37). Wylie and colleagues (37) mutations that have been identified in cancers. þ demonstrated that WT p53 through its interaction with com- We propose that after cancer establishment, the p53 or p53 ponents of the piRNA (piwi-interacting RNA) pathway sup- status along with cancer therapy may influence the potential for pressed transposon mobility in normal cells, while mutant p53 subsequent A3A and A3B mutagenesis as part of tumor evolution in cancer cells could not, resulting in the activation of LINE and metastasis. Most cancer therapies involve agents such as mobility in cancer cells. This is consistent with depletion of ionizing radiation, doxorubicin, and etoposide that induce WT A3C leading to greater LINE retrotransposition activity in a p53. As described, mutant and WT p53 proteins can strongly cancer cell line (38). influence expression of A3A and A3B as well as other A3 genes. Several studies have shown a correlation between aberrant Even the lack of p53 can lead to upregulation of A3B. Given that expression of A3B mRNA in multiple tumors and a specific more than 50% of cancers (some >90%) have altered p53, we mutation signature (15, 17, 39). We have established that WT suggest that specific p53 mutation status should be considered in p53 represses A3B expression while p53 hotspot tumor-asso- treatments. Along this line, it would be interesting to address p53 ciated mutants can lead to upregulation of A3B.Thesep53 status and mutagenesis patterns in secondary tumors as well as hotspot mutants have lost the wild-type ability to interact with cancers that arise in association with Li–Fraumeni disease (50), p53RE sequences, opening the possibility that upregulation of which is due to a germinal defect in TP53. A3B induced by p53 mutants could be through gain-of-func- Overall, our results provide molecular insights into p53 tion activities that enhance transactivation activities of other direct control of immunosurveillance under stress conditions, transcription factors, such as NF-kB (40). Consistent with this integrating p53 tumor suppressor activities with innate immune hypothesis, Maruyama and colleagues recently found that the stimuli. Further studies are needed to establish the functional classical NF-kB pathway is responsible for activation of A3B impact of A3 transcriptional regulation by p53. For example, mRNA in cancer cells (41). under conditions of viral infection how does the p53–A3 axis A recent study found that A3B mRNA expression and enzy- impact the host immune function and the likelihood of muta- matic activity were upregulated following transfection of a tion in the host and the pathogen. The integration of immune high-risk HPV genome. This effect was abrogated by inactiva- response and DDR may have implications for understanding tion of viral E6, a protein that causes degradation of p53 (42). therapeutic approaches. Understanding the relationship could Notably, in breast tumor samples and derived cancer cell lines provide novel ways to incorporate cell-extrinsic and cell-intrin- A3B upregulation correlates with inactivation of TP53,strongly sic defenses against tumorigenesis by pharmacologic activation suggesting that p53 loss and increased A3B could be a tumor- of p53, thereby achieving more effective therapies against initiating event allowing cells to bypass DNA damage check- cancer and other diseases. points triggered by A3B (15). Recently, the A3A protein was found to be a mutator in human Disclosure of Potential Conflicts of Interest cancers (16, 17), as there is a stronger A3A mutagenesis signature No potential conflicts of interest were disclosed. than that associated with A3B (16). Furthermore, A3A itself can generate DNA breaks and activate the DNA damage response Authors' Contributions (DDR; ref. 43, 44) for which p53 is a major downstream regulator Conception and design: D. Menendez, M.A. Resnick and effector (45). In our study, both WT and p53 mutant could Development of methodology: D. Menendez, J. Snipe, M.A. Resnick upregulate A3A in response to DNA damage. Moreover, as chronic Acquisition of data (provided animals, acquired and managed patients, inflammation is commonly related to cancer and as A3A expres- provided facilities, etc.): T.-A. Nguyen sion correlates with inflammatory environments (such as IFN-I), Analysis and interpretation of data (e.g., statistical analysis, biostatistics, A3A-induced damage may contribute to somatic mutation selec- computational analysis): D. Menendez, T.-A. Nguyen tion in cancers (46). Both A3A and A3B are induced in bladder and Writing, review, and/or revision of the manuscript: D. Menendez, T.-A. Nguyen, M.A. Resnick breast cancer cells after bleomycin treatment (47). Consistent Administrative, technical, or material support (i.e., reporting or organizing with our results, the induction of A3B is more robust in cell lines data, constructing databases): D. Menendez, T.-A. Nguyen, J. Snipe, harboring mutant p53 than those with WT p53 (47). M.A. Resnick Among the seven A3 genes, both A3C and A3H were consis- Study supervision: D. Menendez, M.A. Resnick tently upregulated in a p53-dependent manner. However, beyond their immune sentinel functions (19), there is little information Acknowledgments about potential mutagenicity toward host genomes. Elevated The authors thank the following NIEHS core facilities: Molecular Genomics, expression of A3C along with lowered expression of A3B in breast Viral Vector, Flow Cytometry, and Clinical Research Unit. We thank Drs. Carl cancer patients correlate with improved clinical outcome (48), Anderson, Stavros Garantziotis, Dmitry Gordenin, and Shepherd Schurman for consistent with our results where p53 activation promotes expres- critical reviewing and comments. sion of A3C and represses expression of A3B . A role for A3H in A3 cancer-associated mutagenesis has been indicated by an A3H-I Grant Support haplotype and associated A3 mutation pattern in breast tumors This work was supported by the Intramural Research Program of the NIH lacking A3B expression (49). (NIEHS Z01-ES065079; to M.A. Resnick). The costs of publication of this article were defrayed in part by the payment of Thus, it appears that p53 regulation of the A3 genes can have page charges. This article must therefore be hereby marked advertisement in both antitumor and protumor consequences. Our results suggest accordance with 18 U.S.C. Section 1734 solely to indicate this fact. an A3/p53 immune axis where activation of WT p53 in normal cells in response to internally and externally induced lesions Received January 12, 2017; revised December 14, 2016; accepted February 15, (including replication collapse) could also influence A3 expres- 2017; published OnlineFirst February 23, 2017.

OF8 Mol Cancer Res; 2017 Molecular Cancer Research

p53-Dependent Transcriptional Regulation of APOBEC3 Genes

References 1. Aubrey BJ, Strasser A, Kelly GL. Tumor-suppressor functions of the TP53 26. Menendez D, Inga A, Resnick MA. Estrogen receptor acting in cis enhances pathway. Cold Spring Harb Perspect Med 2016;6:pii:a026062. WT and mutant p53 transactivation at canonical and noncanonical p53 2. Allen MA, Andrysik Z, Dengler VL, Mellert HS, Guarnieri A, Freeman JA, target sequences. Proc Natl Acad Sci U S A 2010;107:1500–5. et al. Global analysis of p53-regulated transcription identifies its direct 27. Takaoka A, Hayakawa S, Yanai H, Stoiber D, Negishi H, Kikuchi H, et al. targets and unexpected regulatory mechanisms. Elife 2014;3:e02200. Integration of interferon-alpha/beta signalling to p53 responses in tumour 3. Menendez D, Nguyen TA, Freudenberg JM, Mathew VJ, Anderson CW, Jothi suppression and antiviral defence. Nature 2003;424:516–23. R, et al. Diverse stresses dramatically alter genome-wide p53 binding and 28. Kinomoto M, Kanno T, Shimura M, Ishizaka Y, Kojima A, Kurata T, et al. All transactivation landscape in human cancer cells. Nucleic Acids Res APOBEC3 family proteins differentially inhibit LINE-1 retrotransposition. 2013;41:7286–301. Nucleic Acids Res 2007;35:2955–64. 4. Bieging KT, Mello SS, Attardi LD. Unravelling mechanisms of p53-medi- 29. Koito A, Ikeda T. Intrinsic restriction activity by AID/APOBEC family of ated tumour suppression. Nat Rev Cancer 2014;14:359–70. enzymes against the mobility of retroelements. Mob Genet Elements 5. Martins CP, Brown-Swigart L, Evan GI. Modeling the therapeutic efficacy of 2011;1:197–202. p53 restoration in tumors. Cell 2006;127:1323–34. 30. Pauklin S, Sernandez IV, Bachmann G, Ramiro AR, Petersen-Mahrt SK. 6. Ventura A, Kirsch DG, McLaughlin ME, Tuveson DA, Grimm J, Lintault L, Estrogen directly activates AID transcription and function. J Exp Med et al. Restoration of p53 function leads to tumour regression in vivo. Nature 2009;206:99–111. 2007;445:661–5. 31. Kanu N, Cerone MA, Goh G, Zalmas LP, Bartkova J, Dietzen M, et al. DNA 7. Munoz-Fontela C, Macip S, Martinez-Sobrido L, Brown L, Ashour J, Garcia- replication stress mediates APOBEC3 family mutagenesis in breast cancer. Sastre A, et al. Transcriptional role of p53 in interferon-mediated antiviral Genome Biol 2016;17:185. immunity. J Exp Med 2008;205:1929–38. 32. Sato Y, Tsurumi T. Genome guardian p53 and viral infections. Rev Med 8. Menendez D, Shatz M, Azzam K, Garantziotis S, Fessler MB, Resnick MA. Virol 2013;23:213–20. The Toll-like receptor gene family is integrated into human DNA damage 33. Schoggins JW, Rice CM. Interferon-stimulated genes and their antiviral and p53 networks. PLoS Genet 2011;7:e1001360. effector functions. Curr Opin Virol 2011;1:519–25. 9. Shatz M, Menendez D, Resnick MA. The human TLR innate immune gene 34. Beck CR, Collier P, Macfarlane C, Malig M, Kidd JM, Eichler EE, et al. LINE-1 family is differentially influenced by DNA stress and p53 status in cancer retrotransposition activity in human genomes. Cell 2010;141:1159–70. cells. Cancer Res 2012;72:3948–57. 35. Levine AJ, Ting DT, Greenbaum BD. P53 and the defenses against genome 10. Menendez D, Lowe JM, Snipe J, Resnick MA. Ligand dependent restoration instability caused by transposons and repetitive elements. Bioessays of human TLR3 signaling and death in p53 mutant cells. Oncotarget 2016;38:508–13. 2016;7:61630–42. 36. Symer DE, Connelly C, Szak ST, Caputo EM, Cost GJ, Parmigiani G, et al. 11. Shatz M, Shats I, Menendez D, Resnick MA. p53 amplifies Toll-like receptor Human l1 retrotransposition is associated with genetic instability in vivo. 5 response in human primary and cancer cells through interaction with Cell 2002;110:327–38. multiple signal transduction pathways. Oncotarget 2015;6:16963–80. 37. Wylie A, Jones AE, D'Brot A, Lu WJ, Kurtz P, Moran JV, et al. p53 genes 12. Jarmuz A, Chester A, Bayliss J, Gisbourne J, Dunham I, Scott J, et al. An function to restrain mobile elements. Genes Dev 2016;30:64–77. anthropoid-specific locus of orphan C to U RNA-editing enzymes on 38. Muckenfuss H, Kaiser JK, Krebil E, Battenberg M, Schwer C, Cichutek K, chromosome 22. Genomics 2002;79:285–96. et al. Sp1 and Sp3 regulate basal transcription of the human APOBEC3G 13. Harris RS, Dudley JP. APOBECs and virus restriction. Virology 2015;479– gene. Nucleic Acids Res 2007;35:3784–96. 80:131–45. 39. Leonard B, Hart SN, Burns MB, Carpenter MA, Temiz NA, Rathore A, et al. 14. Stavrou S, Ross SR. APOBEC3 proteins in viral immunity. J Immunol APOBEC3B upregulation and genomic mutation patterns in serous ovarian 2015;195:4565–70. carcinoma. Cancer Res 2013;73:7222–31. 15. Burns MB, Temiz NA, Harris RS. Evidence for APOBEC3B mutagenesis in 40. Muller PA, Vousden KH. Mutant p53 in cancer: new functions and multiple human cancers. Nat Genet 2013;45:977–83. therapeutic opportunities. Cancer Cell 2014;25:304–17. 16. Chan K, Roberts SA, Klimczak LJ, Sterling JF, Saini N, Malc EP, et al. An 41. Maruyama W, Shirakawa K, Matsui H, Matsumoto T, Yamazaki H, Sarca APOBEC3A hypermutation signature is distinguishable from the signature AD, et al. Classical NF-kB pathway is responsible for APOBEC3B expres- of background mutagenesis by APOBEC3B in human cancers. Nat Genet sion in cancer cells. Biochem Biophys Res Commun 2016;478:1466–71. 2015;47:1067–72. 42. Vieira VC, Leonard B, White EA, Starrett GJ, Temiz NA, Lorenz LD, et al. 17. Roberts SA, Lawrence MS, Klimczak LJ, Grimm SA, Fargo D, Stojanov P, Human papillomavirus E6 triggers upregulation of the antiviral and cancer et al. An APOBEC cytidine deaminase mutagenesis pattern is widespread in genomic DNA deaminase APOBEC3B. MBio 2014;5:pii:e02234–14. human cancers. Nat Genet 2013;45:970–6. 43. Landry S, Narvaiza I, Linfesty DC, Weitzman MD. APOBEC3A can activate the 18. Koning FA, Newman EN, Kim EY, Kunstman KJ, Wolinsky SM, Malim MH. DNA damage response and cause cell-cycle arrest. EMBO Rep 2011;12:444–50. Defining APOBEC3 expression patterns in human tissues and hematopoi- 44. Wang Y, Schmitt K, Guo K, Santiago ML, Stephens EB. Role of the single etic cell subsets. J Virol 2009;83:9474–85. deaminase domain APOBEC3A in virus restriction, retrotransposition, 19. Refsland EW, Stenglein MD, Shindo K, Albin JS, Brown WL, Harris RS. DNA damage and cancer. J Gen Virol 2016;97:1–17. Quantitative profiling of the full APOBEC3 mRNA repertoire in lympho- 45. Ciccia A, Elledge SJ. The DNA damage response: making it safe to play with cytes and tissues: implications for HIV-1 restriction. Nucleic Acids Res knives. Mol Cell 2010;40:179–204. 2010;38:4274–84. 46. Nik-Zainal S, Wedge DC, Alexandrov LB, Petljak M, Butler AP, Bolli N, et al. 20. Conticello SG, Thomas CJ, Petersen-Mahrt SK, Neuberger MS. Evolution of Association of a germline copy number polymorphism of APOBEC3A and the AID/APOBEC family of polynucleotide (deoxy)cytidine deaminases. APOBEC3B with burden of putative APOBEC-dependent mutations in Mol Biol Evol 2005;22:367–77. breast cancer. Nat Genet 2014;46:487–91. 21. Komarov PG, Komarova EA, Kondratov RV, Christov-Tselkov K, Coon JS, 47. Middlebrooks CD, Banday AR, Matsuda K, Udquim KI, Onabajo OO, Chernov MV, et al. A chemical inhibitor of p53 that protects mice from the Paquin A, et al. Association of germline variants in the APOBEC3 region side effects of cancer therapy. Science 1999;285:1733–7. with cancer risk and enrichment with APOBEC-signature mutations in 22. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of tumors. Nat Genet 2016;48:1330–8. short interfering RNAs in mammalian cells. Science 2002;296:550–3. 48. Zhang Y, Delahanty R, Guo X, Zheng W, Long J. Integrative genomic 23. Menendez D, Inga A, Resnick MA. The expanding universe of p53 targets. analysis reveals functional diversification of APOBEC gene family in breast Nat Rev Cancer 2009;9:724–37. cancer. Hum Genomics 2015;9:34. 24. Smeenk L, van Heeringen SJ, Koeppel M, van Driel MA, Bartels SJ, Akkers 49. Starrett GJ, Luengas EM, McCann JL, Ebrahimi D, Temiz NA, Love RP, et al. RC, et al. Characterization of genome-wide p53-binding sites upon stress The DNA cytosine deaminase APOBEC3H haplotype I likely contributes to response. Nucleic Acids Res 2008;36:3639–54. breast and lung cancer mutagenesis. Nat Commun 2016;7:12918. 25. Wei CL, Wu Q, Vega VB, Chiu KP, Ng P, Zhang T, et al. A global map of p53 50. Bougeard G, Renaux-Petel M, Flaman JM, Charbonnier C, Fermey P, Belotti transcription-factor binding sites in the human genome. Cell 2006;124: M, et al. Revisiting Li-Fraumeni syndrome from TP53 mutation carriers. J 207–19. Clin Oncol 2015;33:2345–52.

www.aacrjournals.org Mol Cancer Res; 2017 OF9

The Cytidine Deaminase APOBEC3 Family Is Subject to Transcriptional Regulation by p53

Daniel Menendez, Thuy-Ai Nguyen, Joyce Snipe, et al.

Mol Cancer Res Published OnlineFirst February 23, 2017.

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