Fusarium graminearum, a devastating fungal pathogen of small grain cereal crops worldwide. The lack of FgAtm1 reduces the activity of cytosolic Fe-S proteins nitrite reductase and xanthine dehydrogenase, which causes high expression of FgHapX via activating transcription factor FgAreA. FgHapX represses transcription of genes for iron-consuming proteins directly but activates genes for iron acquisition proteins by suppressing another iron regulator FgSreA. In addition, the transcriptional activity of FgHapX is regulated by the monothiol glutaredoxin FgGrx4. Furthermore, the phosphorylation of FgHapX, mediated by the Ser/Thr kinase FgYak1, is required for its functions in iron homeostasis. Taken together, this study uncovers a novel regulatory mechanism of iron homeostasis mediated by an ABC transporter in an important pathogenic fungus.Iron homeostasis is important for growth, reproduction and other metabolic processes in all eukaryotes. However, the functions of ATP-binding cassette (ABC) transporters in iron homeostasis are largely unknown. Here, we found that one ABC transporter (named FgAtm1) is involved in regulating iron homeostasis, by screening sensitivity to iron stress for 60 ABC transporter mutants of  Saccharomyces cerevisiae. Our study found that FgAtm1 regulates iron homeostasis via the transcription factor cascade FgAreA-HapX in F. graminearum and the function of FgHapX is dependent on its interaction with FgGrx4 and phosphorylation by the Ser/Thr kinase FgYak1. This study reveals a novel regulatory mechanism of iron homeostasis in an important plant pathogenic fungus, and advances our understanding in iron homeostasis and functions of ABC transporters in eukaryotes.Essential element iron plays important roles in many cellular processes in all organisms. The function of an ATP-binding cassette (ABC) transporter Atm1 in iron homeostasis has been characterized in  Iron is an essential element for growth, and can be present in various forms such as iron ions, heme and iron sulfur clusters that play critical roles in respiration, DNA synthesis and repair, ribosome biogenesis, metabolism and other cellular processes in all organisms –3. In maCandida albicans  clusters ((GS)4-[2Fe-2S]) from mitochondria to cytosol  clusters onto monothiol glutaredoxins Grx3/4, thus hindering the formation of the complex containing Grx3/4 and the cytosolic proteins Fra1/2. This subsequently enhances retention of the transcription factor Aft1/2 at the promoter of iron acquisition genes, therefore leading to constitutive gene activation  clusters onto monothiol glutaredoxins, subsequently disrupting formation of the Grx3/4-Fra1/2 complex, which results in the failure of Aft1/2 dissociation from the promoters of iron acquisition genes . The precursor of Moco cyclic pyranopterin monophosphate (cPMP) that is synthesized in mitochondria was reported as another substrate of Atm3 [F. graminearum FgAtm1 modulates the assembly of Moco protein nitrate reductase (FgNiaD)   . Furtheroformans , 75, 76.ns [2O2) , since moduction . The pheF. graminearum wild-type strain PH-1 was used as a parental strain for transformation experiments in this study. Mycelial growth of the wild type and the resulting transformants were assayed on potato dextrose agar (PDA) or minimal medium (MM) as described previously [2+, H2O2, or catalytic product of each Fe-S protein, and then incubated at 25°C for 3 days in the dark. Three biological replicates were used for each strain and each experiment was repeated three times independently.eviously , 79. To HPH) driven by the constitutive trpC promoter which was amplified from the pBS-HPH1 vector [F. graminearum was carried out using the protocol described previously [FgATM1 deletion mutation was further confirmed by Southern hybridization assays (The double-joint PCR approach  was used1 vector . Protopleviously . Putativn assays .FgATM1 containing the promoter region and open-reading frame (without the stop codon) was amplified with the relevant primers (NEO) [Escherichia coli strain DH5α for amplification. Using the similar strategy, FgYak1 (FGSG_05418)—and FgTri1 (FGSG_00071) -GFP fusion cassettes were constructed. Using the similar strategy, FgGrx4 (FGSG_01317)—and FgYak1-Flag fusion cassettes were constructed by co-transformation with XhoI-digested PHZ126 vector. Similarly, FgHapX-CYFP and FgGrx4-NYFP fusion cassettes were constructed by co-transformation with XhoI-digested PHZ68 and PHZ65 vectors, respectively.To construct the FgAtm1-GFP cassette,  primers . The resrs (NEO)  into thers (NEO)  using thS245A/S338A- and FgLeu1 (FGSG_09589)-mCherry cassettes. Briefly, the target gene containing the promoter region and open-reading fragment and the geneticin resistance gene (NEO) were amplified, and then fused with mCherry fragment. Before protoplast transformation, each fusion construct was verified by DNA sequencing. The transformation of F. graminearum was carried out using the previously described protocol [The double-joint PCR approach  was alsoprotocol . All theBasidiomycetes sp.  for 4–6 hours. After filtration with funnel and filter paper, the filtrate was centrifuged at 5000 g at 4°C for 10 min. The protoplast was used for total iron determination and mitochondrial extraction with a Cell Mitochondria Isolation Kit . Iron content was determined using the colorimetric ferrozine-based assay previously reported, with ferrozine as chelator and ferric chloride as a standard [3), and 100 μl of the iron-releasing reagent  KMnO4 in H2O). The mixtures were incubated within a fume hood at 60°C for 2 h. After the mixtures had cooled to room temperature, 30 μl of the iron-detection reagent  was added to each tube. After 30 min, 200 μl of the solution in each tube was transferred into a well of a 96-well plate and the absorbance was measured at 550 nm on a microplate reader. The linear range of the ferrozine assay is from 0.2 to 30 nmol.For determination of mitochondrial and total iron content, About 50 mg of fresh mycelia were lysed by 2% cellulase , 2% lysozyme  and 0.2% driselase from standard , 29, 85.4 and amended 0.3 mM iron-specific chelating agent bathophenanthroline disulfonate (BPS) at 25°C for 8 hours. After filtration with funnel and filter paper, the supernatant was used for determining the content of extracellular siderophore, and the mycelia was used for determining the content of intracellular siderophore. Finely ground mycelia (50 mg) were resuspended in 50 mM potassium phosphate buffer. After vigorous vortexing, the cellular debris was pelleted, and the supernatant was determined for the content of intracellular siderophore. An aliquot of supernatant (1 ml) was mixed with 1 ml chrome azurol S (CAS) assay solution that was prepared. After incubation in the dark for 1 hour at room temperature, the absorbance of each sample was measured at 630 nm and the relative content of siderophore was calculated according to previous study [To determine the content of extra- and intracellular siderophores, each strain was cultured in CM for 36 hours, then transferred to MM lack of FeSOus study .For the conidiation assay, fresh mycelia (50 mg) of each strain were inoculated in a 50-ml flask containing 20 ml of carboxymethyl cellulose (CMC) liquid medium. The flasks were incubated at 25°C for 4 days in a shaker (180 rpm). Subsequently, the number of conidia in each flask was determined using a hemacytometer. Three biological replicates were used for each strain and each experiment was repeated three times independently.5 conidia/ml) was injected into a floret in the central section spikelet of a single flowering wheat head of susceptible cultivar Jimai 22. Fifteen days after inoculation, the infected spikelets in each inoculated wheat head were recorded. For virulence on corn silks and wheat seedling leaves, a 5-mm mycelial plug of each strain was inoculated on the middle of corn silks and wheat seedling leaves, and then cultured for 4 days. Ten replicates were used for each strain and each experiment was repeated three times independently. To determine DON production, each strain was grown in liquid trichothecene biosynthesis induction (TBI) medium at 28°C for 3 d in a shaker (150 rpm) in the dark. A DON Quantification Kit  was used to quantify the DON production for each sample. The experiment was repeated three times.Virulence assays on wheat spikelets, corn silks, and wheat seedling leaves were conducted as described previously . For virThe colony edge of each strain cultured on PDA plates at 25°C for 7 days was examined with a Leica TCS SP5 imaging system . Morphology of conidia cultured in CMC liquid medium for 4 days was observed with a Leica TCS SP5 imaging system after staining with the cell wall-damaging agent calcofluor white (CFW) at 10 μg/ml. Germinating conidia in TBI liquid medium or iron-depleted TBI liquid medium were observed with a Leica TCS SP5 imaging system. Fluorescence signals were examined with a Zeiss LSM780 confocal microscope . For the observation of proteins tagged with GFP, mCherry or YFP, each strain was cultured in CM at 25°C for 24 h in a shaker (180 rpm) before staining with MitoTracker or 4’,6-diamidino-2-phenylindole (DAPI) . For exag for 10 min at 4°C. Each protein sample (50 μg) was used for FgLeu1 activity determination. DL-threo-3-isopropylmalic acid  was used as the substrate and β-isopropylmalate formation was measured by monitoring absorbance at 235 nm for 10 minutes [The wild type, ΔFgAtm1 and the ΔFgAtm1-C were grown in CM for 36 hours, and then were harvested after rinsing 3 times with sterile water. To determine FgLeu1 activity in cytosol, 50 mg of finely ground mycelia was resuspended in 1 ml of lysis buffer  for 10 min. The lysate was centrifuged at 14,000  minutes , 93. To  minutes . FgFum1 g in a microcentrifuge for 10 min at 4°C. An aliquot of 10 μl supernatant was used for ROS determination with ROS ELISA Kit . Meantime, to examine ROS, each strain cultured on PDA plates at 25°C for 3 days was stained with 0.05% (wt/vol) nitroblue tetrazolium (NBT) for 2 h. These experiments were all repeated three times.To examine ROS content, fresh mycelia of each strain grown in CM liquid medium for 36 hours were harvested after rinsing 3 times with sterile water. 50 mg of finely ground mycelia were resuspended in 1 ml of extraction buffer. After homogenization with a vortex shaker, the lysate was centrifuged at 14,000 FgACTIN gene was performed as a reference. The experiment was repeated three times.Total RNA isolation from mycelia of each sample with the TaKaRa RNAiso Reagent , and reverse transcription was performed with a HiScript II 1st Strand cDNA Synthesis Kit . The expression level of each gene was determined by qRT-PCR with HiScript II Q RT SuperMix . The expression of the FgACTIN as an internal control, and then by normalizing the value for the immunoprecipitated sample against that for the input. The ChIP-qPCR was independently repeated three times.ChIP was performed as previously described , 95 withN1–230) was amplified and cloned into pGEX-4T-3 vector to generate GST-tagged protein. The resulting construct was transformed into the E. coli strain BL21 (DE3) after verifying the cDNA sequence. The recombinant GST-FgHapXN1–230 was purified by Ni sepharose beads and eluted by reduced glutathione. Promter DNAs were amplified using the primers in Table S1. For EMSA, Reaction mixtures containing purified GST -FgHapXN1–230, promter DNAs and 10×Binding buffer (100 mM Tris-HCl (PH 7.5), 0.5 M NaCl, 10 mM DTT, 10 mM EDTA, 50% glycerol) were incubated for 20 min at 25°C. The purified GST was used as negative controls. The reactions were electrophoresed on 1.2% agarose gel in 0.5×TAE for 45 min in 80 V under low temperature. Signals were detected in J3-3000 imaging system after dying DNA dye ethidium bromide (EB) for 15 min. The experiment was conducted independently three times.The cDNA encoding the N terminal 1–230 amino acids of FgHapX  lacking Leu and Trp, and then transferred to SD stripped of Ade, His, Trp and Leu to assess binding activity. Three independent experiments were performed to confirm yeast two-hybrid assay results.To construct plasmids for Y2H analyses, the coding sequence of each tested gene was amplified from the cDNA of PH-1 with primer pairs indicated in F. graminearum cDNA library was constructed in the Y2H vector pGADT7 using total RNA extracted from mycelia and conidia. The Y2HGold that was co-transformed with the cDNA library as well as FgHapX-pGBKT7 were directly selected using SD-Trp-Leu-His. Approximately 150 potential yeast transformants containing cDNA clones interacting with FgHapX were further confirmed in selection medium SD-Trp-Leu-His-Ade.To search for FgHapX-interacting proteins, we performed Y2H screens. FgHapX was cloned into the yeast vector pGBKT7. A For western blotting assay, protein samples of strains were prepared and extracted as described previously . ProteinThe mCherry and Flag fusion constructs were verified by DNA sequencing and transformed in pairs into PH-1. Transformants expressing the fusion constructs were confirmed by western blot analysis. In addition, the transformants bearing a single fusion construct were used as references. For Co-IP assays, total proteins were extracted and incubated with the anti-Flag  agarose overnight at 4°C as described previously . ProteinThe FgHapX-CYFP and FgGrx4-NYFP fusion constructs were generated by cloning the related fragments into pHZ68 and pHZ65 vectors, respectively. FgHapX-CYFP and FgGrx4-NYFP constructs were co-transformed into PH-1 and ΔFgAtm1. In addition, a pair of constructs, FgHapX-CYFP and NYFP and another pair of constructs, FgGrx4-NYFP and CYFP were used as negative controls. Transformants resistant to both hygromycin and zeocin were isolated and confirmed by PCR. YFP signals were examined with a Zeiss LSM780 confocal microscope .4OH and 80% ACN/2% FA (formic acid). All of the eluted fractions were combined, vacuum-dried and cleaned with C18 ZipTips  according to the manufacturer’s instructions, followed by LC-MS/MS analysis according to a previous study [Proteins of the wild-type PH-1 were extracted in the lysis buffer . For trypsin digestion, the protein sample was diluted in 0.1 M TEAB (triethylammonium bicarbonate), and digested with 1/25 trypsin  for 12 h at 37°C. The digestion was terminated with 1% TFA (trifluoroacetic acid), and the resulting peptides were cleaned with a Strata X C18 SPE column  and vacuum-dried in a scanvac maxi-beta . Then, the resulting peptides (2 mg per sample) were reconstituted in 120 μl 0.5 M TEAB, and treated with a TMTsixplex label reagent kit . Both fractionations were performed with an XBridge Shield C18 RP column  in a LC20AD HPLC system . For phosphorylation enrichment, peptides were dissolved in 80% ACN/6% TFA and then incubated with IMAC-Ti4+ beads  at room temperature. The beads were washed once with 50% ACN/0.5%TFA/200 mM NaCl and once with 50% ACN/0.1% TFA. The bound peptides were then eluted with 10% NHus study . The datus study .S245A/S338A-mCherry fusion construct was transferred into the ΔFgHapX mutant. Protein samples were prepared and extracted as described above. Each resulting protein sample was loaded on 8% SDS-polyacrylamide gels prepared with 25 μM Phos binding reagent acrylamide  and 100 μM ZnCl2. Gels were electrophoresed at 20 mA/gel for 3–5 h. Prior to protein transfer, gels were first equilibrated three times in transfer buffer containing 5 mM EDTA for 5 min and further equilibrated in transfer buffer without EDTA for 5 min for two times. Protein transfer from the Zn2+-phos-tag acrylamide gel to the PVDF membrane was performed for 4–5 h at 100 V on ice, and finally the membrane was analyzed by western blotting with the monoclonal anti-mCherry ab125096  antibody.For Phos-tag assay, the FgHapX-mCherry fusion construct was transferred into the ΔFgHapX and ΔFgYak1 mutants and the FgHapXFgYak1-Flag and FgHapX-mCherry were co-transformed into the wild type. The nuclei of corresponding strains were extracted as described previously  and thenS1 Fig4, and then incubated at 25°C for 3 days (A) or 7 days (B).A 5-mm mycelial plug of each strain was inoculated on MM without or with 8 mM FeSO(TIF)Click here for additional data file.S2 Figin situ complemented strains. (B) Colocalization of FgAtm1- or FgAtm1N1-111-GFP with mitochondrial dye MitoTracker. The plasmid FgAtm1- or FgAtm1N1-111-GFP was transformed in situ into ΔFgAtm1 and the resulting strain was then examined with a fluorescent microscope after MitoTracker staining. Bar = 10 μm.(A) PCR analyses of the wild type, ΔFgAtm1 and the ectopically and (TIF)Click here for additional data file.S3 FigFgATM1 deletion mutant ΔFgAtm1. The hygromycin resistance cassette (HPH) is denoted by the large grey arrow. (B) PCR analyses of the wild type, ΔFgAtm1 and the complemented strain ΔFgAtm1-C. (C) Southern blot hybridization analysis of the wild type and ΔFgAtm1 using an upstream fragment of FgATM1 as a probe (left panel). Southern blot hybridization analysis of the wild type and the complemented strain ΔFgAtm1-C using a G418 fragment as a probe (right panel).(A) Gene replacement strategy for the (TIF)Click here for additional data file.S4 Fig(A) Comparison in fluorescence intensity of the cytoplasmic Fe-S protein isopropyl malate isomerase FgLeu1 between the wild type and ΔFgAtm1. Bar = 10 μm. (B) Comparison in FgLeu1 quantity between the wild type and ΔFgAtm1. Western blots of proteins obtained from transformants of PH-1 and ΔFgAtm1 bearing the FgLeu1-mCherry were detected with the monoclonal anti-mCherry antibody. Detection with the anti-GAPDH antibody was the loading reference. Band intensities were quantified with the program IMAGE QUANT TL. The intensity of FgLeu1 band for each strain is relative to that of the GAPDH band.(TIF)Click here for additional data file.S5 Fig4. A 5-mm mycelial plug of each strain was inoculated on PDA without or with 3 mM FeSO4, and then incubated at 25°C for 3 days. Means and standard errors were calculated from three repeats. Significance was measured using an unpaired t-test (n.s. not significant).Sensitivity of the five Fe-S proteins deletion mutants to FeSO(TIF)Click here for additional data file.S6 FigFgHAPX in PH-1 was set to 1. Means and standard errors were calculated from three repeats. Significance was measured using an unpaired t-test (*p < 0.05).The expression level of (TIF)Click here for additional data file.S7 Figt-test (n.s. not significant). (B and C) The localization (B) and protein quantity (C) of FgHapX in the wild type and ΔFgGrx4. Bar = 10 μm.(A) Total iron content of the wild type and ΔFgGrx4 was determined by colorimetric ferrozine-based assay after growth in CM at 25°C for 36 hours. Means and standard errors were calculated from three repeats. Significance was measured using an unpaired (TIF)Click here for additional data file.S8 Fig(A) Identification of two serine phosphorylation sites (S245 and S338) in FgHapX. (B) Sketch map of the above two residues in FgHapX.(TIF)Click here for additional data file.S9 FigS245A/S338A mutated in the two phosphorylation sites and ΔFgYak1. Bar = 10 μm.The localization (A) and protein quantity (B) of FgHapX in the wild type, ΔFgHapX-C(TIF)Click here for additional data file.S10 Fig4, and then incubated at 25°C for 3 days (A) or 7 days (B). (C) Mycelial growth inhibition of seven kinase mutants was calculated after 3-day-incubation at the conditions (A). Means and standard errors were calculated from three repeats. Significance was measured using an unpaired t-test . (D) Phos-tag assays for the seven kinase mutants. Protein extracted from each strain was subjected to Phos-tag SDS-PAGE and normal SDS-PAGE followed by immunoblotting with an anti-mCherry antibody.(A and B) A 5-mm mycelial plug of each strain was inoculated on MM without or with 10 mM FeSO(TIF)Click here for additional data file.S11 Figt-test .Each strain was cultured in CM at 25°C for 36 hours before activity determination. The FgNiaD activity of the wild type was set as 1. Means and standard errors were calculated from three repeats. Significance was measured using unpaired (TIF)Click here for additional data file.S12 Figt-test . (B) Reactive oxygen species (ROS) production in the wild type, ΔFgAtm1 and ΔFgAtm1-C. The 3-day-old colony of each strain was stained with 0.05% (wt/vol) ROS indicator nitroblue tetrazolium (NBT) for 2 h with or without 50 μM DPI and 4 μM antimycin A. (C) Quantitative determination of ROS in the wild type, ΔFgAtm1 and ΔFgAtm1-C with or without the treatment of 50 μM DPI and 4 μM antimycin A. Means and standard errors were calculated from three repeats. Significance was measured using an unpaired t-test . (D) Sensitivity of the wild type, ΔFgAtm1 and ΔFgAtm1-C to hydrogen peroxide (H2O2). A 5-mm mycelial plug of each strain was inoculated on PDA amended without or with 12 mM H2O2 and then incubated at 25°C for 3 days.(A) Sensitivity of the wild type and ΔFgAtm1 to different mitochondrial complex inhibitors. A 5-mm mycelial plug of each strain was inoculated on PDA without or with diphenylene iodonium (DPI) and rotenone (complex I), antimycin A (complex III), malonic acid (complex II) or 6-Hydroxydopamine hydrobromide (6-OHDA) (complex IV) at the indicated concentration, and then incubated at 25°C for 3 days. Mycelial growth inhibition of each treatment was calculated after 3-day-incubation. Means and standard errors were calculated from three repeats. Significance was measured using an unpaired (TIF)Click here for additional data file.S13 Fig(A) Conidial morphology of the wild type, ΔFgAtm1, and ΔFgAtm1-C. The differential interference contrast (DIC) images of conidia from each strain were captured with an electronic microscope. Bar = 20 μm. Conidia were cultured in CMC liquid medium at 25°C for 4 days in a shaker. (B) Conidia of each strain were quantified after incubation in CMC for 4 days. Means and standard errors were calculated from three repeats. Significance was measured using an unpaired t-test . (C and D) Comparisons of conidium length (C) and septum number (D) among the above strains. Conidia were cultured in CMC liquid medium at 25°C for 4 days in a shaker. A total of 200 conidia were examined for each strain. (E) Perithecia of the wild type, ΔFgAtm1 and ΔFgAtm1-C were grown on carrot agar for induction of perithecial formation. Bar = 500 μm. (F) The amount of DON produced by the wild type, ΔFgAtm1 and ΔFgAtm1-C after incubation in trichothecene biosynthesis induction (TBI) medium for 3 days. The dry weight of mycelium is used as an internal reference. Means and standard errors were calculated from three repeats. Significance was measured using an unpaired t-test . (G) Relative expression levels of DON biosynthetic genes FgTRI1, FgTRI5, and FgTRI6 in the wild type, ΔFgAtm1, and ΔFgAtm1-C after growth in TBI at 28°C in the dark for 3 days. Means and standard errors were calculated from three repeats. Significance was measured using an unpaired t-test . (H) The subcellular localization of FgTri1-GFP. FgTri1-GFP localized to “toxisomes” in PH-1, whereas, the Tri1-GFP labelled toxisomes were nearly undetectable in ΔFgAtm1. Bar = 10 μm. Each strain was examined in artificially wounded wheat seedling leaves 48 h after inoculation. (I) FgTri1-GFP was dramatically reduced in ΔFgAtm1 determined by western blotting with the monoclonal anti-GFP antibody. The protein samples were also detected with the monoclonal anti-GAPDH antibody as a reference. (J) Comparison of red pigment production among PH-1, ΔFgAtm1 and ΔFgAtm1-C. Each strain was grown on PDA for 7 days (left panel) or in potato dextrose broth (PDB) for 2 days (right panel).(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


CTG because of its exclusive presence in the CTG clade of ascomycetes, including Candida albicans, a human pathogen. C. albicans grows both as single yeast cells and hyphal filaments in the planktonic mode of growth. It also forms a three-dimensional biofilm structure in the host as well as on human catheter materials under suitable conditions. H3VCTG null (hht1/hht1) cells of C. albicans are viable but produce more robust biofilms than wild-type cells in both in vitro and in vivo conditions. Indeed, a comparative transcriptome analysis of planktonic and biofilm cells reveals that the biofilm circuitry is significantly altered in H3VCTG null cells. H3VCTG binds more efficiently to the promoters of many biofilm-related genes in the planktonic cells than during biofilm growth, whereas the binding of the core canonical histone H3 on the corresponding promoters largely remains unchanged. Furthermore, biofilm defects associated with master regulators, namely, biofilm and cell wall regulator 1 (Bcr1), transposon enhancement control 1 (Tec1), and non-dityrosine 80 (Ndt80), are significantly rescued in cells lacking H3VCTG. The occupancy of the transcription factor Bcr1 at its cognate promoter binding sites was found to be enhanced in the absence of H3VCTG in the planktonic form of growth resulting in enhanced transcription of biofilm-specific genes. Further, we demonstrate that co-occurrence of valine and serine at the 31st and 32nd positions in H3VCTG, respectively, is essential for its function. Taken together, we show that even in a unicellular organism, differential gene expression patterns are modulated by the relative occupancy of the specific histone H3 type at the chromatin level.Histone H3 and its variants regulate gene expression but the latter are absent in most ascomycetous fungi. Here, we report the identification of a variant histone H3, which we have designated H3V A variant histone H3 specific to the CTG clade of ascomycete fungi modulates the expression of the majority of the biofilm genes in the human pathogen Candida albicans by binding differentially to biofilm-relevant gene promoters. Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe  broth at 30 °C) were analyzed. Approximately one-fifth of all C. albicans genes  were found to have an altered expression  when H3VCTG was absent  and 2 biological replicates of H3VCTG null mutant (LR107 and LR108) in Spider medium–induced biofilm conditions. In the biofilm-inducing conditions, 751 genes were found to be differentially expressed  between the wild-type and H3VCTG null mutants (CTG null mutants (p < 0.05) between the null mutants of H3VCTG and the wild type when the cells were grown in conditions that favor biofilm growth , H3VCTG mutants LR107 and LR108 (hht1/hht1), and the H3VCTG complemented strain LR109 (hht1/hht1::HHT1) were allowed to form biofilm in 6-well polystyrene plates in the Spider medium at 37 °C  was performed in vitro on silicone squares grown biofilms , or H3VCTG complemented strain LR109 (hht1/hht1::HHT1) of C. albicans. After 24 hours of biofilm growth, the catheters were removed, and catheter luminal surfaces were imaged by SEM (CTG null mutants (hht1/hht1) compared with the wild-type strain SC5314 or the H3VCTG complemented strain LR109. Therefore, these in vivo as well as in vitro observations together support the conclusion that deletion of H3VCTG results in the formation of more robust biofilms than those formed by wild-type C. albicans.Because of the presence of many uncharacterized host factors, in vivo biofilms may substantially differ from those formed in vitro. To examine whether absence of H3Ver model  was usedd by SEM . SimilarCTG mutant compared with the wild type motivated us to investigate the mechanism by which H3VCTG negatively regulates biofilm growth in C. albicans. To understand this mechanism at the molecular level, the V5 epitope was used to tag either the variant histone H3 (H3VCTG) or the canonical histone H3 (encoded by HHT21 as described above). Based on the complementation assay, the V5-tagged H3VCTG (LR145 and LR146) was proved to be functional when expressed as the only copy in the cell , biofilm-repressed genes (NRG1 and YWP1), and also of an uncharacterized gene (ORF19.7380), to which 5 of the 6 biofilm master regulators bind, were examined to study the occupancy of either canonical (Hht21-V5) or variant histone H3 (H3VCTG-V5). The occupancy of H3VCTG was significantly higher at the promoters of biofilm-related genes compared with that of the canonical histone H3 when the cells were grown in the planktonic mode of growth  digestion was performed to determine the occupancy of these 2 histone H3 on the BMT7 gene. Both total DNA MNase digestion and ChIP results show that both histone H3 can occupy the same regions  expression in the H3VCTG mutant by ChIP-qPCR  in each of the following mutant strains—bcr1/bcr1, brg1/brg1, efg1/efg1, ndt80/ndt80, rob1/rob1, and tec1/tec1—and examined the biofilm-forming ability of these double mutants by growing biofilms in YPD medium. Strikingly, biofilm growth was found to be significantly enhanced in the bcr1/bcr1 hht1/hht1 and tec1/tec1 hht1/hht1 double-mutant strains and also partially in ndt80/ndt80 hht1/hht1 compared with bcr1/bcr1, tec1/tec1, or ndt80/ndt80, respectively  did not show any significant change in biofilm formation compared with the mutants lacking only the master regulator , H3VCTG (hht1/hht1) mutants LR107 and LR108, and the H3VCTG complemented strain LR109 (hht1/hht1::HHT1) on solid media. Colony wrinkling was enhanced in the mutant strains (hht1/hht1) compared with the wild-type (HHT1/HHT1) and the H3VCTG complemented strain LR109 (hht1/hht1::HHT1) at both 30 °C and 37 °C , and the hht21/hht21 null mutant or point-mutant strains  or in the filamentous form (tup1 mutant) show compromised virulence ), resuspended in 0.5 ml of the same buffer, and disrupted using acid-washed glass beads  by vortexing 5 minutes (1 minute vortexing followed by 1 minute cooling on ice) at 4 °C. C. albicans cell lysates were electrophoresed on a 12% SDS-PAGE gel and blotted onto a nitrocellulose membrane in a semidry apparatus (BIO-RAD). The blotted membranes were blocked with 5% skim milk containing 1× phosphate buffered saline  for 1 hour at room temperature and then incubated with the following dilutions of primary antibodies: anti-V5 antibodies at a dilution of 1:5,000 , anti-PSTAIRE antibodies  at a dilution 1:3,000. Anti-PSTAIRE antibodies recognize Cdc28 and are widely used as a protein loading control in C. albicans. Next, membranes were washed 3 times with PBST (0.1% Tween-20 in 1× PBS) solution. Anti-rabbit HRP conjugated antibodies  and anti-mouse IgG-HRP antibodies  were added at a dilution of 1: 1,000 and incubated for 1 hour at room temperature followed by 3 to 4 washes with PBST solution. Signals were detected using the chemiluminescence method .Western blot analysis was performed as described before by Chatterjee and colleagues . C. albiC. albicans cells were fixed with a one-tenth volume of formaldehyde (37%) for 15 minutes at room temperature. Antibodies used were diluted as follows: 1:100 for mouse anti-V5 antibodies . The dilutions for secondary antibodies used were Alexa Fluor 488 goat anti-mouse IgG  diluted 1:1,000. DAPI   was used to stain the nuclei of the cells. Cells were examined under 100× magnifications using a GE Delta vision microscope. The digital images were processed with Adobe Photoshop.Indirect immunofluorescence was performed as described before by Sanyal and Carbon and Chatterjee and colleagues , 56. Asy260 and A280 using a Nanodrop Spectrophotometer (Thermo Scientific). The samples were labeled using Agilent Quick Amp labeling kit (Part number 5190–0442). A total of 500 ng RNA was reverse transcribed using Oligo(dT) primer tagged to the T7 promoter sequence. The cDNA obtained was converted to double stranded cDNA in the same reaction. Further, cDNA was converted to cRNA in the in vitro transcription step using T7 RNA polymerase, and Cy3 dye was added into the reaction mix. During cRNA synthesis, Cy3 dye was incorporated to the newly synthesized strands. cRNA obtained was cleaned up using Qiagen RNeasy columns . Concentration and the amount of dye incorporated in each sample were determined using a Nanodrop. Samples that passed the QC for the specific activity (minimum RNA concentration 500 ng/μl and absorbance 260/280 = 2) were taken for hybridization. A total of 600 ng of labeled cRNA was hybridized on the array (AMADID 29460) using the Gene Expression Hybridization kit  in Sure hybridization Chambers (Agilent) at 65 °C for 16 hours. Hybridized slides were washed using Agilent Gene Expression wash buffers (part no. 5188–5327). The hybridized, washed microarray slides were then scanned on a G 2600 D scanner (Agilent). Data extraction from images was done using Feature Extraction software version 10.7 of Agilent. Microarray data was preprocessed using Limma package  of statistical R language. Briefly, preprocessing includes (a) background correction (b) within array normalization and (c) fitting data to linear model. Finally, an empirical Bayes moderated statistics was applied to find significant changes in the expression levels. We used a cut-off of p < 0.05 and |fold change| > 1.5 for the differential expression of mutant over wild type to define differentially expressed genes. Differentially regulated genes were clustered using hierarchical clustering to identify significant gene expression patterns. All genes represented on the array are annotated using CGD database. The differentially regulated genes (up or down) were categorized under biofilm, cell cycle, transcription, and others based on the gene ontology annotations. Chi-square test was applied to know the significance of the categorized functions. Gene expression data have been deposited into the NCBI Gene Expression Omnibus (GEO) portal under the accession number GSE72824.The concentration of the extracted RNA was evaluated using Bioanalyzer (Agilent), whereas purity of the extracted RNA was determined by the standard procedure for the same by measuring A600) of 0.5 in 3 ml of Spider medium. The 6-well plates had been pretreated overnight with fetal bovine serum (FBS) and washed with 2 ml PBS. The inoculated plate was incubated at 37 °C for 90 minutes at 110 rpm agitation for initial adhesion of cells. Plates were then washed with 2 ml PBS, and 3 ml of fresh Spider medium was added, and plates were at 37 °C for 48 hours. Biofilm growth assays of the wild type, the H3VCTG null mutants, and the HHT1 complemented strain were performed by diluting ON culture to an OD600 of 0.2 in 3 ml Spider medium. To estimate the dry biomass of biofilms, biofilms were scrapped, and the content of each well was transferred to preweighed nitrocellulose filters. Biofilm-containing filters were dried overnight at 60 °C and weighed. The average total biomass for each strain was calculated from 3 independent samples after subtracting the mass of the empty filter.To perform the genome-wide expression analysis in the biofilm-induced condition, biofilms were grown in vitro in Spider medium by growing the biofilms directly on the bottom of 6-well polystyrene plates . Biofilms were developed as described by Nobile and Mitchell . StrainsCTG mutant strains in YPDU medium at 30 °C, in vitro biofilm growth assays were carried out by growing biofilms on either the silicone squares substrate as previously described by Nobile and Mitchell [To examine the biofilm formation ability of H3VMitchell . Biofilm−1 of concanavalin A-Alexa Fluor 594  for 1 hour at 110 rpm. CLSM was performed at PPMS facility of Institut Pasteur using an upright LSM700 microscope equipped with a Zeiss 40X/ 1.0 W plan-Apochromat immersion objective. Silicone squares were placed in a petri dish, and the biofilms were covered with 1× PBS. Images were acquired and assembled into maximum intensity Z-stack projection using ZEN software (Zeiss).Biofilms were imaged as described before by Nobile and Mitchell . BiofilmC. albicans cells (106 cells/ml). After a 4 hour dwelling period, the catheter volume was withdrawn, and the catheter was flushed with heparinized 0.15 M NaCl. Catheters were removed after 24 hours of C. albicans infection to assay biofilm development on the intraluminal surface by SEM. Catheter segments were washed with 0.1 M phosphate buffer (pH 7.2) fixed in 1% glutaraldehyde/4% formaldehyde, washed again with phosphate buffer for 5 minutes, and placed in 1% osmium tetraoxide for 30 minutes. The samples were dehydrated in a series of 10 min ethanol washes  followed by critical point drying. Specimens were mounted on aluminium stubs, sputter coated with gold, and imaged using a Hitachi S-5700 or JEOL JSM-6100 SEM in the high-vacuum mode at 10kV. Images were processed using Adobe Photoshop software.To form biofilm in vivo, the rat central-venous catheter infection model  was used7 cells/ml) or grown in biofilm condition for 24 hours, and cells were cross-linked with 1% final concentration of formaldehyde for 13 minutes. Chromatin was isolated and sonicated to yield an average fragment size of 300 to 500 bp. The DNA was immunoprecipitated with anti-V5 antibodies  or anti H3 antibodies  or anti myc antibodies  and purified. The total, immunoprecipitated (IP) DNA, and beads only material were used to determine the binding of H3VCTG to the promoters of biofilm genes by qPCR, as described before. The template used was as follows: 1 μl of 1:100 dilution for input and 1 μl of 1:2 dilution for IP for H3-V5 ChIP and undiluted for H3 and myc ChIP. The conditions used in qPCR were as follows: 94 °C for 2 minutes; the 40 cycles of 94 °C for 30 seconds, 58 °C for 30 seconds, 72 °C for 45 seconds. The results were analyzed using CFX Manager Software. The graph was plotted according to the percentage input method [Ct input − adjusted Ct of IP) subtracted from the Ct value of diluted input/IP.The ChIP assays were performed as described previously by Chatterjee and colleagues and Mitra and colleagues , 58. Brit method , and theS1 FigHHT2, HHT21, and HHT1, the 3 C. albicans histone H3 encoding genes. (B) Comparative analysis of the amino acid sequences of histone H3 variants of the CTG-clade species. Amino acid sequences were aligned using the Bioedit software. Identical amino acids are indicated as dots and amino acids differing from C. albicans Hht1 are given as single letter symbol: C. albicans (Ca), C. dubliniensis (Cd), C. tropicalis (Ct), C. parapsilosis (Cp), C. orthopsilosis (Co), Lodderomyces elongisporus (Le), Debaryomyces hansenii (Dh), Pichia stipitis (Ps), C. tenuis (Cte), Spathaspora passalidarum (Sp), C. guilliermondii (Cg), C. lusitaniae (Cl). Hht1 is labeled as 1 , whereas Hht2/Hht21 is labeled as 2  for each species.(A) Comparative analysis of nucleotide sequences of (TIF)Click here for additional data file.S2 FigHHT21 or HHT1 genomic DNA sequences as well as upstream and downstream sequences. HHT21 locus was amplified with the primer pair 1061USFP and 1061DSRP (coordinates 881615 to 883328 of Chromosome 1), whereas the HHT1 locus was amplified with the primer pair H3.6791USFP and H3.6791DSRP (coordinates 57523 to 59160 of Chromosome 3). PCR fragments were cloned into TZ57R/T (Thermo Scientific). Given PCR conditions were optimized to selectively amplify either HHT21 or HHT1. (B) RT-PCR was performed with primers selectively amplifying either HHT21 or HHT1. −RT acts as negative control, whereas actin serves as positive control. The 217 bp on +RT lanes represents the canonical/variant histone H3 (HHT21 or HHT1) and the lower band of 110 bp on the same lanes corresponds to actin. (C) Expression levels of V5-tagged C. albicans canonical histone H3 proteins (approximately 17 kDa), namely, Hht2 and Hht21, were monitored by western blot analysis in the yeast form. The parental strain SN148 was used as the untagged control, whereas PSTAIRE (approximately 34 kDa) was used as the loading control. (D) Subcellular localization of the canonical histone H3 (Hht21-V5) or the variant form (Hht1-V5) was performed in the yeast form of C. albicans with anti-V5 antibodies. Nuclei were stained with DAPI. Co-localization of histone H3 with DNA was shown by merging the images. Both interphase (unbudded) and mitotic cells (large budded) are shown. Scale bars: 5 μm. RT-PCR, reverse transcription PCR.(A) Structure of the plasmids carrying (TIF)Click here for additional data file.S3 FigHHT1 locus in the diploid C. albicans strains, namely, the wild-type SC5314 (HHT1/HHT1), heterozygous mutants (HHT1/hht1) LR103 and LR104, and homozygous null mutants LR105 and LR107 (hht1/hht1). Genomic NcoI sites are marked by down arrows. Location of the variant histone H3 gene, HHT1, is shown as a gray box, whereas the SAT1 cassette is shown as a black box. (B) Isolated genomic DNA from each indicated strain was digested with NcoI and Southern hybridized with an upstream probe (shown by the black bar in panel A). Expected results for the correct transformants were obtained. (C) Growth assays were performed by growing SC5314, LR107, LR108, and the H3VCTG complemented strain LR109 in YPDU liquid medium until the stationary phase was reached. Optical density was measured by using Varioskan Flash (Thermo Scientific). The data underlying this figure can be found in CTG-V5) and LR149 (H3VCTG-V5 hht21/hht21) grown as yeast; the expected size of Hht1 is approximately 17 kDa. The parental strain SN148 was used as the untagged control, whereas PSTAIRE (approximately 34 kDa) was used as the loading control. Levels of histone H3 are normalized with the corresponding PSTAIRE levels, and values are indicated below each lane. (E) Wild-type (SC5314) and canonical histone H3 null mutant (LR155) strains were grown in YPD medium, and DIC images were taken at 60×. Scale bars: 2 μm. YPD, yeast peptone dextrose; YPDU, yeast peptone dextrose supplemented with uridine.(A) Structural schematic of the (TIF)Click here for additional data file.S4 Fighht1/hht1) grown in planktonic condition. (B) Comparison of gene expression profiles of wild-type SC5314 and variant histone H3 null mutants (hht1/hht1) grown as biofilm. (C) Comparison of common gene expression profiles between hht1/hht1 null mutant cells grown in planktonic condition and differentially expressed genes in biofilm mode from Nobile and colleagues [hht1/hht1 null mutant cells grown in biofilm condition and wild-type differentially expressed genes grown in biofilm conditions [CTG null mutants grown in planktonic conditions (blue circles) with differentially expressed genes in biofilm conditions  was compared with the previously published biofilm-induced microarray data of the wild type  Comparison of gene expression profiles of wild-type SC5314 and variant histone H3 null mutants (lleagues . (D) Simnditions . (E) Comcircles; ) in the circles; ). A tota(TIF)Click here for additional data file.S5 FigHHT1/HHT1), H3VCTG null mutant, LR108 (hht1/hht1), and the H3VCTG complemented strain LR109 were allowed to form biofilms on human urinary catheters for 48 hours at 37 °C. The catheter luminal surfaces were visualized by SEM. SEM, scanning electron microscopy.(A)Wild-type SC5314 ((TIF)Click here for additional data file.S6 Fighht1 null mutant and H3VCTG-V5/hht1 strains were spotted on Spider medium. (B) ChIP assays with anti-V5 antibodies were performed in the strain LR144 expressing H3VCTG-V5 and grown in planktonic or biofilm conditions. The enrichment of H3VCTG-V5 to the gene bodies of biofilm genes was compared in both planktonic and biofilm conditions. The data underlying this figure can be found in hht1/hht1 null mutant (LR107) grown in planktonic conditions was performed. The MNase digested DNA was precipitated and quantified by qPCR. The normalized Ct values represent the occupancy of nucleosomes at 2 previously known locations  at the promoter of the BMT7 gene. These 2 regions have been predicted to be nucleosome bound. Similarly, MNase ChIP was performed in LR143 (Hht21-V5) and LR144 (H3VCTG-V5) strains. A greater enrichment of H3VCTG-V5 compared to Hht21-V5 was observed at those 2 regions . The data underlying this figure can be found in (A) To examine the functionality of V5 epitope-tagged strains, wild-type, (TIF)Click here for additional data file.S7 FigCTG , and the H3VCTG complemented strain LR109 (hht1/hht1::HHT1) were grown in liquid YPD medium and then spotted on CM and CM-containing N-acetyl glucosamine agar plates and incubated for 2 to 3 days at 30 °C. (B) SC5314, LR108, H3VCTG complemented strain LR109 (hht1/hht1::HHT1), and canonical histone H3 mutant LR153 (hht2/hht2) were grown in liquid YPD and then spotted on plates containing the indicated media and incubated for 2 to 3 days at 37 °C. (C) Similarly, the extent of filamentation was monitored for the indicated strains by growing colonies from single cells on CM medium at 37 °C. (D) Comparative analysis of filamentation-specific genes after excluding genes common between biofilm and filamentation pathways. The Venn diagrams show the comparison of differentially expressed genes between wild-type SC5314 strain grown in filamentation-induced conditions with H3VCTG null mutant grown either in planktonic or biofilm-inducing conditions. An arrow shows the overlapping genes. CM, complete media; YPD, yeast peptone dextrose.(A) SC5314, null mutants of H3V(TIF)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Datahht1 as compared to the wild type during planktonic growth. hht1 null cells during planktonic growth, respectively. hht1 null mutants when grown in the planktonic condition. hht1 as compared to the wild type during biofilm growth. hht1 null cells in biofilm, respectively. hht1 null mutants grown in the biofilm condition. (XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.


APOBEC cytidine deaminases are the second-most prominent source of mutagenesis in sequenced tumors. Previous studies have proposed that APOBEC3B (A3B) is the major source of mutagenesis in breast cancer (BRCA). We show that APOBEC3A (A3A) is the only APOBEC whose expression correlates with APOBEC-induced mutation load and that A3A expression is responsible for cytidine deamination in multiple BRCA cell lines. Comparative analysis of A3A and A3B expression by qRT-PCR, RSEM-normalized RNA-seq, and unambiguous RNA-seq validated the use of RNA-seq to measure APOBEC expression, which indicates that A3A is the primary correlate with APOBEC-mutation load in primary BRCA tumors. We also demonstrate that A3A has >100-fold more cytidine deamination activity than A3B in the presence of cellular RNA, likely explaining why higher levels of A3B expression contributes less to mutagenesis in BRCA. Our findings identify A3A as a major source of cytidine deaminase activity in breast cancer cells and possibly a prominent contributor to the APOBEC mutation signature. APOBEC-signature mutations are over-represented in ~15% of all sequenced tumors and in many tumors constitute more than 50% of all mutations. Furthermore, APOBEC activity likely contributes to the occurrence of primary driver and late sub-clonal driver mutations, and mutations that contribute to metastasis and resistance to chemotherapeutics in cancer. To clarify the roles of individual APOBEC family members to cancer mutagenesis, we compared the expression of each APOBEC3 gene in a panel of breast cancer cell lines to the corresponding amount of APOBEC-induced mutation, finding that only A3A positively correlated. We found that A3A provides a significant amount of the cell’s total cytidine deamination activity in breast cancer cell lines. We also show that unlike other APOBECs, A3A is not inhibited by RNA, which partly explains its disproportionate contribution to cellular APOBEC activity. Further bioinformatics analysis determined that A3A expression is a major correlate with APOBEC-induced mutagenesis in primary tumors from multiple cancer types. This establishes A3A as a likely contributor to cancer mutagenesis. PIK3CA mutations [CW trinucleotide motifs  within single stranded (ss) DNA [CW sequences [kataegis) [Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) cytidine deaminases are a major cause of mutation in approximately 15% of sequenced human tumors . While tutations , and conutations . In addiutations –10. APOB(ss) DNA . Consequequences , 12, whiequences –15, highataegis) , 12, andataegis) , all of ataegis) , 17–21, CW motif that is deaminated by the remaining seven enzymes [Identifying the specific cytidine deaminases responsible for cancer mutagenesis and the relative contribution of each APOBEC to genomic instability in different cancer types is an ongoing area of research with many clinical implications. However, significant homology and functional redundancy between the 11 APOBEC enzymes encoded by the human genome has hindered such efforts. Nine APOBECs display catalytic activity towards ssDNA and only APOBEC3G (A3G) and Activation-Induced Cytidine Deaminase (AID) deaminate cytidines in sequences differing from the canonical T enzymes . Recent  enzymes , 23. Of  enzymes –26. A3B  enzymes . In addi enzymes .APOBEC3B gene [APOBEC3B deletion polymorphism revealed that these tumors frequently contained APOBEC-induced mutations and often at higher abundance than tumors from non-carriers [APOBEC3B is common in human BRCA [Approximately 22% of humans carry a deletion polymorphism resulting in loss of the C3B gene . Sequenccarriers . Additioman BRCA , which rman BRCA , providiman BRCA . Alternaman BRCA , 32. In man BRCA  as well man BRCA , suggestHere, we provide evidence that indicates A3A may be a major cause of APOBEC-induced mutation in BRCA. Using analysis of mutations from 28 whole exome sequenced BRCA cell lines and the corresponding expression of APOBEC3 family members determined by qRT-PCR, we show that only A3A expression was elevated in APOBEC-mutated lines compared to non-APOBEC-mutated lines and that the overall abundance of APOBEC-induced mutations linearly correlates with A3A expression. We also found that an A3B-targetting shRNA, which was used throughout the seminal study showing that A3B activity is responsible for cytidine deamination activity, accumulation of dU, and C-to-T mutations in BRCA , also deTo identify APOBECs besides A3B that produce mutagenesis in BRCA, we evaluated the mutation signatures of a panel of BRCA cell lines to identify A3B null lines that are APOBEC mutated. We found that autologous BRCA cell lines SKBR3 and AU565 display a strong APOBEC mutation signature, as indicated by a high proportion of mutations involving C to T and C to G substitutions in TCW trinucleotide sequences . This siAPOBEC3A gene introduced by CRISPR/Cas9 editing  or a 5’ Cy5-labeled oligonucleotide that self-anneals into an 11 bp stem and 4 nucleotide loop hairpin structure containing a single TCA trinucleotide in the loop. The sequence of this hairpin substrate was previously identified as a hotspot of APOBEC mutagenesis in breast cancers , likely  editing . Both su editing . To conf editing . Extract editing . Similar editing . To dete editing , indicatGiven our results that cytidine deaminase activity in APOBEC-mutated cell lines SKBR3 and AU565 was primarily A3A-dependent, we decided to examine the possibility that A3A may play a greater role in APOBEC-catalyzed mutagenesis than previously thought. We began by classifying a panel of 28 BRCA cell lines as either APOBEC-mutagenized or not-APOBEC-mutagenized based on an over-representation of the APOBEC mutation signature as previously described . Non-APOWe quantified the contribution of A3A and A3B to cytidine deaminase activity in the APOBEC-mutated cell lines BT474, CAMA-1, and MDA-MB-453, which respectively express 243-, 182-, and 269-fold higher A3B than A3A by qRT-PCR  and contin vitro cytidine deaminase assay using BT474, CAMA-1, and MDA-MB-453 extracts that were treated with RNaseA to remove residual RNA. As expected, addition of RNaseA greatly increased overall cytidine deaminase activity in extracts from all three cell lines. Moreover, this increase was completely abolished by shRNA mediated knockdown of A3B  of A3B may function as a negative regulator of A3B activity in human cells . While A E. coli , A3A fro E. coli , the dia E. coli , and the E. coli , a minim E. coli , is also E. coli . The homBecause our data implicates A3A as a major source of cytidine deaminase activity and mutagenesis in many BRCA cell lines, we sought to determine whether A3A also contributed to the mutagenesis of primary BRCA tumors. Prior analyses had observed a positive correlation for both A3A and A3B mRNA expression levels with the number of APOBEC-induced mutations in 483 primary breast cancers characterized by TCGA . HoweverWe note that the strength of the correlation between A3A and APOBEC mutagenesis in primary BRCA tumors is significantly less than observed among BRCA cell lines. Multiple differences in the sample sets may explain this discrepancy, including the existence of modulators of APOBEC-driven mutagenesis and episodic mutation events that are specific to the tumor environment. Furthermore, tumors samples contain subpopulations of tumors cells and contamination with non-tumor cells that increases cellular heterogeneity, which adversely affects correlation analyses for RNA-seq expression data. Alternatively, failure of APOBEC mRNA expression levels to accurately predict the protein level of these enzymes or the presence of additional factors beyond A3A expression level that influence the amount of APOBEC-induce mutation could also weaken the correlation of A3A mRNA level with mutation in primary tumors. Our initial analysis of A3A and A3B activity in BRCA cell lines  supportsHPRT1 from 410 bladder cancers (BLCA), 197 cervical cancers (CESC), and 549 head and neck cancers (HNSC) from the TCGA. The comparison of A3A and A3B expression to the abundance of APOBEC-induced mutations showed significant correlation of both APOBECs with mutagenesis among all assessed tumors  , multiplAPOBEC3 locus associate with reduced A3B expression and RTCA mutagenesis in pan-cancer analyses [APOBEC3B region and modulation of the APOBEC mutation signature in cancers provide evidence for its potential as a cancer mutator and are validated by experimental evidence indicating that reduction of A3B can be anti-mutagenic [et al. manuscript [Modest correlation of A3B expression with the amount of APOBEC-induced mutations in BRCA, BLCA, CESC, and HSNC cancers indicates that along with A3A, A3B likely contributes to the mutagenesis of some human tumors. This is supported by the analysis of the -2 nucleotide preference in whole genome sequenced tumors, which identified a subset of BRCA tumors that were enriched for mutations within the A3B preferred RTCA motif . Howeveranalyses . These sutagenic  and receutagenic . The disnuscript  to decreOur work suggests that A3A may be a better candidate than A3H haplotype I in causing the APOBEC mutation signature in A3B deleted breast cancers. In contrast to A3A, both A3B and A3H haplotype I , 45 are A3A expression significantly correlates with APOBEC-signature mutations in BRCA cell lines and primary tumors, but tumors often significantly deviate from this trend. One possible explanation for variation in the observed frequency of APOBEC signature mutations is the existence of modifiers of APOBEC activity that differ among tumor samples. Identification and characterization of the mechanisms that control APOBEC-activity in tumors and determining the relative contributions of A3A, A3B, and A3H in tumors of different cancer types will be key to facilitating researchers’ and clinicians’ efforts to develop treatments for APOBEC-mutagenized tumors. Our results that RNA binding dramatically inhibits A3B, but not A3A, activity highlights a potential role for RNA binding as key APOBEC regulator. Future studies are needed to determine if A3B is similarly inhibited by RNA in all cellular contexts, or if cellular factors like post-translational modification, alternative splicing, or cell type-specific protein interactions allow A3B to be more active against genomic DNA in the presence of RNA. Other mechanisms that regulate the mutagenic capacity of APOBECs and protect the genome of human cells also likely exist. In addition to its inhibition by RNA, PKA phosphorylation of A3B at residue T214 limits A3B’s ability to bind ssDNA and deaminate the substrate , likely https://data.broadinstitute.org/ccle/CCLE_RNAseq_rsem_transcripts_tpm_20180929.txt.gz) and the Broad Institute Genomic Data Analysis Center , respectively. Raw RNA-seq reads for unambiguous RNA-seq analysis were obtained from the NCBI Short Read Archive for BRCA cell lines in the GEO series “Transcriptional profiling of a breast cancer cell line panel using RNAseq technology” (GEO accession number: GSE48213). Similar raw RNA-seq reads for TCGA sequenced primary BRCA tumors were downloaded as BAM Slices from the controlled-access portion of the NCI Genomics Data Commons . Lists of mutations identified in BRCA cell lines were downloaded from the Cancer Cell Line Encyclopedia (https://data.broadinstitute.org/ccle/CCLE_DepMap_18Q1_maf_20180207.txt) and the COSMIC database (CosmicCLP_MutantExport.tsv.gz from https://cancer.sanger.ac.uk/cell_lines/download), while primary tumor mutations were obtained from http://gdac.broadinstitute.org/runs/stddata__2016_01_28/data/BRCA/20160128/gdac.broadinstitute.org_BRCA.Mutation_Packager_Oncotated_Calls.Level_3.2016012800.0.0.tar.gz; http://gdac.broadinstitute.org/runs/stddata__2016_01_28/data/BLCA/20160128/gdac.broadinstitute.org_BLCA.Mutation_Packager_Oncotated_Calls.Level_3.2016012800.0.0.tar.gz; http://gdac.broadinstitute.org/runs/stddata__2016_01_28/data/CESC/20160128/gdac.broadinstitute.org_CESC.Mutation_Packager_Oncotated_Calls.Level_3.2016012800.0.0.tar.gz; and http://gdac.broadinstitute.org/runs/stddata__2016_01_28/data/HNSC/20160128/gdac.broadinstitute.org_HNSC.Mutation_Packager_Oncotated_Calls.Level_3.2016012800.0.0.tar.gz.RSEM RNA-seq expression values for BRCA cell lines and TCGA sequenced primary tumors were obtained from the Cancer Cell Line Encyclopedia  to create lentiviral shRNA expression plasmids pTM-66 (A3A-shRNA), pTM-382 (A3B-shRNA-1), pTM-383 (A3B-shRNA-2), and pTM-238 (non-targeting shRNA). Plasmids psPAX2 , pMD2.G , and individual lentiviral shRNA plasmids were co-transfected into HEK293T cells for production of lentivirus. All the shRNA target sequences and oligonucleotides used for cloning are listed in Oligonucleotides corresponding to five A3A-targeting gRNA sequences were cloned into the BsaI site of pGL3-U6-sgRNA-PGK-puromycin  to create plasmids expressing sgRNAs targeting A3A. All the sgRNA target sequences and oligonucleotides used to create these plasmids are listed in the 23VN and 3.5 μM random hexamers, 0.5 mM dNTPs, 1x ProtoScript II Buffer, 10 mM DTT, 400U Protoscript II RT (NEB), and 16 U RNase Inhibitor  in a total volume of 40 μL. Reactions mixtures were incubated at 42°C for 1 hr, then 65°C for 20min and cDNA was stored at -20°C. qPCR was performed using 2 μL template with 1x SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and 0.2 μM primers  on a Bio-Rad CFX96. Reactions were incubated at 95°C for 5 mins, then run for 40 cycles of 95°C for 10 s and 62.5°C for 1 min. Analysis of melt curves indicated singular products for all quantified products. Additionally, the qRT-PCR products were separated on an agarose gel to verify the products were of the expected sizes and lacked non-specific amplification  and treated with DNAse I (ThermoFisher). cDNA was generated by combining 2 μg of denatured RNA from each sample with a reaction mixture (2.5 μM oligo dTfication . The idefication . In orde23VN as a primer. A3H was amplified utilizing KOD hot start DNA polymerase (MilliporeSigma) and a nested PCR approach. Products from 34 cycles of PCR with primers oTM-885 and oTM-883 were column purified and amplified for an additional 30 cycles with oTM-196 and oTM-884. The products were purified via PEG precipitation, cloned into pCR-Blunt, and Sanger sequenced with oTM-196 and oTM-884. Haplotypes were assigned as in [cDNA was generated as described under the methods for qRT-PCR, except for the use of 5 μM oligo dTed as in .10 transformed qRT-PCR values, RSEM normalized RNA-seq values, or normalized unambiguous RNA-seq read values were compared to Log10 transformed values of the minimum estimate of APOBEC mutations. The minimum estimate of APOBEC-induced mutations in each sample was defined as the number of TCW to TTW and TCW to TGW (including the complementary mutations WGA to WAA and WGA to WCA) mutations in excess of what would be expected by random mutagenesis. Samples without a statistical over-representation of APOBEC-induced mutations display a minimum estimate of APOBEC-induced mutations equal to zero. Mutations in BRCA cell lines (obtained from the Cancer Cell Line Encyclopedia and COSMIC Cell Line Project) and TCGA-sequenced primary BRCA tumors were also de-convoluted into the COSMIC mutation signatures using the Mutational Patterns module in R [10 transformed to compare to A3A and A3B expression.Mutation lists for BRCA cell lines (from the Cancer Cell Line Encyclopedia and COSMIC) and TCGA-BRCA tumors (from the Broad GDAC) where subjected to statistical analyses similar to  to deterule in R . The numPlasmid pLenti CMV Puro DEST  was modified in a series of cloning steps to create pTM-637, a lentiviral destination vector suitable for gateway cloning. pTM-637 has a modified CMV promoter for reduced expression and contains a pair of TETO sequences, which allows for doxycycline-induced expression in cells expressing TETR, and the CMV promoter and gateway module is orientated opposite the 5’ UTR. These attributes of pTM-637 prevent the expression of transgenes cloned into gateway module from being expressed in HEK-293T-TETR during lentiviral production, which was key for consistent production of high-titer lentivirus for the APOBEC expression vectors described below. Synthetic DNA fragments corresponding to the coding sequence (CDS) of A3A transcript 1 (NCBI accession NM_145699.4) with A3A intron 3, a C-terminal flexible linker and Strep-tag (A3A-geneblock) and the CDS of A3B transcript 1 (NCBI accession NM_004900.5) with A3B intron 6, a C-terminal flexible linker and Strep-tag (A3B-geneblock) were amplified with phosphorylated oligos oTM-58 and oTM-59, digested with EcoRV, and cloned into the HincII and EcoRV sites of pENTR1A no ccDB  to produce plasmids pTM-216 and pTM-218, respectively. LR-Clonase (ThermoFisher) reactions between pTM-637 and pTM-216 or pTM-218 produced lentiviral vectors pTM-664 (A3A expression) and pTM-666 (A3B expression).Bacillus phage PBS2 was cloned into the EcoRV site of pENTR1A no ccDB to create pTM-775. A LR-Clonase reactions between pTM-775 and pLenti CMV Puro DEST or pLenti PGK Hygro DEST  was used to create lentiviral vectors for UGI expression pTM-601 and pTM-553, respectively.A synthetic DNA sequence encoding human-codon optimized Uracil Glycosylase inhibitor (UGI) from Sequences of synthetic DNA sequences used for A3A, A3B, and UGI cloning are provided in , GenBank6 cells in log-phase growth. Prior to lysate generation, cells were washed twice with PBS, flash frozen in liquid nitrogen, and stored at -80°C. Lysates were generated by resuspending cells in 150 μL of lysis buffer , and sonicating for 30 sec on setting “1” using a Misonix Sonicator 3000. Lysates were clarified by centrifugation at 13,000 g for 10 min at 4°C. Protein concentrations of lysates were measured by BCA assay and equal amounts of protein in lysates used for deaminase assays were confirmed by immunoblots as described below.Whole-cell lysates were produced from approximately 5x106 cells in 10 cm dishes in DMEM + 10% FBS + 2 μg/ml doxycycline. Cells were harvested at a density close to 9x106 cells per 10 cm, which corresponded to approximately 84 hours of growth, washed twice with PBS, and frozen in liquid nitrogen.The cell populations used for purification of A3A and A3B proteins were created by sequential transduction of HEK-293T cells with pLenti CMV TetR Blast , pTM-553 (UGI), and either pTM-664 (A3A) or pTM-666 (A3B). Induction of A3A and A3B expression was achieved by seeding 1X102 and Benzonase (MilliporeSigma) were added to 1 mM and 40 U/mL respectively and lysates incubated at 4°C for 30 min. For A3B, RNAseA (Qiagen) was added to 6.6 μg/ml and the lysate was incubated 10 hours at 4°C, which was required to prevent RNA from co-purifying with A3B. To increase binding of the Strep-tagged protein and decrease non-specific protein binding during affinity purification, Tris-HCl pH 8, NaCl, and avidin were added to 15 mM, 50 mM, and 33 μg/mL respectively. Insoluble material was removed via centrifugation at 20,000 RCF for 30 mins and filtration through a 0.2 μM PES membrane.Lysates for A3A and A3B purification were similarly prepared from cell pellets harvested from approximately 60- and 128–10 cm dishes, respectively. Cells lysates were prepared by addition of 30 mL of M-PER buffer (ThermoFisher) supplemented with 1 mM DTT and 2X Protease Inhibitor cocktail  and incubation at 4°C for 30 mins with gentle agitation. Lysates were subjected to three rounds of sonication on ice for 15 s on setting “5.5” using a Misonix Sonicator 3000. In order to reduce the nucleic acid content, MgClA3A was purified as follows. A3A-Strep-tag was batch bound to 1 mL of Strep-Tactin Superflow Plus (Qiagen) at 4°C for 1 hr. The lysate was centrifuged at 400 RCF for 5 min at 4°C for and the supernatant removed from the Strep-Tactin resin. The Strep-Tactin resin was resuspended in 20 mL of buffer-1 (30 mM Tris-HCl (pH 7.9 at 25°C), 5% glycerol, 150 mM NaCl, 0.05% triton-100, 1 mM DTT) and transferred to a 1x20 cm glass econo-column. The Strep-Tactin resin was washed with an additional 40 mL of buffer-1 and A3A-strep-tag was eluted with buffer-1 containing 20 mM d-desthiobiotin. Fractions containing A3A-strep-tag were pooled and loaded to 1 mL Enrich-Q column (Bio-Rad) in buffer-1, washed with buffer-2 (30 mM Tris-HCl (pH 7.5 at 25°C), 5% glycerol, 150 mM NaCl, 0.05% triton-100, 1 mM DTT) and eluted utilizing a 20 mL linear gradient to 100% buffer-3 (30 mM Tris-HCl (pH 7.5 at 25°C), 5% glycerol, 1200 mM NaCl, 0.05% triton-100, 1 mM DTT). Purified A3A-strep-tag was extensively dialyzed in buffer-4 (30 mM Tris-HCl (pH 7.5 at 25°C), 10% glycerol, 150 mM NaCl, 0.05% triton-100, 1 mM DTT), frozen as aliquots in liquid nitrogen, and stored at -80°C.A3B was purified as follows. A3B-Strep-tag containing lysate was loaded to a 1 mL Streptrap HP column  in buffer-5 (30 mM Tris-HCl (pH 7.9 at 25°C), 5% glycerol, 150 mM NaCl, 0.1% CHAPS, 1 mM DTT). The Streptrap HP column was then washed with 5 mL of buffer-5, 10 mL of buffer-6 (30 mM Tris-HCl (pH 7.9 at 25°C), 5% glycerol, 1000 mM NaCl, 0.1% CHAPS, 1 mM DTT) and 5 mL of buffer-7 (30 mM Tris-HCl (pH 7.9 at 25°C), 5% glycerol, 50 mM NaCl, 0.1% CHAPS, 1 mM DTT). A3B-strep-tag was eluted with buffer-7 containing 20 mM d-desthiobiotin. Approximately 6 mL of A3B-strep-tag containing fractions pooled and loaded to 1 mL Enrich-Q column (Bio-Rad) in buffer-1, washed with buffer-8 (30 mM Tris-HCl (pH 7.5 at 25°C), 5% glycerol, 50 mM NaCl, 0.1% CHAPS, 1 mM DTT) and eluted utilizing a 20 mL linear gradient to 100% buffer-9 (30 mM Tris-HCl (pH 7.5 at 25°C), 5% glycerol, 1200 mM NaCl, 0.1% CHAPS, 1 mM DTT) Purified A3B-strep-tag was extensively dialyzed in buffer-4 (30 mM Tris-HCl (pH 7.5 at 25°C), 10% glycerol, 150 mM NaCl, 0.05% CHAPS, 1 mM DTT), frozen as aliquots in liquid nitrogen, and stored at -80°C.TTCAATTGCAAAATTT*G*T*T. In ATCAATTGCAAAATTT*G*T*T . These substrates contain a YTCA, the A3A preferred motif or RTCA, the A3B preferred motif respectively. All other experiments assessing APOBEC cytidine deaminase activity on a hairpin substrate used only oTM-814. Deaminase activity assays with purified APOBEC enzymes , 0.25 μM oligonucleotide substrate, 20 mM Tris HCl pH7.5, 1 mM DTT, and 1 mM EDTA), in a 20 μL volume were incubated for 30 minutes (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer  and incubation at 95°C for 10 min. Deaminase activity assays with whole-cell lysates , in a 20 μL volume were incubated for 24 hrs (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer . Reactions were incubated at 95°C for 10 min and placed on ice prior to electrophoresis. Deaminase activity assays were separated on pre-warmed 15% polyacrylamide gels with 7.9 M urea (7 cm) in 1x TBE buffer at 15 W for 15 min. Gels were imaged on a BioRad Chemidoc using the Cy5 setting. Percent substrate cleaved was determined by quantification of the intensity of the substrate and cytidine deamination cleavage product using BioRad’s Image Lab software version 5.2.Deaminase activity assays were performed with linear oligonucleotide oTM-910, /5Cy5/T*T* T*GTAGATGTAGATGTAGATTCAGTAGTAGAGTATGTAGT* A*T*T* T, and hairpin forming oligonucleotide substrates hairpin forming oligonucleotide substrates oTM-814, /5Cy5/TTTTATTTTGCAATTGNuclear and cytoplasmic RNA was prepared from 20–10 cm dishes of MDA-MB-453 cells. After harvesting the cells using trypsin, the cells were pelleted and washed twice with cold PBS. All subsequent steps were performed at 4°C. The cell pellet was resuspended in 10 mL of buffer CF  containing 25 μg/mL digitonin (MilliporeSigma) and incubated for 10 min with gentle agitation. This resulted in almost no cell lysis. The cells were pelleted by centrifugation at 2000 RCF for 10 minutes. The resulting cell pellet was resuspended in buffer CF supplemented with 0.5 mM NP-40 and incubated for 20 minutes with periodic gentle agitation. The nuclei were pelleted by centrifugation at 5000 RCF for 10 minutes. The supernatant (the cytoplasmic fraction) was removed, of which 1 mL was set aside for validation of fractionation by immunoblot. RNA was extracted from 7 mL of the cytoplasmic fraction by sequential phenol chloroform extraction, ethanol precipitation, and cleaned up using an RNA purification kit. The nuclei were washed 4 times in 40 mL of buffer CF. After the fourth wash, 10 percent of the total resuspended nuclei were transferred to a separate tube, pelleted, resuspended in buffer CF and lysed by sonication for validation of fractionation by immunoblot. The remaining nuclei were pelleted and used for RNA purification using an RNA purification kit.40 μg of protein from each extract or subcellular fraction were separated on pre-made Mini Protean TGX gels (Bio-Rad) and run at 180 V for 35 min in 1xTGS buffer . Gels were transferred via a Mini-Transblot Cell (Bio-Rad), at 55 V for 90 min at 4°C onto Immun-Blot PVDF membrane (BioRad) in 1xTransfer buffer  Methanol). Membranes were blocked in 2% ECL prime blocking reagent  in 1xTBST solution . Anti-GAPDH , anti-TBP , or anti-α-Tubulin  primary antibodies were used at 1:10000, 1:2000, and 1:20000 dilutions, respectively. A 1:20000 dilution of anti-rabbit secondary antibody  in 0.2% ECL Block in 1x TBST was used for all blots. Blots were developed with ECL Prime Western Blotting Detection Reagent  for 2 min before imaging on the BioRad Chemidoc MP Imaging System, using the Blots-Chemi setting.APOBEC3A and APOBEC3B genes  with the Geneious Alignment module in Geneious v10.2.2 using Global alignment with free end gaps and a 93% similarity cost matrix. Regions in the 5’ and 3’ of each transcript that contain low sequence identity were identified. Raw RNA-seq reads were aligned with BWA using default settings to a reference genome containing the APOBEC3A and APOBEC3B transcripts . Reads aligning to regions between nucleotides 1 and 350 in the APOBEC3A transcript and displaying a CIGARSTRING of 75M, 101M or 105M (indicating complete matching of the read to the reference) were counted for each BRCA cell line, increased by 0.5, and compared to the total number of reads within the corresponding sequencing library. Similarly, reads mapping to the 1 to 622 and 1371 to 1560 regions of the APOBEC3B transcript and displaying a CIGARSTRINGs of 75M, 101M or 105M were counted, augmented by 0.5, and normalized to the total number of reads per sample. These values were Log10 transformed for analyses comparing A3A and A3B expression to minimum estimates of APOBEC-induced mutation in BRCA cell lines. Unambiguous RNA-seq for A3A and A3B expression in BRCA tumors was conducted by counting reads mapping to the genomic coordinates chr22:38957522–38957715 and chr22:38962652–38963183 for A3A transcripts and chr22:38982399–38986411 and chr22:38992611–38992779 for A3B transcripts, augmenting the counts by 0.5, and dividing the resulting values by the number of reads mapping to the HPRT1 gene. As with unambiguous RNA-seq of BRCA cell lines, the normalized unambiguous RNA-seq values for BRCA tumors were Log10 transformed prior to statistical comparisons.To measure A3A and A3B expression in BRCA cell lines by RNA-seq without the complication of reads potentially mis-mapping between the highly homologous regions of the two genes, we conducted a pairwise alignment of the major Refseq transcripts for the S1 TextKey features include: EcoRV site used in cloning (bold green text), coding sequence , intronic sequence (red text), AgeI sites that allow for exchanging epitope tags (yellow highlighted text), stop codon . (A) A3A-geneblock (B) A3B-geneblock (C) UGI-geneblock.(DOCX)Click here for additional data file.S1 FigDifferent amounts of purified A3A or A3B were incubated with either a linear DNA substrate (L) or hairpin substrate (H) containing YTCA deamination motifs for 3 hrs. Following addition of UDG to convert APOBEC-induced dU to a heat-liable abasic site, samples were incubated at 95°C and the cleavage products indicative of APOBEC activity (P) were resolved from the unreacted substrate (S) on a polyacrylamide denaturing gel.(TIF)Click here for additional data file.S2 Fig(A) Clonal cell lines were obtained following transfection of AU565 cells with Cas9 expression vector and APOBEC3A targeting guide RNAs. Genomic DNA was isolated from each line and the APOBEC3A gene amplified to identify lines with detectable disruptions in the gene following gel electrophoresis. Wild type APOBEC3A alleles produce an expected 715bp PCR product. CRISPR/Cas9 edited AU565 contains three disrupted APOBEC3A alleles. (B) Sanger Sequencing of the purified PCR products in the A3A deletion line. All three modified alleles generate either a premature stop codon or frameshift for A3A isoforms A and B.(TIF)Click here for additional data file.S3 FigThe mutations utilized in (TIF)Click here for additional data file.S4 FigA3B-shRNA-1  reduced A3A mRNA expression in BT474 and AU565 derived cell populations. Newly derived A3A- and A3B-2-shRNAs are specific for their target genes and minimally impact expression of other APOBEC3 family members.(TIF)Click here for additional data file.S5 FigIn vitro cytidine deaminase assay conducted as (TIF)Click here for additional data file.S6 FigAPOBEC3A and APOBEC3B relative to HPRT1 measured by qRT-PCR in CAMA-1 or MDA-MB-453 cells and the corresponding cells transduced with lentiviral vectors to express scramble control, A3A-targeting, or A3B targeting shRNAs. CAMA-1 cells were also transduced with either vector-only control or UGI expression vectors. (C) In vitro cytidine deaminase assay (conducted similarly to (A) The mutation profile of CAMA-1 and MDA-MB-453 cells. (B) mRNA expression level of (TIF)Click here for additional data file.S7 FigWhole cell extracts were generated from 10 BRCA cell lines  and either untreated or treated with RNAseA to remove RNA from the extracts. These extracts were incubated with our hairpin oligonucleotide substrate containing an YTCA deamination target sequence for 24 hrs. Three independent assays were quantified and the resulting average activities were plotted against the average mRNA expression level of A3A and A3B measured by qRT-PCR. Error bars indicate the standard deviation in the cytidine deaminase activity measurements. Numerical values of the cytidine deaminase activity assays are provided in (TIF)Click here for additional data file.S8 Fig500 nM of A3A was incubated with 0.25 μM of hairpin DNA substrate containing an YTCA deamination target sequence for 30 minutes in the presence of 0, 100, and 2000 ng/μL RNA. Reactions were processed and quantified as in (TIF)Click here for additional data file.S9 Figin vitro cytidine deaminase assays containing 50 nM of either A3A or A3B and 0.25 μM of hairpin DNA substrate to assess the relative impact of RNA on A3A and A3B activity.MDA-MB-453 cells were fractionated into nuclei and cytoplasm and RNA was isolated separately from each compartment. (A) Western blot analysis for the nuclear protein TBP and cytoplasmic protein tubulin confirming effective separation of the nuclei from cytoplasm. Increasing amounts of nuclear RNA (B) or cytoplasmic RNA (C) were added to (TIF)Click here for additional data file.S10 FigThe blue arrows denote the five basic amino acids of A3H, which have been implicated in RNA binding. A3H-R175, R176 have been shown to be the major determinants of RNA-mediated inhibition of A3H activity . A3A has(TIF)Click here for additional data file.S11 FigComparison of RSEM normalized RNA-seq and unambiguous RNA-seq measurements of A3A and A3B expression among 23 BRCA cell lines or 1207 TCGA sequenced primary BRCA tumors was assessed by Pearson correlation test.(TIF)Click here for additional data file.S12 FigThe mutations utilized in (TIF)Click here for additional data file.S13 FigIn primary BRCA tumors and the subset of APOBEC-mutated BRCA tumors, A3A expression positively correlates with A3B expression. However, in tumors restricted to the lowest quartile of A3B expression, the number of APOBEC-induced mutations still correlates with A3A mRNA transcript level. No correlation is observed between A3B expression and APOBEC mutagenesis in BRCA tumors expressing A3A at low levels.(TIF)Click here for additional data file.S14 Fig(A) Endpoint qRT-PCR products for measuring the expression of individual APOBEC3 family members were separated based on size by agarose gel electrophoresis. All reactions produce a single product of the expected size for specific APOBEC3 family member assessed. (B) qRT-PCR products were cloned into the pCR-Blunt DNA vector and 9–10 independent clones were sequenced to verify the products were on-target. All sequences aligned to the target amplification region.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.