RESEARCH ARTICLE SUMMARY◥

CELL DEATH

A nuclease that mediates cell deathinduced by DNA damage andpoly(ADP-ribose) polymerase-1Yingfei Wang,* Ran An, George K. Umanah, Hyejin Park, Kalyani Nambiar,Stephen M. Eacker, BongWoo Kim, Lei Bao, Maged M. Harraz, Calvin Chang,Rong Chen, Jennifer E. Wang, Tae-In Kam, Jun Seop Jeong, Zhi Xie, Stewart Neifert,Jiang Qian, Shaida A. Andrabi, Seth Blackshaw, Heng Zhu, Hongjun Song, Guo-li Ming,Valina L. Dawson,* Ted M. Dawson*

INTRODUCTION: Poly(ADP-ribose) (PAR)polymerase-1 (PARP-1) is a nuclear enzymeresponding to oxidative stress and DNA dam-age. Excessive activation of PARP-1 causes anintrinsic caspase-independent cell death pro-gram designated parthanatos, whichoccurs in many organ systems be-cause of toxic or stressful insults,including ischemia-reperfusion in-jury after stroke and myocardialinfarction, inflammatory injury, re-active oxygen species–induced in-jury, glutamate excitotoxicity, andneurodegenerative diseases. Inhibi-tion or genetic deletion of PARP-1 isprofoundly protective against suchcellular injury in models of humandisease.

RATIONALE:Themolecularmech-anisms underlying parthanatosinvolve release of mitochondrialapoptosis-inducing factor (AIF) andits translocation to the nucleus,which results in chromatinolysis into20- to 50-kb largeDNA fragments—a commitment point for partha-natos. Because AIF itself has noobvious nuclease activity, we pro-pose that AIF recruits a nucleaseor a nuclease complex to the nucleusto trigger DNA cleavage and partha-natos. Although the endonucleaseG (EndoG) homolog may promoteDNA degradation in Caenorhabditiselegans through cooperating with

the AIF homolog, our group and others showedthat EndoG does not have an essential rolein PARP-dependent chromatinolysis and celldeath in mammals. Thus, the identity of thenuclease responsible for large DNA fragmen-

tation following AIF entry to the nucleusduring parthanatos has been a long-standingmystery.

RESULTS: Using two sequential unbiasedscreens, including a human protein array anda small interfering RNA screen, we discoveredthat macrophage migration inhibitory factor

(MIF) binds AIF and isrequired for parthanatos.Three-dimensional mod-eling ofMIF revealed thatthe MIF trimer has thesame core topology struc-ture as PD-D/E(X)K super-

family nucleases. In the presence of Mg2+ orCa2+, MIF has both 3′ exonuclease and endo-nuclease activity. It binds to 5′ unpaired basesof single-stranded DNA with stem loop struc-ture and cleaves its 3′ unpaired bases. Thesenuclease activities allow MIF to cleave genomicDNA into large fragments. Depletion of MIFmarkedly reduced chromatinolysis and celldeath induced byN-methyl-D-aspartate (NMDA)receptor–activated glutamate excitotoxicity in

primary neuronal cultures, DNAdamage caused by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)or focal stroke in mice. Mutatingkey amino acid residues in thePD-D/E(X)K nuclease domain ofMIF eliminated its nuclease acti-vity and prevented parthanatos.Disrupting the AIF and MIF in-teraction prevented the transloca-tion of MIF from the cytosol tothe nucleus and protected againstparthanatos. Moreover, depletionof MIF, disruption of AIF andMIFinteraction, and eliminating MIF’snuclease activity has long-lastinghistological and behavioral res-cue in the focal ischemia modelof stroke.

CONCLUSION: We identifiedMIF as a PARP-1–dependent AIF-associated nuclease that is requiredfor parthanatos. In response tooxidative stress or DNA damage,PARP-1 activation triggers AIF re-lease from the mitochondria. AIFthen recruits MIF to the nucleuswhere MIF cleaves genomic DNAinto large fragments and causescell death. Depletion of MIF, dis-ruption of AIF and MIF interac-tion, or blocking MIF nucleaseactivity inhibited chromatinolysisand parthanatos. Targeting MIFnuclease activity may offer an im-portant therapeutic opportunityfor a variety of disorders with ex-cessive PARP-1 activation.▪

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The list of author affiliations is available in thefull article online.*Corresponding author. Email: tdawson@jhmi.edu (T.M.D.); vdawson1@jhmi.edu(V.L.D.); yingfei.wang@utsouthwestern.edu (Y.W.)Cite this article as Y. Wang et al., Science354, aad6872 (2016). DOI: 10.1126/science.aad6872

Stressors lead to DNA damage, PARP-1 activation, and PAR forma-tion. PAR facilitates the release of AIF from mitochondria where it bindsMIF. This complex translocates to the nucleus to bind DNA; the result isDNA fragmentation and cell death. Interference with this cascade by pre-venting the formation of the AIF-MIF complex or by a nuclease-deficientMIF prevents DNA fragmentation and promotes cell survival.

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RESEARCH ARTICLE◥

CELL DEATH

A nuclease that mediates cell deathinduced by DNA damage andpoly(ADP-ribose) polymerase-1Yingfei Wang,1,2,3,4‡ Ran An,1,2,5 George K. Umanah,1,2 Hyejin Park,1,2,6

Kalyani Nambiar,1,2 Stephen M. Eacker,1,2,6 BongWoo Kim,3 Lei Bao,3

Maged M. Harraz,1,2,7 Calvin Chang,1 Rong Chen,1,2 Jennifer E. Wang,3 Tae-In Kam,1,2,6

Jun Seop Jeong,8,9 Zhi Xie,10* Stewart Neifert,1,2,6 Jiang Qian,10 Shaida A. Andrabi,1,2†Seth Blackshaw,7,9,10 Heng Zhu,8,9 Hongjun Song,1,2,7 Guo-li Ming,1,2,7

Valina L. Dawson,1,2,6,7,11‡ Ted M. Dawson1,2,6,7,8‡

Inhibition or genetic deletion of poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) isprotective against toxic insults in many organ systems. The molecular mechanismsunderlying PARP-1–dependent cell death involve release of mitochondrial apoptosis-inducing factor (AIF) and its translocation to the nucleus, which results in chromatinolysis.We identified macrophage migration inhibitory factor (MIF) as a PARP-1–dependentAIF-associated nuclease (PAAN). AIF was required for recruitment of MIF to the nucleus,where MIF cleaves genomic DNA into large fragments. Depletion of MIF, disruption ofthe AIF-MIF interaction, or mutation of glutamic acid at position 22 in the catalyticnuclease domain blocked MIF nuclease activity and inhibited chromatinolysis, cell deathinduced by glutamate excitotoxicity, and focal stroke. Inhibition of MIF’s nuclease activityis a potential therapeutic target for diseases caused by excessive PARP-1 activation.

Poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) is a nuclear enzyme that is activated byDNA damage and facilitates DNA repair (1).Excessive activation of PARP-1 causes an in-trinsic caspase-independent cell death pro-

gramdesignated parthanatos (2, 3), which occursafter toxic insults in many organ systems (4, 5),including ischemia-reperfusion injury after strokeand myocardial infarction; inflammatory injury;reactive oxygen species–induced injury; glutamateexcitotoxicity; and neurodegenerative diseases,such asParkinson’s disease andAlzheimer’s disease(2, 4, 6). Consistent with the idea that PARP-1 is akey cell-deathmediator, PARP inhibitors or geneticdeletion of PARP-1 protect against such cellularinjury in models of human disease (2, 4, 5, 7).During parthanatos, PAR causes release of

apoptosis-inducing factor (AIF) from the mito-chondria and its translocation to the nucleus, re-sulting in fragmentation of DNA into 20- to 50-kbfragments (2, 8–11). AIF itself has no obvious nucle-ase activity (2). Although it has been suggestedthat CED-3 protease suppressor (CPS)–6, an endo-nuclease G (EndoG) homolog in Caenorhabditis

elegans, cooperates with the worm AIF homolog(WAH-1) to promote DNA degradation (12), mam-malian EndoG does not seem to have an essentialrole in PARP-dependent chromatinolysis and celldeath (13, 14) and after transient focal cerebralischemia in mammals (15). Thus, the nuclease re-sponsible for the chromatinolysis during partha-natos is not known.

PARP-1–dependent cell deathrequires MIF

To confirm that the EndoG is dispensable forparthanatos, the CRISPR-Cas9 system was usedto deplete (knockout) EndoG from human neu-roblastoma cell line (SH-SY5Y) cells (fig. S1A).Wefound that knockout of EndoG failed to block N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)–induced parthanatos (fig. S1B) and large DNAfragmentation (fig. S1C); thus, EndoG is unlikelyto be the main contributor to PARP-1–dependentlarge DNA fragmentation and MNNG-inducedcell death (fig. S1) (13, 15).To identify a PARP-1–dependent AIF-associated

nuclease (PAAN), we probed protein chips con-

taining more than 16,000 and 5000 human re-combinant proteins in duplicate along with severalcontrol proteins (16)with recombinantmouseAIF.The 160 strongest interacting proteins were de-pleted with small interfering RNA (siRNA) in cul-tured human HeLa cells to screen for modifiersof parthanatos induced by MNNG (2, 9, 11) (Fig.1, A and B). We further tested whether depletionof thesepotentialAIF-interactingproteinsprovidedprotection equivalent to that of depletion of PARP-1 andwhether the proteins exhibited sequence andstructure similarity consistent with possible nucle-ase activity. Depletion of AIF interactor 18 wasas protective as depletion of PARP-1 (Fig. 1B).AIF interactor 18 is previously known under var-ious synonyms (glycosylation-inhibiting factor,phenylpyruvate tautomerase, L-dopachrome tau-tomerase, L-dopachrome isomerase), and it iscollectively known as macrophage migration in-hibitory factor (MIF orMMIF) (17, 18). Effects ofthree different short hairpin RNA (shRNA) con-structs against human and mouse MIF con-firmed that depletion of MIF protected againstparthanatos induced byMNNG toxicity inHeLacells or N-methyl-D-aspartate (NMDA) excitotox-icity in mouse primary cortical neurons (fig. S2,A to F). To rule out off-target effects from theshRNA, we prepared MIF constructs that wereresistant to shRNA1 (RshRNA1)and 3 (RshRNA3).Cells expressing these constructswere imperviousto effects of the shRNAs (fig. S2, G and H).MIF contains three PD-D/E(X)K superfamily

motifs that are found in many nucleases (19–21)(Fig. 1, C and D) and are highly conserved acrossmammalian species (fig. S3A). It also contains aCxxCxxHx(n)C zinc finger domain (Fig. 1C and fig.S3B), which is commonly found in DNA damage-response proteins (20). MIF exists as a trimer(22–24). The core PD-D/E(X)K topology structurein the MIF trimer consists of four b strands nextto two a strands (Fig. 1E and fig. S3, C to G, andsupplementary text), which is similar to those ofwell-characterized nucleases, including Eco RI,Eco RV, Exo III, and Pvu II (fig. S3, H to O, andsupplementary text). These sequence analysesand three-dimensional (3D) modeling resultsindicated that MIF belongs to the PD-D/E(X)Knuclease-like superfamily (25, 26).

MIF is a nuclease

To determine whetherMIF has nuclease activity,we incubated a plasmid c–promoterDNA (pcDNA)vector with recombinant human MIF. Super-coiled pcDNA was cleaved by MIF—but not byits nuclease-deficient mutant MIF E22Q [inwhich glutamine (Q) replaces glutamic acid (E)at position 22] identified in the nuclease assaysbelow—into an open circular form and, further,

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1Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. 2Department of Neurology, Johns HopkinsUniversity School of Medicine, Baltimore, MD 21205, USA. 3Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. 4Department of Neurology andNeurotherapeutics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. 5Department of Neurology of Huashan Hospital, State Key Laboratory of Medical Neurobiology,Fudan University, Shanghai 200032, China. 6Adrienne Helis Malvin Medical Research Foundation, New Orleans, LA 70130-2685, USA. 7Solomon H. Snyder Department of Neuroscience, JohnsHopkins University School of Medicine, Baltimore, MD 21205, USA. 8Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205,USA. 9Center for High-Throughput Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. 10Department of Ophthalmology, Johns Hopkins University School ofMedicine, Baltimore, MD 21205, USA. 11Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.*Present address: Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China. †Present address: Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL35294, USA. ‡Corresponding author. Email: tdawson@jhmi.edu (T.M.D.); vdawson1@jhmi.edu (V.L.D.); yingfei.wang@utsouthwestern.edu (Y.W.)

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to a linear form (Fig. 2A). Moreover, MIF cleavedhuman genomicDNA in a concentration- and time-dependent manner (fig. S4, A and B). Addition of10 mM Mg2+, 2 mM Ca2+, or 1 mM Mn2+ wasrequired for MIF nuclease activity (fig. S4C),consistent with the divalent cation concentra-tions required for in vitro activity of other similarnucleases (27). EDTA blocked MIF’s nucleaseactivity against human genomic DNA (Fig. 2B).In the absence of the divalent cation or with

the cation at 2 to 10 mM, MIF had no nucleaseactivity (fig. S4C). Addition of 200 mM Zn2+ pre-cipitated genomic DNA in the presence of MIF,whereas 2 mM Zn2+ had no effect. Na+ had noeffect onMIF’s nuclease activity (fig. S4C). Pulsed-field gel electrophoresis indicated thatMIF cleaveshuman genomic DNA into large fragments com-parable to those of DNA purified from HeLacells treated with MNNG (Fig. 2B, lane 8). De-pletion of MIF with shRNA prevented MNNG-

induced DNA cleavage, which was similar to theeffect of PARP inhibition by 3,4-dihydro-5[4-(1-piperindinyl)butoxy]-1(2H)-isoquinoline (DPQ) (Fig.2C). Because MIF has been reported to havetautomerase activity, we tested the effects of theMIF tautomerase inhibitor ISO-1 (28). ISO-1 failedto preventMNNG-inducedDNAdamage (Fig. 2C).Moreover, the MIF P2G (also known as the P1G)tautomerase mutant, which lacks tautomeraseactivity (29), had no effect on MIF’s nuclease

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Fig. 1. Identification of MIF as a key cell-death effector mediatingPARP-1–dependentcell death. (A) Strategy for identifying AIF-associatedproteins involved in PARP-1–dependent cell death. (B) siRNA-based PARP-1–dependent cell viability high-throughput screening in HeLa cells 24 hoursafterMNNG treatment (50 mM, 15min); n= 8.The experiments were repeatedin four independent tests ***P < 0.001, one-way ANOVA. (C) Schematicrepresentation of MIF’s PD-D/E(X)K domains. Single-letter abbreviationsfor the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F,Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg;S, Ser; T,Thr; V, Val; W,Trp; Y, Tyr; and X, any amino acid. (D) Alignment ofthe nuclease domain of human MIF and other nucleases. Arrows abovethe sequence indicate b strands and rectangles represent a helices.Amino acid residues that were mutated are indicated with an arrowand number (see Results). Nuclease and CxxCxxHx(n)C domains arehighlighted in green and pink, respectively. (E) Crystal structure of MIFtrimer (pdb:1GD0) (left) and MIF PD-D/E(x)K motif in trimer (right).

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activity (fig. S4D). These data indicate that MIFis a nuclease that functions in PARP-1–dependentDNA fragmentation.To identify amino acid residues critical forMIF’s

nuclease activity, wemutated key aspartate, gluta-mate, and proline residues within the PD-D/E(X)Kdomains of MIF. E22Q, but not Glu replaced byAsp (E22D), inhibited MIF’s nuclease activity,whereas replacement with Ala (E22A) partiallyreduced MIF’s nuclease activity (Fig. 2D; fig. S4,E to H; and supplementary text). Thus, this glu-tamic acid residue (E22) in the first a helix ofMIF is critical for its nuclease activity, which isconsistent with reports that this glutamic acid inthe firsta helix ofmany exonuclease-endonuclease-phosphatase (EEP) domain superfamily nucleasesis highly conserved and that it is the active sitefor nuclease activity (25, 26). MIF has both oxido-reductase and tautomerase activities (28, 30, 31).MIF active site mutants E22Q and E22A had noeffect on MIF’s oxidoreductase or tautomeraseactivities (fig. S5, A and B, and supplementarytext). The lack of effect indicated that MIF nu-clease activity is independent of its oxidoreductaseand tautomerase activities. Moreover, MIF’s pro-tein conformationwas unaffected by the E22QandE22Amutations as determined by far-ultraviolet(UV) circular dichroism (CD) and near UV CDspectroscopy (fig. S5, C to M, and supplementarytext). The purity of MIF proteins was confirmedby Coomassie blue staining, fast protein liquidchromatography (FPLC), and mass spectrometry(MS) assays (fig. S4G and fig. S5, C and D; Mate-rials and methods; and supplementary text). Noadventitious nuclease contaminationwas observed.

MIF preferentially binds to stem-loopsingle-stranded DNA

Todetermine the characteristics ofDNA sequencesbound byMIF in an unbiasedmanner, HeLa cellswere treated with dimethyl sulfoxide (DMSO) orMNNG (50 mM, 15 min), followed by anti-MIFchromatin immunoprecipitation (ChIP) assays anddeep sequencing (fig. S6 and supplementary text).

We used the multiple Em for motif elicitation(MEME) program, which performs comprehen-sive motif analysis on large sets of nucleotide se-quences (32), and we identified two classes ofMIF-binding motifs (Fig. 3A). The first class (se-quences 1 through 3) represents a highly relatedfamily of overlapping sequences (Fig. 3A and fig.S7A). The sequence features of this family are bestcaptured in sequence 1 with 30 nucleotides anddesignated PS30, the most statistically significantmotif identified, as determined by theMEMEpro-gram (E-value= 1.4e-051) (Fig. 3A and fig. S7A). Thesecond class identified was a poly(A) sequence.We performed 3Dmodeling to determine likely

points of DNA interaction withMIF’s PD-D/E(X)Kmotif. Within the PD-D/E(X)K motif, P16 andD17 onMIF are predicted to be positioned close todouble-stranded DNA (dsDNA), whereas E22 isclose to ssDNA, indicatingMIFmight bind single-stranded DNA (ssDNA), dsDNA, or both (fig. S7B).We examined both single-stranded and double-stranded forms of MIF DNA substrates for MIFbinding and cleavage specificity. We synthesizedthe ssPS30 sequence with a 5′ biotin label and sub-jected it to an electrophoretic mobility shift assay(EMSA) (fig. S7C).MIFbound to the biotin-labeledssPS30, forming onemajor complex in the presenceof 10 mMMg2+ (fig. S7C), which was completelydisrupted by the addition of excess unlabeledDNA substrate (PS30) or a polyclonal antibody toMIF (fig. S7C). MIF E22Q, E22A, P16A, P17A, andP17Qmutants still formedMIF/ssPS30 complexes(fig. S7C).Because ssPS30 has the potential to form a stem-

loop structure with unpaired bases at the 5′ and 3′ends, we tested whether MIF binds to ssDNAwithsequence or structure specificity.We used 5′ biotin-labeled ssPS30 and sequence-related substrateswith different structures created by removing un-paired bases at the 5′ end, 3′ end, or both 5′ and 3′ends, or by eliminating the stem loop in the EMSA(Fig. 3B and fig. S8). Completely removing the 3′unpaired bases (5′bLF) had no effect on the DNA-MIF complex formation (Fig. 3B). In contrast,

removing the 5′ unpaired bases (5′bRF) reduced,but did not abolish DNA-MIF binding. Similarresults are observed when both 5′ and 3′ unpairedbases were removed (5′bSL). ThusMIF appears tomainly bind to 5′ unpaired bases in ssDNA withstem-loop structures. We also used a poly(A) se-quence that has no stem loop (5′bPA30) and ashort poly(A) sequence at the 5′ end of a stem-loopstructure (5′b3F1) as the substrates. MIF failed tobind to 5′bPA30 but did bind to 5′b3F1. Theseresults indicated that a stem loop is required forMIF-ssDNA binding (Fig. 3B and fig. S8).We alsotested a substrate unrelated in sequence but thathad a stem loop–like structure (5′bL3). MIF boundweakly to 5′bL3. But its binding efficiency wasmuch lower than that of 5′bPS30. These data in-dicate that MIF preferentially binds to ssDNAwith a stem loop and that its specificity is notentirely determined by the sequence.We also testedwhether MIF bound to dsDNA with PS30; poly(A);substrates with sequence similarity to PS30 (5′bPS30,5′bSL, 5′bLF, 5′bRF, 5′bPA30, and 5′bPA5E); andothers with nonrelated sequences (PCS and 5′bL3)(Fig. 3B and fig. S8). MIF failed to bind to any ofthese double-stranded substrates (Fig. 3B).

MIF cleaves 3′ unpaired bases ofstem-loop ssDNA

To determine whether MIF cleaves ssDNA ordsDNA,we added 35 randomnucleotides to boththe 5′ and 3′ ends of the PS30 DNA binding motif(designated PS100) and, under identical conditions,measured cleavage of ssDNA (ssPS100) or dsDNA(dsPS100) .MIF cleaved ssPS100 and its complemen-tary strand ssPS100R, but not dsPS100 (fig. S9, Aand B). The MIF DNA binding motif identifiedby ChIP sequencing (PS30) appeared to be suffi-cient forMIF cleavage becauseMIF cleaved ssPS30

in a concentration-dependent manner (fig. S9C).MIF cleavage of ssPS30 required Mg2+, and MIFE22Q and E22A mutations blocked the cleavageof ssPS30 (fig. S9D). MIF cleaved ssPS30 in a time-dependent manner with a t1/2 of 12 min, and itcleaved ssPS30 in a concentration-dependent

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Fig. 2. MIF is a nuclease that cleaves genomic DNA. (A) In vitro MIF (2 mM) nuclease assay with pcDNA as substrate. (B) In vitro pulsed-field gelelectrophoresisMIF (4 mM)nuclease assaywith human genomicDNA (hgDNA)as a substrate in buffer containingMg2+ (10mM)with or without EDTA (50mM) orCa2+ (2 mM) with or without EDTA (25mM). (C) Pulsed-field gel electrophoresis assay of MNNG-induced DNA damage in MIF-deficient HeLa cells and wild-type(WT)HeLa cells treatedwith orwithout DPQ (30 mM) or ISO-1 (100 mM). NT/MNNG (nontargeting shRNA/MNNG). (D) Nuclease assayofMIFWTandmutants (4 mM)using human genomic DNA as the substrate.

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mannerwith an affinity for substrate (Km) of 2 mMand a maximum initial velocity (Vmax) of 41.7 nM/min (fig. S9, E to G). These kinetic properties aresimilar to those of other PD-D/E(X)K nucleases,such as Eco RI (27, 33). MIF also cleaved dsPS30

(fig. S9H), but required at least 4 times as highMIF concentrations and a four-fifths reductionin substrate concentration (compare lane 2 offig. S9C to lane 2 of fig. S9H). MIF failed to cleaveits related sequence dsRF, or the nonrelated se-quence dsL3 (fig. S9H). MIF’s preference forssDNA is consistent with the 3Dmodel of ssDNAbinding toMIF’s active site (fig. S7B) and ourMIF-DNA binding assays (Fig. 3B). In the presenceof AIF, MIF more efficiently cleaved genomicDNA and dsPS30 (fig. S10, A to C), which mightbe because of the observation that AIF enhancedMIF binding to dsDNA (fig. S10D).To determine whether MIF has sequence- or

structure-specific endonuclease or exonuclease ac-tivity, we synthesized a series of variants labeled atthe 5′ and 3′ ends with biotin, on the basis of thesecondary structure of the DNA substrate ssPS30,and measured their cleavage by MIF (Fig. 3C andfig. S8). MIF had 3′ exonuclease activity and pref-erentially recognized and degraded unpaired basesat the 3′ end of ssPS30. This was blocked by biotinmodification at the 3′ end (lanes 2 to 5 in Fig. 3C,fig. S8, and tables S1 and S2). MIF’s 3′ exonu-clease activity was also supported by cleavageassays in which the 5′bRF or 5′b3E substrates wereused (Fig. 3C, fig. S8, and tables S1 and S2). More-over, we used poly(A) (PA30), which lacks second-ary structure and cannot be stained by ethidiumbromide (EtBr) (Fig. 3C, top). We found that MIF’s3′ exonuclease activity allowed it to cleave 5′biotin–poly(A) (5′bPA30), butnot3′biotin–poly(A) (3′bPA30),so that MIF’s 3′ exonuclease activity can occurindependently of secondary structure (Fig. 3C,bottom, and fig. S8). MIF endonuclease activitywas also influenced by secondary structure, be-cause it cleaved short unpaired bases of ssDNAat the 3′ end adjacent to the stem loop (5′bPS40,3′bPS40, 5′b3F1, 3′b3F1, and 5′bL3), as well as 3′-OH or 3′-biotin adjacent to the stem loop (3′bSLand 3′bLF) (Fig. 3C and fig. S8). In contrast to itsexonuclease activity, MIF’s endonuclease activitywas not blocked by biotin modification at thesubstrate’s 3′ end (3′bSL, 3′bLF, 3′bPS40, and3′b3F1).However, 5′bL3, a sequence not related to PS30 butwith a similar stem-loop structure, was cleavedby MIF, but with less efficiency (Fig. 3C and fig.S8). These results indicate that MIF has both 3′exonucleaseandendonucleaseactivities andcleavesunpaired bases of stem-loop ssDNA at the 3′ end. Inthe presence of AIF, AIF also increased the bindingof MIF to ssDNAs, including 5′bPS30, as well as5′bSL, which has no 5′ unpaired bases (fig. S10D).Nevertheless, we found that AIF increased bothexonuclease and endonuclease activities of MIF(0.5 mM) on 5′bPS30, 3′bPS30, and 3′bSL (fig. S10E).However, AIF has a rather weak effect, if any, onthe nuclease activity of MIF at 4 mM (fig. S10F). Atthis higher concentration,MIF itself can efficientlybind and cleave ssDNAs. These data suggest thatAIF may enhance MIF nuclease activity by in-creasing its binding to ssDNAs.

To further study where MIF cleaves DNA andto avoid the potential interference of biotin label-ing, we used non-labeled PS30 and 3F1, which hasonly one unpaired base at the 3′ end of the stem-loop structure as substrates and customized two

different DNA ladders based on PS30. After incu-bation of these substrates with MIF (2 mM) for2 hours, two major products of 20 and 22 nucleo-tidesweredetected (Fig. 3D). Faint bands of highermolecular mass were also observed. These bands

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Fig. 3. MIF binds and cleaves ssDNA. (A) MIF DNA binding motif determined by ChIP-seq. (B) Bindingof MIF to biotin-labeled small DNA substrates with different structures or different sequences in an EMSAassay (see fig. S8 for illustrations of substrates, and tables S1 and S2 for sequences). Arrow indicatesthe DNA-MIF complex. Asterisk indicates nonspecific bands. PCS, positive control substrate from theLightShift Chemiluminescent EMSA Kit (Thermo Scientific) containing 60 base pairs (bp) of 5′ biotin-labeled duplex.With or without BSA, bovine serum albumin; PC, Epstein-Barr nuclear antigen extract fromthe LightShift Chemiluminescent EMSA Kit or MIF. (C) MIFcleavage of unpaired bases at the 3′ end of thestem loop of 5′ or 3′ biotin-labeled small DNA substrates with various structures or sequences in a nucleaseassay (see fig. S8 for illustrations of substrates, and tables S1 and S2 for sequences). Experiments werereplicated four times with three independent preparations of MIF protein. (D) MIF cleavage of 3′ unpairedbases from nonlabeled PS30 and 3F1 substrates. DNA ladders 1 and 2 were customized with PS30 and itscleavage products by removing its 3′ nucleotides one by one. DNA ladder 1 was prepared using PS30, PS28,PS26, PS24, PS22, and PS20. DNA ladder 2 was prepared using PS29, PS27, PS25, PS23, and PS21. (E) MIFcleavage sites on nonlabeled PS30 and 3F1 substrates.

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Fig. 4. Requirement of AIF for the recruitment of MIF to the nucleus inNMDA excitotoxicity. (A) Binding of immobilized GST-MIF WT and GST-MIFvariants to AIF. (B) Nuclease activity andAIF-bindingactivity ofMIFWTandMIFvariants. (C andD) Coimmunoprecipitation (IP) ofMIFand AIF in control (CSS)and NMDA-treated cortical neurons. Asterisk indicates IgG. Ab, antibody.(D) Intensity of treated versus untreated cultures. *P < 0.05, Student’s t test.(E) Images of nuclear translocation of AIFandMIFafter NMDA treatment inWT,AIF knockdown, and MIF knockdown cortical neurons. AIF shRNA (AIF sh) andMIF shRNA (MIF sh) caused a 71.3 ± 5.2% and 73.3 ± 6.1% protein reduction,respectively.White color indicates the overlayof AIF,MIF, and 4′,6′-diamidino-2-phenylindole (DAPI), showing the nuclear translocation of AIF and MIF. Purplecolor indicates the overlay of AIF and DAPI, showing the nuclear translocation

of AIF. Z stacks illustrating the x,z and y,z axis are provided to demarcate thenucleus. Arrowheads indicate cells with the high magnification. (F) Quantifica-tion of the percentage of cells with nuclear translocation of MIF and AIF afterNMDA treatment in WT, AIF knockdown, and MIF knockdown cortical neurons.CSS, control salt solution. ****P < 0.0001, versus its CSS control; ####P <0.0001,versus itsWT treatedwithNMDA,one-wayANOVA. (G) Immunoblots ofnuclear translocation of AIF and MIF after NMDA treatment in WT, AIF knock-down, and MIF knockdown cortical neurons. Compare total protein (T), post-nuclear fraction (PN), nuclear fraction (N), and Mito, mitochondrial antibody.(H and I) Relative levels of AIF and MIF in T, PN, and N. Means ± SEM. Exper-iments were replicated at least three times; ****P < 0.0001, versus its CSScontrol; ##P < 0.01, ###P < 0.0001, versus its total protein, one-way ANOVA.

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were more obvious in the experiment in whichPS30 was biotin labeled and the incubation timewas 1 hour (Fig. 3D). MIF cleavage of the 3F1 sub-strate yielded only a 29-nucleotide (nt) band con-sistent with cleavage of one unpaired base at the3′ end of the stem-loop structure (Fig. 3, D andE).These data indicate that PS30 is initially cleaved byMIF after “A23↓T24↓T25↓” (arrow indicates cleav-age) by both 3′ exonuclease and endonucleaseactivity (Fig. 3E, left). Then the resulting productappears to form a new stem-loop structure, aspredicted by the online RNA/DNA structure pre-diction software (http://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1/Predict1.html)(Fig. 3E, right). MIF then cleaves at the new un-paired bases at the 3′ end of this stem-loop struc-ture after “G20↓G21↓G22↓”.We conclude thatMIFcleaves unpaired bases at the 3′ end adjacent tothe stem loop at the +1 to ∼+3 positions throughboth 3′ exonuclease and endonuclease activities.

AIF interacts with MIF and recruits MIFto the nucleus

Wild-type (WT) glutathione S-transferase–taggedAIF (GST-AIF) associatedwithMIF, andwild-typeGST-MIF associated with AIF in GST pulldownanalyses from cell lysates (Fig. 4A; fig. S11, A to D;and supplementary text). We mapped the MIF-AIF binding domain. MIF bound to AIF at aminoacids 567 to 592 (fig. S11, A toC, and supplementarytext). Conversely, the MIF E22A mutant showedreduced binding to GST-AIF, whereas the E22DandE22Qmutants still bound to GST-AIF (Fig. 4,A and B, and fig. S11D). The other PD-D/E(X)Kand C57A;C60A mutations still bound GST-AIF(fig. S11D). Thus, MIF E22 appears to be criticalfor AIF binding. Endogenous AIF also coimmu-noprecipitated with MIF from cortical neuronstreated with NMDA (500 mM) but was barely de-tectable in untreated cultures (Fig. 4, C and D).MIF was localized predominantly to the cyto-

sol of both cortical neurons (Fig. 4E) and HeLacells (fig. S12A). Both MIF and AIF translocatedto the nucleus in cortical neurons treated withNMDA (Fig. 4, E and F) andHeLa cells stimulatedwith MNNG (fig. S12A). Depletion of AIF withshRNA led to a loss of MIF translocation to thenucleus, but depletion of MIF did not preventtranslocation of AIF to the nucleus in cells exposedto NMDA (Fig. 4, E and F). Subcellular fractiona-tion into nuclear and postnuclear fractions con-firmed the translocation of MIF and AIF to thenucleus in cultured cortical neurons exposed toNMDA (Fig. 4, G to I). AIF was required for MIFtranslocation (Fig. 4, E to I). DPQ prevented ac-cumulation of both MIF and AIF in the nucleusin HeLa cells treated withMNNG (fig. S12, A to C)and cortical neurons treatedwithNMDA (fig. S13,A to C). Consistent with the notion that NMDAexcitotoxicity involves nitric oxide production,the nitric oxide synthase inhibitor nitro-arginine(N-Arg) prevented accumulation of bothMIF andAIF in the nucleus (fig. S13, A to C).We transduced primary cortical cultures from

WT MIF knockout mice with lentivirus carryingFlag-taggedMIF (MIF-WT-Flag) orMIFmutants(MIF-E22Q-Flag and MIF-E22A-Flag) to confirm

that AIF and MIF binding is required for MIFnuclear accumulation after NMDA administra-tion (Fig. 5, A and B). Wild-type MIF and E22Qinteracted with AIF, but MIF E22A did not bindto AIF (Fig. 5B). In nontransducedMIF knockoutcultures and inMIF knockout cultures transducedwithMIF-WT-Flag,MIF-E22Q-Flag, andMIF-E22A-Flag, AIF translocated to the nucleus when cellswere exposed to NMDA (Fig. 5, C and D). BothMIFwild-type andMIFE22Qalso translocated tothe nucleus; however, the MIF E22A mutant,which is deficient in AIF binding, failed to do so(Fig. 5, C and D). Separation of nuclear and post-nuclear fractions confirmed the observationsmadeby immunofluorescence (Fig. 5, E to G). Theseresults indicate that MIF’s interaction with AIFis required for the nuclear translocation of MIF.

MIF nuclease activity is required forchromatinolysis and parthanatos

To determinewhetherMIF’s nuclease activity andAIF-mediated recruitment are required for par-thanatos, we transduced MIF knockout cultureswith the nuclease-deficient MIF E22Q mutantand the AIF binding–deficient MIF E22Amutant.Consistent with the shRNA experiments, corticalcultures lacking MIF were resistant to NMDA ex-citotoxicity (Fig. 6, A and B). Transduction of cellswith wild-type MIF or the tautomerase-deficientmutant MIF P2G fully restored NMDA excitotox-icity; conversely, neitherMIFE22QnorMIFE22ArestoredNMDA excitotoxicity (Fig. 6, A and B). Bythe comet assay, a method to measure DNA dam-age, we found that NMDAadministration inwild-type cortical neurons resulted in substantial numbersof neurons with DNA damage, whereas no suchdamage was detected in MIF knockout neurons(Fig. 6, C to F). Transduction of knockout neu-rons with wild-typeMIF, but not withMIF E22Qor MIF E22A, restored DNA damage in cellstreated with NMDA (Fig. 6, C to F). Depletion ofMIFwith shRNA inHeLa cells with two differentshRNAs resulted in a reduced number of cellsshowing damaged DNA after treatment withMNNG compared with DNA in cells treated withnontargeted shRNA (fig. S14, A to D). A pulsed-field gel electrophoresis assay of genomic DNAconfirmed thatNMDAadministration caused largeDNA fragments in wild-type cortical neurons butnot inMIF knockout cortical neurons (Fig. 6G).No obvious large DNA fragments were observedin MIF knockout neurons transduced with MIFE22Q or MIF E22A (Fig. 6G). Transduction ofknockout neurons with wild-type MIF or MIFP2G restored NMDA-induced formation of largeDNA fragments (Fig. 6G). HeLa cells lackingMIFafter we used CRISPR-Cas9 were resistant toMNNG toxicity (fig. S15, A to C). Transduction ofknockout HeLa cells with wild-type MIF or MIFP2G restored MNNG-induced formation of largeDNA fragments and toxicity (fig. S15). These re-sults indicate that MIF is the major nuclease in-volved in large-scale DNA fragmentation duringMNNG- or NMDA-induced parthanatos, which isindependent from MIF’s tautomerase activity.To evaluate the requirement of MIF nuclease

activity andMIFbinding toAIF in cell death due to

parthanatos in vivo, we transducedMIF knockoutmice with adeno-associated virus serotype 2 virus(AAV2) containing wild-type MIF, or the nuclease-deficient MIF E22Q mutant or the AIF-binding-deficient MIF E22A mutant by injecting thedifferent AAV2 MIFs into the intracerebroventri-cular zone of newborn mice. The effectiveness oftransductionwas confirmedby immunostaining forMIF-Flag in the cortex, striatum, and hippocam-pus in adultmice (fig. S16, A andB). Two-month oldmalemice were then subjected to 45-min transientocclusion of the middle cerebral artery (MCAO).Despite the similar intensity of the ischemic insult(fig. S16C), infarct volume as previously reported(34) was reduced in MIF knockout mice in thecortex, striatum, and hemisphere by about 75%compared to that in their wild-type counterparts(Fig. 7, A to D). Moreover, the neuroprotection inMIF knockout mice remained for at least 7 days(Fig. 7, C andD). Expression of wild-typeMIF, butnotMIFE22QorMIFE22A, in theMIF knockoutmice restored infarct volume to that observed inwild-type animals (Fig. 7, A to D). We assessedbehavior by spontaneous activity in the open fieldtask on days 1, 3, and 7 after MCAO. Consistentwith the infarct data, MIF knockout mice hadimproved behavioral scores compared to thoseofwild-typemice.MIF knockoutmice expressingwild-type MIF had behavioral scores equivalent tothose of wild-typemice whereas expression ofMIFE22Q or MIF E22A had no effect (Fig. 7, E and F).Over 3 and 7 days, the behavioral scores of MIFknockout mice remained higher than those ofwild-type treated mice (Fig. 7, F and G). A cornertest measuring sensorimotor function showed thatall mice do not show a side preference beforeMCAOsurgery. However, wild-typemice andMIF knockoutmice expressing wild-type MIF had significantly(P < 0.05 to P < 0.001, one-way analysis of var-iance (ANOVA)] increased turning toward the non-impaired side at days 1, 3, and 7 afterMCAO (Fig.7G), indicating these mice have more severe sen-sory and motor deficits. No preference was ob-served in MIF knockout mice andMIF knockoutmice with expression of MIF E22Q or MIF E22A(Fig. 7G).Significant (P < 0.0001, one-way ANOVA) DNA

damage as assessed by pulse field gel electropho-resis was observed at days 1, 3, and 7 after MCAOinwild-typemice orMIFknockoutmice expressingwild-type MIF (Fig. 7, H and I). DNA damage wasreduced in the MIF KO mice and MIF knockoutmice expressing E22Q or E22A MIF (Fig. 7, H andI). We examined the localization of AIF and MIFby confocal microscopy in the penumbra regionof the stroke (fig. S17, A and B). Consistent withthe observation in cultured cortical neurons, AIFsignificantly (P < 0.001, one-way ANOVA) translo-cated to the nucleus at 1, 3, and 7 days afterMCAOin wild-type animals. InMIF knockout animals aswell as MIF knockout mice injected with MIFwild-type, E22Q, and E22A AIF significantly (P <0.001, one-way ANOVA) translocated to the nucleusat 1 and 3 days after MCAO and there was reducedtranslocationofAIFat 7days (fig. S17,AandB).BothMIF wild-type and MIF E22Q also significantly(P < 0.001, one-way ANOVA) translocated to the

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nucleus at 1 and 3 days after MCAO and therewas reduced translocation at 7 days; however, theAIF binding–deficient mutant MIF E22A failed todo so (fig. S17, A and B). These data indicate thatMIF is required for AIF-mediated neurotoxicityand DNA cleavage and that AIF is required forMIF translocation in vivo.

Conclusion

We identifiedMIF as a PAAN. Prior crystallizationstudies of MIF allowed us to show via 3-D mod-

eling thatMIF is structurally similar toPD-D/E(x)Knucleases (25, 26). TheMIFmonomer, which haspseudo 2-fold symmetry does not contain thecore PD-D/E(X)K structure since the MIF mono-mer has four b strands next to the two a helices,and the orientations of the b-strands within anisolatedmonomer do not fit the requirement of thePD-D/E(x)K topology (23). However, our structure-activity analyses based on the MIF trimer, whichhas 3-fold symmetry, indicated that the interac-tions of the b strands of each monomer with the

othermonomers results in aMIFPD-D/E(x)K struc-ture that consists of four b strands next to two astrands (23). Two of the b strands are parallel (b-4and b-5), whereas the other two strands (b-6 andb-7) (from the adjacent monomer) are antiparallel.This topology exquisitely supports the idea thatMIF’s nuclease activity requires the trimer as themonomers do not support the required topologyand is consistentwithMIF existing as a trimer. ThePD-D/E(X)K domains inMIF are highly conservedin vertebrates. The glutamic acid residue (E22) in

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Fig. 5. MIF E22A mutant prevents AIF’s recruitment of MIF to thenucleus in NMDA-excitotoxicity. (A) Expression of MIF in WTand knockout(KO) neurons. (B) Coimmunoprecipitation of Flag-tagged MIF variants andAIF in cortical neurons after NMDA treatment. (C) Images of nuclear trans-location of AIFand exogenousMIFWTandMIFvariants after NMDA treatmentin MIF KO cortical neurons. Scale bar, 20 mm. White color indicates theoverlay of AIF, MIF, and DAPI, showing the nuclear translocation of AIF andMIF. Purple color indicates the overlay of AIF and DAPI, showing the nucleartranslocation of AIF. Z stacks illustrating the x,z and y,z axis are provided todemarcate the nucleus. (D) Quantification of the percentage of cells with

nuclear translocation of AIF and exogenous MIF WT and MIF variants afterNMDA treatment in MIF KO cortical neurons. ****P < 0.0001, versus KOgroup; ###P < 0.001, versus KO-WTgroup, one-way ANOVA. (E) Immunoblotsof nuclear translocation of AIF and exogenous MIF WT and MIF variantsafter NMDA treatment in MIF KO cortical neurons. H4, histone H4; mito,mitochondrial antibody. (F and G) Relative levels of AIF and MIF in totalprotein (T), postnuclear fraction (PN) and nuclear fraction (N). Means ± SEM.Experiments were replicated at least three times. **P <0.01, ****P < 0.001,versus KO control group; ##P < 0.01, ####P < 0.0001, versus its total protein,one-way ANOVA.

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the first a helix of MIF is critical for its nucleaseactivity, which is consistent with prior reports thatthis glutamic acid in the first a helix of manyexonuclease-endonuclease-phosphatase (EEP)domain superfamily nucleases is highly conserved,and it is the active site for nuclease activity (25, 26).MIF has both 3′ exonuclease and endonuclease

activity. It preferentially binds to 5′ unpaired basesof ssDNA with the stem-loop structure and cleavesits 3′ unpaired bases. AIF interacts withMIF andrecruitsMIF to the nucleuswhereMIF binds andcleaves genomicDNA into large fragments similarto the size induced by stressors that activateparthanatos. MIF binding to AIF facilitates itscleavage of double-stranded genomic DNA, and,based on the chromatin immunoprecipitationsequencing ChiP-seq data, the average distanceof MIF binding is about 15 to 60 kb, which iscomparable to the size of large DNA fragmentscaused by MIF. MIF’s cleavage of genomic DNAinto 20- to 50-kb fragments is likely due to itsrare binding on genomicDNA. Knockout ofMIFreduces DNA fragmentation induced by stimulithat activate PARP-1–dependent cell death. Mu-tating a key amino acid residue, glutamic acidresidue (E22), in the PD-D/E(X)K motif elim-inates MIF’s nuclease activity and protects cellsfrom parthanatos both in vitro and in vivo. Dis-ruption of the AIF and MIF protein-protein in-teraction prevents the translocation of MIF fromthe cytosol to the nucleus, which also protectsagainst PARP-1–dependent cell death both in vitroand in vivo. Neither MIF’s thiol-protein oxido-reductase activity nor tautomerase activity areinvolved in its actions as a nuclease. Knockoutof MIF, a MIF nuclease–deficient mutant and aMIF AIF binding–deficient mutant all reduceinfarct volume and have long-lasting behavioralrescue in the focal ischemia model of stroke inmice. Thus, MIF is a PAAN that is important incell death because of activation of PARP-1 and therelease of AIF (2). Future studies are required tofurther determine whether the stem-loop–ssDNAbinding activity or the 3′ exonuclease and endo-nuclease activities of MIF, is important for its invivo PAAN activity. In addition, our stroke datafromMIF knockout mice indicate that other nu-cleases other than MIF might be involved in is-chemic neuronal cell death. However, how thesenucleases interact with MIF and contribute toPARP-1–induced cell death requires future studies.MIF has a variety of pleiotropic actions. It is

widely distributed throughout the brain (35, 36).It functions as a nonclassically secreted cytokineandmay play important roles in cancer biology,immune responses, and inflammation (18, 37). MIFalso has important roles in cellular stress andapoptosis (34, 38, 39). How MIF’s nuclease ac-tivity relates to its role in the immune system andits other actions requires future studies.Like PARP, inhibition of MIF nuclease activity

is an attractive target for acute neurologic dis-orders. However, it may have advantages overPARP inhibition in chronic neurodegenerativediseases where long-term inhibition of PARPcould impair detection and repair ofDNAdamage.Inhibition of MIF’s nuclease activity could bypass

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Fig. 6. MIF nuclease activity is critical for DNA damage and PARP-1–dependent cell death in corti-cal neurons. (A) Representative images and (B) quantification of NMDA-induced (500 mM for 5 min)excitotoxicity in MIF WT, KO, and KO cortical neurons expressing MIF WT, E22Q, E22A, or P2G. Scale bar,200 mm. (C) Representative images and (D to F) quantification of NMDA-induced DNA damage 6 hoursafter treatment determined by the comet assay in MIFWT, KO, and KO neurons expressingMIFWT, E22Q,E22A, or P2G. Dashed lines indicate the center of the head and tail. Scale bar, 20 mm. (G) Pulsed-field gelelectrophoresis assay of NMDA-induced DNA damage 6 hours after treatment in MIFWTand KO neuronsand KO neurons expressingMIFWT, E22Q, E22A, or P2G. Means ± SEM are shown in (B), (D), (E), and (F).*P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA; ns, nonsignificant.

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this potential concern and offer a therapeuticopportunity for various disorders.

Materials and methodsHuman protein chiphigh-throughput screening

Human protein chips (16K and 5K), which wereprepared by spotting more than 16,000 or 5000highly purified proteins onto special nitrocellulose-coated slides (16), were incubated in renaturationbuffer containing 50mMTris-HCl, pH8.0, 100mM

NaCl, 1 mMDTT, 0.3% Tween 20 for 1 hour at 4°C.After Blocking with 5% non-fat dry milk for1 hour at room temperature, protein chips wereincubatedwith purifiedmouseAIF protein (50 nM,NP_036149) in 1% milk for 1 hour. Protein inter-actionwas then determined either by sequentiallyincubating with rabbit anti-AIF antibody (JH532,JHU) (9, 11) andAlexaFluor 647donkey anti-rabbitIgG, or Alexa Fluor 647 donkey anti-rabbit IgGonly as negative control. ProteinmicroarrayswerescannedwithGenePix4000BMicroscanner (Tecan)

using the Cy5 image and the median fluores-cence of each spot was calculated. We used thesame procedure described previously to identifyinteracting proteins (16).

Reverse transfection formatsiRNA-based screen forPARP-1–dependent cell viability

On-Target plus SMARTpool siRNAs targeting AIF-interacting proteins resulting from human proteinchip high throughput screening were customized

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Fig. 7. MIF nuclease activity iscritical for DNA damage andischemic neuronal cell deathin vivo. (A) Representativeimages of triphenyl tetrazoliumchloride staining of MIF WT, KO,and KO mice that wereinjected with AAV2-MIF WT,E22Q, or E22A 24 hours after45 min of middle cerebral arteryocclusion (MCAO). (B to D)Quantification of infarction volumein cortex, striatum, and hemi-sphere 1 day or 7 days after45-min MCAO.WT MCAO (n =29); KO MCAO (n = 20); KO-WTMCAO (n= 23). KO-E22Q (n= 22)andKO-E22AMCAO (n= 19). *P<0.05, ***P < 0.001, versus KOgroup at the same time point;##P < 0.01, ###P < 0.001, thesame group at 7 days versus at1 day after 45-min MCAO, one-wayANOVA. (E toG) Neurologicaldeficit was evaluated by [(E) and(F)] open field on a scale of 0 to 5and (G) corner test evaluated bypercentage of right turns at 1 day,3 days, or 7 days after MCAOsurgery.WT MCAO (n = 16); KOMCAO(n= 12); andKO-WTMCAO(n = 16). KO-E22QMCAO (n = 16)and KO-E22A MCAO (n = 16).Means ± SEM. *P < 0.05, ***P <0.001, one-way ANOVA in (E) and(G). **P < 0.01, two-way ANOVAin (F),WTand KO-WTversus KO,KO-E22Q,andKO-E22Aatdifferenttime points. (H) DNA frag-mentation determined by pulsed-field gel electrophoresis in thepenumbra 1 day, 3 days, or 7 daysafter 45-min MCAO surgery inMIFWT, KO, andKOmutantmice,which were injected with AAV2-MIF WT, E22Q, or E22A.WTMCAO (n = 15); KO MCAO (n =15); and KO-WT MCAO (n = 15).KO-E22Q (n = 15) and KO-E22AMCAO (n = 15). (I) Quantificationof noncleaved genomic DNA.Means ± SEM. ****P < 0.0001,versus its sham treatment group,one-way ANOVA.

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in 96-well plates fromDharmacon. The plateswererehydrated using DharmaFECT 1 transfectionreagent at room temperature for 30 min. HeLacells were then seeded in the plates with the celldensity at 1 × 104/well. 48 hours after transfec-tion, cells were treated with MNNG (50 mM) orDMSO for 15 min and then incubated in normalcompletemedium for 24hours. After addingAlamarBlue for 1-4 hours, cell viability was determinedby fluorescence at excitation wavelength 570 nmandEmissionwavelength 585nm.PARP-1 siRNAswere used as the positive control and non-targetsiRNAs as the negative control.

Nuclease assays

Human genomic DNA (200 ng/reaction, Pro-mega), pcDNA (200 ng/reaction) or PS30 and itsrelated and non-related substrates (1 mM) wasincubated with wild-type MIF or its variants at afinal concentration of 0.25-8 mM as indicated in10mMTris-HCl buffer (pH 7.0) containing 10mMMgCl2 or specific buffer as indicated, for 1 hour(with pcDNAand smallDNA substrates) or 4 hours(with human genomicDNA) at 37°C. The reactionwas terminated with loading buffer containing10 mMEDTA and incubation on ice. The humangenomic DNA samples were immediately sep-arated on a 1.2% pulse field certified agarose in0.5 X TBE buffer with initial switch time of 1.5 sand a final switch time of 3.5 s for 12 hours at6 V/cm. pcDNA samples were determined by 1%agarose gel. Small DNA substrates were sepa-rated on 15% or 25% TBE-urea polyacrylamide(PAGE) gel or 20% TBE PAGE gel. The gel wasthen stained with 0.5 mg/ml Ethidium Bromide(EtBr) followed by electrophoretic transfer to anylonmembrane. Biotin-labeledDNAwas furtherdetected by chemiluminescence using the Chemi-luminescent Nucleic Acid Detection Module(Thermo Scientific).

Electrophoretic mobility shift assay (EMSA)

EMSA assays were performed using the Light-Shift Chemiluminescent EMSA kit (Thermo Sci-entific) following the manufactures instruction.Briefly, purifiedMIF protein (2 mM)was incubatedwith biotin-labeled DNA substrates (10 nM) inthe binding buffer containing 10mMMgCl2 for30min on ice. Then sampleswere separated on6%retardationpolyacrylamide followedbyelectropho-retic transfer to a nylonmembrane. Biotin-labeledDNA was further detected by chemiluminescenceusing the Chemiluminescent Nucleic Acid Detec-tion Module (Thermo Scientific).

Comet assay

Comet assays were conducted following proto-cols provided by Trevigen (Gaithersburg, MD).Briefly, HeLa cells with or without MNNG treat-ment and cortical neurons with or without NMDAtreatment were washed with ice-cold PBS 6 hoursafter the treatment, harvested by centrifugation at720 g for 10 min and re-suspended in ice-cold PBS(Ca2+ and Mg2+ free) at 1 × 105 cells/ml. Cells werethen combined with 1% low melting point agarosein PBS (42°C) in a ratio of 1:10 (v/v), and 50 ml of thecell–agarose mixture was immediately pipetted

onto the Comet Slide and placed flatly at 4°C inthe dark for 30 min to enhance the attachment.After being lysed in lysis buffer, slides were im-mersedwith alkaline unwinding solution (200mMNaOH, pH >13, 1 mM EDTA) for 1 hour at RT.The comet slides were transferred and electro-phoresedwith 1 L of alkaline unwinding solutionat 21Volts for 30min in ahorizontal electrophoresisapparatus. After draining the excess electrophoresisbuffer, slides were rinsed twicewith dH2O and thenfixed with 70% ethanol for 5 min and stained withSYBR Green for 5 min at 4°C. Cell images werecaptured using a Zeiss epifluorescentmicroscope(Axiovert 200M) and image analysis was performedwith CASP software (version 1.2.2). The length ofthe “comet tail,”which is termedas the length fromthe edge of the nucleus to the end of the comet tail,for each sample, was measured.

Protein expression and purification

Human EndoG (NM_004435), cyclophilin A(NM_021130), mouse AIF (NM_012019), humanMIF (NM_002415) cDNA and their variants weresubcloned into glutathione S-transferase (GST)-tagged pGex-6P-1 vector (GE Healthcare) by EcoRIand XhoI restriction sites and verified by sequenc-ing. The protein was expressed and purified fromEscherichia coli by glutathione sepharose. The GSTtag was subsequently proteolytically removed forthe nuclease assay. MIF point mutants were con-structed by polymerase chain reaction (PCR) andverified by sequencing. The purity of MIF proteinsthat were used in the nuclease assays was furtherconfirmed by mass spectrometry. MIF proteinspurified by FPLC were also used in the nucleaseassays and no obvious difference was observedbetween FPLCMIF and non-FPLCMIF proteins.GST protein was used as a negative control in thenuclease assay.

Middle cerebral artery occlusion (MCAO)

Cerebral ischemia was induced by 45 min of re-versibleMCAO as previously described (40). Adultmale MIF knockout (KO) mice (2 to 4 monthsold, 20 to 28 g) were anesthetizedwith isofluraneand body temperature was maintained at 36.5 ±0.5°C by a feedback-controlled heating system. Amidline ventral neck incision wasmade, and un-ilateral MCAO was performed by inserting a 7.0nylon monofilament into the right internal ca-rotid artery 6-8 mm from the internal carotid/pterygopalatine artery bifurcation via an externalcarotid artery stump. Sham-operated animals weresubjected to the same surgical procedure, but thesuture was not advanced into the internal carotidartery. After 1 day, 3 days or 7 days of reperfusion,mice were perfused with PBS and stained withtriphenyl tetrazolium chloride (TTC). The brainswere further fixed with 4% PFA and sliced forimmunohistochemistry (9, 11, 41).

ChIP-seq

We preformed ChIP-seq as previously described(42, 43). Briefly, HeLa Cells were first treatedwith DMSO or MNNG (50 mM, 15 min). 5 hoursafter MNNG treatment, cells were cross-linkedwith 1% formaldehyde for 20 min at 37°C, and

quenched in 0.125 M glycine. Chromatin extrac-tion was performed before sonication. The anti-MIF antibody (ab36146, Abcam) was used andDNA was immunoprecipitated from the soni-cated cell lysates. The libraries were prepared ac-cording to Illumina’s instructions accompanyingthe DNA Sample kit and sequenced using anIllumina HiSEq. 2000 with generation of 50 bpsingle-end reads. Detailed procedures are asfollows. HeLa cells were treated with DMSO orMNNG (50 mM) for 15 min and cultured in thefresh medium for an additional 5 hours. Cellswere then cross-linked with 1% formaldehyde for10min at 37°C, and the reactionwas quenched in0.125 M glycine for 20min at room temperature.Chromatin was extracted using SimpleChIP En-zymatic Chromatin IP kit from Cell SignalingTechnology (Cat# 9003), and sonicated 30 s onand 30 s off for 15 cycles using a Bioruptor Twin(Diagenode). The quality and size of sheared chro-matin DNA were examined on an agarose gel byDNA electrophoresis. 10% of chromatin was keptas input and the rest of the chromatinwas dilutedand pre-cleared using 10 ml Magnetic protein Gagarose slurry for 30 min at 4°C to exclude non-specific binding to proteinG agarose beads directly.The pre-cleared chromatin was incubated over-nightwith an anti-MIF antibody (3 mg/ml, ab36146,Abcam) or control IgG (3 mg/ml) in the presence ofMagnetic protein G agarose slurry (30 ml) at 4°C.After washing the protein G agarose beads for3 times, half of the protein G agarose/antibodycomplex was subjected to immunoblot assaysto check the quality of the immunoprecipitation.Another half of the protein G agarose/antibodycomplex was eluted in 170 ml of elution buffercontaining 1% SDS, 0.1 M NaHCO3 at 65°C. Theeluates aswell as the chromatin inputwere treatedwith 1 mg/ml RNase A at 37°C for 30 min, andreverse-crosslinked by incubating at 65°C for4 hours after adding 3 ml of 5 M NaCl and 1 ml of10mg/mlproteinaseK. Finally the chromatinDNAwas purified using phenol/chloroform/isoamylalcohol and precipitated by ethanol. TheChIP andinputDNA librarieswere prepared using Illumina’sTruseq DNA LT Sample Prep Kit according to theinstructions. The final product was amplified for15 cycles. The quality and the size of the insert wasanalyzed using a bioanalyzer. Sequencing was per-formed in theNext Generation Sequencing Centerat Johns Hopkins using an Illumina HiSEq. 2000with generation of 50 bp single-end reads. TheChIP-seq raw data have been deposited in theGEO database accession #: GSE65110.

ChIP-seq data analysis

Rawdata from theHiSEq. 2000was converted toFASTQ using CASAVA v1.8 and demultiplexed.Reads weremapped to the human genome (hg19)using Bowtie2 (v2.0.5) using the default parame-ters. Converted SAM files were passed to MACS(v1.4.1) for peak calling using the default param-eters. Peaks from DMSO- and MNNG-treated li-braries were reported in .bed format and areprovided in GEO. Peaks differentially identified inthe DMSO- andMNNG-treated groupswere parsedby a custom R script. Sequence corresponding to

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peaks identified in only MNNG-treated, but notDMSO-treated librarieswere fed into SeSiMCMC_4_36, Chipmunk v4.3+, andMEMEchip v4.9.0 formotif discovery using default parameters.Data transfer: The CASAVAv1.8 software was

used to convert the raw files into FASTQ files aswell demultiplex the lanes.

MIF-DNA docking methods

A DNA duplex structure (44) (PDB accession1BNA) and a single-stranded DNA structure [PDBaccession 2RPD (45)] were docked onto the surfaceof MIF [PDB accession 1FIM (24)] using Hex-8.0.protein-DNA docking program (46, 47). The HEXprogramuses a surface complementarity algorithmto identify contact between protein and DNA.MIFsurfaces were generated using Pymol. All imageswere viewed and labeled with pdb viewer, Pymol.The MIF-DNA docked models are shown as ob-tained from the HEX program.

Lentivirus, adeno-associated virus (AAV)construction and virus production

Mouse MIF-WT-Flag (NM_010798), MIF-E22Q-Flag and MIF-E22A-Flag were subcloned into alentiviral cFugw vector by AgeI and EcoRI rest-riction sites, and its expression was driven by thehuman ubiquitin C (hUBC) promoter. HumanMIF andmouseMIF shRNAswere designed usingthewebsite <http://katahdin.cshl.org/siRNA/RNAi.cgi?type=shRNA>. The program gave 97 nt oligosequences for generating shRNAmirs. Using PacISME2 forward primer 5’ CAGAAGGTTAATTAA-AAGGTATATTGCTGTTGACAGTGAGCG 3’ andNheI SME2 reverse primer 5’ CTAAAGTAGC-CCCTTGCTAGCCGAGGCAGTAGGCA 3’, we thenPCR amplified them to generate the second strandand added PacI and NheI restriction sites to clonethe products into pSME2, a construct that insertsan empty shRNAmir expression cassette in thepSM2 vector with modified restriction sites intothe cFUGw backbone. This vector expresses GFP.The lentivirus was produced by transient transfec-tion of the recombinant cFugw vector into 293FTcells together with three packaging vectors: pLP1,pLP2, and pVSV-G (1.3:1.5:1:1.5). The viral super-natants were collected at 48 and 72 hours aftertransfection and concentrated by ultracentrifuga-tion for 2 hours at 50,000 g. MIF-WT-Flag, MIF-E22Q-Flag and MIF-E22A-Flag were subclonedinto a AAV-WPRE-bGH (044 a.m./CBA-pI-WPRE-bGH) vector by BamHI andEcoRI restriction sites,and its expression was driven by chicken b-actin(CBA) promoter. All AAV2 viruses were producedby the Vector BioLabs.

Sequences of MIF substrates, templates,and primers

Sequences of MIF substrates, templates and pri-mers used for shRNA constructs and point mu-tation constructs are provided in Table S1.

Cell culture, transfection, lentiviraltransduction, and cytotoxicity

HeLa cells were cultured in Dulbecco’s modifiedEagle’s medium (Invitrogen) supplemented with10% fetal bovine serum (HyClone). V5-tagged MIF

was transfected with Lipofectamine Plus (Invitro-gen). Primary neuronal cultures from cortex wereprepared as previously described (9). Briefly, thecortexwas dissected and the cellsweredissociatedby trituration in modified Eagle’s medium (MEM),20% horse serum, 30 mM glucose, and 2 mML-glutamine after a 10-min digestion in 0.027%trypsin/saline solution (Gibco-BRL). Theneuronswere plated on 15-mm multiwell plates coatedwith polyornithine or on coverslips coated withpolyornithine. Neurons were maintained in MEM,10% horse serum, 30 mM glucose, and 2 mML-glutamine in a 7% CO2 humidified 37°C in-cubator. The growth mediumwas replaced twiceper week. In mature cultures, neurons represent70 to 90% of the total number of cells. Days invitro (DIV) 7 to 9, neurons were infected by len-tivirus carrying MIF-WT-Flag, MIF-E22Q-Flag,or MIF-E22A-Flag [1 × 109 units (TU)/ml] for72hours. Parthanatoswas inducedby eitherMNNG(Sigma) in HeLa cells or NMDA (Sigma) in neu-rons. HeLa cells were exposed toMNNG (50 mM)for 15 min, and neurons (DIV 10 to 14) werewashed with control salt solution [CSS, contain-ing 120 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2,25 mM tris-Cl, and 20 mM glucose (pH 7.4)], ex-posed to 500 mM NMDA plus 10 mM glycine inCSS for 5 min, and then exposed to MEM con-taining 10% horse serum, 30 mM glucose, and2 mM L-glutamine for various times before fixa-tion, immunocytochemical staining, and confocallaser scanning microscopy. Cell viability was de-termined the following day by unbiased objectivecomputer-assisted cell counting after staining ofall nuclei with 7 mM Hoechst 33342 (Invitrogen)and dead cell nuclei with 2 mM propidium iodide(Invitrogen). The numbers of total and dead cellswere counted with the Axiovision 4.6 software(Carl Zeiss). At least three separate experimentsusing at least six separate wells were performedwith a minimum of 15,000 to 20,000 neurons orcells counted per data point. For neuronal toxicityassessments, glial nuclei fluoresced at a differentintensity than neuronal nuclei and were gatedout. The percentage of cell death was determinedas the ratio of live to dead cells comparedwith thepercentage of cell death in control wells to accountfor cell death attributed to mechanical stimulationof the cultures.

Pull-down, coimmunoprecipitation,and immunoblotting

For the pull-down assay, GST-tagged MIF or AIFproteins immobilized glutathione Sepharose beadswere incubated with 500 mg of HeLa cell lysates,washed in the lysis buffer, and eluted in the proteinloading buffer. For coimmunoprecipitation, 1 mgwhole-cell lysates were incubated overnight withAIF antibody (1 mg/ml) in the presence of proteinA/G Sepharose (Santa Cruz Biotechnology), fol-lowed by immunoblot analysis withmouse anti-Flag antibody (CloneM1, Sigma), mouse anti-V5(V8012, Sigma) or Goat anti-MIF (ab36146, Abcam).The proteins were separated on denaturing SDS-PAGE and transferred to a nitrocellulose mem-brane. Themembranewas blocked and incubatedovernightwithprimary antibody (50ng/ml;mouse

anti-Flag; rabbit anti-AIF; or goat anti-MIF) at4°C, followed by horseradish peroxidase (HRP)–conjugated donkey anti-mouse, anti-rabbit or anti-goat for 1 hour at RT. After washing, the immunecomplexes were detected by the SuperSignalWestPico Chemiluminescent Substrate (Pierce).

Subcellular fractionation

The nuclear extracts (N) and postnuclear cell ex-tracts (PN), which is the fraction prepared fromwhole-cell lysates after removing nuclear proteins,were isolated in hypotonic buffer (9, 11). The in-tegrity of the nuclear and postnuclear subcellularfractions was determined by monitoring histoneH3 or H4 and MnSOD or mitochondria antibody(MTC02) (Mito) immunoreactivity (9, 11).

Immunocytochemistry,immunohistochemistry, andconfocal microscopy

For immunocytochemistry, cellswere fixed 4hoursafter MNNG or NMDA treatment with 4% para-formaldehyde, permeabilizedwith 0.05%TritonX-100, and blocked with 3% BSA in PBS. AIF wasvisualized by Donkey anti-Rabbit Cy3 or donkeyanti-rabbit 647. MIF was visualized by donkeyanti-mouse cy2 (2 mg/ml), donkey anti-goat Cy2or donkey anti-goat 647. Immunohistochemistrywas performed with an antibody against Flag.Immunofluorescence analysis was carried out withan LSM710 confocal laser scanning microscope(Carl Zeiss) as described (9).

Quantification of relative percentagelevels of AIF and MIF insubcellular fractions

The relative levels of AIF and MIF in different frac-tions were quantified and calculated as the percent-age of their total proteins based on the intensityof protein signals relative to the protein amountprepared from the same number of cells (6million).The detailed calculation is as follows: 1) The signalintensity of each interestedbandwasmeasured andnormalized to their mitochondrial and nuclearmarkers, with the total proteins of CSS in Fig. 4,G to I and knockout neurons treatedwithNMDAin Fig. 5 E to G. A volume factor was used tocalculate the relative amount of total protein (T),post-nuclear protein (PN) and nuclear protein(N) prepared from the same number (6 million)of cells. As such, the relative ratio of different sam-ples in the same fractions and the same sample indifferent fractions will be calculated as the relativeintensity of total protein (Ti), post-nuclear fraction(PNi) and nuclear fraction (Ni). 2) A Z factor for theadjusted total proteins for each sample was deter-mined via Z = (PNi + Ni)/Ti. 3) Relative proteinlevels in PN and N fractions were calculated asfollows: PN%= (PNi x Z)/ Ti × 100%;N%= (Ni x Z)/Ti × 100%; T% = (Ti x Z)/ Ti × 100%.

FPLC

The native state and purity of the purified re-combinant MIF were determined using stan-dard calibration curve between elution volumeand molecular mass (kD) of known molecularweightnativemarker proteins in Akta Basic FPLC

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(Amersham-Pharmacia Limited) using Superdex200 10/300GL column (GE Healthcare, Life Scien-ces). The gel filtration column was run in standardPBS buffer at a flow rate of 0.5 ml/min. The fol-lowing molecular weight standards were used:Ferritin (440 kD), aldolase (158 kD), conalbumin(75 kD), ovalbumin (43 kD), carbonic anhydrase(29 kD), and ribonuclease (13.7 kD) respectively(GE Healthcare, Life Sciences). Eluted fractionscontaining MIF were resolved on 12%SDS-PAGEand stained with commassie blue to check thepurity of the protein.

Mass spectrometry analysis for MIFprotein purity

MIF proteins used for nuclease assays were alsoexamined by mass spectrometry in order to ex-clude any possible contamination from otherknown nucleases. We performed analyses usingdifferent criteria at a 95% and lower confidencelevels in order to capture any remote possibilityof a nuclease. Analysis and search of the NCBIdatabase using all species reveal that no knownnuclease that can digest single or double-strandedDNA was detected in the MIF protein that wasused in the nuclease assays.

Circular dichroism (CD) spectroscopy

CD spectroscopy was performed on a AVIV 420CD spectrometer (Biomedical Inc., Lakewood, NJ,USA). Near-UV CD spectra were recorded between240–320 nm using a quartz cuvette of 0.5 cm pathlength with protein samples at a concentration of2mg/ml at room temperature. Far UV CD spectrawere also recorded at room temperature between190–260 nm using quartz sandwich cuvettes of0.1 cm path length with protein samples at a con-centration of 0.2 mg/ml (48). The proteins weresuspended in PBS buffer with or without magne-sium chloride (5.0 mM) and/or zinc chloride(0.2 mM). The CD spectra were obtained from0.5 nm data pitch, 1 nm/3 s scan speed and 0.5 sresponse time were selected for the recordings.

Oxido-reductase activity assay

The thiol-protein oxidoreductase activity of MIFwas measured using insulin as the substrate asdescribed previously (30). Briefly, the insulin as-say is based on the reduction of insulin and sub-sequent insolubilization of the insulin b-chain.The time-dependent increase in turbidity is thenmeasured spectrophotometrically at 650 nm. Thereaction was started by adding 5 mM MIF to WT,E22A, E22Q, C57A;C60A or and P2G mutants dis-solved in 20 mM sodium phosphate buffer (pH7.2), and 200 mM reduced glutathione (GSH) toice-cold reaction mixture containing 1 mg/ml in-sulin, 100 mM sodium phosphate buffer (pH 7.2)and 2 mMEDTA.MIF insulin reduction was mea-sured against the control solution (containing GSH)in the same experiment.

Tautomerase activity assay

Tautomerase activity was measured using D-dopachrome tautomerase as the substrate asdescribed previously (49). Briefly, a fresh solutionof D-dopachrome methyl ester was prepared by

mixing 2mM L-3,4 dihydroxyphenylalaninemeth-yl ester with 4 mM sodium peroxidate for 5 minat room temperature and then placed directlyon ice before use. The enzymatic reaction was ini-tiated at 25°C by adding 20 ml of the dopachromemethyl ester substrate to 200 ml of MIFWT, E22A,E22Q, C57A;C60A (final concentration 5 mM) orand P2Gmutants prepared in tautomerase assaybuffer (50mMpotassiumphosphate, 1 mMEDTA,pH 6.0). The activity was determined by thesemi-continuous reduction of OD 475 nm usinga spectrophotometer.

Quantification of noncleavedgenomic DNA

Noncleaved genomic DNAwas quantified as per-centage (%) of the total genomic DNA that in-cluded both noncleaved genomicDNAand cleavedgenomic DNA in each individual group.

Quantification of cells with AIF and MIFnuclear translocation

Nuclear translocation of AIF and MIF was cal-culated as the percentage of total cells in eachindividual immunostained image. At least 5 to 12images were quantified for each group. 500 ormore neurons were counted for each condition.White indicates theoverlay ofAIF (red),MIF (green)and 4′,6′-diamidino-2-phenylindole (DAPI) (blue)suggesting the nuclear translocation of both AIFand MIF. Pink indicates the overlay of AIF (red)and DAPI (blue) suggesting the nuclear trans-location of AIF only. Cyan indicates the overlayof MIF (green) and DAPI (blue) suggesting thenuclear translocation of MIF. Representative im-munostaining images of MIF and AIF nucleartranslocation were shown in Figs. 4E and 5C andfig. S17A.

Intracerebroventricular (ICV) injection

Three microliters of AAV2-MIF WT, E22Q andE22A (1 × 1013 GC/ml, Vector BioLabs) were in-jected into both sides of intracerebroventricularof the newborn MIF KO mice (41). The expres-sion of MIF and its variants were checked byimmunohistochemistry after MCAO surgery at 8–16 weeks of age.

Neurobehavioral activity

Spontaneous motor activity was evaluated 1 day,3 days and 7 days after MCAO by placing theanimals in amouse cage for 5min. A video camerawas fittedon topof the cage to record the activity ofa mouse in the cage. Neurological deficits wereevaluated by an observer blinded to the treatmentand genotype of the animals with a scale of 0-5 (0,no neurological deficit; 5, severe neurological de-ficit). The following criteria were used to scoredeficits: 0 = mice appeared normal, explored thecage environment andmoved around in the cagefreely; 1 = mice hesitantly moved in cage butcould occasionally touch thewalls of the cage, 2 =mice showed postural and movement abnormal-ities, and did not approach all sides of the cage,3 = mice showed postural and movement ab-normalities and made medium size circles in thecenter of cage, 4 = mice with postural abnormal-

ities and made very small circles in place, 5 =mice were unable tomove in the cage and stayedat the center. Recordings were evaluated by ob-servers blinded to the treatment and genotype ofthe animals.The corner test was performed 1 day, 3 days

and 7 days after MCAO to assess sensory andmotor deficits following both cortical and striatalinjury. A video camera was fitted on top of thecage to record the activity of a mouse in the cagefor 5 min. The mice were placed between twocardboards each with a dimension of 30 cmX 20cm X 0.5 mm attached to each other from theedges with an angle of 30°. Once in the corner,the mice usually rear and then turn either left orright. Before stroke mice do not show a side pref-erence.Mice with sensory andmotor deficits fol-lowing stroke will turn toward the non-impairedside (right). Percent of right turn = right turns/total turns × 100 was calculated and compared.Recordings were evaluated by observers blindedto the treatment and genotype of the animals.

Animals

The JohnsHopkinsMedical Institutions are fullyaccredited by the American Association for the Ac-creditation of Laboratory Animal Care (AAALAC).All research procedures performed in this studywere approved the Johns HopkinsMedical Insti-tutions Institutional Animal Care and Use Com-mittee (IACUC) in compliance with the AnimalWelfare Act regulations and Public Health Service(PHS) Policy. All animal studies were performedin a blinded fashion. Mouse genotype was deter-mined by K.N. Stroke surgery was performed byR.A. Mouse genotypes were decoded after thestroke surgery, mouse behavior tests and dataanalysis. Based on their genotype, mice weregrouped asWT, KO, KO-WT, KO-E22Q and KO-E22A. Within each group, mice were randomlyassigned to subgroups including sham, 1 day-poststroke, 3 days- or 7 days-post stroke.

Statistical analysis

Unless otherwise indicated, statistical evaluationwas carried out by Student’s t test between twogroups andbyone-wayanalysis of variance (ANOVA)followed by post hoc comparisons with theBonferroni test using GraphPad Prism softwarewithinmultiple groups.Data are shownasmeans±SEM. P <0.05 is considered significant. Experi-ments for quantification were performed in ablinded fashion. In order to ensure adequatepower to detect the effect, at least 3 independenttests were performed for all molecular biochem-istry studies and at least 5 mice from 3 differentlitters were used for animal studies.

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ACKNOWLEDGMENTS

This work was supported by NIH grant K99/R00 NS078049,American Heart Association (AHA) National ScientistDevelopment Grant 12SDG11900071, and the Universityof Texas Southwestern Medical Center Department of PathologyStartup funds and UT Rising Stars to Y.W.; and grants fromNational Institute on Drug Abuse, NIH, DA000266; and NationalInstitute of Neurological Disorders and Stroke, NIH, R01NS067525, R37 NS067525, and NS38377 to T.M.D. and V.L.D.T.M.D. is the Leonard and Madlyn Abramson Professor inNeurodegenerative Diseases. The authors acknowledge the jointparticipation by the Adrienne Helis Malvin Medical ResearchFoundation through its direct engagement in the continuousactive conduct of medical research in conjunction with the JohnsHopkins Hospital and the Johns Hopkins University School ofMedicine and the foundation’s Parkinson’s Disease ProgramM-2016. ChIP-seq data are archived in National Center forBiotechnology Information, NIH, GEO, GSE65110. Y.W. contributedto all aspects of the project. R.A., G.K.U., K.N., H.P., B.K., L.B.,M.M.H., C.C., R.C., S.M.E., M.M.H., T.-I.K., S.N., G.M., and H.S.helped with some experiments. J.S.J., S.B., and H.Z. providedprotein chips. Z.X. and J.Q. helped with the bioinformatics analysis.Y.W., V.L.D., and T.M.D. designed experiments and wrote the paper.The study was conceived and scientifically directed by V.L.D.and T.M.D. Y.W., H.P., V.L.D., and T.M.D. are inventors on apatent application submitted by Johns Hopkins University thatcovers the use of inhibitors of MIF nuclease activity to treatstroke and other disorders. The supplementary materials containadditional data.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/354/6308/aad6872/suppl/DC1Supplementary TextFigs. S1 to S17Tables S1 and S2References (50, 51)

2 November 2015; accepted 22 August 201610.1126/science.aad6872

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polymerase-1A nuclease that mediates cell death induced by DNA damage and poly(ADP-ribose)

Shaida A. Andrabi, Seth Blackshaw, Heng Zhu, Hongjun Song, Guo-li Ming, Valina L. Dawson and Ted M. DawsonM. Harraz, Calvin Chang, Rong Chen, Jennifer E. Wang, Tae-In Kam, Jun Seop Jeong, Zhi Xie, Stewart Neifert, Jiang Qian, Yingfei Wang, Ran An, George K. Umanah, Hyejin Park, Kalyani Nambiar, Stephen M. Eacker, BongWoo Kim, Lei Bao, Maged

DOI: 10.1126/science.aad6872 (6308), aad6872.354Science 

, this issue p. 82; see also p. 36Sciencecould thus provide a therapeutic strategy against diseases in which PARP-1 activation is excessive.mouse neurons in culture, loss of MIF protected neurons from cell death caused by excessive stimulation. Targeting MIFAIF-associated endonuclease that contributes to PARP-1-induced DNA fragmentation (see the Perspective by Jonas). In

found that macrophage migration inhibitory factor (MIF) is anet al.activates one or more nucleases to cleave DNA. Wang nuclear enzyme known as PARP-1. During this process, apoptosis-inducing factor (AIF) translocates to the nucleus and

Cells that experience stresses and accumulate excessive damage to DNA undergo cell death mediated by aDNA damage-activated nuclease identified

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REFERENCES

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