RESEARCH ARTICLE

Loss of DNA methylation in zebrafish embryos activatesretrotransposons to trigger antiviral signalingYelena Chernyavskaya1,2,3, Raksha Mudbhary1,2,4, Chi Zhang1,2,3, Debra Tokarz5,6, Vinitha Jacob1,2,4,Smita Gopinath1,2, Xiaochen Sun1,7, Shuang Wang1,2, Elena Magnani1,2,3, Bhavani P. Madakashira3,Jeffrey A. Yoder5,6, Yujin Hoshida1,3,7 and Kirsten C. Sadler1,2,3,4,*

ABSTRACTComplex cytoplasmic nucleotide-sensing mechanisms canrecognize foreign DNA based on a lack of methylation and initiatean immune response to clear the infection. Zebrafish embryos withglobal DNA hypomethylation caused bymutations in the ubiquitin-likewith PHD and ring finger domains 1 (uhrf1) orDNAmethyltransferase1 (dnmt1) genes exhibit a robust interferon induction characteristic ofthe first line of defense against viral infection. We found that thisinterferon induction occurred in non-immune cells and examinedwhether intracellular viral sensing pathways in these cells were thetrigger. RNA-seq analysis of uhrf1 and dnmt1 mutants revealedwidespread induction of Class I retrotransposons and activation ofcytoplasmic DNA viral sensors. Attenuating Sting, phosphorylatedTbk1 and, importantly, blocking reverse transcriptase activitysuppressed the expression of interferon genes in uhrf1 mutants.Thus, activation of transposons in cells with global DNAhypomethylation mimics a viral infection by activating cytoplasmicDNA sensors. This suggests that antiviral pathways serve assurveillance of cells that have derepressed intragenomic parasitesdue to DNA hypomethylation.

KEY WORDS: Transposon, DNA methylation, Uhrf1, Interferon,Antiviral

INTRODUCTIONCytosine methylation promotes heterochromatin formation onterminally repressed regions, such as repeats (Biscotti et al., 2015;Yoder et al., 1997), imprinted genes (Li et al., 1993) andtransposable elements (TEs) (Yoder et al., 1997). These canonicalroles for DNA methylation are well established; however, little isknown about the physiological responses to epigenetic stress whenthese functions fail. DNA hypomethylation is associated withdiseases such as cancer and autoimmune disorders, making thistopic both important and relevant. We used zebrafish embryos

with mutations in the central DNA methylation machinery toidentify the cellular and physiological responses to global DNAhypomethylation.

Despite intense work in the DNA methylation field, its role inregulating differentially expressed genes is controversial (Gutierrez-Arcelus et al., 2013). Many studies in organisms across theevolutionary spectrum have demonstrated that DNA methylationdoes not serve as a universal mechanism of repressing geneexpression (Feng et al., 2010; Grow et al., 2015; Gutierrez-Arceluset al., 2013; Jackson-Grusby et al., 2001; Jacob et al., 2015; Potoket al., 2013; Qi et al., 2015; Zemach et al., 2010; Zhang et al., 2016).In fact, most data indicate that relatively few genes are induced whenmethylation is lost: there is a lack of global gene induction inmammalian cells deficient in the proteins required for DNAmethylation, namely ubiquitin-like with PHD and ring fingerdomains 1 (Uhrf1) (Qi et al., 2015) or DNA methyltransferase 1(DNMT1) (Jackson-Grusby et al., 2001), or during the stage of earlyembryogenesis when the parental methylome is nearly completelyerased (Grow et al., 2015; Potok et al., 2013). Similarly, we foundno evidence of widespread genome activation in zebrafish uhrf1mutants (Jacob et al., 2015). Instead, we found that genesupregulated in uhrf1 mutants fell essentially into two majorcategories: cell cycle and immunity (Jacob et al., 2015). Ourprevious work examined the functional relevance of cell cycle geneactivation (Jacob et al., 2015). Here, we investigate the basis forimmune gene induction in zebrafish mutants that lack DNAmethylation due to uhrf1 (Sadler et al., 2007) or dnmt1 (Andersonet al., 2009) mutations.

Although DNA methylation contributes to the regulation of somegenes,many studies have led to the conclusion that this function is theexception rather than the rule. Indeed, we find no evidence that thegenes induced in uhrf1 mutants are directly regulated by DNAmethylation (Jacob et al., 2015). What, then, is the primary purposeof DNA methylation? In most organisms, transposons are the mostheavily methylated regions of the genome. Several recent examplesshow widespread induction of retrotransposon expression in cancercells (Chiappinelli et al., 2015; Leonova et al., 2013; Roulois et al.,2015), embryonic stem cells (Sharif et al., 2016), neural stem cells(Ramesh et al., 2016) and in human embryos (Grow et al., 2015)when the genome becomes hypomethylated, indicating thattransposon repression is the central repressive role of DNAmethylation. Interestingly, in several of these cases, the same set ofimmune genes is upregulated in uhrf1 mutants. Thus, transcriptomedata highlight the conserved function of DNAmethylation to represstransposons and prevent activation of the immune system.

The relationship between TE induction and immune activationhas recently been investigated. Prokaryotic and viral genomes arenot methylated, and this ʻnon-self’ signal triggers the host immunesystem to detect and clear infected cells (Singer et al., 2015; ZhongReceived 29 November 2016; Accepted 2 July 2017

1Division of Liver Diseases, Department of Medicine, Icahn School of Medicine atMount Sinai, 1 Gustave L. Levy Place, Box 1020, New York, NY 10029, USA.2Department of Developmental and Regenerative Biology, Icahn School ofMedicine at Mount Sinai, 1 Gustave L. Levy Place, Box 1020, New York, NY 10029,USA. 3Program in Biology, NewYork University Abu Dhabi, Abu Dhabi, United ArabEmirates. 4Graduate School of Biomedical Sciences, Icahn School of Medicine atMount Sinai, 1 Gustave L. Levy Place, Box 1020, New York, NY 10029, USA.5Department of Molecular Biomedical Sciences, North Carolina State University,Raleigh, NC 27607, USA. 6Center for Comparative Medicine and TranslationalResearch, North Carolina State University, Raleigh, NC 27607, USA. 7Liver CancerProgram, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, 1Gustave L. Levy Place, Box 1020, New York, NY 10029, USA.

*Author for correspondence (kirsten.edepli@nyu.edu)

K.C.S., 0000-0002-1100-4125

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et al., 2006). Unmethylated DNA in the cytoplasm elicits anucleotide detection signaling pathway involving DAI (ZBP1),cGAS (MB21D1), DDX41 and AIM2 and a complementary RNA-sensing pathway that involves RIG-1/MDA5 (IFIH1) (Dempsey andBowie, 2015). The DNA- and RNA-sensing pathways activatestimulator of interferon genes (STING, or TMEM173) andmitochondrial antiviral signaling protein (MAVS), respectively,and these both converge on TANK-binding kinase 1 (TBK1) totranslate the non-self signal into interferon production andactivation of a systemic antiviral response (Dempsey and Bowie,2015). The model that emerges is that loss of DNA methylationleads to derepression of endogenous retrotransposable elements,mimicking a viral infection. The response activated as a result canserve to clear these pseudo-infected cells. Thus, TE derepressionmight be a mechanism to flag cells with epigenetic stress(Milutinovic et al., 2003; Timp and Feinberg, 2013) for immunesurveillance.We used uhrf1 and dnmt1 mutant zebrafish with global DNA

hypomethylation (Anderson et al., 2009; Jacob et al., 2015; Sadleret al., 2007) to determine how loss of genomic methylation leads toinduction of the innate immune system in the developing embryo.We identified a robust activation of type I interferon, the first line ofaction in antiviral signaling, and an expanded population of immunecells, which could not be explained by loss of DNA methylation inthe promoter of the upregulated genes. Instead, we found that Stingand phosphorylated Tbk1 (pTbk1) were induced in uhrf1 and dnmt1mutants accompanied by widespread induction of retrotransposons.Blocking either Sting, pTbk1 or production of cytoplasmic DNA byinhibiting retrotranscription repressed the interferon response inuhrf1mutants.We conclude that TE repression is a primary functionof DNAmethylation during vertebrate development and suggest thatantiviral pathways serve to mark epigenetically damaged cells forclearance by the immune system.

RESULTSUhrf1 loss activates immune genesUhrf1 recognizes hemi-methylated DNA (Arita et al., 2008;Avvakumov et al., 2008; Hashimoto et al., 2008; Qian et al.,2008) and recruits Dnmt1 during DNA replication (Bostick et al.,2007; Sharif et al., 2007). Thus, total cytosine methylation[5-methylcytosine (5MeC)] levels in uhrf1 mutant embryos arereduced to less than half of wild-type (WT) levels (Feng et al., 2010;Jacob et al., 2015; Tittle et al., 2011). Transcriptome analysis ofuhrf1mutants at 120 h post fertilization (hpf ), a time point when themorphological phenotype of these mutants is fully evident, wascarried out using both microarray and RNA-seq. These approachesrevealed that genes annotated as having a function in the immunesystem dominated the category of upregulated genes (Fig. 1A,Tables S1 and S2), and that this pattern was mirrored in dnmt1mutants analyzed by RNA-seq at 120 hpf (Fig. 1A, Table S1). Geneset enrichment analysis (GSEA) using the Gene Ontology (GO)database (geneontology.org) showed that immune-related genesand apoptosis are two of the most robust and significantly enrichedpathways in uhrf1mutants (Fig. 1B, Table S2). We reasoned that theapoptosis genes are largely related to the high degree of cell deathobserved in uhrf1 mutants (Jacob et al., 2015; Sadler et al., 2007;Tittle et al., 2011). Nearly all the immune genes identified from themicroarray were confirmed as significantly upregulated in uhrf1(Fig. 1C, Fig. S1, Table S3) and dnmt1 (Fig. S1B) mutants at120 hpf. On the other hand, genes significantly downregulated inuhrf1 mutants correlated with reduced organs and tissues thataccompany the mutant phenotype (Table S4).

Interferon signaling is the first line of defense to viral infection,and all of the interferons, their receptors and other key players in theinterferon response are well conserved across vertebrates (Briolatet al., 2014; Hamming et al., 2011; Robertsen, 2006; Zou andSecombes, 2011). Genes dysregulated in uhrf1mutants were highlycorrelated with those classified as involved in the type I and type IIinterferon response in human cells (Fig. 2A, Table S2). Many of theupregulated genes in uhrf1 mutants were also enriched in gene setsfrom human cells treated with interferons (Fig. 2B, Table S2).Zebrafish infected with the chikungunya virus (CHIKV) mount atype I interferon response (Briolat et al., 2014; Palha et al., 2013),whereas those infected with the hematopoietic necrosis virus(IHNV) do not (Briolat et al., 2014). We compared the previouslypublished gene expression pattern from zebrafish infected withthese two viruses (Briolat et al., 2014) with the gene expression inuhrf1mutants and found that the pattern of expression of the top tensignificantly upregulated and downregulated genes from CHIKV-infected embryos was similar to that of uhrf1 and dnmt1 mutants(Fig. 2C). However, there was not a strong correlation with the geneexpression pattern induced by the IHNV, which does not elicit arobust interferon response (Briolat et al., 2014) (Fig. 2C). A similarpattern was observed in RNA-seq analysis of 120 hpf dnmt1mutants (Fig. 2C). Furthermore, ifnphi1, the zebrafish ortholog ofmammalian type I interferon (Aggad et al., 2009), was significantlyupregulated in uhrf1 mutants at 120 hpf (Fig. 2D). Therefore,although the embryos in our study were not infected, the geneexpression pattern indicates that uhrf1 and dnmt1mutants mount aninterferon response as a result of loss of genome-wide DNAmethylation similar to that observed upon viral infection.

We next investigated the timecourse of expression of selectinterferon-response genes and ifnphi1 by RT-qPCR (Fig. 3A) inparallel with assessment of global DNA methylation status usingslot blotting of genomic DNA for 5MeC (Fig. 3B). We find thatsome genes (ifnphi1, nfkb2 and irf1b) are detected as upregulated byRT-qPCR as early as 55 hpf (Fig. 3A) and became furtheroverexpressed in older uhrf1 mutant larvae (Fig. 3A, Fig. 1C,Fig. S1, Table S3). These genes were also detected as significantlyupregulated by RNA-seq analysis of uhrf1 and dnmt1 mutants at120 hpf (Fig. 3A, Table S1). Although RNA-seq analysis of 55 hpfuhrf1 mutant embryos failed to detect robust gene expressionchanges (Fig. 3A, Table S1), this is likely to reflect differences inassay sensitivity between RT-qPCR and RNA-seq (Seqc/Maqc-IiiConsortium, 2014). Regardless, the finding that some interferon-response genes are upregulated as early as 55 hpf (by RT-qPCR) issignificant, as this immediately follows the global depletion of uhrf1maternal mRNA (at 48 hpf) and DNA hypomethylation (Fig. 3B)(Jacob et al., 2015). Importantly, these early responses precede anydetectable phenotypic features of uhrf1mutants (Jacob et al., 2015).Taken together, these data show that induction of type I interferonexpression is the earliest detectable change in uhrf1 mutantsfollowing DNA hypomethylation.

uhrf1 mutation and hypomethylated DNA expand theleukocyte populationInterferons are produced by a variety of cell types to recruit andactivate leukocytes (Le Page et al., 2000; Palha et al., 2013). Innateimmunity is functional at the developmental stages studied here,whereas adaptive immunity is not yet fully developed (Masud et al.,2017), and previous studies found that neutrophils and hepatocytesaccount for most ifnphi1 production in virally infected zebrafish(Palha et al., 2013). We asked whether an expanded population ofleukocytes caused the induction of interferon genes in uhrf1

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mutants. Transgenic markers of macrophages [Tg(mpeg:mCherry)(Ellett et al., 2011)] and neutrophils [Tg(lysZ:dsRed) (Hall et al.,2007)] revealed expanded populations of these cell types in uhrf1mutants as early as 80 hpf, and this further increased by 120 hpf(Fig. 4A-D) when leukocytes were spread throughout the larvae(Fig. S2). These results were confirmed using Neutral Red to stainfor macrophages (Herbomel et al., 2001), showing that the head andjaw of uhrf1 (Fig. S3A,B, arrows) and dnmt1 (Fig. S3C) mutantshad significantly increased Neutral Red staining. Together, theseresults illustrate that the population of macrophages is expanded inboth uhrf1 and dnmt1 mutants.

Hypomethylated DNA from pathogens can be animmunostimulant (Hemmi et al., 2000; Yeh et al., 2013), and weasked if extracellular hypomethylated DNA could contribute to theexpanded leukocyte population in uhrf1 mutants. We injected theotic vesicle of 72 hpf Tg(mpeg:mCherry) embryos with shearedgenomic DNA isolated from 120 hpf uhrf1mutants or WT siblings.Both sources of DNA increased the number of macrophagesrecruited to the injection site (Fig. S4A), and we reasoned thatgenomic DNA from whole embryos contains cells with varyingamounts of DNAmethylation and thus the genomic DNA from uhrf1mutants was not fully unmethylated. To test this, Tg(mpeg:mCherry)

Fig. 1. uhrf1 loss induces immune gene expression. (A) Heat map of the top 50 upregulated genes in uhrf1 zebrafish mutants at 120 hpf rank ordered by theirexpression analysis via microarray and expression in uhrf1 and dnmt1 via RNA-seq at the same time point. 39 of the top 50 genes (76%) are annotated as having afunction in the immune system (red text). (B) Gene ontology (GO) pathway enrichment analysis of biological processes based on differentially expressed genesidentified through microarray analysis, rank ordered by normalized enrichment score. Red, blue and gray bars denote immune, apoptotic and other pathways,respectively. (C) RT-qPCR validation of microarray results for genes randomly chosen from among those significantly upregulated genes and categorized as havingan immune function, in addition to key components of the immune response. Gene symbols that are marked with an asterisk were identified as upregulated in uhrf1mutants as compared with WT by microarray. *P<0.05, **P<0.005 by t-test (see Table S3 for P-values and fold changes). Error bars represent s.d.

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embryos were injected with either completely unmethylated orcompletely (CpG) methylated oligodeoxynucleotide. Theunmethylated oligo recruited a significantly greater number ofmacrophages (Fig. S4B). This shows that unmethylated DNA is astrong immunostimulant, as viral and microbial DNA is entirelyunmethylated at CpGs and this feature of DNA can be used by theimmune system to distinguish foreign nucleic acid from self. Wehypothesize that dying cells in uhrf1 mutants release DNA into theextracellular space, stimulating recruitment of macrophages andneutrophils. Another intriguing possibility is that uhrf1 loss in innate

immune cells promotes their activation, as has been recently suggestedin humans (Yao et al., 2016).

The interferon induction in uhrf1 mutants is independent ofimmune cellsWe tested whether the expanded population of immune cells was thesource of interferon and immune gene expression in uhrf1 mutantsby blocking all leukocyte development using a morpholinotargeting pu.1 (spi1b) (Rhodes et al., 2005). This achievedmarked reductions in mpeg:mCherry-positive and lysZ:dsRed-

Fig. 2. Induction of a type I interferon response in uhrf1mutant embryos. (A) Gene set enrichment analysis (GSEA) reveals enrichment of type I (alpha, beta)and type II (gamma) interferon pathways in 120 hpf uhrf1 embryos. Normalized enrichment score (NES) and false discovery rate (FDR) are indicated. (B) Leadingedge analysis comparing the uhrf1 interferon gene signature with those reported from human cells exposed to interferon (Table S2). (C) Heat map comparingexpression of the top ten upregulated (red) and downregulated (blue) genes in zebrafish embryos infected with CHIKV or IHNV virus (Briolat et al., 2014) with theexpression of these genes in uhrf1 and dnmt1 mutants (log2 fold change). Note that expression was assessed by RT-qPCR in infected embryos by Briolat et al.(2014) and byRNA-seq in uhrf1 and dnmt1mutants. (D) Expression of zebrafish interferon genes inwhole 120 hpf uhrf1mutants assayed byRT-qPCR. Fold changein individual clutches compared with WT siblings is indicated (black dots). Boxes indicate the 25th and 75th percentile of the range, with whiskers marking the 10thand 90th percentile values.

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positive cells in both WT and uhrf1 mutant embryos at 80 hpf(Fig. 4E), but surprisingly had no effect on the morphologicalphenotype of uhrf1 mutants (Fig. 4E) or the induction of theinterferon gene panel (Fig. 4F, Fig. S5). Similarly, depletingmacrophages using a splice-blocking morpholino that effectivelytargeted irf8 (Fig. S6A-C) had no effect on the morphologicalphenotype of uhrf1 mutants (Fig. S6C) or on the expression ofinterferon genes (Fig. S6D,E). These data demonstrate thatneutrophils and macrophages are not required for robust interferongene expression, nor for the gross morphological defects observedin uhrf1 mutants.Next, we determined which tissues respond to interferons in

uhrf1 mutants, using in situ hybridization to detect interferon targetgene expression. irf1b and isg15 were not detected in WT embryosbut were highly expressed in the jaw, head, eye, liver and gut ofuhrf1 mutants at 80 and 120 hpf (Fig. 5). This differs from thepattern of leukocytes in uhrf1 mutants (Fig. 4A,B), further

supporting the hypothesis that immune cells are not the primarysource of the interferon signaling in uhrf1 mutants.

To ask directly whether loss of Uhrf1 could induce immunegene expression in the absence of immune cells, we used siRNA toknock down UHRF1 in cultured human hepatoma cells (HuH7).Both total and phosphorylated STAT1 protein (Fig. S7A) and theexpression of interferon-response genes were increased in UHRF1-depleted cells (Fig. S7B). Taken together, these data demonstratethat Uhrf1 loss induces the interferon-response genes in non-immune cells.

Changes in ifnphi1/4 transcript levels occur independentlyof changes in local DNA methylationIf DNA methylation changes in uhrf1 mutants directly contributeto the induction of immune gene expression then a difference inthe methylation pattern should be observed in the regulatoryregions of these genes. Whole-genome bisulfite sequencing ofuhrf1 mutants revealed that all genomic elements evaluated hadreduced 5MeC levels (Feng et al., 2010); however, this low-coverage approach to measure DNA methylation was insufficientto evaluate the methylation profile at specific loci. We used Sangersequencing of bisulfite-treated DNA to examine the CpG-richregions in the promoter (−5 kb to +2 kb relative to the transcriptionstart site) of ifnphi1, which is upregulated in uhrf1 mutants, ascompared with ifnphi4, which is not (Fig. 2D). As a positivecontrol, we assessed KenoDr1, a long interspersed nuclear element(LINE) that is heavily methylated in control embryos (Feng et al.,2010). All CpGs analyzed in KenoDr1 were over 90% methylatedin WT embryos and significantly hypomethylated in uhrf1 mutantsat 120 hpf (Fig. S8A), confirming that TE methylation issignificantly reduced in this model. By contrast, all CpGsanalyzed in the promoters of ifnphi1 and ifnphi4 werecompletely unmethylated in WT embryos, and remainedunmethylated in uhrf1 mutants (Fig. S8B). Methylome datasetsfrom WT embryos (Lee et al., 2015; Zhou et al., 2014) showed thatthese genes lack methylation at 24 hpf as well (Fig. S8C). Thus,there is no correlation between the expression of interferon genesand methylation of their promoters. Although we cannot excludethe possibility that other regulatory regions are affected by globalloss of DNA methylation, as suggested by recent studies (Leeet al., 2015), our data argue against a direct regulatory role ofpromoter DNA methylation in the upregulation of interferon genesin uhrf1 mutants.

Cytosolic DNA signaling is required for interferon geneinduction in uhrf1 mutantsAll cells have the potential to sense and respond to foreign cytosolicnucleic acids, and there are multiple sensors for non-self RNA andDNA (Dempsey and Bowie, 2015). Phosphorylation of Tbk1(pTbk1) serves as an indicator that viral sensor signaling is activated(Ma et al., 2012). pTbk1 was barely detectable in WT larvae at120 hpf, but was significantly induced in uhrf1mutants (Fig. 6A,B).Since multiple antiviral pathways converge on pTbk1, we reasonedthat inhibiting its activity would have a greater effect on downstreamsignaling than inhibiting one sensor at a time. A previous studyreported that 1 µM BX795 suppressed pTBK1 signaling byinhibiting its ability to phosphorylate IRF3 (Clark et al., 2009).Here, we found that uhrf1 mutant embryos treated with 1 µMBX795 from 48-120 hpf had significantly reduced expression ofnearly all of the interferon-response genes in our panel (Fig. 6C),whereas BX795 treatment had no effect on immune gene expressionin WT larvae (Fig. S9A).

Fig. 3. Immune gene induction occurs as an early response to loss ofDNA methylation. (A) Transcript levels examined by RT-qPCR of a panel ofimmune genes in 55, 72, 80 and 120 hpf uhrf1 mutants relative to expressionin WT siblings, as compared with RNA-seq from 120 hpf uhrf1 and dnmt1mutants. Expression is depicted as log2 fold change, with significanceindicated by an asterisk (individual P-values are listed in Tables S1 and S3).(B) Slot blot analysis of global 5MeC in uhrf1 mutants at the same timepoints as assayed for gene expression. Error bars represent s.d.

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To determine whether the RNA- or DNA-sensing arm wasactivated in uhrf1 mutants, we analyzed our RNA-seq data andfound upregulation of gene expression in both arms. For instance,mavs was unregulated 1.4-fold (log2) in both uhrf1 and dnmt1mutants at 120 hpf, whereas sting (tmem173) was significantlyupregulated by 3.97-fold and 2.14-fold (log2), respectively. Weinvestigated the contribution of Sting signaling to the immune generesponse in uhrf1 mutants using a previously characterized stingmorpholino (Ge et al., 2015). This significantly suppressed all the

interferon genes in uhrf1mutants to nearly WT levels (Fig. 6D, Fig.S9B). We conclude that signaling via the cytoplasmic DNA-sensingpathway is required for induction of the interferon response in uhrf1mutants. Further work is needed to assess the contribution of theviral RNA-sensing pathway in this system.

Retrotranscribed DNA in the cytoplasm serves as a signal toactivate the STING/TBK pathway. We asked whether thiscontributed to immune induction in uhrf1 mutants usingFoscarnet (Fos), an inhibitor of reverse transcriptases and

Fig. 4. Leukocyte depletion does not reduce the induction of immune genes in uhrf1 mutants. Macrophages (A) and neutrophils (B) are enriched inthe head (magnified in inset) of Tg(mpeg:mCherry;uhrf1) (A) and Tg(lysZ:dsRed;uhrf1) (B) andWTembryos at 80 and 120 hpf. Macrophages (C) and neutrophils(D) in the head region (inset in A,B) are significantly increased in uhrf1mutants at 80 and 120 hpf. The number of embryos and clutches analyzed is indicated foreach sample. (E) 0.5 mM pu.1morpholino (MO) effectively reduces the leukocyte population. (F) RT-qPCR analysis reveals fold change in expression of immunegenes from five clutches of uhrf1 and uhrf1 mutant/pu.1 morphants at 80 hpf (expression in WT embryos is shown in Fig. S5). A line connecting two dotsrepresents one paired sample, which are siblings from a single clutch. P-values were determined by t-test (C,D) or paired t-test (F).

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polymerases that is an effective antiviral agent against HIV andHerpes virus (Crumpacker, 1992; Das et al., 2016; Marchand et al.,2007; Vashishtha and Kuchta, 2016) and has also been shown toreduce the activity of retrotransposons in yeast (Hage et al., 2014).Consistently, Fos treatment significantly reduced the expressionof the genes that were most highly upregulated in uhrf1mutants (ifnphi1, irf1b, isg15; Fig. 6E, Fig. S9C). Importantly,Fos treatment also reduced the enrichment of macrophages in thehead of uhrf1 mutants (Fig. 6F). This points to the intriguingpossibility that cytoplasmic DNA produced via retrotranscriptionserves as the trigger signal for cytoplasmic antiviral signaling inuhrf1 mutants.

Retrotransposable elements are activated in uhrf1 mutantsActivation of cytosolic viral pathways coupled with a stronginduction of type I interferon expression suggest the presence ofan infectious agent, yet the uhrf1 mutants used in this study werenot selectively infected. An alternative explanation is thatintragenomic parasites, i.e. TEs, had become activated andmimicked viral infection in mutants lacking DNA methylation.We tested this by first examining the expression of zferv, a TEpreviously reported to be expressed specifically in the thymus ofWT zebrafish larvae at 5 days post fertilization (dpf ) (Shen andSteiner, 2004). We discovered that 55% of uhrf1 mutantsexpressed zferv in the head (Fig. 7A,B), similar to where irf1band isg15 were upregulated (Fig. 5), indicating that TE expressionand immune induction occur at the same time and place in uhrf1mutants.

To examine TE expression more broadly, we generated an RNA-seq library that was not poly(A) selected to allow us to capture TEexpression from uhrf1 mutants at 55 and 120 hpf and from dnmt1mutants at 120 hpf (Fig. 7C,D, Table S1). There were 90 and 92transposons significantly differentially expressed in 55 and 120 hpfuhrf1mutants, respectively (Fig. 7C, Table S1). The majority of theupregulated TEs belonged to class I retrotransposons, whereasdownregulated transposons were enriched for class II DNAtransposons (84%) (Fig. 7D). Genomic TE abundance in eachcategory cannot account for this distribution, as DNA transposons(class I) dominate, occupying 38% of the zebrafish genome ascompared with 10% of the genome occupied by retrotransposons(class II) (Howe et al., 2013). The pattern of TE induction was thesame in dnmt1 mutants (Fig. 7D, Fig. S10B, Table S1). These datademonstrate that TE dysregulation is a transcriptional response toDNA hypomethylation during development.

If the model that DNA methylation causes derepression of TEsleading to the induction of immune gene expression is valid, thenthese events should occur in that chronological sequence. RNA-seqanalysis of uhrf1 mutants at 55 hpf revealed a marked upregulationof TEs: out of the 90 TEs differentially expressed at this time point,the majority were upregulated (69%), with nearly all (95%) of theupregulated TEs annotated as retrotransposons (Fig. 7D), mirroringfindings in uhrf1 and dnmt1mutants at 120 hpf (Fig. 7D, Table S1).Although the induction of retrotransposons in uhrf1 mutants at55 hpf (Fig. 7E) is lower than that detected at 80 hpf (Fig. S10A) or120 hpf (Fig. 7E, Fig. S10B), it occurs soon after global DNAhypomethylation is detected (Fig. 3B) and is accompanied by a

Fig. 5. Interferon target genes are induced in the head, jawand liver in uhrf1mutants. ISH for irf1band isg15, two of themost upregulated interferon genes, in 80and 120 hpf uhrf1 and WT siblings (3-6 embryos per group/per time point). Staining is particularly intense along the caudomedial edge of the optic tectum (whitearrowhead), ear (asterisk) and liver (black arrowhead) at 120 hpf for both genes. n=15, clutches=3.

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modest interferon response that becomes amplified as timeprogresses. This suggests that TE induction is the trigger forimmune gene induction in embryos with global DNAhypomethylation.We further evaluated the relationship between TE methylation

and expression through bisulfite Sanger sequencing. Analysis oflocus-specific transposon methylation of many TEs wasconfounded by the high divergence between transposon

sequences across their multiple sites of integration in the genome,divergence in TE populations between individuals and thedivergence in the TE sequences between the strains of zebrafishused in this study (AB, TAB14 and TAB5), as compared with thereference genome that we used to design primers and analyze thesequencing results (Tubingen). Nevertheless, we were able toamplify and sequence two TEs that were overexpressed in uhrf1mutants at different time points – Gypsy-21 at 55 hpf and Gypsy-10

Fig. 6. Cytosolic antiviral signaling is activated in uhrf1 mutants. (A) Change of pTbk1 protein levels quantified in three clutches of 120 hpf uhrf1 mutantsand their siblings and (B) a representative western blot. (C) Embryos treated with 1 µM BX795 from 48-120 hpf were assayed for immune gene expression at120 hpf by RT-qPCR in at least three clutches. Relative percentage change in gene expression is shown in treated uhrf1 compared with 0.1% DMSO-treateduhrf1 embryos (n=4). (D) Immune gene expression in uhrf1 mutant embryos injected with sting morpholino (MO) compared with uninjected uhrf1 mutants(n=4). (E) Examination of the expression of a limited panel of immune genes before and after 500 µg/µl Foscarnet (Fos) treatment in uhrf1mutants (n=8). A lineconnecting two dots represents one paired sample of siblings from a single clutch. Significance was determined by t-test (A) or paired t-test (C-E).(F) Assessment of macrophage number in the head of embryos treated with 500 µg/µl Fos. The area quantified in WT and uhrf1 mutants is indicated(red dashed box). Error bars represent s.d.

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at 120 hpf – and found both to have significantly reducedmethylation in uhrf1 mutants (Fig. 7F,G). Since retrotransposonsare both hypomethylated and overexpressed at 55 hpf, preceding thefull activation of the interferon gene panel in uhrf1 mutants(Fig. 8A), and since preventing retrotransposon expression viainhibition of retrotranscription blocked interferon gene induction,we conclude that retrotransposon derepression and

retrotranscription trigger an interferon response in embryos withDNA hypomethylation (Fig. 8B).

DISCUSSIONThe data here show that loss of DNA methylation in developingembryos is the first step in a series of events (Fig. 8A) that leads tothe derepression of retrotransposons, which become retrotranscribed

Fig. 7. Endogenous retrotransposon expression is upregulated in uhrf1 mutants. (A) ISH of zferv expression in representative 120 hpf larvae. Staining isapparent in the eye and optic tectum (arrowheads). (B) ISH quantification of total number of embryos from two clutches scored for zferv expression in the head.(C) Expression of individual TEs, as normalized read counts, is plotted against log2 fold change of analogous TEs in uhrf1 relative toWT siblings as identified throughRNA-seq. Red dots indicate TEs with significantly altered expression (P<0.05) and include the highly expressed Gypsy-10 and Gypsy-21. (D) TEs showingsignificant changes in C and two additional RNA-seq datasets from 55 hpf uhrf1 and 120 hpf dnmt1 mutants were grouped by expression and subdivided intotransposon classes depending on their mechanism of mobility. (E) Expression comparison of select significantly induced TEs shared between 55 and 120 hpf uhrf1mutant RNA-seq datasets. (F,G) Sanger sequencing of BS-PCR products from theGypsy-21 (F) orGypsy-10 (G) locus at 55 and 120 hpf inWT and uhrf1mutants.Bottom panel shows differential methylation.

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and activate the cytoplasmic DNA sensor Sting and its partnerpTbk1 (Fig. 8B) to induce interferon expression. The associationbetween DNA hypomethylation and the induction of the interferonresponse has recently been reported in several systems (Chiappinelliet al., 2015; Jackson-Grusby et al., 2001; Leonova et al., 2013;Matthews et al., 2011; Ramesh et al., 2016; Roulois et al., 2015;Sharif et al., 2016); our work showing that both retrotranscriptionand Sting are required to induce interferon genes by DNAhypomethylation are novel findings. We speculate that globalDNA hypomethylation activates the interferon response as amechanism to prune cells with widespread epigenetic aberrationsfrom the developing embryo. As such, the immune system mightfunction both to flag and eliminate cells with foreign pathogens andthose with other alterations that pose a danger to the organism.The relationship between the methylation status of self DNA and

immune activation is not well understood. We identified TEderepression and the activation of cytosolic antiviral signaling as aresponse mechanism to stimulate interferon production in cells withhypomethylated self DNA. Transcriptome analysis of uhrf1 anddnmt1 mutants uncovered a gene expression profile highlyreminiscent of those caused by viral and bacterial infections(Briolat et al., 2014; Levraud et al., 2007; Meijer and Spaink, 2011;Palha et al., 2013). This was surprising, because nearly all of the

work in the field linking hypomethylated DNA to the interferonresponse has focused on exogenous DNA from either an infectiousor experimental source or has suggested that DNA methylationdirectly regulates the expression of interferon genes. In the firstscenario, engulfment of hypomethylated DNA through theendosomal pathway triggers TLR9, which activates the immunesystem and interferon production (Kužnik et al., 2011; Yasuda et al.,2009). Our attempts to block Tlr9 signaling in uhrf1 mutants havenot been successful and thus it remains possible that hypomethylatedself DNA, perhaps from dead cells, is engulfed and activates Tlr9signaling in neighboring cells. Although the expansion of leukocytesis a prominent phenotype of embryos with DNA hypomethylation,this does not account for the induction of immune genes in uhrf1mutants. Instead, we speculate that leukocytes may be recruited byhypomethylated DNA and debris released from dead cells. This isconsistent with recent findings that uhrf1 and dnmt1 mutants haveincreased neutrophils and tnfa induction in the intestine (Marjoramet al., 2015), where there is an abundance of dead and dying cells.Indeed, our finding that blocking reverse transcription reducesmacrophage expansion suggests that the macrophages are activatedin response to TE activation.

Our findings indicate that DNA methylation causes induction ofimmune gene transcription in uhrf1 mutant cells independently of

Fig. 8. Model for uhrf1 loss and DNAhypomethylation driving immune induction.(A) Summary of phenotype onset in uhrf1 mutantembryos. Phenotypes are subdivided into events thatoccur early (1st wave) and late (2nd wave) indevelopment. (B) uhrf1 mutation results in global DNAhypomethylation and apoptosis. Release of cellulardebris and hypomethylated DNA into the environment,coupled with secretion of immune factors, can recruitimmune cells. Increase in expression of interferon andimmune genes can occur either directly by derepressionof gene promoters or indirectly through cytosolic viralsensors, reflecting reactivation of repressedretrotransposable elements. Arrow colors are indicativeof confirmed (black) or proposed (gray) contributions ofeach pathway to the uhrf1 phenotype.

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any direct regulatory function of DNAmethylation in the expressionof these genes. Indeed, there are few, if any, CpG islands inproximity to any interferon genes in zebrafish, mouse and human;those that are present are largely unmethylated, consistent with theconsensus in the field that CpG islands are protected frommethylation (Jones, 2012). The possibility exists that methylation-sensitive putative enhancers (Lee et al., 2015) exert some level ofcontrol over the genes that we assayed. Although we cannot rule outthat enhancers or other cis-acting regulatory elements of ifnphi1outside of what we examined here are differentially methylated inuhrf1mutants, thus far there is no support for this idea in our model.Instead, our data agree with studies showing that most genes are notrepressed by DNA methylation, since fewer than 1% of all geneswere significantly induced in uhrf1 or dnmt1 mutants, or in othersystems in which DNA methylation is globally reduced (Gutierrez-Arcelus et al., 2013; Jackson-Grusby et al., 2001; Xie et al., 2011).By contrast, our data are consistent with the overwhelming

evidence that repression of potentially mutagenic and genotoxicretrotransposons is a central and conserved function of DNAmethylation (Chiappinelli et al., 2015; Coit et al., 2013; Hutnicket al., 2010; Roulois et al., 2015; Wen et al., 2007; Yeh et al., 2013;Yoder et al., 1997; Grow et al., 2015). Although transcriptionof endogenous retrovirus (ERV) long terminal repeats (LTRs)has previously been reported in association with DNAhypomethylation, only functional retrotransposons would be ableto reverse transcribe their RNA in the cytoplasm. We propose thatuhrf1mutants undergo reactivation of latent TEs, which contributesto the expansion of macrophages and potentially could alsocontribute to other phenotypes. For instance, it is possible thatonce active and retrotranscribed, intact retrotransposons couldreintegrate into the genome, causing genomic instability.A similar mechanism of ERV activation has recently been

reported to cause an interferon response via viral mimicry in cancercells; however, that study did not explore activation of the DNA-sensing arm of the cytoplasmic antiviral cascade nor the possibilityof active ERV reverse transcription (Chiappinelli et al., 2015;Roulois et al., 2015). Our study confirms and extends this bydemonstrating that an analogous mechanism operates in non-transformed cells and in a whole organism, both features that areessential for translating these findings to clinically relevant fields.Moreover, its possible that some of the immune gene induction weobserve in uhrf1 mutants is in response to translated viral proteinsencoded by the overexpressed TEs. The finding that activelyexpressed and translated ERVs in human blastocysts serve as aprotective mechanism against viral infection (Grow et al., 2015)suggests the potential to utilize this response to promoteimmunoprotection in normal cells.How much methylation is required to repress TE expression is a

key unanswered question in the field. We observe 5MeC reductionof as little as 8.4%, as in the case ofGypsy-21, and conclude that thisis sufficient to derepress this transposon. However, our method ofbulk methylation analysis cannot address the cell-specific changesin 5MeC that could be impacting TE expression. Thus, the level ofhypomethylation can vary greatly from cell to cell. For example, inone study that used a transgenerationally silenced CpG-rich UASpromoter in zebrafish, a reduction to 69-79% from the original 90%CpG methylation was correlated with derepression (Goll et al.,2009). Given that TE expression also depends on the age of the TE,which is inversely correlated with both methylation (Hutnick et al.,2010; Jackson-Grusby et al., 2001) and the capacity for activation(Huang et al., 2012), the presence of functional regulatorysequences, which are largely uncharacterized in zebrafish, and the

presence of other repressive epigenetic marks, a simple correlationbetween methylation and expression is difficult to decipher.

This work has relevance beyond infection, as autoimmunediseases and cancer are characterized by heightened immuneresponses. Interestingly, both of these diseases are alsocharacterized by DNA hypomethylation: T-cells from humansystemic lupus erythematosus patients have extensivelyhypomethylated DNA and a hyperactive type I interferonresponse (Absher et al., 2013; Brooks et al., 2010; Volkman andStetson, 2014; Wen et al., 2007) and treating T-cells in vitro withDNA demethylating agents phenocopies some lupus symptoms(Wen et al., 2007). In cancer, the entire genome becomeshypomethylated, which both promotes malignancy and induces animmune response against cancer cells. We identified DNAhypomethylation as a driver of liver cancer in zebrafish withhepatocyte-specific UHRF1 overexpression (Mudbhary et al.,2014). Since chromosomal instability is a hallmark of manycancers, it is possible that reactivation of functional TEs leads to anincreased rate of mutation and contributes to the development ofaggressive cancers. Indeed several studies point in this direction(Honda, 2016; Kemp and Longworth, 2015; Solyom and Kazazian,2012). Therefore, the canonical role of DNA methylation tosuppress TE activation is essential for both normal vertebratedevelopment and for preventing disease.

MATERIALS AND METHODSZebrafish maintenance and generation of transgenic linesFish were raised in accordance with the policies of the Mount SinaiInstitutional Animal Care and Use Committee (IACUC) on a 14:10 h light:dark cycle at 28°C. Embryos carrying the hi272 allele of uhrf1mutants wereused as described (Jacob et al., 2015; Sadler et al., 2007). Embryoshomozygous for the hi272 allele (referred to as uhrf1 mutants) (Amsterdamet al., 2004) were generated from an incross of uhrf1hi272/+ parents. Allanimal research involving zebrafish was approved by the IACUC of IcahnSchool of Medicine and of New York University. The Tg(mpeg:mCherry)and Tg(lysZ:dsRed) lines were described previously (Ellett et al., 2011;Hall et al., 2007). dnmt1s904/s904 mutant embryos were obtained fromcrossing heterozygous parents (Anderson et al., 2009) and sorted based onmorphology. All morphological, histological and gene expression assays fordnmt1 mutants were performed in the same manner as for uhrf1 mutants.

GenotypingIndividual embryos were genotyped at 48-60 hpf, which is before uhrf1mutants can be identified based on phenotype, using DNA from wholeembryos if they had previously been fixed in 4% paraformaldehyde (PFA)or on tails if further tissue collection was required for RNA isolation. Tissuewas collected into 30 µl genotyping DNA lysis buffer (10 mM Tris-HCl pH7.5, 50 mM KCl, 0.3% Tween 20, 0.3% NP-40) per embryo and genotypedusing the primers listed in Table S5 as described (Amsterdam et al., 2004).

RNA and DNA extractionRNAwas extracted from three to ten embryos using Trizol (Thermo Fisher,15596026) and precipitated with ethanol. For embryos younger than 5 dpf,only the anterior half was used for RNA extraction to enrich for tissue mostaffected by uhrf1 loss. RNA used to assess expression of TEs was DNasetreated and re-extracted in Trizol as above. For DNA preparation, five toten embryos were incubated overnight at 55°C in DNA lysis buffer (10 mMTris-HCl pH 7.5, 5 mM EDTA, 1% SDS) and extracted withphenol/chloroform followed by ethanol precipitation. 1 μg RNA was usedwith qScript (QuantaBio, 95048-025) to generate cDNA.

Quantitative reverse-transcription PCR (RT-qPCR)RT-qPCR was performed on cDNA from embryos at 1-5 dpf using primers(Table S5) designed for immune genes or to verify some of the top50 upregulated genes identified from the microarray. Expression in all

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samples was normalized to rplp0. All genes were analyzed in at least threeclutches.

Gene expression profilingGenome-wide expression profiling was performed using the ZebrafishGenome Array (Affymetrix) according to the manufacturer’s instructions.Scanned raw data were normalized using robust multiarray analysis (RMA)algorithm implemented in the GenePattern genomic analysis toolkit(broadinstitute.org/cancer/software/genepattern/) (Reich et al., 2006).Multiple probes corresponding to a single gene were collapsed into theofficial gene symbol provided by NCBI by extracting a probe with maximalvariation. Orthologous conversion to human genes was performed based onthe mapping table provided by Ensemble Biomart (http://www.ensembl.org/biomart/martview/da9e5e05cf511b144e8e81ed57fcfe80). Differentiallyexpressed genes were determined using Bayesian t-test implemented inCyber-T software from the top 1000 genes with the largest coefficient ofvariation. Posterior probability of differential expression (PPDE) >0.95was regarded as statistically significant. As described by Jacob et al. (2015),117 upregulated and 131 downregulated genes were detected in uhrf1mutants compared with WT siblings. Induced or suppressed molecularpathways from the microarray dataset described by Jacob et al. (2015)(GSE55339) were determined using GSEA (Subramanian et al., 2005)implemented in the GenePattern genomic analysis toolkit and MolecularSignatureDatabase (MSigDB, broadinstitute.org/cancer/software/genepattern/)(Jacob et al., 2015).

RNA-seq libraries were prepared according to the Illumina TruSeq RNAsample preparation version 2 protocol with Ribo-Zero Gold. RNA from a poolof embryos (between 10 and 20) were used to generate libraries, which wereanalyzed on an Agilent 2100 Bioanalyzer. We analyzed three clutches of uhrf1mutants and their phenotypicallyWT siblings at 55 hpf, two clutches at 120 hpfand three clutches of dnmt1mutants and siblings at 120 hpf. cDNA libraries for120 hpf uhrf1mutants were sequenced on the IlluminaNextSeq500platform toobtain 75 bp single-end reads, while 55 hpf uhrf1 and 120 hpf dnmt1 mutantswere run on the HiSeq platform to obtain 100 bp paired-end reads. Sequencingquality was assessed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) and the reads were quality trimmed using Trimmomatic(Bolger et al., 2014) to remove low Q scores, adapter contamination andsystematic sequencing errors. Reads were aligned to the Danio rerio GRCz10reference genome assembly with TopHat 2.0.9 (Trapnell et al., 2009). Toestimate gene expression, both FPKM and read counts were calculated withEnsembl annotation (Aken et al., 2016; Anders et al., 2015; Trapnell et al.,2009).Annotation of TEs is according to RepeatMasker track in the UCSCtable browser. Reads of each TE were counted by HTseq (Anders et al.,2015) and normalized to specific sample size. Test of differentialexpression uses a generalized linear model. Counts of each TE weremodeled as negative binomial distributions, which were bothimplemented in DESeq2 in Bioconductor (Gentleman et al., 2004). Anadjusted P-value [false discovery rate (FDR)] <0.05 was considered assignificantly different expression. Transposon families were identifiedvia RepBase (Jurka et al., 2005).

Macrophage stainingWe treated embryos with 20 µg/ml Neutral Red stain in embryowater for 1 hfollowed by a 1 h washout in embryo water to allow the background dye todiffuse out. We increased the concentration of dye and decreased theincubation time from that previously reported in order to obtain maximumintensity of macrophage staining while only staining the most activepopulation of macrophages, since that population would take up the dyemore quickly.

Bisulfite conversion and PCRBisulfite (BS) conversion of 500 ng genomic DNAwas carried out using theEpiJET Bisulfite Conversion Kit (Thermo Scientific, K1461) as permanufacturer’s instructions. As is the case with BS conversion, extensivedegradation and loss of DNA prevents accurate concentration measurementsusing conventional spectrophotometers. Instead, 1 μl of each BS-DNAsample was pipetted onto a thin layer (0.5 cm) of 0.8% agarose containing a

1:1000 dilution of GelStar (Lonza, 50535) and allowed to be absorbed. Thefluorescence from each sample was compared with serially diluted DNAstandards pipetted onto the same plate and the amount of each sample used inPCR was adjusted accordingly. Bisulfite-specific PCR (BS-PCR) primers(Table S5) were designed usingMethPrimer software (Li andDahiya, 2002).

In situ hybridization (ISH)Antisense probes recognizing a 350 bp region of the irf1b transcript and500 bp of the isg15 transcript were generated by PCR from 80 hpf uhrf1mutant cDNA using gene-specific primers (Table S5) with a T7 promotersequence at the 5′ end and a T3 promoter at the 3′ end. A 1.9 kb fragment ofzferv was amplified from the zferv PCRII plasmid (Addgene, 22399) andin vitro transcribed from the T7 promoter. ISH was carried out as previouslydescribed (Thisse et al., 2004).

Slot blotuhrf1 mutants were identified based on phenotypic features after 80 hpf orwere individually genotyped at earlier stages and the DNA extracted using astandard protocol as above and probed for 5MeC by slot blot essentially asdescribed (Jacob et al., 2015; Mudbhary et al., 2014). Briefly, 3 ng DNAwas denatured in 0.4 M NaOH/10 mM EDTA, neutralized with 2 mMammonium acetate and loaded in duplicate onto a nitrocellulose membraneusing a slot blot apparatus. Membranes were baked at 80°C, blocked with5% milk followed by incubation in either anti-5MeC (Eurogentec, BI-MECY-100; 1:2000) or anti-dsDNA (Abcam, ab27156; 1:8000) overnight,washed in TBST (37 mM NaCl, 20 mM Tris pH 7.5, 0.1% Tween 20) andprobed with anti-mouse HRP secondary antibody (Promega; 1:5000) for 1 hat room temperature followed by development in ECL (Thermo Scientific).Total 5MeC and dsDNA was averaged between duplicates, and the 5MeC:dsDNA ratio was calculated for at least three clutches at each time point andaveraged.

Otic vesicle injectionsAt 72 hpf, Tg(mpeg:mCherry) larvae were anesthetized in 0.016% tricaineand microinjected in the left otic vesicle with 1 nl PBS containing eitherCpG oligodeoxynucleotide (15 µM, 100 ng/µl), methylated CpG (mCpG)oligodeoxynucleotide (15 µM) or no oligodeoxynucleotide (control). Thesequence of the oligos is 5′-TCGTCGTTGTCGTTTTGTCGTT-3′, with theCpG oligo unmethylated and the mCpG oligo methylated at all cytosineresidues (Yeh et al., 2013). Macrophage presence in the otic vesicle wasassessed by fluorescent confocal microscopy at 2 and 4 h post injection inagarose-mounted larvae.

Drug treatmentWT and uhrf1 mutant embryos were incubated in 5 ml 1 µM BX795,500 ng/ml Foscarnet or 0.1% DMSO from 48-120 hpf. Owing to instability,a fresh 1 µM BX795 solution was used each day. At 120 hpf, five to tenlarvae were collected for RNA extraction.

Morpholino injectionMorpholinos (Table S5) were obtained from GeneTools and injected into1-cell embryos at an average of 4 nl per embryo using stock concentrationsof 0.6 mM, 0.5 mM or 0.3 mM.

ImmunodetectionEmbryos were collected in 10 µl protein lysis buffer per embryoand homogenized by sonication. Samples were run on an 8% SDS-PAGE gel,transferred to PVDF membrane and blotted with anti-pTBK1 (Cell Signaling,5483; 1:1000). Anti-tubulin (Developmental Studies Hybridoma Bank; 1:5000)and anti-β-actin (Sigma, A2228; 1:5000) were used as loading controls.

ImagingWhole-mount embryos were anesthetized and mounted in 3% methyl-cellulose. Imaging was carried out using a Nikon SMZ25 stereomicroscope.

Statistical analysis and quantificationAnalyses of continuous and categorical variables between groups werecompared using the Wilcoxon rank-sum test and Fisher’s exact test,

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respectively. Two-tailed P<0.05 was regarded as statistically significant. Inexperiments involving treatment of embryos to abrogate the immuneresponse, statistical significance was determined based on the percentageresidual gene induction between the treated (either drug or morphant) andcontrol embryos. Multiple hypothesis testing was adjusted using FDR atP<0.05 or Bonferroni correction as appropriate. Band intensity andmacrophage activity quantifications were performed using GelAnalyzer(http://www.gelanalyzer.com/) and ImageJ (NIH), respectively. CpGmethylation analysis was carried out using QUMA (Kumaki et al., 2008).Prism6 software (GraphPad) was used for figure generation and analysis.Venn diagrams and analysis of overlapping genes between the microarrayand RNA-seq datasets were generated using BioVenn (Hulsen et al., 2008).

AcknowledgementsWe thank Patrick Bradley andMatthew Nash for expert fish care; Amaia Lujambio forresearch insight and editorial critique; Jessica Lau for technical contributions; andJill Gregory for scientific illustration.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: Y.C., R.M., S.G., J.A.Y., K.C.S.; Methodology: Y.C., R.M., D.T.,V.J., S.G., C.Z., X.S., K.C.S.; Software: C.Z.; Validation: Y.C., S.W., E.M., B.P.M.;Formal analysis: Y.C., C.Z., X.S., Y.H.; Investigation: Y.C., R.M., D.T., V.J., S.G.,S.W., E.M., B.P.M., K.C.S.; Resources: J.A.Y., K.C.S.; Data curation: C.Z., X.S.;Writing - original draft: Y.C., K.C.S.; Writing - review & editing: Y.C., S.W., J.A.Y.,Y.H., K.C.S.; Supervision: J.A.Y., Y.H., K.C.S.; Project administration: K.C.S.;Funding acquisition: Y.H., K.C.S.

FundingThis work was supported by grants from the National Institutes of Health[6R01DK080789 to K.C.S., R01DK099558 to Y.H., F30DK094503 to V.J. andT32CA078207-14 to support Y.C.] and the European Commission FrameworkProgramme 7 [Heptromic, proposal number 259744 to Y.H.]. Deposited in PMCfor release after 12 months.

Data availabilityRNA-seq data generated in this study are deposited in NCBI Gene ExpressionOmnibus with accession number GSE91024.

Supplementary informationSupplementary information available online athttp://dev.biologists.org/lookup/doi/10.1242/dev.147629.supplemental

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