Abstract
Pericentromeric satellite repeats are enriched in 5-methylcytosine (5mC). Loss of 5mC at these sequences is common in cancer and is a hallmark of Immunodeficiency, Centromere and Facial abnormalities (ICF) syndrome. While the general importance of 5mC is well-established, the specific functions of 5mC at pericentromeres are less clear. To address this deficiency, we generated a viable animal model of pericentromeric hypomethylation through mutation of the ICF-gene ZBTB24. Deletion of zebrafish zbtb24 caused a progressive loss of 5mC at pericentromeres and ICF-like phenotypes. Hypomethylation of these repeats triggered derepression of pericentromeric transcripts and activation of an interferon-based innate immune response. Injection of pericentromeric RNA is sufficient to elicit this response in wild-type embryos, and mutation of the MDA5-MAVS dsRNA-sensing machinery blocks the response in mutants. These findings identify activation of the innate immune system as an early consequence of pericentromeric hypomethylation, implicating derepression of pericentromeric transcripts as a trigger of autoimmunity.
Introduction
In vertebrate genomes, the majority of cytosine residues within CpG dinucleotides are methylated at the 5 position of the cytosine ring (5-methylcytosine, 5mC) (Suzuki and Bird, 2008). 5mC is established by the de novo DNA methyltransferases of the Dnmt3 family, and is propagated by the maintenance DNA methyltransferase, Dnmt1 (Goll and Bestor, 2005). In mice, frogs and zebrafish, mutation or morpholino-mediated depletion of Dnmt1 results in extensive genome-wide methylation loss and embryonic lethality (Anderson et al., 2009; Lei et al., 1996; Rai et al., 2006; Stancheva and Meehan, 2000). In these species, global methylation deficiencies are linked to a variety of adverse outcomes including deregulation of gene expression, derepression of transposons, elevated levels of DNA damage and increased genome instability during mitosis (Smith and Meissner, 2013). Recent studies have further linked global hypomethylation to activation of antiviral signaling pathways in zebrafish mutated for dnmt1 and in cancer cells treated with the DNA methyltransferase inhibitor 5-azacytidine (Chernyavskaya et al., 2017; Chiappinelli et al., 2015; Roulois et al., 2015). While these studies reinforce the general importance of DNA methylation in vertebrate development and tissue homeostasis, the extensive genome-wide loss of methylation in these models makes it difficult to assign significance to methylation deficiencies at any particular subclass of sequence.
The pericentromeric satellite sequences that juxtapose chromosome centromeres represent an essential structural component of chromosomes and a significant source of 5mC in vertebrate genomes. These highly repetitive sequences appear particularly susceptible to methylation loss in cancer and senescent cells, although the consequences of this hypomethylation are not well understood (Enukashvily et al., 2007; Fanelli et al., 2008; Nakagawa et al., 2005; Narayan et al., 1998; Qu et al., 1999; Suzuki et al., 2002; Tsuda et al., 2002). In contrast to global hypomethylation, loss of 5mC at pericentromeric repeats is compatible with human development. Individuals with the rare, autosomal recessive disorder Immunodeficiency, Centromere and Facial anomalies (ICF) syndrome show extensive hypomethylation of pericentromeric repeats, while methylation across the rest of the genome is relatively intact (Tuck-Muller et al., 2000; Weisenberger et al., 2005). Affected individuals usually die in late childhood or early adulthood, and exhibit variable symptoms including immunoglobulin deficiency, facial dysmorphism, growth retardation and a generalized failure to thrive (Ehrlich et al., 2008). Chromosome anomalies including whole-arm deletions and multiradial chromosomes have also been reported in mitogen-stimulated lymphocytes from ICF-patients. However, similar chromosome anomalies are not observed in primary tissues from affected individuals (Ehrlich, 2003).
Homozygosity mapping and whole-exome sequencing have separately implicated four genes in ICF syndrome: DNA Methyltransferase 3B (DNMT3B, ICF type-1), Zinc-finger and BTB domain containing 24 (ZBTB24, ICF type-2), Cell division cycle associated 7 (CDCA7, ICF type-3) and Helicase, lymphoid-specific (HELLS, ICF type-4) (de Greef et al., 2011; Thijssen et al., 2015; Xu et al., 1999). Most of the described mutations in DNMT3B cause amino acid substitutions within the C-terminal catalytic domain, suggesting they may be hypomorphic. In contrast, the majority of mutations in ZBTB24, CDCA7 and HELLS are predicted to cause loss of function. Mechanistically, ZBTB24, CDCA7 and HELLS are thought to converge in a singular pathway that facilitates DNMT3B access to pericentromeric DNA (Jenness et al., 2018; Wu et al., 2016).
To date, most studies of pericentromeric 5mC loss have been performed using transformed B-cell lines derived from ICF patients carrying mutations in DNMT3B (Ehrlich et al., 2008). Attempts to generate viable mouse models of pericentromeric hypomethylation through mutation of ICF genes have had limited success. Mice harboring ICF-like mutations in Dnmt3b exhibit some characteristics of ICF syndrome including small size and facial anomalies. However, most mice die within 24 hours of birth (Ueda et al., 2006). Global methylation profiles were not assessed in these mutants; but significant hypomethylation was reported at both pericentromeric repeats and retroviral sequences. Similar perinatal lethality was observed following deletion of the mouse HELLS orthologue. In this case, mutations were accompanied by roughly 50% reductions in 5mC, and methylation loss was noted at pericentromeres, retroviruses and some single copy sequences (Tao et al., 2011). Deletion of the mouse Zbtb24 gene was reported to cause embryonic lethality; but methylation changes in these mutants have not been investigated (Wu et al., 2016).
Here, we describe a viable model of pericentromeric methylation loss, generated through mutation of the zebrafish zbtb24 gene. Homozygous mutant adults exhibited key phenotypic hallmarks of ICF syndrome including hypomethylation of pericentromeric satellite repeats. Hypomethylation of these repeats was first detected in mutants at 2 weeks post fertilization (wpf) and became more severe as animals matured. This progressive methylation loss allowed us to investigate the primary consequences of pericentromeric hypomethylation in the context of a vertebrate animal. Using this model, we link derepression of transcripts from hypomethylated pericentromeres to activation of an interferon-based innate immune response, and we demonstrate that this response is mediated through the MDA5-MAVS dsRNA sensing machinery. Our findings provide insights into the earliest consequences of pericentromeric hypomethylation, demonstrating an unappreciated function for methylation of pericentromeric repeats in protecting against autoimmunity.
Results
Mutation of zebrafish zbtb24 causes ICF syndrome-like phenotypes
The zebrafish genome encodes a single, well-conserved orthologue of ZBTB24, which we mutated using TAL effector nucleases (TALENs) (Figure 1A and Figure 1-figure supplement 1 and 2). The recovered 7.9 kb deletion allele (zbtb24mk22; here after referred to as zbtb24Δ), eliminates coding sequence between exons 2 and 5 (Figure 1B). Animals that were homozygous for this deletion lacked detectable zbtb24 transcripts, suggesting zbtb24Δ is a null allele (Figure 1-figure supplement 2D). Zbtb24Δ/Δ embryos were born to heterozygous parents at the expected Mendelian ratios and had no obvious morphological abnormalities during the first two weeks of development (Figure 1C). Phenotypes that were reminiscent of ICF syndrome emerged as animals matured. Consistent with the small stature observed in ICF syndrome, by 3-4 weeks post fertilization (wpf), zbtb24Δ/Δ mutant zebrafish were smaller than wild-type siblings raised under identical conditions, and this size reduction persisted into adulthood (Figure 1D-F). As adults, zbtb24Δ/Δ mutants exhibited facial anomalies that were characterized by a quantifiable elongation of the snout (Figure 1G-H). We also noted evidence of hypogammagloblulinemia in the presence of normal lymphoid cell numbers, which is an immunological hallmark of ICF syndrome (Figure 1I-J). Significant death was noted among homozygous mutants at 4 months of age and fewer than 10% of zbtb24Δ/Δ animals survived beyond 8 months (Figure 1K). Similar phenotypes were observed in zebrafish that were homozygous for a second independently-isolated mutant allele of zbtb24 (zbtb24mk19) (Figure 1-figure supplement 3). Taken together, these findings identify zbtb24 homozygous mutant zebrafish as a faithful animal model of ICF syndrome phenotypes.
Progressive methylation loss at pericentromeric repeats in zbtb24 mutants
Pericentromeric satellite type-1 (Sat1) repeats are found on all zebrafish chromosomes and comprise 5-8% of the zebrafish genome (Phillips and Reed, 2000). As expected, we found that Sat1 sequences from wild-type adults were resistant to digestion with the methylation sensitive restriction enzyme HpyCH4IV, indicating that these pericentromeric repeats were heavily methylated. In contrast to wildtype, Sat1 sequences from zbtb24Δ/Δ and zbtb24mk19/mk19 mutant adults were readily digested with HpyCH4IV, indicating extensive loss of methylation at these repeats (Figure 2A-B and Figure 2-figure supplement 1A). Comparable Sat1 methylation deficiencies were observed when DNA was isolated from dissected adult brain, skin, muscle and fin, suggesting that these sequences were similarly hypomethylated in most adult tissues (Figure 2-figure supplement 1B and Figure 2-figure supplement 2A).
Somewhat unexpectedly, we found that pericentromeric methylation loss in zbtb24Δ/Δ mutants was progressive. While extensive hypomethylation of Sat1 sequences was detected in adults lacking zbtb24, similar hypomethylation was not observed in mutants at 1 wpf (Figure 2C-D). At 2 wpf, zbtb24 mutants exhibited roughly 3-fold increases in HpyCH4IV digestion, and sensitivity to digestion became increasingly pronounced in older animals (Figure 2C-D). By 32 weeks, Sat1 sequences from zbtb24 mutants exhibited a 23-fold increase in HpyCH4IV digestion compared to wildtype, suggesting a greater than 95% reduction in methylation of these repetitive sequence blocks.
Zbtb24 mutants exhibit modest reductions in 5mC at non-pericentromeric sequences
To clarify whether additional sequences were hypomethylated in zbtb24 mutants, we performed Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) using genomic DNA isolated from the fins of three zbtb24Δ/Δ mutant adults and three wild-type siblings at 25 wpf (Garrett-Bakelman et al., 2015). At this stage, Sat1 sequences from isolated fins were 20-fold more sensitive to HypCH4IV in zbtb24 mutants compared to controls, indicating extensive loss of DNA methylation at pericentromeric repeats (Figure 2-figure supplement 2A-B). We then used ERRBS data to interrogate the methylation status of 979,971 non-pericentromeric CpG sites across the genome in the same tissue samples. Our analysis revealed a strong correlation between genome wide 5mC levels in wild-type and zbtb24Δ/Δ mutant adults (Pearson’s correlation value of 0.928), although overall methylation levels were reduced by ∼10% in mutants (Figure 2E and Figure 2-figure supplement 2C). This 10% methylation reduction in mutants consisted primarily of small-magnitude changes in 5mC across the genome, with only 1.3% (13,205) of examined CpG dinucleotides exhibiting methylation differences of greater than 20%. Consistent with this finding, at a threshold of 20% change (p-value<0.01), only 55 differentially methylated regions (DMRs) were identified between wild-type and zbtb24Δ/Δ adults (Supplementary File 4). Methylation levels at endogenous retroviruses and other transposable elements were also examined by methylation sensitive restriction digest. All tested elements were similarly resistant to digestion in zbtb24Δ/Δ mutant adults and wild-type siblings, indicating that these sequences are comparably methylated in both genotypes (Figure 2-figure supplement 3). Collectively, these data reveal limited methylation changes at non-pericentromeric CpG sites across the genome.
Mutation of zbtb24 causes activation of innate immune response genes
To gain insights into the early consequences of methylation loss in zbtb24 mutants, we performed transcriptome analysis on RNA isolated from wild-type and zbtb24Δ/Δ zebrafish at 2 wpf. At this stage, zbtb24Δ/Δ mutants remain morphologically indistinguishable from wildtype, but show clear hypomethylation of pericentromeric sequences. RNA-seq identified 58 genes that were downregulated by more than 2-fold in zbtb24Δ/Δ larvae at 2 wpf, while 119 were upregulated by 2-fold or more (Figure 3A). No gene enrichment signature was observed among downregulated genes. However, roughly 30% of upregulated genes were associated with activation of the innate immune system. In particular, we noted that upregulated transcripts included those associated with interferon stimulated genes (ISGs) and inflammatory cytokines (Figure 3B). Consistent with these observations, Gene Set Enrichment Analysis (GSEA) identified significant enrichment of genes involved in viral response, a key function of innate immune pathways (Figure 3C). Upregulation of ISGs was also observed in zbtb24Δ/Δ and zbtb24mk19/mk19 mutants by qRT-PCR at 3 wpf, whereas the same genes were expressed at wild-type levels at 1 wpf (Figure 3D-E and Figure 3-figure supplement 1). No immune-related genes (and only 1 gene differentially upregulated in the RNA-Seq) were found within 100kb of identified DMRs, suggesting that direct loss of methylation at these sequences was unlikely to cause the response (Figure 2-figure supplement 2D and Supplementary File 4).
The innate immune response in zbtb24 mutants is mediated by sensors of cytosolic RNA
The innate immune system represents an ancient defense system in which pathogen-associated molecular patterns (PAMPs) are recognized by pattern recognition receptors (PRRs). These PRRs induce signaling cascades that drive the production of interferons and other inflammatory cytokines with antiviral and immune modulating functions (Schneider et al., 2014). In addition to extracellular pathogens, PRRs also recognize PAMPs associated with cell-intrinsic stimuli including DNA damage, endogenous retroviral RNA and RNA-DNA hybrids (Chiappinelli et al., 2015; Hartlova et al., 2015; Mankan et al., 2014; Roulois et al., 2015).
To clarify the origin of the response in zbtb24 mutants, we examined the major families of PRRs involved in innate immunity. These include the Toll-like receptors (TLRs), which have broad functions in detecting PAMPs, the RIG-I like receptors (RLRs), which are involved in the detection of cytosolic RNA and cGAMP synthase (cGAS), which functions as a cytosolic sensor of DNA and RNA/DNA hybrids (Crowl et al., 2017). Mutations in key mediator proteins required to propagate interferon signaling from each PRR family were introduced onto the zbtb24 mutant background and we tested the effect on ISG expression. Mutations in the zebrafish orthologs of mitochondrial antiviral-signaling protein (mavs), which is an intermediate in RLR signaling and stimulator of interferon genes (sting), which is involved in cGAS signaling were generated using CRISPR/Cas9 technology (Figure 4-figure supplement 1A-B). The mutant allele of Myeloid differentiation primary response 88 (myd88), which is required for signaling through most TLRs, was previously described (van der Vaart et al., 2013).
As in prior experiments, significant increases of the ISGs, signal transducer and activator of transcription 1b (stat1b) and interferon regulatory factor (irf7) were observed in zbtb24Δ/Δ larvae at 3 wpf by qRT-PCR (Figure 4A-C). Introduction of myd88 or sting mutations had little impact on expression of these ISGs, as similar transcript levels were detected in zbtb24Δ/Δ single mutant animals compared to myd88hu3568/hu3568; zbtb24Δ/Δ or stingmk30/mk30; zbtb24Δ/Δ double mutants (Figure 4A-B). Sustained ISG expression in these double mutants suggests limited roles for TLR and cGAS PRRs in mediating the interferon response in zbtb24 mutants. In contrast to myd88 and sting, mutation of mavs suppressed stat1b and irf7 upregulation in zbtb24Δ/Δ mutant animals. Expression levels of irf7 and stat1b were reduced 2- and 4-fold respectively in mavsmk28/mk28; zbtb24Δ/Δ double mutants when compared to zbtb24Δ/Δ single mutant zebrafish, indicating a requirement for mavs in the upregulation of these ISGs (Figure 4C). This finding implicates RLR signaling in the activation of the innate immune system in zbtb24 mutants and suggests a cytosolic RNA trigger for the response.
Pericentromeric RNA transcripts are sufficient to trigger the interferon response in zbtb24 mutants
Given known roles for DNA methylation in transcriptional repression, we next tested whether loss of methylation at pericentromeric sequence resulted in increased levels of Sat1 transcripts that could trigger the RNA mediated interferon response. Consistent with this model, strong derepression of Sat1 RNA from hypomethylated pericentromeres was noted in zbtb24 mutant adults (Figure 5A and Figure 5-figure supplement 1A), whereas transcripts for other dispersed repetitive elements remained unchanged between mutants and wildtype (Figure 5-figure supplement 1B). Increases in Sat1 transcripts correlated with levels of irf7 expression in adult zebrafish (r=0.77), and upregulation of Sat1 transcripts coincided with the window of ISG induction during development (Figure 5B-C). Both sense and antisense transcripts were detected in mutants using TAG-aided sense/antisense transcript detection (TASA-TD) strand-specific PCR (Henke et al., 2015), suggesting the potential for derepressed Sat1 transcripts to form double stranded RNAs (Figure 5D-E).
To determine whether Sat1 transcripts were sufficient to activate an innate immune response, in vitro synthesized RNA corresponding to Sat1 sense and antisense sequence was injected into wild-type embryos at the 1-cell stage. Expression of the ISGs stat1b, irf7, irf1b and mxa was then assessed at 8 hours post fertilization. Injection of Sat1 RNA was sufficient to cause a 3 to 4-fold upregulation in expression of these ISGs, whereas injection of control transcripts encoding the dsRed fluorophore had no effect on expression of these genes (Figure 5F). These results functionally link the derepression of Sat1 transcripts to the activation of the innate immune response in zbtb24 mutants.
The cytosolic dsRNA helicase MDA5 is required for the interferon response in zbtb24 mutants
Finally, we sought to identify the specific PRR required for the interferon response in zbtb24 mutants. The RLR family of PRRs consists of two RNA helicases that signal through Mavs: Melanoma Differentiation-Associated protein 5 (Mda5) and Retinoic acid-inducible gene I (Rig-I). Rig-I binds 5’ triphosphorylated RNA molecules, whereas Mda5 recognizes long double-stranded RNAs in the cytosol (Crowl et al., 2017). Given that 5’ triphosphorylation of RNAs is a typical viral signature that is unlikely to be present on endogenous RNA transcripts, we reasoned that Mda5 was a more likely candidate for the receptor. To test the requirement for mda5, we generated a 7 base-pair deletion in this gene that disrupted the DEAD box helicase domain (Figure 4-figure supplement 1C). This mda5mk29 allele was then introduced onto the zbtb24 mutant background, and expression of the ISGs stat1b and irf7 was examined at 3 wpf. Homozygous mutation of mda5 was sufficient to restore stat1b and irf7 expression to wild-type levels in zbtb24Δ/Δ mutant larvae, suggesting that Mda5 is the primary PPR required for the response (Figure 6A). This requirement was further validated by RNA-seq, which revealed that a broad panel of ISGs that showed elevated expression in zbtb24 single mutants were no longer upregulated in mda5mk29/mk29; zbtb24Δ/Δ double mutants (Figure 6B-C).
Taken together, these results support a model in which derepression of transcripts from hypomethylated pericentromeres triggers activation of the innate immune system through the Mda5/Mavs viral RNA recognition pathway (Figure 6D). These findings identify roles for pericentromeric RNA as a trigger of autoimmunity and reveal important functions for pericentromeric methylation in suppressing the generation of these immunostimulatory transcripts. Based on these results, we propose that induction of the innate immune system is one of the earliest in vivo consequences of pericentromeric methylation loss.
Discussion
In this study, we describe a viable animal model of ICF syndrome which recapitulates key phenotypic hallmarks of the disease including slow growth, facial anomalies, immunoglobulin deficiencies and reduced lifespan. Given that previous attempts to model ICF syndrome have resulted in perinatal or embryonic lethality (Geiman et al., 2001; Ueda et al., 2006; Wu et al., 2016), this zebrafish model provides an important new resource for understanding ICF disease etiology during juvenile and adult life stages. In particular, zbtb24 mutant zebrafish will be useful for understanding phenotypes such as immunoglobulin deficiency, which have not been observed in mouse models and are difficult to study in cell culture systems.
Methylation analysis of zbtb24 mutant zebrafish suggests that the general methylation landscape in these mutants is comparable with that in ICF syndrome. Recent methylome analysis of primary blood from ICF patients identified methylation changes of greater than 20% at roughly 3% of examined CpG dinucleotides. Significant changes in methylation of retroviruses and other dispersed repeats were not observed in these patients (Velasco et al., 2018). Consistent with these findings, our ERRBS analysis revealed methylation changes of greater than 20% at roughly 1.3% of assayed CpG dinucleotides and found methylation of dispersed repeats to be similar between wildtype and in zbtb24 mutant zebrafish.
In the current study, methylation levels at pericentromeric Sat1 sequences could not be quantified by ERRBS, as this technique relies on Msp1 restriction digest to enrich for CpG containing sequences, and zebrafish Sat1 repeats are lacking in this restriction site. Instead we employed digestion with the methylation sensitive restriction enzyme HpyCH4IV to measure methylation at these repeats. This approach remains the most effective way to assess methylation at highly repetitive sequences. At 32 wpf, we observed increases in sensitivity to HpyCH4IV digestion that are consistent with up to 95% reductions in methylation at Sat1 pericentromeric repeats in zbtb24 mutants, while methylation of these sequences was similar to wildtype at 1 wpf. This progressive loss of 5mC implicates Zbtb24 in regulating the long-term maintenance of methylation at pericentromeric repeats. We note that the onset of ICF-like growth defects in zbtb24 mutant zebrafish emerged in the weeks following Sat1 methylation loss. In at least one case of ICF syndrome type 2, growth reductions and immunodeficiency were also reported to develop with age, raising the possibility that similar progressive methylation loss may impact ICF etiology in humans (von Bernuth et al., 2014). It is also possible that Zbtb24 functions in both maintenance and establishment of pericentromeric methylation, but that requirements for establishment are masked by maternally deposited RNA in zbtb24 mutant zebrafish lines. Unfortunately, zbtb24 homozygous mutant zebrafish are sterile, preventing the generation of the maternal-zygotic mutants required to address this question.
In the current study, we take advantage of the progressive Sat1 methylation loss in zbtb24 mutants to identify activation of interferon signaling as one of the earliest in vivo consequences of pericentromeric hypomethylation. This phenotype cannot be attributed to defects in adaptive immunity, as the zebrafish adaptive immune system is not functional until roughly 4 wpf (Trede et al., 2004). Induction of an interferon response has been reported in the context of global hypomethylation in cancer cell lines treated with the DNA methyltransferase inhibitor 5-azacytidine and in zebrafish mutated for the maintenance DNA methyltransferase machinery (Chernyavskaya et al., 2017; Chiappinelli et al., 2015; Roulois et al., 2015). In each of these cases induction of the interferon response was attributed to massive derepression of endogenous retroviral elements.
Our results are distinguished from these earlier studies in that we identify hypomethylation of pericentromeric sequences and subsequent derepression of associated satellite transcripts as a previously unappreciated trigger of innate immunity. Immunostimulatory motifs have been noted in pericentromeric RNAs derived from mouse and humans, and transcripts derived from these repeats have been observed in p53 null mouse fibroblasts following global methylation loss (Leonova et al., 2013; Tanne et al., 2015). However, while these studies suggest the potential for pericentromeric hypomethylation to drive an interferon response in diverse vertebrate species, experimental evidence in support of this model has been lacking. Here we demonstrate a causative link between derepression of pericentromeric RNAs and the interferon response, and identify a requirement for Mda5/Mavs in mediating the response. Our findings suggest that aberrant upregulation of both sense and antisense transcripts derived from pericentromeric repeats creates an abundance of double stranded RNAs within the cytosol, which mimic features of double stranded RNA viruses. This finding raises the possibility that this pathway may also recognize additional endogenous long dsRNAs that lack viral origin.
While mutation of mda5/mavs rescued the interferon response in zbtb24 mutants, mda5/mavs mutation had little impact on other ICF phenotypes observed in zbtb24 mutants. Therefore, we find it unlikely that the interferon response drives ICF etiology. Rather this response represents an additional consequence of pericentromeric hypomethylation. Hypomethylation of pericentromeric sequences is compatible with human viability and is observed in abnormal cell contexts including cancer and senescence. Massive increases in pericentromeric transcripts and upregulation of interferon genes have both been noted in cancer (Cheon et al., 2014; Ting et al., 2011). Our data raise the possibility that pericentromeric hypomethylation and subsequent derepression of associated RNAs represents an important but underappreciated trigger of autoimmunity in a variety of disease states.
Materials and Methods
Zebrafish husbandry
Zebrafish husbandry and care were conducted in full accordance with animal care and use guidelines with approval by the Institutional Animal Care and Use Committees at Memorial Sloan Kettering Cancer Center and the University of Georgia. Zebrafish were raised under standard conditions at 28° C. Wild-type lines were of the AB background. All mutant alleles are summarized in Supplementary File 1.
TALEN and CRISPR mutagenesis
TALEN sequences were selected using Targeter 2.0 software (Doyle et al., 2012). TAL repeat assembly was achieved using the Golden Gate assembly method, and assembled repeats were integrated into the GoldyTALEN scaffold (Bedell et al., 2012; Cermak et al., 2011). Assembled vectors served as templates for in vitro mRNA transcription using the T3 mMessage mMachine kit (Ambion) according to manufacturer’s instructions. 50–100pg mRNA was injected into wild-type embryos at the one-cell stage. Injected embryos were raised to adulthood and F1 progeny were screened for germline transmission of mutations as previously described (Li et al., 2015). Primers used for detection of mutations and subsequent genotyping are included in Supplementary File 1.
Target selection for CRISPR/Cas9 mediated mutagenesis was performed using CHOPCHOP (Labun et al., 2016). sgRNA templates were generated either by cloning into pT7-gRNA as described by (Jao et al., 2013) or using the oligo-based approach described in (Gagnon et al., 2014) and (Burger et al., 2016). All template oligos are listed in Supplementary File 3. sgRNAs were in vitro transcribed from their respective templates using T7 RNA polymerase (Promega) as per manufacturer protocol. Cas9 RNA was in vitro transcribed from the pT3TS-nls-zCas9-nls plasmid (Jao et al., 2013) using the T3 mMessage mMachine Kit (Ambion). For mutagenesis, 200-400ng of sgRNA and ∼500ng of Cas9 mRNA were co-injected into wild-type embryos at the one-cell stage. Injected embryos were raised to adulthood, and F1 progeny were screened for germline transmission of mutations as previously described (Li et al., 2015). Primers used for detection of mutations and subsequent genotyping are included in Supplementary File 1.
Zebrafish imaging and length measurements
All bright field imaging of zebrafish larvae and adult was performed using Olympus MVX10 with CellSens Standard software. Standard-length was documented using ImageJ as defined in (Parichy et al., 2009). Photoshop (Adobe) adjustments to brightness and contrast were equally applied to all images of whole zebrafish in order to improve visualization.
FACS Analysis of Whole Kidney Marrow
Adult zebrafish at 6 months were sacrificed with a combination of tricaine (Sigma-Aldrich, CAS number 886-86-2) and rapid chilling. Whole kidneys were dissected using forceps and placed in 0.9× PBS/5% FCS. Manual disaggregation using a P1000 pipette resulted in single cell suspensions. Cells were filtered over a 40 μm nylon mesh filter, and resuspended in PBS/FCS to give a final concentration of 100,000 cells/μl. FACS sorting of single cells were analyzed for forward/side scatter profiles. FACS data were analyzed using FloJo software.
DNA Methylation Analysis
For Southern blot analysis, 1 μg of purified total genomic DNA was digested with the indicated methylation sensitive restriction enzyme, fractionated by electrophoresis through a 0.9% agarose gel and transferred to nylon membrane. Probes were PCR amplified using primers in Supplementary File 2 and radiolabeled with 32P-dCTP using RediprimeTM II Random Prime Labelling System (Amersham) according to manufacturer protocol. Hybridization signals were imaged and analyzed using a Typhoon phosphorimager (GE Life Sciences). Signal intensities were measured using ImageJ. Methylation changes at Sat1 was quantified as a ratio of the intensity of the unmethylated / methylated blot regions as indicated in the respective blot.
HypCH4IV was selected for Sat1 methylation analysis over the more traditional Msp1/HpaII isoschizomer pair because Sat1 sequences lack the CCGG sites that are recognized by these enzymes.
Enhanced Reduced Representation Bisulfite Sequencing (ERRBS)
50 ng of high quality genomic DNA was prepared from fin tissue from adult zebrafish at 24 wpf as previously described (Garrett-Bakelman et al., 2015). DNA was digested with Msp1. Bisulphite conversion rates (calculated using non-CpG methylation conversion rates) ranged from 99.6 to 99.7% for all samples (Figure 2-figure supplement 2C). Amplified libraries were sequenced on the Hiseq2000 platform for 50 cycles single end read runs. ERRBS data were filtered for sequence adapters, limited to the first 29 bp of the read (Boyle et al., 2012), and mapped to the zebrafish genome (danRer7) using BSmap (v 2.90) (Xi and Li, 2009). Methylation scores were calculated as the number of unconverted reads divided by the number of total reads at each CpG site. DMRs with at least a 0.2 change in methylation were determined using DSS (delta=0.2, p.threshold=0.01) (Park and Wu, 2016). Sat1 sequences are deficient in Msp1 sites, and are therefore not included in ERRBS data.
RNA Expression Analysis
For qRT-PCR, total RNA was isolated using Trizol (Invitrogen) and precipitated with isopropanol. RNA used for assaying expression of repeat sequences subsequently was treated with DNase using TURBO DNA-free(tm) Kit (Ambion) prior to analyses. RNA was converted to cDNA using GoScriptTM Reverse Transcriptase Kit (Promega) and Real Time PCR was performed using an Applied Biosystems 7500 PCR Machine. Analysis was performed using the 2 − ΔΔCt method, with relative mRNA levels of all transcripts normalized to β-actin1. All primer sequences are listed in Supplementary File 2.
For Northern blot analysis, total RNA was extracted with using Trizol (Invitrogen). 2 μg of RNA was subjected to electrophoresis on 1% agarose gel and transferred to Amersham Hybond-N+ membrane (GE Healthcare). The membrane was probed with 32P-dCTP radiolabeled Sat1 DNA probe at 42°C. Hybridization signals were imaged and analyzed using a Typhoon phosphorimager (GE Life Sciences).
TAG-aided sense/antisense transcript detection (TASA-TD) strand-specific PCR was performed as described by (Henke et al., 2015). Oligos used are listed in Supplementary File 3.
Transcriptome sequencing
After RiboGreen quantification and quality control by Agilent BioAnalyzer, 500 ng of total RNA underwent polyA selection and TruSeq library preparation according to instructions provided by Illumina (TruSeq Stranded mRNA LT Kit), with 8 cycles of PCR. Samples were barcoded and run on a HiSeq 2500 High Output in a 50bp/50bp paired end run, using the TruSeq SBS v4 Kit (Illumina). An average of 45.3 million paired reads was generated per sample. The percent of mRNA bases averaged 62.8%.
For single-mutant RNA-seq analysis presented in Fig 3, reads were mapped to the Zebrafish genome (danRer7) using the rnaStar aligner v2.5.0a (Dobin et al., 2013). We used the two pass mapping method outlined in (Engstrom et al., 2013). The first mapping pass used a list of known annotated junctions from Ensemble. Novel junctions found in the first pass were then added to the known junctions and a second mapping pass was done (on the second pass the RemoveNoncanoncial flag was used). Expression counts (counts per million, cpm) were computed from the mapped reads using HTSeq v0.5.3 (Anders et al., 2015) and Ensemble D.rerio v79 gene annotations. Normalization and differential expression was performed using DESeq (Anders and Huber, 2010).
For RNA-seq analysis presented in Fig 6, raw RNA-seq FASTQ reads were trimmed for adapters and preprocessed to remove low-quality reads using Trimmomatic v0.33 (arguments: LEADING:3 TRAILING:3 MINLEN:36) (Bolger et al., 2014) prior to mapping to the Danio rerio GRCz10 reference genome assembly. Reads were mapped using TopHat v2.1.1 (Kim et al., 2013) supplied with a reference General Features File (GFF) to the Danio rerioGRCz10 reference genome assembly, and with the following arguments: - i 10 -I 5000 --library-type fr-firststrand. Gene expression was estimated using Cuffquant (a tool from Cufflinks v2.2.1), with following arguments --library-type fr-firststrand. Expression level were normalized in FPKM units by Cuffnorm (a tool from Cufflinks v2.2.1), with following arguments --library-type fr-firststrand.
RNA synthesis and injections
Sat1 RNA and dsRed RNAs were in vitro transcribed using Riboprobe® in vitro transcription systems (Promega). Oligos to amplify the DNA template for in vitro transcription are included in Supplementary File 3. Sense and anti-sense transcripts were transcribed in vitro using the T3 and T7 RNA polymerases respectively. RNA was purified illustra MicroSpin G-50 Columns (GE Healthcare) and 50 ng of sense and antisense RNA was co-injected into zebrafish embryos at the 1-cell stage.
Statistical Analysis
The Student unpaired 2-tailed t-test was used for statistical analysis unless specified otherwise. Statistical analysis was performed using GraphPad PRISM software.
Accession Number
All ERRBS and RNA-Seq data reported in this paper have been deposited in GEO under the accession GSE116360. The data is currently private and will be released at publication.
Acknowledgements
This research was supported by a grant from the National Institutes of Health (R01GM11009) to M.G.G. We thank the Goll laboratory for helpful discussions and critical reading of the manuscript. We acknowledge the use of the Integrated Genomics Operation Core of MSKCC, funded by the NCI Cancer Center Support Grant (CCSG, P30 CA08748), Cycle for Survival, and the Marie-Josée and Henry R. Kravis Center for Molecular Oncology. We also acknowledge the use of the Bioinformatics core of MSKCC for support with sequence analysis. ERRBS was performed in the Weill Cornell Medicine Epigenomics Core Facility.