DNMT1 in Six2 Progenitor Cells Is Essential for Transposable Element Silencing and Kidney Development

BASIC RESEARCH www.jasn.org

Szu-Yuan Li,1,2,3,4 Jihwan Park ,1,2 Yuting Guan,1,2 Kiwung Chung,1,2 Rojesh Shrestha,1,2 Matthew B. Palmer,5 and Katalin Susztak 1,2

1Renal-Electrolyte and Hypertension Division, Department of Medicine, 2Department of Genetics, and 5Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; 3Division of Nephrology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan; and 4School of Medicine, National Yang-Ming University, Taipei, Taiwan

ABSTRACT Background Cytosine methylation of regulatory regions, such as promoters and enhancers, plays a key role in regulating gene expression, however, its role in kidney development has not been analyzed. Methods To identify functionally important epigenome-modifying enzymes and genome regions where methylation modifications are functionally important for kidney development, we performed genome- wide methylation analysis, expression profiling, and systematic genetic targeting of DNA methyltransferases (Dnmt1, Dnmt3a,andDnmt3b) and Ten-eleven translocation methylcytosine hydroxylases (Tet2) in nephron progenitor cells (Six2Cre)inmice. Results Genome-wide methylome analysis indicated dynamic changes on promoters and enhancers during development. Six2CreDnmt3af/f, Six2CreDnmt3bf/f, and Six2CreTet2f/f mice showed no significant structural or functional renal abnormalities. In contrast, Six2CreDnmt1f/f mice died within 24 hours of birth, from a severe kidney developmental defect. Genome-wide methylation analysis indicated a marked loss of methylation of transposable elements. RNA sequencing detected endogenous retroviral transcripts. Expression of intracellular viral sensing pathways (RIG-I), early embryonic, nonrenal lineage genes and increased cell death contributed to the phenotype development. In podocytes, loss of Dnmt1, Dnmt3a, Dnmt3b,orTet2 did not lead to functional or structural differences at baseline or after toxic injury. Conclusions Genome-wide cytosine methylation and gene expression profiling showed that by silencing embryonic, nonrenal lineage genes and transposable elements, DNMT1-mediated cytosine methylation is essential for kidney development.

J Am Soc Nephrol 30: 594–609, 2019. doi: https://doi.org/10.1681/ASN.2018070687

DNA cytosine methylation (5mC) is largely erased and because of active and passive demethylation. During cell then reestablished between generations in mammals. At type diversiﬁcation and differentiation, CpG methyl- the blastocyst stage, most cytosines are unmethylated ation increases.1 DNA methylation is accomplished by

Received July 5, 2018. Accepted February 3, 2019.

S.-Y.L., J.P., and Y.G. contributed equally to this work. Correspondence: Dr. Katalin Susztak, University of Pennsylvania, Perelman School of Medicine, Room 12-123, Smilow Trans- Published online ahead of print. Publication date available at lational Center, 3400 Civic Center Boulevard, Philadelphia, PA www.jasn.org. 19104. Email: [email protected]

594 ISSN : 1046-6673/3004-594 J Am Soc Nephrol 30: 594–609, 2019 www.jasn.org BASIC RESEARCH the de novo DNA methyltransferases DNMT3A and DNMT3B.2,3 Significance Statement DNMT3A and DNMT3B have been proposed to be critical for establishing methylation of enhancers that are important for cell Although cytosine methylation plays a key role in regulating gene type–specific transcription factor binding and cell differentiation. expression, including expression of transposable elements such as DNMT1 is the key hemimethylase responsible for methylating the endogenous retroviruses, its role in kidney development is un- known. Using genome-wide methylation analysis in a mouse model, 2,4 newly synthesized DNA strand during replication. The combi- the authors showed dynamic changes in methylation of gene pro- nation of cytosine methylation, histone modification, and chro- moters and enhancers in developing kidneys. Genetic deletion of de matin structure defines genome accessibility and allows the differ- novo methyltransferases in nephron progenitor cells did not lead to entiation of multiple cell types from a single fertilized egg.5 De novo developmental alterations, whereas deletion of Dnmt1, which cytosine methylation of promoters and enhancers plays a key role encodes a maintenance hemimethylase, caused a severe kidney developmental defect. Dnmt1 deletion resulted in a marked loss of in establishing gene expression therefore it is key for cellular dif- methylation of transposable elements, accumulation of endoge- ferentiation. Although this model of gene expression and differen- nous retroviral transcript, and activation of viral sensing pathways tiation is widely accepted, the direct evidence linking promoter and and cell death. These findings indicate that DNMT1-mediated enhancer methylation to gene expression in vivo is limited because methylation to silence embryonic nonrenal lineage genes and of technical difficulties in manipulating cytosine methylation in a transposable elements is essential for kidney development. temporal and site-specific manner. Animalmodelswithgeneticdeletion ofDnmtsindifferent cells during development have been proposed to be the mediators andorganshavebeenusedtounderstandtheroleofmethylation of fetal programming that eventually lead to kidney disease in regulating gene expression and cellular differentiation. In em- development.19–22 bryonic stem cells, pancreatic, gut epithelium, and skin, Dnmt1 This study aimed to identify functionally important epige- depletion leads to dramatic organ development failure.6–9 The nome-modifyingenzymesandregionsinthegenomewheremeth- mechanism of Dnmt1 loss-induced organ defect has been attrib- ylation modifications are functionally important for kidney uted to differential methylation of cell type–specific genes leading developmentbygenome-widemethylationandexpressionanalysis. to cell cycle arrest, premature differentiation, and a failure of tissue self-renewal.10,11 Deletion of de novo methyltransferases (Dnmt3a or Dnmt3b) in different compartments have been as- METHODS sociated with milder phenotypes.12 Transposable elements (TEs) control is one of the most ancient Mice and fundamental function of cytosine methylation. TEs are gen- Dnmt1f/f mice were obtained from Mutant Mouse Regional erally fully methylated in all cells.13 Transposons are mobile genetic Resource Center (MMRRC_014114-UCD). Dnmt3af/f and elements found in every eukaryotic genome sequenced to date and Dnmt3bf/f mice were obtained from the Jackson Laboratory.23,24 account for at least half of the mammalian genome.14 In most Tet2f/f mice were purchased from the Jackson Laboratory (stock mammals, retrotransposons are the predominant TEs. These can number 017573). Dnmts and Tet2 mice were crossed to transgenic be divided into endogenous retroviruses (ERVs), long interspersed mice carrying the PodCre 25 (Jackson Laboratory stock number elements (LINEs), and short interspersed elements (SINEs).13,15 008205) or Six2Cre (Jackson Laboratory stock number 009606). Because of the low methylation state of the blastomere, some ERVs Double transgenic mice and animal gender were identified are highly expressed at this stage. Although some believe ERV by genomic PCR analysis.26 Podocyte injury was induced in expression is a simple byproduct of epigenetic reprogramming, 6-week-old male mice by doxorubicin (20 mg/kg, intravenous; studies indicate ERVs could contribute to multipotency. Recent Pfizer) or streptozotocin injection (50 mg/kg administered intra- studies also found significantERVexpressioninsomecancercells peritoneally, for 5 days; Sigma-Aldrich). due to epigenetic misregulation. Release of TE silencing in cancer cells induces the cytosolic double-stranded RNA (dsRNA) sens- Histologic Procedures and Staining ing pathway that triggers a type 1 IFN response.16,17 Renal histologic changes were examined by PAS stained sec- Nephronnumberinhumansishighlyvariable.Six2- tions. Immunostaining were performed using the follow- expressing cap mesenchymal cells represent a multipotent ing primary antibodies: DNMT1 (ab188453; Abcam), SIX2 nephron progenitor population, which undergo self-renewal (11562–1-AP; Proteintech), p-53 (#2526; CST), fluorescein and give rise to the functioning nephron epithelium.18 labeled Peanut agglutinin (PNA), Lotus tetragonolobus lectin Nephron number at birth shows strong association with hy- (LTL), and Dolichos biflorus agglutinin (DBA) (FL-1071, pertension and kidney disease development later in life. FL-1321, FL-1031; Vector) after heat-induced antigen retrieval Studies have shown that intrauterine nutritional and envi- by Tris-EDTA buffer (pH 9.0 or 6.0). ronmental alterations are key determinants of nephron en- dowment. Epigenome-modifying enzymes use products of Isolation of Six2-Positive Cells intermediate metabolism (methyl or acetyl groups) as their Kidneys from Six2Cre control and Six2Cre Dnmt1f/f mice were substrates. Variations in substrate availability can lead to dissected under a stereo microscope and placed in RPMI, changes in the epigenome. Therefore, epigenetic changes briefly dissociated using 18G and 21G needles. Kidneys were

J Am Soc Nephrol 30: 594–609, 2019 DNMT1 Is Critical for Nephrogenesis 595 BASIC RESEARCH www.jasn.org incubated in collagenase for 10 minutes at 37°C. After being Statistical Analyses passed through a 40 mm cell strainer, 10% serum was added to Statistical analyses were performed using GraphPad Prism neutralize collagenase. After red blood cell lysis, single-cell software (GraphPad Software Inc., La Jolla, CA). All values suspension was collected by centrifugation and suspended in are expressed as mean and SD. Two-tailed t test was used to 500 ml of PBS containing 2% serum. FACS analysis was per- compare two groups. One-way ANOVA with post hoc Tukey formed using BD FACSAria II. test was used to compare multiple groups. A P value ,0.05 was considered statistically significant. Reduced Representation Bisulfite Sequencing Genome-wide DNA methylome analysis was performed us- Study Approval ing the Premium Reduced Representation Bisulfite Se- All experiments in animals were reviewed and approved by the quencing (RRBS) Kit (Diagenode) following manufacturer’s Institutional Animal Care and Use Committee of University instruction. In brief, genomic DNA was extracted from of Pennsylvania and were performed in accordance with the wholekidneysusingPuregeneCoreKitA(Qiagen),100ng institutional guidelines. genomic DNA was then digested using the methylation- insensitive enzyme MspI. Fragment ends were filled in, and Data Access adapters were ligated. After size selection, fragments were All sequencing data (RRBS and RNA sequencing) have been subjected for bisulfite conversion. Libraries were sequenced deposited in NCBI’s Gene Expression Omnibus and are acces- on Illumina Hiseq 50 bp SE configuration. The adapter se- sible through GEO accession number GSE110481. quences and low-quality base calls (,30) were trimmed from the 39 end using Trim_Galore program with–rrbs option. The bisulfite converted sequencing reads were then RESULTS aligned to the mouse reference genome (mm10) using Bis- mark program (default parameters). Differentially methyl- Genome-Wide Methylation During Kidney ated regions (DMRs) were determined using methylKit in R Development package with definition number of CpG $3andDMC$1 We first analyzed methylation changes in developing (newborn) and q ,0.05. Chromatin states of DMRs were annotated and differentiated (adult) kidneys (Figure 1A) by genome-wide using mouse kidney specificChromHMMmaps.27,28 Tran- RRBS. 5mC levels were lower in adult kidneys when compared scription factor motif were identified in the DMRs using with newborn kidneys that are still undergoing nephrogenesis. Homer program. Upon mapping 5mC to RefSeq-based regulatory annotation, we found low 5mC levels around transcription start sites (Figure 1B). RNA Sequencing We identified 4178 DMRs with higher methylation and 4167 RNAwas isolated usingRNeasy mini kit (Qiagen). First, 200 ng total regionswithlowermethylationinadultkidneys(Figure1C). RNA was used to isolate poly A purified mRNA using the Illumina Next, we mapped DMRs onto kidney specific functional reg- TruSeq RNA Preparation Kit. Sequencing was performed in Illu- ulatory regions,definedbycombination ofhistone modifications mina by paired-end 150 bp, and the annotated RNA counts (fastq) and ChromHMM maps.27,28 Promoters and enhancers were were calculated by Illumina’s CASAVA 1.8.2. Sequence quality was significantly enriched in regions showing lower methylation lev- surveyed with FastQC. Adaptor and lower-quality bases were trim- els in adult samples (Figure 1D), whereas 5mC levels were in- med with Trim-galore. Reads were aligned to the Gencode mouse creased in heterochromatin regions. Regions with increased genome (GRCm38) using STAR-2.4.1d. The aligned reads were methylation levels were enriched for developmental transcrip- mapped to the genes (GRCm38; version 7 Ensembl 82) using tion factors, such as LHX binding sites (Supplemental Figure 1). HTSeq-0.6.1. DESeq2 was used to test for differential gene expres- On the other hand, methylation levels were lower at HNF bind- sion between control and knockout groups. ing motifs (Supplemental Figure 1). Gene ontology analysis for To analyze the TEs, the sequencing reads aligned to the mouse DMRs with increased methylation indicated an enrichment genes were firstly removed. First, the aligned reads were mapped to around pathways involved in embryonic morphogenesis and the genes (GRCm38; version 7 Ensembl 82) using HTSeq-0.6.1 development, and a decrease in developmental and metabolic with intersection-nonempty parameter for the mode option. processes (Figure 1E). We showcase some of the DMRs at Slc3a1, Then, the reads which were mapped to the Ensembl genes were Hnf4a,andHoxc9 gene loci (Figure 1F). In summary, our results filtered out from the sam files. Finally, the rest of the sequencing show dynamic methylation changes of regulatory regions (pro- reads (marked as __no_feature) were aligned to the Repeatmasker moter and enhancer) in the developing kidney. which was downloaded from the UCSC genome browser. In re- garding to qRT-PCR, 1 mg RNA was reverse transcribed using the Changes in Dnmt and Tet Expression in Developing cDNA Archive kit (Life Technology), and qRT-PCR was run in the Kidneys ViiA7 System (Life Technology) machine using SYBR Green Mas- Next, we investigated the temporal course and relative expres- ter Mix and gene-specific primers (Supplemental Table 1). The sion of methylome editing enzymes, Dnmt and Tet,inmouse data were normalized and analyzed using the DDCt method. kidneys. Gene expression analysis (by qRT-PCR and RNA

596 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 594–609, 2019 www.jasn.org BASIC RESEARCH

AB C 1 25 P0 adult 20 RRBS 0.5 RNA seq 15 Methylation 10

0 -log10 (q-value) TSS TES 5 Relative Position 4,167 4,178 0 Adult -100 -50 0 50 100 P0 Methylation diffference

D E active promotor weak transcription Hyper-DMRs with q-val<1.0E-10 poised promotor repressed pattern specification process strong enhancer insulator weak enhancer heterochromatin negative regulation of developmental... transcription regulation of cell proliferation 3.1% 1.6% organ morphogenesis embryonic morphogenesis 16.4% embryo development 33.9% Hyper- 1.7% DMRs 020 16.9%

0.8% 16.8% Hypo-DMRs with q-val<1.0E-10 8.9% ornithine metabolic process

apoptotic process involved in... 0.8% 16.6% 16.2% 0.9% 3.5% regulation of fatty acid metabolic... Hypo- 5.6% regulation of glucose metabolic process DMRs 32.2% 20.4% anatomical structure regression

0 5 10 3.7% -log10(binom p-val)

F Slc3a1 Hnf4a Hoxc9 DMR DMR DMR Scale 2 kb mm10 Scale 2 kb mm10 Scale 5 kb mm10 chr17: 85,030,000 chr2: 163,550,000 chr15: 102,980,000 102,985,000 100 100 100 P0-1 0 0 0 100 100 100 P0-2 0 0 0 100 100 100 P0-3 0 0 100 100 0 Adult1 100 0 0 100 100 0 Adult2 100 0 0 100 100 0 Adult3 100 0 0 Slc3a1 Hnf4a CpG Islands Hnf4a 0 Kidney Hnf4a Hoxc9 Hnf4a CpG Islands Hnf4a Kidney Hnf4a CpG Islands Kidney

Figure 1. Methylation changes during kidney development. (A) RRBS and RNA sequencing of P0 and adult mice. (B) Methylation profiles near and within all mouse genes. TES, transcription end site; TSS, transcription start site. (C) Volcano plot showing the DMRs between kidneys of control P0 and adult mice. (D) Annotation of DMRs by kidney-specific chromatin states. (E) Functional annotation analysis for the DMRs between P0 and adult mice. (F) UCSC genome browser screenshot showing the representative DMRs. DMRs are marked by red boxes. y-axis represents methylation level from 0% to 100%. Chromatin states are shown at the bottom (color scheme as above; red promoter, yellow enhancer). sequencing) indicated the expression of Dnmt1, Dnmt3a, Immunofluorescence staining indicated that in the P0 control Dnmt3b, Tet1,andTet3 were higher at birth (P0) when com- mouse kidney cortex, DNMT1 was highly expressed in the cap pared with adults, whereas Tet2 expression did not change mesenchyme and nephron precursors such as renal vesicle and (Figure 2, A and B). S-shaped body (Figure 3B). Although we observed some regional

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A Dnmt1 Dnmt3a Dnmt3b B 25 *** *** *** 1.5 1.5 1.5 1.0 1.0 1.0 20 0.5 0.5 0.5 Relative Relative Relative 15 expression expression expression 0.0 0.0 0.0 P0 3wks P0 3wks P0 3 wks

FPKM 10 Tet1 Tet2 Tet3 p=ns * 5 1.5 *** 2.0 1.5 1.5 1.0 1.0 1.0 0 0.5 0.5

0.5 Relative Relative Relative expression expression expression 0.0 0.0 0.0 Tet1 Tet2 Tet3 Dnmt1 P0 3 wks P0 3 wks P0 3 wks Dnmt3aDnmt3b

CDSix2Cre Six2Cre Dnmt3af/f

Six2 CreGFP loxP Dnmt loxP

Six2+

Six2Cre Dnmt3bf/f Six2Cre Tet2f/f UB

Six2 GFP Cre

loxP Dnmt1 loxP Delete Dnmt exclusively in Six2+ cells

50µm

Figure 2. Differences in expression of methylome editing enzymes in developing mouse kidneys. Expression of Dnmt1, Dnmt3a, Dnmt3b, Tet1, Tet2,andTet3 in newborn (P0) and 3-week-old mouse kidneys analyzed by (A) qRT-PCR and (B) in P0 mouse kidneys analyzed by RNA sequencing. *P,0.05 and ***P,0.001. (C) Breeding scheme of generating Six2Cre Dnmt1f/f mice. (D) Representative PAS-stained sections of control, Six2Cre Dnmt3af/f, Six2Cre Dnmt3bf/f,andSix2Cre Tet2 f/f kidneys in 3-week-old mice. FPKM, fragments per kilobase of transcript per million mapped reads. differences in DNMT1 expression, the double-immunoﬂuorescence functionally signiﬁcant methylome editing enzymes, we crossed staining of DNMT1 with differentiated kidney segment markers Dnmt1, Dnmt3a, Dnmt3b,andTet2 conditional mice with Six2- LTL (proximal tubule), PNA (loop of Henle and distal tubule), EGFPCre mouse 18 (Figure 2C). Six2CreDnmt3af/f, Six2CreDnmt3bf/f, and DBA (collecting duct) showed that the expression of DNMT1 and Six2CreTet2f/f mice showed no clear structural and functional was much lower in differentiated epithelial cell (Figure 3B). renal abnormalities (Figure 2D), indicating that Dnmt3a, Dnmt3b, and Tet2 are dispensable for kidney development. Dnmt3a, Dnmt3b, and Tet2 Are Dispensable for Kidney Development Loss of Dnmt1 in Six2-positive Cells Results in Severe In mice, the progeny of Six2 positive cells give rise to the functional Kidney Developmental Defect nephron epithelium from the glomerulus to distal tubule, but not To study the role of Dnmt1 in kidney development, we created to the last tubule segment, the collecting duct. To determine the Six2CreDnmt1f/f mice. Dnmt1 transcript level was lower in Six2Cre

598 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 594–609, 2019 www.jasn.org BASIC RESEARCH

A B 25 Dnmt1f/f Six2Cre Dnmt1f/f ** Dnmt1f/f 20 Six2Cre Dnmt1f/f 15

FPKM 10

0 DNMT1 / LTL DAPI

Tet1 Tet2 Tet3 Dnmt1 Dnmt3a Dnmt3b C f/f Dnmt1 DNMT1 / PNA DAPI f/f Dnmt1 DNMT1 / DBA DAPI Cre

Six2 50µm EFWT1/DAPI *** 50µm 50

D f/f Dnmt1f/f Six2Cre Dnmt1f/f 40 30 Dnmt1 20

Gloms per slide 10

0 f/f f/f f/f

Dnmt1 Dnmt1 Cre Dnmt1 Six2 Cre Six2

50µm 50µm G Nphs1 Aqp1 Umod Slc12a3 Aqp2 1.5 *** 1.5 *** 1.5 *** 1.5 *** 1.5

1.0 1.0 1.0 1.0 1.0 Dnmt1f/f Cre f/f 0.5 0.5 0.5 0.5 0.5 Six2 Dnmt1

0.0 0.0 0.0 0.0 0.0 Relative expression Relative expression Relative expression Relative expression Relative expression

Figure 3. Dnmt1 is critical for kidney development. (A) Expression of Dnmt1, Dnmt3a, Dnmt3b, Tet1, Tet2,andTet3 in Dnmt1f/f and Six2Cre Dnmt1f/f mouse kidneys (P0) as analyzed by RNA sequencing. Asterisks represent FDR significant differences calculated by DESeq2. **P,0.01. (B) Immunofluorescence staining of DNTM1 (red) and kidney segment markers LTL, PNA, and DBA (green) in P0 kidneys from Dnmt1f/f and Six2Cre Dnmt1f/f mice. (C and D) Representative PAS-stained kidney sections from control (Dnmt1f/f)and Six2Cre Dnmt1f/f kidneys (P0). Specific kidney structures are highlighted. (E) Immunofluorescence staining of Dnmt1f/f and Six2Cre Dnmt1f/f kidneys with WT1 (podocyte marker). (F) Total number of glomeruli per longitudinal kidney sections in Dnmt1f/f and Six2Cre Dnmt1f/f kidney sections at P0 (n=5 per group). (G) Expression of differentiated kidney segment markers in Dnmt1f/f and Six2Cre Dnmt1f/f kidneys by qRT-PCR. FPKM, fragments per kilobase of transcript per million mapped reads. ***P,0.001.

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Dnmt1f/f kidneys compared with controls, whereas no transcrip- that level of Hgf, Fgf10,andRet were increased in Dnmt1 tion change of Dnmt3a and Dnmt3b was detected (Figure 3A). null kidneys (Figure 4G). These genes are usually expressed Immunofluorescence staining of Six2CreDnmt1f/f samples con- before Six2, as they are critically involved in the ureteric bud firmed the lower expression of DNMT1 in Six2-positive cell line- and metanephric mesenchyme interaction before nephron ages such as LTL- and PNA-positive tubule segment, but not in progenitors establishment. Genes that play a role in elongation ureteric bud lineage (DBA-positive segment) (Figure 3B). and segmentation of the renal epithelial vesicles or markers Six2CreDnmt1f/f mice were born at expected Mendelian ratio, of differentiated glomerular and proximal tubule cells were but they died within 24 hours of birth. Animals had visibly smaller almost uniformly decreased in Dnmt1 knockout mice kidneys and their bladders were void of urine (Supplemental Fig- (Figure 4G). Markers of collecting duct cell fate, such as ure 2). Structural analysis indicated significant defect in nephron Aqp2 and Hsd11b2,werenotchangedinSix2CreDnmt1f/f differentiation. In control P0 mouse kidneys, we observed multi- mice, as this segment develops from a ureteric bud and not ple layers of nascent nephrons as they formed in the nephrogenic from a Six2-positive population. These results indicate an ab- zone, followed by a thick layer of renal cortex (Figure 3C). How- sence of differentiation of Six2 progenitor cells and reexpres- ever, Six2CreDnmt1f/f kidneys showed lack of tubule structures, sion of early nephric duct markers. expanded interstitium, and very few comma and S-shape bodies, We found male primordial germ cell–specific genes, such or fully differentiated nephrons (Figure 3, C and D). The expres- as Dazl and Sohlh2, showed one of the highest increases in sion of Ccnd1 (marker of renal vesicle) was slightly increased in Dnmt1 null kidneys (Supplemental Figure 7, A and B). There Dnmt1 knockout kidneys, which is consistent with a differen- was an enrichment for genes with function in homologous tiation defect (Supplemental Figure 3). Expression of WT1, a recombination and meiosis in absence of Dnmt1 (Figure 4F, podocyte marker, was decreased (Figure 3E) and very few LTL- Supplemental Figure 6). We observed the expression of even positive and PNA-positive segments were found in knockout kid- earlier markers such as Zscan4b and Zscan4d that are normally neys (Figure 3B), which confirmed that Six2CreDnmt1f/f mice only expressed in 2–4 cell-stage embryos.31 The full list of had a broad differentiation defect (Figure 3F). On the other DEGs can be found in Supplemental Table 2. In summary, hand, DBA-positive collecting duct cells, which are differentiated genome-wide expression analysis not only indicated the lack from ureteric bud, appeared to be unaffected (Figure 3B). Gene of differentiated epithelial cell markers but also the expression expression analysis showed lower levels of Nphs1 (podocyte), of nonlineage and early embryonic markers in absence of Aqp1, Slc34a1, Hnf4a (proximal tubule), Umod (loop of Henle), Dnmt1. and Slc12a3 (distal convoluted tubule) in Six2CreDnmt1f/f mice,29 but conserved Aqp2 level (Figure 3G, Supplemental Figure 4). In Dnmt1 Loss Results in Broad Cytosine Methylation summary, our studies indicate that very few, if any fully differen- Changes tiated nephrons are formed in the absence of Dnmt1 from Six2- Next, we analyzed the DNA methylomes of control and knock- positive progenitor cells. out kidneys (Figure 5A). Genome-wide RRBS analysis revealed a significant decrease in overall CpG methylation Dnmt1 Depletion Did Not Reduce Six2-Positive Cell (Figure 5B). A bimodal distribution pattern of CpG methyl- Population ation was observed in controls. There was a marked decrease To understand the mechanism of Dnmt1 depletion-induced in the number of fully methylated loci in knockout kidneys defect in nephrogenesis, we first quantified the number of (Figure 5C). Most of the DMRs were hypo-DMRs (45,026 Six2-positive cell population. Through fluorescence-activated hypo versus 88 hyper) (Figure 5D). Next, we mapped DMRs cell sorting, we found no significant differences in GFP- onto kidney-specific regulatory regions, defined by histone positive (Six2-positive) cell number between control P0 modifications and ChromHMM maps. Surprisingly, only a kidneys and Six2CreDnmt1f/f samples (Figure 4, A and B). Im- few promoters or enhancers showed differential methylation munofluorescence staining with SIX2 antibody showed that in Dnmt1 null kidneys (Figure 5D). Transcription factor mo- Six2CreDnmt1f/f mice had slightly, but not significantly higher tifs analysis of the DMRs revealed enrichment for ELK4, SIX2-positive staining (Supplemental Figure 5). We also found MEF2C, and GATA2 binding motifs (Supplemental Figure 8). no change in Six2 transcript levels (Figure 4, C and D), al- Lower methylated regions in Dnmt1 knockout mice were en- though Cited1 level was decreased.30 riched at genes with blastocyst development and formation Next, we performed comprehensive gene expression anal- function (Figure 5E). ysis by RNA sequencing. We identified 1829 differentially ex- More than 50% of the observed DMRs in the Dnmt1 pressed genes (DEGs) when we compared Dnmt1 knockout knockout mice were on regions annotated as repressed region P0 kidneys to controls (FDR,0.05) (Figure 4E, Supplemental or heterochromatin (Figure 5D). These regions are normally Table 2). Genes that showed significantly lower expression in fully methylated. In absence of Dnmt1, methylation differ- knockout kidneys were related to ion transport and renin- ences of intergenic regions, including LINE, SINE, and ERV angiotensin pathways (Figure 4F, Supplemental Figure 6), in- were the most prominent (Figure 5F). LTR retrotransposons dicating the lack of renal tubule epithelia in the knockout include ERVs derived from infectious retroviruses that inte- mice. Although Six2 expression was unaffected, we found grated in the germline. Most of DMRs detected in ERV were

600 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 594–609, 2019 www.jasn.org BASIC RESEARCH

Cre Cre f/f A Six2 Six2 Dnmt1 B 10 p=ns 250K 250K 60000 8 50000 200K GFP+ cells 200K GFP+ cells 6 4.35 6.43 40000 4 150K 150K 30000

GFP+ cell (%) 2 FSC-H FSC-H 20000 100K 100K

0 GFP+ cells / Kidney 10000 f/f Cre f/f 50K 50K Cre

Six2 Dnmt1 Six2 Cre Dnmt1 0 0 Cre 3 3 4 5 3 3 4 5 -10 010 10 10 -10 010 10 10 Six2 Six2

C Six2 D Cited1 E 1.5 2.0 *** f/f 1.5 1.0 -2 1.0 Dnmt1 0.5 0.5 0

0.0 Z-score

0.0 Relative expression Relative expression f/f f/f f/f f/f f/f Cre 2 Adult Adult Six2 Dnmt1 Dnmt1 Dnmt1 Dnmt1 Dnmt1 Cre Cre

Six2 Six2

2 G *** * 1 * F * 0 negative regulation of apoptotic... up-regulated -1 positive regulation of cell proliferation -2 male meiosis

Log2 fold change -3 cellular response to interferon-beta -4 Hgf Ret Six2 Osr1 negative regulation of cell... Gdnf Sall1 Eya1 Pax2 Fgf10 Cited1

meiotic cell cycle Hoxa11 Hoxd11 0369 0.5

excretion down-regulated 0.0 *** fatty acid metabolic process -0.5 lipid metabolic process

regulation of pH -1.0 oxidation-reduction process Log2 fold change -1.5 Wt1

metabolic process Lef1 Lhx1 Jag1 Pax8 Hey2 Wnt4 Hes1 Ccnd1 Ncam1 Notch1 transmembrane transport Notch2 sodium ion transport 0 * ***** ion transport transport -2 0 2 4 6 8 101214 -log10(p value) -4 Log2 fold change

-6 Nrp1 Aqp1 Aqp2 Umod Nphs1 Slc3a1 Slc12a1 Slc12a3 Hsd11b2

Figure 4. Dnmt1 is indispensable for renal epithelial cell differentiation. (A) Representative flow cytometry images of GFP-positive cells in Six2Cre and Six2Cre Dnmt1f/f kidneys. (B) Quantification of GFP-positive cells. (C and D) Expression of Six2 and Cited1 in Dnmt1f/f P0, Six2Cre Dnmt1f/f P0, and wild-type adult kidneys, as analyzed by qRT-PCR. (E) Heatmap of the DEGs between Dnmt1f/f and Six2Cre Dnmt1f/f mice. Color scheme uses z-score distribution. (F) Functional annotation analysis (DAVID) of the differentially expressed (up- and downregulated) genes. (G) Log2 fold change of expression genes involved in kidney development. Asterisks represent FDR significant differences calculated by DESeq2. *P,0.05 and ***P,0.001.

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A B P=0.0004 80

f/f Dnmt1 60 RRBS RNA seq 40 Six2Cre 20 f/f Dnmt1 CpG methylation (%) f/f f/f C Dnmt1 Dnmt1 Cre

Six2 Dnmt1f/f D hyper-DMR 0.1% 0.2% 0.9% 7.3% 0.3%

Cre Six2 Fractiom of CpGs 47.0% 21.9% Dnmt1f/f hypo-DMR 99.8% 17.4% 050100 050100 050100 Methylation level 0.9% 4.1% DMRs with q-val<1.0E-10 E genetic imprinting transcription active promoter lymph vessel development weak transcription poised promoter trophectodermal cell differentiation repressed strong enhancer blastocyst formation dicarboxylic acid transport insulator weak enhancer axis elongation heterochromatin transcription initiation from RNA... blastocyst development 01234567 F Exon lntron TSS ± 3kb 5’ UTR SINE LINE ERV lntergenic 16 50 25 1.2 8 9 12 50

12 40 20 0.9 6 9 40 6 30 15 30 8 0.6 4 6 20 10 20 3 4 10 5 0.3 2 3 10 % of differentially methylated regions 0 0 0 0 0 0 0 0 CTL vs KO P0 vs adult G SINE LINE ERV H Duxf3 (Dux) 1.5 Alu * L1 ERVK B2 1.0 L2 ERVL-MaLR MIR

CR1 ERV1 Relative 0.5

B4 expression RTE-X ERVL ID 0.0 CTL KO

Figure 5. Dnmt1 loss results in broad cytosine methylation changes. (A) RRBS and RNA sequencing analysis of kidneys of Dnmt1f/f and Six2Cre Dnmt1f/f mice. (B) Global CpG methylation in Dnmt1f/f and Six2Cre Dnmt1f/f mouse kidneys. (C) Methylation distributions of CpGs in Dnmt1f/f and Six2Cre Dnmt1f/f mouse kidneys. Histogram plots showing counts by CpG methylation level (x-axis: 0%–100%) distributed across 20 bins of 5% intervals. (D) Annotation of DMRs by kidney-speciﬁc chromatin states. (E) Gene ontology annotation analysis for the DMRs between Dnmt1f/f and Six2Cre Dnmt1f/f mouse kidneys. (F) Distribution of DMRs in different genomic elements. Blue bar represents DMRs detected between Dnmt1f/f and Six2Cre Dnmt1f/f mice. Orange bar represents DMRs detected between Dnmt1f/f P0 and wild-type adult mice. (G) Distribution of DMRs in different transposable elements. (H) Relative expression level of Duxf3 (Dux). y-axis is normalized expression level determined by RNA sequencing. Asterisks represent signiﬁcant differences calculated by DESeq2. *P,0.05.

602 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 594–609, 2019 www.jasn.org BASIC RESEARCH hypo-DMRs and more than half of them were observed in IFN and retinoid acid inducible gene I (RIG-I) signaling was ob- ERVK family (Figure 5G). In contrast, transcription start sites served in Dnmt1 null kidneys (Figure 7, A and B, Supplemental and 59 UTR showed differential methylation during develop- Figure 11). RIG-I-like receptor is a dsRNA helicase that functions ment (Figure 5F). Furthermore, in Dnmt1 knockout mice, we as a pattern recognition receptor and viral sensor. In addition, detected a significant increase in the expression level of the increased p53 signaling was observed in Dnmt1 knockout mice Dux transcription factor, which is a known activator of ERV (Figure 7, C and D, Supplemental Figure 11). We quantitatively elements (Figure 5H).32,33 In summary, loss of Dnmt1 was confirmed the increase in protein expression of P53 in Dnmt1 associated with significantly lower methylation levels in the knockout mice (Figure 7E). Furthermore, the cell death rate was kidney, which specifically affected the normally fully methyl- higher in knockout animals (Figure 7E), whereas cell proliferation ated heterochromatin regions (LINE, SINE, and ERV). rate was lower (Figure 7F). Altogether, our data indicates that Dnmt1 deletion in the progeny of Six2-positive cells leads to re- Dnmt1 Maintains Silencing of TEs in Six2-Positive lease of TEs, silencing ERVs transcription, and subsequent RIG-I Progenitor Cells and P53 activation, contributing to the severe kidney develop- Next, we integrated methylation changes with transcriptome mental defect, but the contribution of other factors such as in- differences. In Six2CreDnmt1f/f kidneys, 1829 DEGs and 25,722 creased senescence cannot be excluded (Figure 7G). DMRs within promoter or gene body regions were identified (Figure 6A). Large numbers of DEGs (749 out of 1829) were in Dnmts and Tets Are Dispensable in Podocytes the vicinity of DMRs. A total of 415 genes showed a decrease To study the role of 5mC in differentiated podocytes, we crossed both in methylation and gene expression levels. Genes in this Dnmt1 f/f, Dnmt3a f/f, Dnmt3b f/f,andTet2 f/f animals with the group mostly included renal tubule epithelial–specific genes Podcre mice 25.MicewerebornatexpectedMendelianfre- such as Slc3a1, Slc23a1,andUmod. Expression of these genes quency and we did not observe significant functional or struc- were lower despite the low methylation in their regulatory re- tural renal abnormalities even at 20 weeks of age (Figure 8A). gions. These results are consistent with dropout of differentiated Next, we tested their response to podocyte injury. To induce epithelial cells in Six2CreDnmt1f/f kidneys (Figure 6B, Supple- diabetes we injected podocyte-specificknockoutmiceand mental Figures 9 and 10A). A limitation of our work is the lack control littermates with streptozotocin. Phenotype analysis of lineage enriched transcriptome analysis, as whole tissue performed 24 weeks after the initiation of diabetes showed analysis identified not only cell-specific but also cell composi- no differences in albuminuria level (Figure 8B). Doxorubicin tion changes (dropout). A small fraction (18%) of all DEGs is known to be toxic to podocytes and can induce FSGS. Even (334) followed a classic pattern of lower methylation and in- after careful characterization of control and Dnmt1, Dnmt3b, crease in gene expression. Gene ontology analysis showed this and Tet2 knockout animals, we failed to observe differences in cluster of genes are enriched for meiotic cell cycle, and sper- albuminuria level in knockout animals (Figure 8, C–E). These matogenesis. (Supplemental Figure 10B). Dnmt1 knockout finding indicated that Dnmt1, Dnmt3b,andTet2 proteins are kidneys expressed high levels of primordial germ cell–specific dispensable in podocytes, even under injury conditions. genes, including Dazl,34,35 Sohlh2,36,37 Tex19.1,38 Pet2,39 and also genes exclusively expressed in 2–4 cell-stage embryos, Zscan4b and Zscan4d.31 Such genes were undetectable in con- DISCUSSION trol P0 kidneys (Figure 6B, Supplemental Figure 7). Tissue enrichment analysis of the DEGs indicated enrichment for Overall, this is the first study to define the methylome at base- brain, testis, and embryo genes (Figure 6C), highlighting the pair resolution in the developing and adult mouse kidneys. To expression nonlineage specific genes and a broad dysregulation define functionally critical methylation changes, we have com- in cell identity in Dnmt1 null kidneys. plemented the methylome atlas by deleting most epigenome Next, we wanted to understand whether reduced methyl- editing enzymes. Methylome analysis of the wild-type devel- ation on TE was associated with increased ERV transcription. oping mouse kidney indicateddynamicchangesof methylation Among 619 LINE, SINE, and ERV families, we have identified levels enriched in gene promoters and enhancers, which is 43 differentially expressed TEs, of which 38 ERV families consistent with the consensus model of cell differentiation were increased and only two ERV families were decreased in where loss of methylation was associated with expression of knockout kidneys (Figure 6D, Supplemental Table 3). These cell type–specific genes and nonlinage gene silencing was as- increased ERV families included all three ERV classes. On the sociated with increased methylation of cis-regulatory ele- other hand, three LINE families (L1Md_Gf, L1Md_T, and ments. It was, however, to our surprise that genetic deletion L1Md_F) were increased in knockout kidneys. Figure 6E il- of de novo methyltransferases Dnmt3a or Dnmt3b, which are lustrates that Dnmt1 depletion–induced hypomethylation was supposed to be responsible for regulatory region methylation, associated with increase in RNA levels of three different ERVs, did not result in observable phenotype development. It is pos- MMERGLN, IAPLTR2b, and IAPEY4. siblethatnofurtherde novo methylation is needed for kidney An increase in ERV in cancer cells triggers cytosolic viral sen- development at this stage; however, this is not consistent with sors, IFN release, and immune activation. Increased expression of our methylome data. Further studies are needed to determine

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A DMR DEG DMR Up DEG f/f Down DMRs 24,779 943 DMRs 25,029 693 Dnmt1 DEGs 390 DEGs 334 690 415 f/f Cre Six2 Dnmt1

Embryonic, ectopic lineage, TE Cell loss

Dazl B DMR Slc3a1 Scale 2kb mm10 DMR chr17: 50,295,000 100 DEG Scale 2 kb mm10 DEG chr17: 85,030,000 85,035,000 100

f/f 0 100

f/f 0 100 0 100 Dnmt1 0 100 Dnmt1 0 100 0 100

f/f 0 100 Cre

f/f 0 100 Cre 0

Six2 100 Dnmt1 0 100 Six2 Dnmt1 0 Dazl Dazl 0 Dazl Slc3a1 Dazl 100 20 30 CpG Islands 0 25 50 CpG Islands FPKM FPKM

Z-score C D Dnmt1f/f Six2Cre Dnmt1f/f Embryo Liver 0-2 2 Cerebellum Testis Brain 0 50 100 150

Gene count Transposable elements

E MMERGLN-int IAPLTR2b IAPEY4_I-int

Scale 200 bases mm10 Scale 50 bases mm10 Scale 200 bases mm10 chrX: 161,101,500 161,102,000 chrX: 61,413,100 61,413,150 61,413,200 chr16: 3,633,000 100 100 100

f/f 0 0 0 100 100 100

0 0 0 100 100 100 Dnmt1

0 0 0 100 100 100

f/f 0 0 0 100 100 100 Cre

0 0 0 100 100 100 Six2 Dnmt1 0 0 0 SINE SINE SINE LINE 0 20000 LINE 0 500 LINE 0 5000 LTR Normalized LTR Normalized LTR Normalized reads reads reads

Figure 6. Methylation changes in Six2Cre Dnmt1f/f mice are associated with embryonic, ectopic lineage and transposable element expression. (A) The number of identiﬁed DMR and DEGs and their consistency and directionality in control and Six2Cre Dnmt1f/f mice. (B) Representative DMRs and their target gene expression changes. DMRs are marked by red boxes. Left panel shows a UCSC genome browser screenshot with DNA methylation patterns and y-axis represents methylation level from 0% to 100%. Right panel shows gene expression level and x-axis represents normalized sequencing reads. (C) Tissue enrichment analysis of the target genes (DAVID). (D) Heatmap of the differentially expressed TEs between Dnmt1f/f and Six2Cre Dnmt1f/f mice. Color scheme uses z-score distribution. (E) Representative DMRs and their target TE expression changes. DMRs are marked by red boxes. Left panel shows a UCSC genome browser screenshot with DNA methylation patterns and y-axis represents methylation level from 0% to 100%. Right panel shows TE expression level and x-axis represents normalized sequencing reads.

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A RIG-I-Like signaling B 0.00 40 -0.05 30 * -0.10 -0.15 20 -0.20 10 * * -0.25 Dnmt1f/f -0.30 3 * Cre f/f -0.35 * Six2 Dnmt1 -0.40 2 Enrichment score (ES) -0.45 * Relative expression 1

0 Dnmt1f/f Six2Cre f/f Irt7 Tnf Tank

Dnmt1 Isg15 CxcI10 D1Pas1

p53 signaling pathway C D 20 0.05 * 0.00 15 * -0.05 10 -0.10 * -0.15 5 * f/f * Dnmt1 -0.20 -0.25 2.0 * Six2Cre Dnmt1f/f -0.30 1.5 * * * * -0.35

Enrichment score (ES) 1.0 -0.40 Relative expression 0.5 0.0 Sfn

f/f Cre Atm

Dnmt1 Six2 Rprm Apaf1

f/f Ccnd2 Ccng1 Ccnb3 Pmaip1 Dnmt1 Cdkn2a Serpinb5

E F Ki67/DAPI Dnmt1f/f Six2Cre Dnm1f/f

0.05 *** 0.8 *** 0.04

f/f 0.6 0.03 0.02 Dnmt1 0.4 per field P53 / DAPI

P53+/DAP1 0.01 0.2 0.00 Ki67+/DAPI per field 0.0 0.04 *** f/f f/f 0.03

f/f Dnmt1 0.02 Six2Cre Dnmt1 per field TUNEL / DAPI 0.01 Dnmt1 Cre TUNEL+/DAPI 0.00 50μm Six2 Dnmt1f/f Six2Cre Dnmt1f/f 50μm

CM Embryonic genes G Six2+ Dnmt1 KO dsRNA sensing (RIG-I, IFN↑)

Transposable UB elements Cell death (p53 ↑)

Cell division

Figure 7. RIG-I-Like signaling and apoptosis are increased in kidneys of Six2Cre Dnmt1f/f mice. (A) The GSEA enrichment plots of RNA sequencing data showing enrichment for RIG-I–like signaling in Six2Cre Dnmt1f/f mice. (B) Relative expression level of genes involved in RIG-I–like signaling. y-axis is normalized expression level determined by RNA sequencing. Asterisks represent significant differences calculated by DESeq2. (C) The GSEA enrichment plots of RNA sequencing data of P53 signaling in Six2Cre Dnmt1f/f mice. (D) Relative expression level of genes involved in p53 signaling. y-axis is normalized expression level determined by RNA sequencing. Asterisks represent significant differences calculated by DESeq2. (E) Left panel: immunofluorescence staining of P53 (red) and TUNEL assay (red) in P0 Dnmt1f/f and Six2Cre Dnmt1f/f mice kidneys; right panel: quantification of P53- and TUNEL-positive signaling according to the staining. (F) Left panel: immunofluorescence staining of Ki67 (red) in P0 Dnmt1f/f and Six2Cre Dnmt1f/f mice kidneys; right panel: quantification of Ki67-positive cells. (G) Schematic of our model which Dnmt1 deletion in progeny of Six2-positive cells affects kidney development. *P,0.05 and ***P,0.001.

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A DnMt1f/f PodCre Dnmt1f/f Dnmt1f/f PodCre Dnmt3af/f

10μm

Dnmt3a3bf/f PodCre Dnmt3a3bf/f Tet2f/f PodCre Tet2f/f

10μm Dnmt1f/f PodCre Dnmt3bf/f B 400 p=NS

300

200

100 Urine albumin/ Creatinine (ug/mg) 0 μ 10 m f/f f/f f/f f/f

Dnmt1 Dnmt1 Dnmt1 Cre Cre STZ Dnmt1 Pod

STZ Pod CDE p=NS p=NS 2000 2000 p=NS 2000 1500 1500 1500

1000 1000 1000

500 500 500

200 Urine albumin/ 200 200 Urine albumin/ Urine albumin/ Creatinine (ug/mg) Creatinine (ug/mg) Creatinine (ug/mg) 100 100 100 0 0 0

f/f f/f f/f f/f f/f f/f f/f f/f f/f f/f f/f f/f

Tet2 Tet2 Tet2 Dnmt1 Dnmt1 Dnmt1 Dnmt3b Cre Cre Cre Cre Dnmt3b Dnmt3b Cre Cre doxo Tet2 doxo Dnmt1 Pod Pod doxo Dnmt3b Pod doxo Pod doxo Pod doxo Pod

Figure 8. Epigenetic editing enzymes are dispensable in mature podocytes. (A) Representative PAS-stained mouse kidney sections of control (Dnmt1f/f) and mice with podocyte-speciﬁc deletion of Dnmt1, Dnmt3a, Dnmt3b, Dnmt3a/3b,andTet. (B) Albuminuria (urine albumin-to-creatinine, micrograms per milligram) of Dnmt1f/f and PodCre Dnmt1f/f mice at baseline and 20 weeks after streptozotocin- induced diabetes. (C–E) Albuminuria (urine albumin-to-creatinine, micrograms per milligram) of control, PodCre Dnmt1f/f, PodCre Dnmt3bf/f,andPodCre Tet2f/f mice at baseline and 7 days after doxorubicin-induced glomerular injury.

606 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 594–609, 2019 www.jasn.org BASIC RESEARCH base resolution methylation and gene expression levels in In summary, to understand the role of 5mC in kidney de- Dnmt3a/3b animals. velopment, we performed genome-wide methylation and gene Once differentiation completed, such as in terminally dif- expression analysis coupled with comprehensive genetic de- ferentiated (nondividing) podocytes, deletion of Dnmt1, letion of methylome editing enzymes. We observed dynamic Dnmt3a, Dnmt3b,andTet2 did not lead to phenotypic alter- methylation changes on promoters and enhancers in kidney ations at baseline and even after injury. These results indicate development and show that DNMT1-mediated DNA methyl- that either methylation changes play only a minor role in po- ation is essential for kidney development by silencing embry- docyte diseases or only minimal remodeling of the podocyte onic, nonlineage specific genes and TEs. methylome occurs in disease states. Dnmt1 plays a critical and nonredundant role in the differentiation of the Six2-positive progenitor population by main- taining methylation. Dnmt1 loss was associated with marked ACKNOWLEDGMENTS decrease in global methylation levels and resulted in very severe kidney developmental defect with barely any fully differ- S.-Y.L., K.C., and Y.G. created transgenic mouse models, S.-Y.L. entiated nephrons. On the other hand, in contrast to the devel- performed animal experiments. J.P. analyzed the RRBS and RNA oping mice, more than 50% of the observed DMRs in the Dnmt1 sequencing data. R.S. and Y.G. performed FACS analysis and im- knockout mice were on regions annotated as repressed region or munostaining. M.B.P. performed histopathological analysis. S.-Y.L. heterochromatin, in addition to promoters and enhancers. The and K.S. conceptualized the work and wrote the manuscript. broad loss of methylation leads to expression of early embryonic S.-Y.L. received research support from Taipei Veterans General genes and nonrenal linage genes (brain, testes, and early embry- Hospital–National Yang-Ming University Excellent Physician Scientists onic genes). This effect was marked and progressive in the prog- Cultivation Program (grant 104-V-A-003) and Ministry of Science and eny of the Six2-positive cells and resulted in a broad and severe Technology (grants 107-2314-B-075-037-MY3 and 105-2314-B-075 differentiation defect that was not specific for a developmental -050 -MY3). J.P.is supported by American Diabetes Association training stage, whereas the Six2-positive cell number was not seemed to grant 1-17-PDF-036. Work in the Susztak laboratory is supported by the be altered. This is consistent with Dnmt1’s function, hemime- National Institutes of Health grants R01 DK076077, DK087635, and thylating the newly synthesized DNA strand during cell division. DP3 DK108220. While our work was under consideration, a study by Wanner The work was presented at the annual meeting of the American et al.40 was published also reporting on the critical role of Dnmt1 Society of Nephrology on November 4, 2017 in New Orleans, LA and in progenitor cell renewal and differentiation of the kidney. This October 26, 2018 in San Diego, CA and deposited in BioRx on June 29, paper reported statistically significant differences in Six2-posi- 2018 (doi: https://doi.org/10.1101/359448). tive cell number at embryonic day 19.5 (P=0.03, one-tailed t test) by immunostaining studies. Using gene expression analysis and sensitive FACS-based cell counting, we could not confirm a de- DISCLOSURES fect in the Six2-positive cell number. The Susztak laboratory receives research support from Biogen, Boehringer Our data suggest that the most profound effect on phenotype Ingelheim, GSK, Merck, Regeneron, Gilead, and ONO Pharma for work not developmentintheSix2CreDnmt1f/f mice was caused by lowering related to this manuscript. the methylation of TEs, regions that are fully methylated in the genome. Methylation is essential for TE silencing, and the lower methylation was associated in an increase in transcriptional ac- SUPPLEMENTAL MATERIAL tivity of these regions, such as ERV expression.11,41,42 Although ERV activation is important in pluripotent stem cells,43 it seems This article contains the following supplemental material that Six2-positiveprogenitorcellsareunabletotoleratecellular online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ dsRNA. The presence of cellular dsRNA induces expression of ASN.2018070687/-/DCSupplemental. viral sensing pathways, such as an IFN response,44 which was Supplemental Figure 1. Transcription factor motif enrichment observed in the Six2CreDnmt1f/f mice. In our review of the liter- analysis of DMRs. ature, this is the first study describing ERVactivation precipitated Supplemental Figure 2. Gross morphology of Dnmt1f/f and Six2Cre by loss of Dnmt1 in tissue progenitor cells. Release of TEs has Dnmt1f/f mice. recently been identified as major mechanism of the antitumor Supplemental Figure 3. Immunofluorescence staining of CCND1 effect of azacytidine in various cancer models.16,17 Ultimately, in Dnmt1f/f and Six2Cre Dnmt1f/f kidneys. ERV activation is associated with reduction in cell proliferation Supplemental Figure 4. Expression of proximal tubule markers in and increased cell death, such as DNA damage related-Trp53 ac- Dnmt1f/f and Six2Cre Dnmt1f/f kidneys. tivation, in the progeny of Six2-positive cells. These downstream Supplemental Figure 5. Immunofluorescence staining of SIX2 in pathways are consistent with previous descriptions in other organ Dnmt1f/f and Six2Cre Dnmt1f/f kidneys. systems.45 Haploinsufficiency of p53 has partially protected from Supplemental Figure 6. Heatmap of representative target genes phenotype development in the Dnmt1-depleted pancreas.8 showing gene expression differences.

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Supplemental Figure 7. Gene expression patterns in Dnmt1f/f and 15. Singer MF: SINEs and LINEs: Highly repeated short and long in- Six2Cre Dnmt1f/f mice. terspersed sequences in mammalian genomes. Cell 28: 433–434, 1982 Supplemental Figure 8. Transcription factor motif enrichment 16. Chiappinelli KB, Strissel PL, Desrichard A, Li H, Henke C, Akman B, et al.: Inhibiting DNA methylation causes an interferon response in cancer via analysis of DMRs. dsRNA including endogenous retroviruses. Cell 169: 361, 2017 Supplemental Figure 9. Representative DMRs and their target gene 17. Roulois D, Loo Yau H, Singhania R, Wang Y, Danesh A, Shen SY, et al.: expression changes. DNA-demethylating agents target colorectal cancer cells by inducing Supplemental Figure 10. Functional annotation analysis (DAVID). viral Mimicry by endogenous transcripts. Cell 162: 961–973, 2015 Supplemental Figure 11. KEGG pathways for RIG-I–like receptor 18. Kobayashi A, Valerius MT, Mugford JW, Carroll TJ, Self M, Oliver G, et al.: Six2 defines and regulates a multipotent self-renewing nephron signaling pathway and P53 signaling pathway. progenitor population throughout mammalian kidney development. Supplemental Table 1. Primer sequences used in this study. Cell Stem Cell 3: 169–181, 2008 Supplemental Table 2. List of DEGs between P0 kidneys of Dnmt1 19. Chu AY, Tin A, Schlosser P, Ko YA, Qiu C, Yao C, et al.: Epigenome-wide knockout mice and controls. association studies identify DNA methylation associated with kidney Supplemental Table 3. List of differentially expressed trans- function. Nat Commun 8: 1286, 2017 posable elements between P0 kidneys of Dnmt1 knockout mice and 20. Susztak K: Understanding the epigenetic syntax for the genetic al- phabet in the kidney. J Am Soc Nephrol 25: 10–17, 2014 controls. 21. Beckerman P, Ko YA, Susztak K: Epigenetics: A new way to look at kidney diseases. Nephrol Dial Transplant 29: 1821–1827, 2014 22. Ko YA, Mohtat D, Suzuki M, Park AS, Izquierdo MC, Han SY, et al.: REFERENCES Cytosine methylation changes in enhancer regions of core pro-fibrotic genescharacterizekidneyfibrosis development. Genome Biol 14: R108, 2013 1. Weber M, Hellmann I, Stadler MB, Ramos L, Pääbo S, Rebhan M, et al.: Distribution, silencing potential and evolutionary impact of promoter 23. Kaneda M, Okano M, Hata K, Sado T, Tsujimoto N, Li E, et al.: Essential DNA methylation in the human genome. Nat Genet 39: 457–466, role for de novo DNA methyltransferase Dnmt3a in paternal and ma- – 2007 ternal imprinting. Nature 429: 900 903, 2004 2. Okano M, Bell DW, Haber DA, Li E: DNA methyltransferases Dnmt3a 24. Lin H, Yamada Y, Nguyen S, Linhart H, Jackson-Grusby L, Meissner A, and Dnmt3b are essential for de novo methylation and mammalian et al.: Suppression of intestinal neoplasia by deletion of Dnmt3b. Mol – development. Cell 99: 247–257, 1999 Cell Biol 26: 2976 2983, 2006 3. Jeltsch A: Molecular enzymology of mammalian DNA methyl- 25. Sweetwyne MT, Gruenwald A, Niranjan T, Nishinakamura R, Strobl LJ, transferases. Curr Top Microbiol Immunol 301: 203–225, 2006 Susztak K: Notch1 and Notch2 in podocytes play differential roles 4. Li E, Bestor TH, Jaenisch R: Targeted mutation of the DNA methyl- during diabetic nephropathy development. Diabetes 64: 4099–4111, transferase gene results in embryonic lethality. Cell 69: 915–926, 1992 2015 5. Palacios D, Summerbell D, Rigby PW, Boyes J: Interplay between DNA 26. McFarlane L, Truong V, Palmer JS, Wilhelm D: Novel PCR assay for methylation and transcription factor availability: Implications for de- determining the genetic sex of mice. Sex Dev 7: 207–211, 2013 velopmental activation of the mouse Myogenin gene. Mol Cell Biol 30: 27. Bogu GK, Vizán P, Stanton LW, Beato M, Di Croce L, Marti-Renom MA: 3805–3815, 2010 Chromatin and RNA maps reveal regulatory long noncoding RNAs in 6. Elliott EN, Sheaffer KL, Schug J, Stappenbeck TS, Kaestner KH: Dnmt1 mouse. Mol Cell Biol 36: 809–819, 2015 is essential to maintain progenitors in the perinatal intestinal epithe- 28. Ernst J, Kellis M: ChromHMM: Automating chromatin-state discovery lium. Development 142: 2163–2172, 2015 and characterization. Nat Methods 9: 215–216, 2012 7. Sen GL, Reuter JA, Webster DE, Zhu L, Khavari PA: DNMT1 maintains 29. Luyckx VA, Bertram JF, Brenner BM, Fall C, Hoy WE, Ozanne SE, et al.: progenitor function in self-renewing somatic tissue. Nature 463: Effect of fetal and child health on kidney development and long-term 563–567, 2010 risk of hypertension and kidney disease. Lancet 382: 273–283, 2013 8. Georgia S, Kanji M, Bhushan A: DNMT1 represses p53 to maintain 30. Little M, Georgas K, Pennisi D, Wilkinson L: Kidney development: Two progenitor cell survival during pancreatic organogenesis. Genes Dev tales of tubulogenesis. Curr Top Dev Biol 90: 193–229, 2010 27: 372–377, 2013 31. Nakai-Futatsugi Y, Niwa H: Zscan4 is activated after telomere short- 9. Liao J, Karnik R, Gu H, Ziller MJ, Clement K, Tsankov AM, et al.: Tar- ening in mouse embryonic stem cells. Stem Cell Reports 6: 483– 495, geted disruption of DNMT1, DNMT3A and DNMT3B in human em- 2016 bryonic stem cells. Nat Genet 47: 469–478, 2015 32. Young JM, Whiddon JL, Yao Z, Kasinathan B, Snider L, Geng LN, et al.: 10. Sheaffer KL, Kim R, Aoki R, Elliott EN, Schug J, Burger L, et al.: DNA DUX4 binding to retroelements creates promoters that are active in methylation is required for the control of stem cell differentiation in the FSHD muscle and testis. PLoS Genet 9: e1003947, 2013 small intestine. Genes Dev 28: 652–664, 2014 33. Geng LN, Yao Z, Snider L, Fong AP, Cech JN, Young JM, et al.: DUX4 11. Karimi MM, Goyal P, Maksakova IA, Bilenky M, Leung D, Tang JX, et al.: activates germline genes, retroelements, and immune mediators: Im- DNA methylation and SETDB1/H3K9me3 regulate predominantly plications for facioscapulohumeral dystrophy. Dev Cell 22: 38–51, 2012 distinct sets of genes, retroelements, and chimeric transcripts in 34. Tsui S, Dai T, Roettger S, Schempp W, Salido EC, Yen PH: Identification mESCs. Cell Stem Cell 8: 676–687, 2011 of two novel proteins that interact with germ-cell-specific RNA-binding 12. Sproul D, Nestor C, Culley J, Dickson JH, Dixon JM, Harrison DJ, et al.: proteins DAZ and DAZL1. Genomics 65: 266–273, 2000 Transcriptionally repressed genes become aberrantly methylated and 35. Chen HH, Welling M, Bloch DB, Muñoz J, Mientjes E, Chen X, et al.: distinguish tumors of different lineages in breast cancer. Proc Natl Acad DAZL limits pluripotency, differentiation, and apoptosis in developing Sci U S A 108: 4364–4369, 2011 primordial germ cells. Stem Cell Reports 3: 892–904, 2014 13. Gifford WD, Pfaff SL, Macfarlan TS: Transposable elements as genetic reg- 36. Shin YH, Ren Y, Suzuki H, Golnoski KJ, Ahn HW, Mico V, et al.: Tran- ulatory substrates in early development. Trends Cell Biol 23: 218–226, 2013 scription factors SOHLH1 and SOHLH2 coordinate oocyte differentia- 14. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, et al.: tion without affecting meiosis I. JClinInvest127: 2106–2117, 2017 International Human Genome Sequencing Consortium: Initial sequencing 37. Ballow DJ, Xin Y, Choi Y, Pangas SA, Rajkovic A: Sohlh2 is a germ cell- and analysis of the human genome. Nature 409: 860–921, 2001 specific bHLH transcription factor. Gene Expr Patterns 6: 1014–1018, 2006

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38. Crichton JH, Playfoot CJ, MacLennan M, Read D, Cooke HJ, Adams IR: to epigenetic reprogramming in the mouse germline. Development 139: Tex19.1 promotes Spo11-dependent meiotic recombination in mouse 3623–3632, 2012 spermatocytes. PLoS Genet 13: e1006904, 2017 42. Walter M, Teissandier A, Pérez-Palacios R, Bourc’his D: An epigenetic 39. Bergsagel PL, Timblin CR, Eckhardt L, Laskov R, Kuehl WM: Sequence switch ensures transposon repression upon dynamic loss of DNA and expression of a murine cDNA encoding PC326, a novel gene ex- methylation in embryonic stem cells. ELife 5, 2016 pressed in plasmacytomas but not normal plasma cells. Oncogene 7: 43. Robbez-Masson L, Rowe HM: Retrotransposons shape species-speciﬁc 2059–2064, 1992 embryonic stem cell gene expression. Retrovirology 12: 45, 2015 40. Wanner N, Vornweg J, Combes A, Wilson S, Plappert J, Rafﬂenbeul G, 44. Chuong EB, Elde NC, Feschotte C: Regulatory evolution of innate immunity et al.: DNA methyltransferase 1 controls nephron progenitor cell re- through co-option of endogenous retroviruses. Science 351: 1083–1087, 2016 newal and differentiation. J Am Soc Nephrol 30: 63–78, 2019 45. Jackson-Grusby L, Beard C, Possemato R, Tudor M, Fambrough D, 41. Hackett JA, Reddington JP, Nestor CE, Dunican DS, Branco MR, Reichmann Csankovszki G, et al.: Loss of genomic methylation causes p53-dependent J, et al.: Promoter DNA methylation couples genome-defence mechanisms apoptosis and epigenetic deregulation. Nat Genet 27: 31–39, 2001

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