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Viewed and Approved by the Quencing (RRBS) Kit (Diagenode) Following Manufacturer’S Institutional Animal Care and Use Committee of University Instruction BASIC RESEARCH www.jasn.org DNMT1 in Six2 Progenitor Cells Is Essential for Transposable Element Silencing and Kidney Development 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 methyltrans- ferases (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 dur- ing development. Six2CreDnmt3af/f, Six2CreDnmt3bf/f, and Six2CreTet2f/f mice showed no significant struc- tural 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] Present address: Dr. Jihwan Park, School of Life Sciences, Gwangju Copyright © 2019 by the American Society of Nephrology Institute of Science and Technology, Gwangju, South Korea. 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
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