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• W3115449903 abstract "•TET mutants that stall oxidation at 5hmC uncouple active DNA demethylation pathways•TET-mediated oxidation to 5fC/5caC, but not 5hmC, promotes iPSC reprogramming efficiency•Novel base resolution-sequencing methods reveal distinctive roles for 5hmC from 5fC/5caC•5fC/5caC is the major driver of DNA demethylation during iPSC reprogramming Active DNA demethylation via ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming in cell state transitions. TET enzymes catalyze up to three successive oxidations of 5-methylcytosine (5mC), generating 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). Although these bases are known to contribute to distinct demethylation pathways, the lack of tools to uncouple these sequential oxidative events has constrained our mechanistic understanding of the role of TETs in chromatin reprogramming. Here, we describe the first application of biochemically engineered TET mutants that unlink 5mC oxidation steps, examining their effects on somatic cell reprogramming. We show that only TET enzymes proficient for oxidation to 5fC/5caC can rescue the reprogramming potential of Tet2-deficient mouse embryonic fibroblasts. This effect correlated with rapid DNA demethylation at reprogramming enhancers and increased chromatin accessibility later in reprogramming. These experiments demonstrate that DNA demethylation through 5fC/5caC has roles distinct from 5hmC in somatic reprogramming to pluripotency. Active DNA demethylation via ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming in cell state transitions. TET enzymes catalyze up to three successive oxidations of 5-methylcytosine (5mC), generating 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). Although these bases are known to contribute to distinct demethylation pathways, the lack of tools to uncouple these sequential oxidative events has constrained our mechanistic understanding of the role of TETs in chromatin reprogramming. Here, we describe the first application of biochemically engineered TET mutants that unlink 5mC oxidation steps, examining their effects on somatic cell reprogramming. We show that only TET enzymes proficient for oxidation to 5fC/5caC can rescue the reprogramming potential of Tet2-deficient mouse embryonic fibroblasts. This effect correlated with rapid DNA demethylation at reprogramming enhancers and increased chromatin accessibility later in reprogramming. These experiments demonstrate that DNA demethylation through 5fC/5caC has roles distinct from 5hmC in somatic reprogramming to pluripotency. As a key regulator of tissue-specific gene expression patterns and chromatin organization, DNA methylation presents a significant epigenetic barrier in the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) (Gao et al., 2013Gao Y. Chen J. Li K. Wu T. Huang B. Liu W. Kou X. Zhang Y. Huang H. Jiang Y. et al.Replacement of Oct4 by Tet1 during iPSC induction reveals an important role of DNA methylation and hydroxymethylation in reprogramming.Cell Stem Cell. 2013; 12: 453-469Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar; Nashun et al., 2015Nashun B. Hill P.W. Hajkova P. Reprogramming of cell fate: epigenetic memory and the erasure of memories past.EMBO J. 2015; 34: 1296-1308Crossref PubMed Scopus (103) Google Scholar; Takahashi and Yamanaka, 2006Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (18206) Google Scholar). Erasure of DNA methylation proceeds through one of two distinct mechanisms: (1) passive loss of 5mC during DNA replication via suppression of DNA methyltransferase (DNMT) activity or (2) active demethylation by ten-eleven translocation (TET) enzymes (Figure 1A) (Hill et al., 2014Hill P.W.S. Amouroux R. Hajkova P. DNA demethylation, Tet proteins and 5-hydroxymethylcytosine in epigenetic reprogramming: an emerging complex story.Genomics. 2014; 104: 324-333Crossref PubMed Scopus (112) Google Scholar; Tahiliani et al., 2009Tahiliani M. Koh K.P. Shen Y. Pastor W.A. Bandukwala H. Brudno Y. Agarwal S. Iyer L.M. Liu D.R. Aravind L. Rao A. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.Science. 2009; 324: 930-935Crossref PubMed Scopus (4087) Google Scholar). Active demethylation is initiated through the progressive oxidation of 5mC to 5hmC, 5fC, or 5caC (Ito et al., 2011Ito S. Shen L. Dai Q. Wu S.C. Collins L.B. Swenberg J.A. He C. Zhang Y. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine.Science. 2011; 333: 1300-1303Crossref PubMed Scopus (2366) Google Scholar), after which demethylation is achieved through one of two potential pathways. 5hmC is not well recognized by the DNMT1 maintenance methylation machinery (Hashimoto et al., 2012Hashimoto H. Liu Y. Upadhyay A.K. Chang Y. Howerton S.B. Vertino P.M. Zhang X. Cheng X. Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation.Nucleic Acids Res. 2012; 40: 4841-4849Crossref PubMed Scopus (318) Google Scholar), allowing for its passive loss over several rounds of DNA replication and cellular division (here called the “hmC pathway”; Figure 1A). Although 5fC and 5caC are also subject to passive loss (Inoue et al., 2011Inoue A. Shen L. Dai Q. He C. Zhang Y. Generation and replication-dependent dilution of 5fC and 5caC during mouse preimplantation development.Cell Res. 2011; 21: 1670-1676Crossref PubMed Scopus (210) Google Scholar), their steady-state levels are many orders of magnitude lower than 5hmC, suggesting that their involvement in this pathway is limited (Wagner et al., 2015Wagner M. Steinbacher J. Kraus T.F.J. Michalakis S. Hackner B. Pfaffeneder T. Perera A. Müller M. Giese A. Kretzschmar H.A. Carell T. Age-dependent levels of 5-methyl-, 5-hydroxymethyl-, and 5-formylcytosine in human and mouse brain tissues.Angew. Chem. Int. Ed. Engl. 2015; 54: 12511-12514Crossref PubMed Scopus (88) Google Scholar; Wu et al., 2014Wu H. Wu X. Shen L. Zhang Y. Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing.Nat. Biotechnol. 2014; 32: 1231-1240Crossref PubMed Scopus (95) Google Scholar). Instead, 5fC and 5caC can be targeted for base excision by thymine DNA glycosylase (TDG) (He et al., 2011He Y.F. Li B.Z. Li Z. Liu P. Wang Y. Tang Q. Ding J. Jia Y. Chen Z. Li L. et al.Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA.Science. 2011; 333: 1303-1307Crossref PubMed Scopus (1935) Google Scholar; Maiti and Drohat, 2011Maiti A. Drohat A.C. Thymine DNA glycosylase can rapidly excise 5-formylcytosine and 5-carboxylcytosine: potential implications for active demethylation of CpG sites.J. Biol. Chem. 2011; 286: 35334-35338Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar; Zhang et al., 2012Zhang L. Lu X. Lu J. Liang H. Dai Q. Xu G.L. Luo C. Jiang H. He C. Thymine DNA glycosylase specifically recognizes 5-carboxylcytosine-modified DNA.Nat. Chem. Biol. 2012; 8: 328-330Crossref PubMed Scopus (236) Google Scholar). In this mode of active demethylation (here called the “fC/caC pathway”; Figure 1A), subsequent steps of base-excision repair (BER) restore an unmodified cytosine at the former abasic site (Kohli and Zhang, 2013Kohli R.M. Zhang Y. TET enzymes, TDG and the dynamics of DNA demethylation.Nature. 2013; 502: 472-479Crossref PubMed Scopus (1001) Google Scholar). TET enzymes and TDG are critical for iPSC reprogramming. TET1 has been proposed to have a vitamin C-dependent role in promoting iPSC formation through a positive feedback loop with Pou5f1 (Oct4) and Nanog (Chen et al., 2013Chen J. Guo L. Zhang L. Wu H. Yang J. Liu H. Wang X. Hu X. Gu T. Zhou Z. et al.Vitamin C modulates TET1 function during somatic cell reprogramming.Nat. Genet. 2013; 45: 1504-1509Crossref PubMed Scopus (218) Google Scholar, Chen et al., 2015Chen J. Gao Y. Huang H. Xu K. Chen X. Jiang Y. Li H. Gao S. Tao Y. Wang H. et al.The combination of Tet1 with Oct4 generates high-quality mouse-induced pluripotent stem cells.Stem Cells. 2015; 33: 686-698Crossref PubMed Scopus (31) Google Scholar; Costa et al., 2013Costa Y. Ding J. Theunissen T.W. Faiola F. Hore T.A. Shliaha P.V. Fidalgo M. Saunders A. Lawrence M. Dietmann S. et al.NANOG-dependent function of TET1 and TET2 in establishment of pluripotency.Nature. 2013; 495: 370-374Crossref PubMed Scopus (317) Google Scholar; Olariu et al., 2016Olariu V. Lövkvist C. Sneppen K. Nanog, Oct4 and Tet1 interplay in establishing pluripotency.Sci. Rep. 2016; 6: 25438Crossref PubMed Scopus (38) Google Scholar). TET2, furthermore, can directly interact with KLF4 or PARP1 to drive site-specific demethylation of reprogramming enhancers and promoters, and Tet2-depleted cells have reduced reprogramming potential (Doege et al., 2012Doege C.A. Inoue K. Yamashita T. Rhee D.B. Travis S. Fujita R. Guarnieri P. Bhagat G. Vanti W.B. Shih A. et al.Early-stage epigenetic modification during somatic cell reprogramming by Parp1 and Tet2.Nature. 2012; 488: 652-655Crossref PubMed Scopus (295) Google Scholar; Sardina et al., 2018Sardina J.L. Collombet S. Tian T.V. Gomez A. Di Stefano B. Berenguer C. Brumbaugh J. Stadhouders R. Segura-Morales C. Gut M. et al.Transcription Factors Drive Tet2-Mediated Enhancer Demethylation to Reprogram Cell Fate Article Transcription Factors Drive Tet2-Mediated Enhancer Demethylation to Reprogram Cell Fate.Cell Stem Cell. 2018; 23: 1-15Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Importantly, both Tet triple-knockout and Tdg null mouse embryonic fibroblasts (MEFs) fail to undergo iPSC reprogramming, suggesting that the fC/caC pathway may be essential to the process (Hu et al., 2014Hu X. Zhang L. Mao S.Q. Li Z. Chen J. Zhang R.R. Wu H.P. Gao J. Guo F. Liu W. et al.Tet and TDG mediate DNA demethylation essential for mesenchymal-to-epithelial transition in somatic cell reprogramming.Cell Stem Cell. 2014; 14: 512-522Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). However, given the multifunctional roles of TDG in DNA repair, transcriptional activation, and histone modification, as well as the relatively early stage in reprogramming at which Tdg null MEFs arrest, the contribution of the hmC and fC/caC pathways to epigenetic reprogramming remains poorly defined (Neddermann et al., 1996Neddermann P. Gallinari P. Lettieri T. Schmid D. Truong O. Hsuan J.J. Wiebauer K. Jiricny J. Cloning and expression of human G/T mismatch-specific thymine-DNA glycosylase.J. Biol. Chem. 1996; 271: 12767-12774Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar; Tini et al., 2002Tini M. Benecke A. Um S.J. Torchia J. Evans R.M. Chambon P. Association of CBP/p300 acetylase and thymine DNA glycosylase links DNA repair and transcription.Mol. Cell. 2002; 9: 265-277Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar; Um et al., 1998Um S. Harbers M. Benecke A. Pierrat B. Losson R. Chambon P. Retinoic acid receptors interact physically and functionally with the T:G mismatch-specific thymine-DNA glycosylase.J. Biol. Chem. 1998; 273: 20728-20736Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar; Cortázar et al., 2007Cortázar D. Kunz C. Saito Y. Steinacher R. Schär P. The enigmatic thymine DNA glycosylase.DNA Repair (Amst.). 2007; 6: 489-504Crossref PubMed Scopus (157) Google Scholar, Cortázar et al., 2011Cortázar D. Kunz C. Selfridge J. Lettieri T. Saito Y. MacDougall E. Wirz A. Schuermann D. Jacobs A.L. Siegrist F. et al.Embryonic lethal phenotype reveals a function of TDG in maintaining epigenetic stability.Nature. 2011; 470: 419-423Crossref PubMed Scopus (283) Google Scholar; Cortellino et al., 2011Cortellino S. Xu J. Sannai M. Moore R. Caretti E. Cigliano A. Le Coz M. Devarajan K. Wessels A. Soprano D. et al.Thymine DNA glycosylase is essential for active DNA demethylation by linked deamination-base excision repair.Cell. 2011; 146: 67-79Abstract Full Text Full Text PDF PubMed Scopus (595) Google Scholar). Furthermore, although differential accumulation of 5hmC and 5fC/5caC across the genome suggests that these pathways may have distinct roles, functional studies into differences in their epigenetic reprogramming potential have been hindered by a lack of molecular tools to distinguish between the pathways in vivo (Shen et al., 2013Shen L. Wu H. Diep D. Yamaguchi S. D’Alessio A.C. Fung H.L. Zhang K. Zhang Y. Genome-wide analysis reveals TET- and TDG-dependent 5-methylcytosine oxidation dynamics.Cell. 2013; 153: 692-706Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar; Wu et al., 2011Wu H. D’Alessio A.C. Ito S. Wang Z. Cui K. Zhao K. Sun Y.E. Zhang Y. Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells.Genes Dev. 2011; 25: 679-684Crossref PubMed Scopus (444) Google Scholar). We recently identified a threonine residue (T1372) in the active site of the human TET2 catalytic domain (CD) that can be mutated to alter the catalytic processivity of the enzyme (Liu et al., 2017Liu M.Y. Torabifard H. Crawford D.J. DeNizio J.E. Cao X.J. Garcia B.A. Cisneros G.A. Kohli R.M. Mutations along a TET2 active site scaffold stall oxidation at 5-hydroxymethylcytosine.Nat. Chem. Biol. 2017; 13: 181-187Crossref PubMed Scopus (41) Google Scholar). While some substitutions reduced the overall activity in each oxidative step, others elicited a “5hmC-stalling” phenotype whereby 5hmC is efficiently generated but not further oxidized, thus depleting the cell of downstream 5fC and 5caC. We posited that mutants with altered processivity can be used to inform the importance of the 5hmC-driven mode of DNA demethylation versus the fC/caC pathway. Here, we introduce TET2 mutants into Tet2-depleted cells, in concert with a novel chemoenzymatic sequencing approach, to investigate the specific role of the fC/caC pathway in iPSC formation and chromatin reorganization. To determine the role of the fC/caC pathway in epigenetic reprogramming, we developed an allelic series of mouse TET mutants exhibiting diverse catalytic capacities. Because the human TET2 T1372 residue is conserved in mouse TET1 (T1642) and TET2 (T1285) (Figure S1A), we tested whether corresponding mutations elicited similar changes in catalytic activity. We transfected HEK293T cells with candidate FLAG-Tet1-CDT1642 or FLAG-Tet2-CDT1285 mutants and collected DNA after 48 h to measure the effect on global modified cytosine levels. Slot blot analysis of 5hmC and 5caC levels recapitulated the previously reported 5hmC-stalling phenotype through the T1642V or T1285E substitution of mouse TET1-CD and TET2-CD, respectively (Figures 1B and S1B). Furthermore, T > A substitutions resulted in a phenotype whereby the enzymes produced both 5hmC and 5caC, but at a reduced rate compared to their wild-type (WT) counterparts, which has previously been called a “low-efficiency” variant (Liu et al., 2017Liu M.Y. Torabifard H. Crawford D.J. DeNizio J.E. Cao X.J. Garcia B.A. Cisneros G.A. Kohli R.M. Mutations along a TET2 active site scaffold stall oxidation at 5-hydroxymethylcytosine.Nat. Chem. Biol. 2017; 13: 181-187Crossref PubMed Scopus (41) Google Scholar). Importantly, western blots of transfected cellular lysates indicated that TET protein levels were unaffected by T1642 or T1285 substitution in HEK293T cells (Figure 1C). To quantify the catalytic activity of our TET mutants more rigorously, we next analyzed DNA from transfected HEK293T cells by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In the absence of transfected TET, 5mC accounted for the majority of total modified cytosines (99.74% ± 0.02%; n = 4) (Figure 1D). When WT TET1-CD or TET2-CD was transfected, however, we observed a robust increase in 5hmC, 5fC, and 5caC levels. By contrast, TET 5hmC-stalling and low-efficiency mutants exhibited a range of oxidative potential that largely matched their expected activity based on slot blot analysis. From the LC-MS/MS data, we quantified TET catalytic activity in two ways: “total activity,” referring to the combined percentage of 5hmC, 5fC, and 5caC genome wide, and “fC/caC activity,” referring to the combined percentage of 5fC/5caC (Figure 1E). Because total activity is driven largely by 5hmC production, these levels were only modestly affected in our catalytic mutants. Conversely, fC/caC activity was strongly affected in all of the catalytic mutants, with low-efficiency mutants exhibiting ∼45% of WT fC/caC activity versus 20% for 5hmC-stalling mutants. To confirm that the observed phenotypes were reproducible in additional cell types, we repeated our mouse TET1-CDT1642 mutant transfections in mouse NIH 3T3 fibroblasts, and found that low efficiency and 5hmC-stalling phenotypes were recapitulated (Figures S1C and S1D). These experiments demonstrate that the human TET2-CDT1372 mutant phenotypes were conserved for the mouse TET2-CD ortholog as well as its TET1-CD isoform. To elucidate the mechanism by which TET enzymes promote epigenetic reprogramming in cell state transitions, we performed iPSC induction on Tet2−/− MEFs transduced with our TET2 catalytic mutants. These Tet2−/− MEFs carry a single-copy insertion of the STEMCCA OKSM reprogramming cassette in a Rosa26:M2rtTA background (OKSM-rtTA), allowing for doxycycline (dox)-inducible expression of the four Yamanaka factors (Figures S2A–S2D) (Stadtfeld et al., 2010Stadtfeld M. Maherali N. Borkent M. Hochedlinger K. A reprogrammable mouse strain from gene-targeted embryonic stem cells.Nat. Methods. 2010; 7: 53-55Crossref PubMed Scopus (143) Google Scholar). Before dox induction, Tet2−/−; OKSM-rtTA MEFs were retrovirally transduced with either empty vector or one of four Tet2-CD constructs: WT Tet2-CD (Tet2-WT), low-efficiency Tet2-CDT1285A (Tet2-A), 5hmC-stalling Tet2-CDT1285E (Tet2-E), or catalytically inactive Tet2-CDH1295Y,D1297A (Tet2-HxD) (Ko et al., 2010Ko M. Huang Y. Jankowska A.M. Pape U.J. Tahiliani M. Bandukwala H.S. An J. Lamperti E.D. Koh K.P. Ganetzky R. et al.Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2.Nature. 2010; 468: 839-843Crossref PubMed Scopus (1003) Google Scholar). Two days after infection, MEFs were seeded onto feeder cells and placed in 2i/LIF (leukemia inhibitory factor) media + dox for iPSC induction (Figure 2A). After 5 days of dox treatment, we evaluated samples from each group by qRT-PCR to confirm proper Tet2 expression levels (Figure S2E). Furthermore, we verified that expression of Tet1, Tet3, Dnmt1, Dnmt3a, and Dnmt3b was unaffected by Tet2 depletion or mutant overexpression (Figure S2E). After 10 days of dox treatment, we performed alkaline phosphatase (AP) staining to determine the relative proportion of iPSC-like colonies prior to dox withdrawal (e.g., while exogenous OKSM factors are still expressed). Interestingly, all Tet2−/− cultures, regardless of Tet2-CD overexpression, exhibited ∼30% fewer AP+ colonies relative to WT reprogramming MEFs (Figure 2B). Because cellular proliferation rates are positively correlated with iPSC reprogramming efficiency, we performed 5-ethnyl-2′-deoxyuridine (EdU) staining (Hanna et al., 2009Hanna J. Saha K. Pando B. van Zon J. Lengner C.J. Creyghton M.P. van Oudenaarden A. Jaenisch R. Direct cell reprogramming is a stochastic process amenable to acceleration.Nature. 2009; 462: 595-601Crossref PubMed Scopus (779) Google Scholar). Although untreated Tet2−/− MEFs exhibited a slightly slower rate of EdU incorporation relative to WT MEFs, this effect disappeared after 3 days of dox treatment (Figure S2F). Likewise, we observed no differences in the rate of apoptosis or cell death between WT and Tet2−/− MEFs as assayed by annexin V and propidium iodide staining (Figure S2G). As a more rigorous assay of stable iPSC formation, we removed dox from the media after 11 days of treatment, requiring cells to use endogenous OKSM expression to maintain pluripotency. One week after dox withdrawal, we examined NANOG expression as a marker of stable iPSC colonies. In agreement with the previous literature, reprogramming Tet2−/− cultures exhibited on average 40% fewer NANOG+ colonies relative to WT cultures (Figure 2C) (Doege et al., 2012Doege C.A. Inoue K. Yamashita T. Rhee D.B. Travis S. Fujita R. Guarnieri P. Bhagat G. Vanti W.B. Shih A. et al.Early-stage epigenetic modification during somatic cell reprogramming by Parp1 and Tet2.Nature. 2012; 488: 652-655Crossref PubMed Scopus (295) Google Scholar; Sardina et al., 2018Sardina J.L. Collombet S. Tian T.V. Gomez A. Di Stefano B. Berenguer C. Brumbaugh J. Stadhouders R. Segura-Morales C. Gut M. et al.Transcription Factors Drive Tet2-Mediated Enhancer Demethylation to Reprogram Cell Fate Article Transcription Factors Drive Tet2-Mediated Enhancer Demethylation to Reprogram Cell Fate.Cell Stem Cell. 2018; 23: 1-15Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Strikingly, this reduced reprogramming efficiency was fully rescued by either Tet2-WT or Tet2-A overexpression, but not Tet2-E or Tet2-HxD. Together with the AP staining results, these data suggest that although entry into the early iPSC state is generally reduced in a Tet2−/− background, the fC/caC activity of TET2 can rescue reprogramming efficiency during the later maturation phase as iPSC colonies transition to stable pluripotency. Furthermore, given the comparable potential of TET2-E and TET2-A to generate 5hmC, we can exclude that this effect may be due to a general loss of activity relative to TET2-WT. To test the generality of our results, we repeated our experiments in Tet2−/− MEFs with retrovirally transduced WT Tet1-CD (Tet1-WT), 5hmC-stalling Tet1-CDT1642V (Tet1-V), or catalytically inactive Tet1-CDH1672Y,D1674A (Tet1-HxD), and observed that only TET1-WT rescued NANOG+ iPSC colony counts to WT levels (Figures S2H and S2I). These experiments suggest that fC/caC generative potential, as opposed to TET isoform identity, is more critical for the rescue of Tet2−/− reprogramming. Previous research identified the role of TET proteins in regulating early transcriptional changes in MEF reprogramming, particularly the mesenchymal-to-epithelial transition (MET) (Hu et al., 2014Hu X. Zhang L. Mao S.Q. Li Z. Chen J. Zhang R.R. Wu H.P. Gao J. Guo F. Liu W. et al.Tet and TDG mediate DNA demethylation essential for mesenchymal-to-epithelial transition in somatic cell reprogramming.Cell Stem Cell. 2014; 14: 512-522Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). To test the influence of fC/caC activity on the transcriptome, we performed RNA sequencing (RNA-seq) on Tet2−/− MEFs retrovirally transduced with empty vector, Tet2-WT, or Tet2-E after 5 days of dox treatment. Relative to untreated Tet2−/− MEFs, there was a 72% overlap in differentially regulated genes (2,956 upregulated, 3,165 downregulated) among the 3 conditions, indicating that the general reprogramming trajectory is unaltered by TET2 expression (Figure S2J; Table S4). However, we identified 1,044 genes (409 upregulated, 635 downregulated) with significantly altered expression in Tet2-WT cells relative to vector control at iPSC day 5 (Figure 2D). Gene Ontology (GO) enrichment analysis identified several pathways critical for early MEF reprogramming, including cell proliferation, extracellular matrix (ECM) reorganization, immune regulation, and mesenchymal fate repression. This effect was strongly attenuated in Tet2-E cells (26 upregulated, 35 downregulated relative to vector control), suggesting that the fC/caC activity of TET2 is essential to promote rapid transcriptional changes during early MEF reprogramming (Figures 2D and 2E). A direct comparison of the Tet2-WT and Tet2-E day 5 transcriptomes identified 452 genes with significantly altered expression (fold change > 1.5; false discovery rate [FDR] < 0.05) (Figure 2F). Upregulated genes included several signaling factors known to promote cell growth and survival (Peg10, Itgb4, Epgn, Spock2, Fgfbp1, and Bmp6), as well as Zfp961, a KRAB-zinc finger protein implicated in retrotransposon silencing that may influence iPSC reprogramming efficiency (Friedli et al., 2014Friedli M. Turelli P. Kapopoulou A. Rauwel B. Castro-Díaz N. Rowe H.M. Ecco G. Unzu C. Planet E. Lombardo A. et al.Loss of transcriptional control over endogenous retroelements during reprogramming to pluripotency.Genome Res. 2014; 24: 1251-1259Crossref PubMed Scopus (76) Google Scholar; Wolf et al., 2020Wolf G. de Iaco A. Sun M.A. Bruno M. Tinkham M. Hoang D. Mitra A. Ralls S. Trono D. Macfarlan T.S. KRAB-zinc finger protein gene expansion in response to active retrotransposons in the murine lineage.eLife. 2020; 9: 1-22Crossref Scopus (26) Google Scholar). Among the downregulated genes were several involved in mesenchymal ECM organization, cell adhesion, and motility (Ndnf, Postn, Egfl6, Fbn2, Fat4, and Coll11a1). Aberrant expression of these key factors may contribute to the diminished reprogramming potential of Tet2-E cells relative to Tet2-WT. Because the rescue of Tet2−/− iPSC reprogramming was dependent on TET fC/caC activity, we next tested whether this result correlated with increased DNA demethylation. Bisulfite (BS) sequencing is commonly used to assess DNA demethylation; treatment of DNA with BS deaminates unmodified C, 5fC, and 5caC, giving a readout of 5mC + 5hmC. However, because the hmC-stalling variants were unable to rescue reprogramming, it was necessary to distinguish 5hmC alone. We therefore used BS-assisted APOBEC-coupled epigenetic (bACE) pyrosequencing (Schutsky et al., 2018Schutsky E.K. DeNizio J.E. Hu P. Liu M.Y. Nabel C.S. Fabyanic E.B. Hwang Y. Bushman F.D. Wu H. Kohli R.M. Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase.Nat. Biotechnol. 2018; 36: 1083-1090Crossref Scopus (85) Google Scholar). Briefly, DNA is treated with BS and then the DNA deaminase APOBEC3A. The enzyme deaminates residual 5mC, but cytosine 5-methylenesulfonate (CMS), the product of 5hmC reaction with BS, is resistant to deamination, providing a specific readout of 5hmC. Pyrosequencing of a control oligonucleotide confirmed the sensitive detection of 5mC + 5hmC by standard BS and 5hmC alone by bACE pyrosequencing (Figure S3A). We focused our analysis on TET2 target enhancers. A recent study identified a subset of enhancers targeted by the TET2-KLF4 complex, whose active DNA demethylation early in reprogramming (iPSC days 2–4) contributes to increased chromatin accessibility at later stages (Sardina et al., 2018Sardina J.L. Collombet S. Tian T.V. Gomez A. Di Stefano B. Berenguer C. Brumbaugh J. Stadhouders R. Segura-Morales C. Gut M. et al.Transcription Factors Drive Tet2-Mediated Enhancer Demethylation to Reprogram Cell Fate Article Transcription Factors Drive Tet2-Mediated Enhancer Demethylation to Reprogram Cell Fate.Cell Stem Cell. 2018; 23: 1-15Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Selecting four enhancers highlighted by the authors as representative of this effect (Sall4 intragenic, Smarcd2, Tet2 intragenic, and Ebf3), we measured the changes in 5mC and 5hmC levels relative to untreated control Tet2−/− MEFs after 5 days of dox treatment (Figures 3A–3D). 5hmC levels at all 4 enhancers were significantly augmented by the overexpression of catalytically active Tet2. Most notably, Tet2-E generated levels of 5hmC at least as high as Tet2-WT, yet this was insufficient to rescue reprogramming. Given that unmodified cytosine and 5fC/5caC are indistinguishable by BS, we also performed M.SssI methylation-assisted pyrosequencing (MAB-seq) of these 4 loci. However, 5fC/5caC levels were below our detection limit (<5% of cytosines) for all of the tested conditions, suggesting that these bases are still efficiently removed by TDG in reprogramming Tet2−/− MEFs (Figure S3A; Table S5). Thus, by subtracting the BS and bACE signals, we can attribute the remaining signal to unmodified cytosine. For all 4 enhancers, we observed a strong linear correlation between the fC/caC activity of a given TET2 mutant (defined in Figure 1E) and the proportion of unmodified cytosine generated (lack-of-fit F-test p > 0.05; n = 4–5). This relationship was not observed when unmodified cytosine levels were instead plotted against total TET2 catalytic activity (Figure S3B; lack-of-fit F-test p < 0.05; n = 4–5). A similar effect was also observed at the miR200b cluster promoter, whose activities are thought to regulate MET during iPSC reprogramming (Figures S3C and S3D) (Hu et al., 2014Hu X. Zhang L. Mao S.Q. Li Z. Chen J. Zhang R.R. Wu H.P. Gao J. Guo F. Liu W. et al.Tet and TDG mediate DNA demethylation essential for mesenchymal-to-epithelial transition in somatic cell reprogramming.Cell Stem Cell. 2014; 14: 512-522Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Our data therefore suggest that the degree of DNA demethylation at TET2 target regions during iPSC reprogramming correlated most strongly with the ability of the TET mutant to generate the higher-order oxidation products 5fC and 5caC. To test the effect of TET2 catalytic activity on the methylation status of known pluripotency loci, we performed BS sequencing of the Oct4 promoter and Nanog intron 1 (Figure S3E). Although the average DNA methylation at these loci was unaffected after 5 days of dox treatment, we observed a trending increase in lowly methylated clones among Tet2-WT cells. This effect likely reflects an expansion of rare, stably pluripotent cells in the Tet2-WT population, which is consistent with increased reprogramming efficiency. In addition to DNA demeth" @default.
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• W3115449903 date "2021-02-01" @default.
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• W3115449903 title "Functionally distinct roles for TET-oxidized 5-methylcytosine bases in somatic reprogramming to pluripotency" @default.
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• W3115449903 doi "https://doi.org/10.1016/j.molcel.2020.11.045" @default.
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