Research Article TET2 Inhibits Differentiation of ...downloads.hindawi.com/archive/2014/986571.pdf · HG) which competes with -ketoglutarate to inhibit TET activity [ ]. Various similarities

Research ArticleTET2 Inhibits Differentiation of Embryonic Stem Cells but DoesNot Overcome Methylation-Induced Gene Silencing

Louis Norman1 Paul Tarrant1 and Timothy Chevassut12

1 Medical Research Building Brighton and Sussex Medical School Sussex University Falmer Brighton BN1 9PS UK2 Royal Sussex County Hospital Brighton and Sussex University Hospitals NHS Trust Brighton BN2 5BE UK

Correspondence should be addressed to Timothy Chevassut tchevassutbsmsacuk

Received 6 February 2014 Accepted 18 July 2014 Published 25 August 2014

Academic Editor Issa F Khouri

Copyright copy 2014 Louis Norman et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

TET2 is a methylcytosine dioxygenase that is frequently mutated in myeloid malignancies notably myelodysplasia and acutemyeloid leukemia TET2 catalyses the conversion of 51015840-methylcytosine to 51015840-hydroxymethylcytosine within DNA and has beenimplicated in the process of genomic demethylation However the mechanism by which TET2 loss of function results inhematopoietic dysplasia and leukemogenesis is poorly understood Here we show that TET2 is expressed in undifferentiatedembryonic stem cells and that its knockdown results in reduction of 51015840-hydroxymethylcytosine in genomic DNA We also presentDNA methylation data from bone marrow samples obtained from patients with TET2-mutated myelodysplasia Based on thesefindings we sought to identify the role of TET2 in regulating pluripotency and differentiation We show that overexpressionof TET2 in a stably integrated transgene leads to increased alkaline phosphatase expression in differentiating ES cells andimpaired differentiation in methylcellulose culture We speculate that this effect is due to TET2-mediated expression of stemcell genes in ES cells via hydroxylation of 51015840-methylcytosines at key promoter sequences within genomic DNA This leads torelative hypomethylation of gene promoters as 51015840-hydroxymethylcytosine is not a substrate for DNMT1-mediated maintenancemethylationWe sought to test this hypothesis by cotransfecting the TET2 gene with methylated reporter genesThe results of theseexperiments are presented

1 Introduction

11 TETGenes Function and Background The family ofTET(ten-eleven translocations) genes comprise three membersTET1 TET2 and TET3 each with high level of homologyto one another TET2 at locus 4q24 has three distinctvariant forms from alternative splicing each of which pos-sesses 2-oxoglutarate- (2OG-) Fe(II) dependent oxygenaseactivity that is dependent on 120572-ketoglutarate and Fe2+ ionas cofactors [1 2] TET2 acts functionally to catalyse thebase conversion of 5-methylcytosine (5mC) DNA to 5-hydroxymethyl-cytosine (5hmC) [3ndash7] Other 2OG-Fe(II)-dependent enzymes are common in humans with a broadspectrum of functions including DNA repair hypoxia sens-ing histone demethylation fatty-acid metabolism and col-lagen stabilization [8] In recent studies He et al and Ito etal used thin layer chromatography (TLC) and mass spec-trometry to show that TET2 has further oxidative activity to

convert 5hmC to 5-carboxylcytosine (5caC) in HEK293T cellsalthough the other expected intermediate 5-formylcytosine(5fC) was only observed in mouse embryonic stem (ES) cellsin the study by Ito et al [9] and He et al [10] 5caC is arelatively stable base modification and an active demethyla-tion process was seen involving thymine-DNA-glycosylase(TDG) base excision repair [10] Recent evidence suggeststhat 5hmC can also be dehydroxymethylated to cytosine by thede novomethyltransferase proteins DNMT3A and DNMT3Balthough this needs to be confirmed by other studies [11]Thus TET2 plays a central role in the biology of cytosinemethylation However the functional role of TET2 in the celland the importance of its product 5hmC to transcriptionalactivity of genes remain to be elucidated

12 TET2 in ES Cells and Mutations in MDSAML TET2mutation is enriched amongst cases of myeloid malignancy

Hindawi Publishing CorporationBone Marrow ResearchVolume 2014 Article ID 986571 9 pageshttpdxdoiorg1011552014986571

2 Bone Marrow Research

occurring in approximately 12 of acute myeloid leukemia(AML) cases and up to 58 of mixed myeloproliferativeneoplasms (MPN) and myelodysplastic syndromes (MDS)[12ndash14] Therefore it is likely that TET2 plays an importantrole in regulation of hematopoietic differentiation and mayeven serve as a prognostic tool inmyeloidmalignancies AMLis characterized by enhanced proliferation and impaireddifferentiation of myeloid progenitors resulting in clonalpopulations of immature hematologic blasts Furthermorespecific AML subgroups show patterns of dysregulated DNAmethylation patterns which can be correlated with patientsurvival [4] The majority of TET2 mutations recorded inpatients with myeloid malignancy are within regions criticalfor enzymatic activity suggesting they are loss of functionmutations and hence TET2 is seen to be having a tumoursuppressor role [1 3]

The clinical outcome of patients with a mutation in TET2remains contentious In AML patients one study found adecreased survival rate in TET2 mutants when comparedto a wild-type group [15] whereas another study found nosignificant impact of mutation status on clinical outcomebut mutation was strongly associated to a mutation in theNPM1 gene which is also implicated in AML [16 17] InMDSpatients the 5-year survival rate was 769 for TET2mutantsagainst 183 for wild-type indicating amutation as amarkerfor good prognosis [18] Additionally Smith et al couldfind no prognostic significance for TET2 mutation whenpatients were clustered into WHO subtypes scored usingthe International Prognostic Scoring System or grouped intocytogenic status or progression to AML [19]

The isocitrate dehydrogenase enzyme IDH1 and its mito-chondrial homologue IDH2 are responsible for convertingisocitrate to 120572-ketoglutarate in the KrebsTCA cycle Het-erozygous gain of function mutations of IDH1 and IDH2 hasbeen shown to convert isocitrate into 2-hydroxyglutarate (2-HG) which competes with 120572-ketoglutarate to inhibit TETactivity [4] Various similarities exist between TET2 mutantand IDH12 mutant phenotypes including increased 5mC incultured cells and an increase in progenitor cell numbersand stem cells markers [4] These findings provide importantinsights into a possible common mechanism of epigeneticdisruption that may lead to AML or precursor diseases suchas MDS

13 Methylcytosine Hydroxymethylcytosine and Its Effect onPromoter Activity The total 5mC content of the humangenome is 1ndash3 of all cytosine bases [3 20ndash22] occurringalmost exclusively at CpG dinucleotides Clusters of CpGsites occurring at higher than expected frequency withingenomic regions are termed CpG islands (CGIs) which aregenerally hypomethylated within cells Around 65 of CGIslocalise around the point of gene promoter regions includingall constitutively expressed genes [23] Aberrant methylationof CGIs leads to stable transcriptional repression and isthought to be important in silencing tumour suppressorgenes in some conditions such as MDS CGI methylation atpromoter regions upstream of genes has the greatest effect ontranscription via prevention of elongation of the transcript[20] Some evidence suggest that CGI methylation within

exonic regions of genes is permissive of transcription anddoes not block initiation or elongation [24] The recentdiscovery of cytosine hydroxymethylation raises the possi-bility that methylation at CGIs can be reversed via activecytosine demethylation involving TET proteins leading to thereactivation of transcriptionally silenced promoters [9 10]Furthermore new evidence on induced pluripotent stem cells(iPSCs) suggests that the 5hmC base modification not only isan intermediate within cytosine demethylation but acts asan epigenetic mark in itself possibly aiding in recruitmentof factors that promote chromatin remodeling [25 26]

We show here that levels of 5hmC are reduced by TET2knockdown in ES cells and in TET2 mutated MDS bonemarrow samples We investigated the phenotypic effects ofTET2 overexpression on differentiation of murine ES cellsusing stable transfectants Finally using reported genes weshow that transient overexpression of TET2 has no significanteffect on the transcriptional activity ofmethylated promoters

2 Results

21 Differentiation of ES Cells Leads to Decreased 5ℎ119898C Wesought to investigate the levels of 5mC and 5hmC in undif-ferentiated murine ES cells using HPLC mass spectrometryWe found levels of 75 for 5mC and 015 for 5hmC relativeto guanosine bases demonstrating that 5mC is present inES cells at approximately 50-fold greater levels than 5hmC(Figure 1(a))

Next we sought to investigate what happens to levels of5hmCduring induction of differentiationWe compared levelsof 5hmC in undifferentiated ES cells grown in the presence ofleukemia inhibitory factor (LIF) and in differentiated cells 5days post-LIF withdrawal (Figure 1(b)) We found that levelsof 5hmC fall by approximately 50 from 0155 to 0072relative to guanosine bases upon differentiation This resultsuggests that LIF withdrawal induces downregulation of oneor more of the TET family genes leading to a reduction inconversion of 5mC to 5hmC

22 Knockdown of TET2 in ES Cells Causes Reduced Lev-els of 5ℎ119898C Based on this finding we performed siRNAknockdown experiments of the TET2 gene Using two dif-ferent gene-specific siRNA oligos in triplicate knockdownexperiments we measured reductions in 5hmC levels of 5and 20 respectively compared with a scrambled control(Figure 1(c)) These results provide evidence that TET2 likeTET1 catalyses the conversion of 5mC to 5hmC

23 Measurement of 5ℎ119898C Levels in TET2MutatedMDSAMLPatient Marrow Samples Genomic DNA analysis usingmassspectrometry of five patients with myeloid malignancy failedto identify a clear association between the TET2 mutationalstatus and 5hmC levels (Figure 1(d)) Patient samples weregenotyped to identify mutations within the TET2 gene locusany patients found to have a mutation were heterozygouswild-type While this result is based on a small number ofsamples and was not correlated with the size of the malignant

Bone Marrow Research 3

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Figure 1Hydroxymethylation status in ES cells under differentiation andTET2 knockdown and in patient samplesHPLCmass spectrometrywas used to determine the levels of methylcytosine (5mC) and hydroxymethylcytosine (5hmC) expressed as a percentage of total guanosinebases All experiments were in triplicate and averaged unless otherwise stated (a) 51015840-hydroxymethylcytosine is 015 of wild-type ES cellsLevels of 5hmC were compared to levels of 5mC in undifferentiated E14 ES cells grown in the presence of LIF to maintain an undifferentiatedstate levels were 015 and 75 respectively (b) 51015840-hydroxymethylcytosine levels fall in differentiated ES cells 5hmC levels dropped byapproximately 50 from 015 to 0076 relative to guanosine bases upon differentiation via the removal of LIF from the growthmedia 5hmClevels were determined on day 5 after removal of LIF from the growth medium (c) Effect of TET2 knockdown on 51015840-hydroxymethylcytosinelevels Small inhibitory RNA (siRNA) knockdown of TET2 using a control scrambled oligonucleotide (wild-type ES) and two specific TET2oligonucleotides (Oligo 1 and Oligo 2) Oligo 1 showed no significant knockdown of 5hmC levels compared to the control whereas Oligo 2showed reduced levels of 5hmC upon HPLCmass spectrometric analysis of around 18 compared to control (d) Bone marrow samples frompatients with myelodysplasia Marrow samples from myelodysplastic syndrome (MDS) patients were analysed with the mass spectrometerfor 5hmC status Patients were genotyped for mutations in the TET2 gene locus and any mutants were found to be heterozygous for the TET2gene

clone or the nature of the genetic mutation it nonethelessshows that global deficiency of 5hmC is not sufficient onits own to cause disease However it does not preclude thepossibility that localised loss of 5hmC at specific gene sites hasoccurred in TET2mutated disease

24 Stable Transfection of Murine ES Cells with a TET2-Containing Plasmid Vector Leads to Slight Increased 5ℎ119898CLevel Immunostaining E14murine ES cells were stably trans-fected with TET2-pCDNA3 plasmid vector (TET2 stabletransfects) or empty pCDNA3 plasmid as a control (EVstable transfects) in triplicate Selection in G418 antibioticconfirmed the presence of the incorporated plasmid DNAand surviving colonies were grown to confluency Figure 2shows that TET2 was present to a relatively similar degree

in both EV and TET2 stable transfects and was stronglyassociated to the nuclear region of cells 5hmC levels wereslightly raised in TET2 stable transfects

25 Stable Transfection of TET2-pCDNA3 Vector Reduces ESCellular Differentiation as Observed by Leukocyte AlkalinePhosphatase Analysis A differentiation assay was conductedby removing LIF from the media and relative differenti-ation measured using alkaline phosphatase (AP) analysiswhere each colony is attributed to a leukocyte AP analysis(LAPA) score Reducing alkaline phosphatase activity is anindicator of increasing differentiation LAPA scoring of 7day differentiating cells revealed colonies from the TET2-pCDNA3 condition having an average LAPA score of 09compared to 045 for the control group showing that the


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Figure 2 Stable transfection ofmurine ES cells with a pCDNA-TET2plasmid vector leads to slight increased immunostaining for 5hmC Stablytransfected E14 murine ES cells were grown to confluency and analysed by immunocytochemistry for 5hmC TET2 and 410158406-diamidino-2-phenylindole (DAPI) as a nuclear-identifying control Empty vector (EV) stable transfect cells are shown in (a) and TET2 stable transfect cellsare shown in (b)

TET2-pCDNA3 transfect colonies were on average 50 lessdifferentiated than the control transfect colonies at the timeof analysis (Figure 3(a)) A total of 262 colonies were analysedfor AP activity across the two conditions Figure 3(b) showstypical colonies observed at 7 days of differentiation post-LIFwithdrawal and stained for AP activity

26 Stable Transfection of Murine ES Cells with a TET2-Containing Plasmid Vector Leads to a Slightly Reduced Abilityto Differentiate under Methylcellulose + G-CSF Followingthis we conducted a further differentiation experiment usingmethylcellulose with granulocyte-colony stimulating factor(G-CSF) At 22 days under MethoCult + G-CSF differenti-ation cells showed a slight difference in extent of differen-tiation with an indication of a reduction in the percentageof cell colony differentiation seen with the TET2-pCDNA3vector transfects compared to controls (Figure 4(a)) A totalof 412 colonies were scored based on the MethoCult GFH4034 protocol and observed under a light microscopeacross triplicate experiments of both TET2-pCDNA3 vectorand pCDNA3 control stable transfects respectively Typicalcolonies observed are illustrated in Figure 4(b)

27 Murine ES Cell Differentiation Fails to Overcome Methy-lation Induced Silencing pRL (Renilla) and pGL3 (Firefly)Luciferase reporter plasmids were then used to determine theability of E14 cells to demethylate a previously methylatedreporter plasmid Transient transfections were carried outusing methylated or unmethylated pRL reporter plasmid indifferentiating (minusLIF) or undifferentiating (+LIF) conditionsand relative luminescence was compared to the secondarypGL3 reporter that was not methylated (Figure 5(a)) Nosignificant difference was seen between the minusLIF and +LIFconditions however the methylated pRL condition leads to

a significant reduction (of around 96) in relative Renillaluminescence compared to Firefly This shows the methy-lation had successfully silenced the reporter by reducingthe cells ability to express the Renilla protein but upondifferentiation there was no difference in the cells ability todemethylate and hence reexpress the reporter gene

28 TET2 Fails to Overcome Silencing of a MethylatedLuciferase GeneWhen Cotransfected into HEK Cells In orderto determine if the concentration of TET2 protein within thecell impacts DNA methylation transient transfection exper-iments were performed on HEK293T cells using the pRLreporter plasmid (either methylated or unmethylated) plusdiffering concentrations of the TET2-pCDNA3 plasmid con-struct (Figure 5(b)) The unmethylated pRL plasmid controlshowed high luminescence in comparison with methylatedpRL with relative expression reduced by gt98 Howeverincreasing amounts of TET2-pCDNA3plasmid cotransfectedwith the pRL reporter failed to demonstrate any significantdifference in Renilla expression levels

3 Discussion

Our findings reveal that ES cell differentiation induced byLIF-removal is associated with a 50 reduction in levels ofhydroxymethylcytosine that is likely to be due to downregu-lation of TET genes We also demonstrate that knockdownof TET2 in ES cells leads to reduced levels of 5hmC evenin the presence of LIF consistent with the finding of otherssuggesting that TET2 normally converts 5mC to 5hmC [3ndash7] Our observations suggest that TET2 gene expression isimportant for maintenance of ES cell pluripotency althoughthemechanismwhereby this occurs is not clearWe speculate


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Figure 3 Alkaline phosphatase analysis of stably transfected ES cells at 7 days post-LIF removal Once at confluency stably transfectedpCDNA3 and TET2-pCDNA3 E14 ES cells were plated at 1000 cells per plate on gelatin coated 10 cm tissue culture plates in triplicate 72 hoursafter plating LIF was removed from the media and colonies were left to grow for a further 7 days Analysis of alkaline phosphatase activitywas conducted using the Alkaline Phosphatase Leukocyte kit (Sigma-Aldrich) and each colony was given a leukocyte alkaline phosphatase(LAPA) score (a) Average LAPA scores of stably transfected ES cells at 7 days post-LIF removal A total of 159 colonies were analysed forthe TET2-pCDNA3 condition and 128 for the pCDNA3 condition The LAPA score for TET2-pCDNA3 was 09 (inverse was 107) whereaspCDNA3 stable transfects had an average LAPA score of 045 (inverse was 222) showing that the TET2-pCDNA3 colonies were 50 lessdifferentiated than the control colonies (b) shows colonies at 7 days post-LIF removal that have been stained for alkaline phosphatase (AP)activity

that TET proteins prevent silencing of stem cell genes bymaintaining promoters in a hypomethylated stateThismightalso explain why loss of function of TET2 contributes tomyeloidmalignancies such asMDS andAMLdue to silencingof tumour suppressor genes via aberrant promoter methy-lation In support of this possibility mutations of IDH1 andIDH2 also occur in myeloid malignancies where neomorphicactivity of the enzyme results in aberrant generation of 2-hydroxyglutarate which inhibits the 2-oxoglutarate depen-dent conversion of 5mC to 5hmC by TET2 [4 7]

We studied the association between the TET2mutationalstatus and 5hmC levels in bonemarrow samples obtained frompatients with myelodysplasia We found no clear associationbetween 5hmC levels and TET2 mutational status althoughimportantly the IDH12 mutational status of these samples

was not known Whilst our analysis of patient samples herewas limited due to small numbers we note with interest thatother investigators have indeed found that TET2 and IDH12mutated bone marrow samples typically have reduced levelsof 5hmC compared with nonmutated samples from patientswith MDS or AML [4]

We sought next to test the hypothesis that TET2 expres-sion promotes ES cell pluripotency We find that ES cellscarrying a stably integrated TET2 transgene expressed morealkaline phosphatase following LIF removal than controlES cells carrying the empty vector suggesting that aberrantexpression ofTET2may impair normal ES cell differentiationConsistent with this result we find that ES cells carryingtheTET2 transgene yieldedmore undifferentiated colonies inmethylcellulose culture than did ES cells carrying the empty


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Figure 4 Methylcellulose differentiation of stably transfected ES cells in the presence of hematopoietic cytokines E14 ES cells were stablytransfected as described and grown to confluency Triplicate 10 cm gelatin coated tissue culture plates containing 4000 cells each were platedof both the TET2-pCDNA3 (TET2 stable transfect) and pCDNA3 control (EV stable transfect) Cells were differentiated using MethoCultGF H4034 (Stem Cell Technologies) a combination of methylcellulose and granulocyte-colony stimulating factor (G-CSF) in the media andcolonies analysed according to the manufacturerrsquos instructions at day 22 after plating for differentiation A total of 229 colonies were classifiedin the TET2 stable transfect condition and 173 colonies for the EV stable transfect condition (a) Relative differentiation levels of TET2 stabletransfects and EV stable transfects at 22 days under methylcellulose condition TET2 stable transfects were on average 8 less differentiatedat the time of analysis (b) Photomicrographs of embryoid bodies derived from stably transfected ES cells

vector suggesting again that TET2 expression promotes apluripotent ES cell state

Based on our findings we hypothesised that TET2 func-tions by maintaining gene promoters in a hypomethylatedstate and that this is important for ES cell pluripotency Ourresults failed to demonstrate any significant effect of TET2cotransfection on luciferase expression suggesting that theTET2 enzyme is unable to reactivate gene silencing due topromoter methylation However we were unable to confirmexpression of the TET2 transgene over and above the endoge-nous gene Moreover TET2-mediated ldquodemethylationrdquo mayoccur via passive loss of methylcytosines during cell divisionrather than active demethylation of methylated promoters[23] Transfection experiments in undifferentiated and differ-entiated ES cells once again failed to demonstrate differential

expression of the luciferase reporter genes according to themethylated state of their promoters Hence we conclude thatTET2 does not directly affect transcriptional activity of genesat methylated promoters However it remains distinctly pos-sible that TET2 functions to maintain unmethylated genes inan active state by maintenance of promoter hypomethylationvia hydroxylation of methylcytosines leading to passive lossIf so then we speculate that this action may be important forthe role of TET2 in ES cell pluripotency and myeloid tumoursuppression

4 Materials and Methods

41 Cell Culture Human embryonic kidney (HEK)293T cells were cultured in Dulbeccorsquos modified eagle


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Figure 5 (a) Expression of methylated reporter plasmids is identical in differentiated and undifferentiated ES cells A Dual LuciferaseReporter Assay (Promega) was modified to determine if E14 murine ES cells undergoing differentiation were more able to demethylate(unsilenced) a reporter gene plasmid Methylation of the pRL reporter by SssI methylase (Promega M0226S) showed optimal gene silencingand relative expression of the Renilla Luciferase was compared to the unmodified pGL3 plasmid expression of Firefly Luciferase via aluminometer E14 cells were plated at 1 times 105 cells per well in a 24-well plate in triplicate experiments and induced to differentiation onday 1 posttransfection by removal of leukemia inhibitory factor (LIF) and transient transfection of both the pGL3 and methylated pRLwas conducted using the Lipofectamine LTX (Invitrogen) reagent as per the manufacturerrsquos instructions on day 5 after plating Analysisof Renilla and Firefly expression was conducted on day 6 Transient transfection of unmodified pRL and pGL3 was used as a control (a)shows that although methylation clearly had a significant silencing effect on pRL there was no significant difference seen between the minusLIF(differentiating) or +LIF (undifferentiated) conditions on relative expression of pRL (b) TET2 is unable to overcome silenced expression ofa cotransfected methylated Renilla plasmid Human embryonic kidney (HEK) 293T cells were plated at 3 times 104 cells per well in a 24-wellplate in triplicate and transiently transfected using the Lipofectamine LTX (Invitrogen) reagent as per the manufacturerrsquos instruction onday 1 after plating with both Dual Luciferase reporter plasmids and a TET2-pCDNA3 construct plasmid Control experiments were run inparallel with either no pRLmethylation no pCDNA-TET2 cotransfect or a pCDNA3 (empty vector) cotransfect Dual Luciferase experimentswere modified by methylation of the pRL reporter by SssI methylase (Promega M0226S) which showed optimal gene silencing and relativeexpression of the Renilla Luciferase was compared to the unmodified pGL3 plasmid expression of Firefly Luciferase via a luminometerExperiments were conducted in triplicate and averaged Cotransfection of a TET2-pCDNA3 plasmid vector at varying concentrations withthe Dual Luciferase plasmids does not lead to changes in the unsilencing (demethylation) of the methylated reporter Control 1 shows againthat unmethylated pRL plasmid has much higher expression levels than the methylated form There was no dose response even when 2120583gof TET2-pCDNA3 plasmid DNA was added to each well and there was no difference in Renilla expression between controls 2 and 3 andbetween those cotransfections that contained the TET2-pCDNA3 plasmid

medium (DMEM) supplemented with 10 fetal calf serum100 120583gmL penicillin 100 120583gmL streptomycin and 100mML-glutamine Murine E14 ES cells were cultured in GlasgowMinimum Essential Medium (GMEM) supplemented with10 fetal calf serum 100 120583gmL penicillin 100 120583gmLstreptomycin 100mM L-glutamine 100 120583gmL nonessentialamino acids (NEAAs) 10 120583gmL 2-mercaptoethanol (2-ME)and 10 120583gmL leukemia inhibitory factor (LIF) (SigmaL5158) Both cell types were cultured in T25 or T75 tissueculture flasks in tissue incubators with 5 CO

2at 37∘C

42 siRNA siRNA knockdown experiments of TET geneswere conducted using Dharmacon siGENOME siRNAduplexes (Thermo Fisher Scientific Inc) according tothe manufacturerrsquos instructions siRNA molecules againstmurine TET1 (Cxxc6 1) and TET2 (E130014J05Rik 5 andE130014J05Rik 6) were obtained and the DharmaconsiGENOME nontargeting siRNA number 2 (D-001210-02)was used as a negative control Knockdown experimentswere performed on E14 ES cells cultured in LIF-containingmedium on gelatin coated 12-well plates at a density of 1times105cells per well Serial siRNA transfections were performed

using 50 nM siRNA and Lipofectamine RNAiMAX reagent(Invitrogen) Retransfectionswere performedonpreadherentcells at day 2 at a split of 1 4 and finally at day 4 at a split of1 2 in 6-well plates Cells were harvested at day 5 for HPLCmass spectrometry analyses

43 HPLC Mass Spectrometry Analysis To measure levelsof 5hmC and 5mC a high-performance liquid chromatog-raphy (HPLC) mass spectrometry technique was usedmeasuring levels of 5-hydroxymethyl-deoxycytidine (5hmdC)and 5-methyl-deoxycytidine (5mdC) respectively DNA wasextracted and digested using benzonase and alkaline phos-phatase and a 5120583L sample used for analysis The HPLC con-figuration was as follows Phenomenex Gemini C18 column(150 times 20mm ID) with 3120583m size particle guard cartridgemobile phase A 01 formic acid B acetonitrile01 formicacid

0ndash2mins 1 B

2ndash7mins 1 to 40 B

7ndash14m mins 1 B


The Applied Biosystems QTrap MSMS detector config-uration was as follows orthogonal electrospray ion sourcewith parent ionproduct ion in the following ratios

dGndash268115215mdCndash242112625ohmdCndash25821422

Source temperature was 450∘C and a spray voltage of 3 kVwas used Collision gas pressure was 15mTorr Data was thengraphed as a percentage of deoxyguanosine bases

44 Immunocytochemistry Cells were passaged into wellsof a 24-well plate at 3000 cells per well containing poly-L-lysine (PLL) coverslips 24 hours prior to immunolabelingCells were indirectly labelled using the standard procedureby removing coverslips and washing in phosphate bufferedsaline (PBS) fixing cells in 4 paraformaldehyde for 20 min-utes rewashing in PBS and permeabilising cells in 04 Tri-ton for 5 minutes before washing again in PBSThe followingantibodieswere used as per themanufacturerrsquos recommendeddilutions and instructions TET2 protein was labelled withTET2 rabbit IgG (S-13 Santa Cruz Biotechnology Inc) withAlexa488 goat anti-rabbit (Invitrogen) secondary antibody5hmC was labelled with 5hmC rabbit (Active Motif) withAlexa488 goat anti-rabbit secondary antibody Coverslipswere rinsed again in PBS and then costained with 410158406-diamidino-2-phenylindole (DAPI) for 5 minutes and thenrinsed again DAPI acts as a nuclear identifying controlCoverslips were placed on Citifluor AF1 mounting mediumon glass slides and sealed with nail varnish Cells were visual-ized under a DM5000B (Leica) fluorescence microscope andimages captured using an attached DPC300FX (Leica) digitalcamera

45 Dual Luciferase Reporter Assay Dual LuciferaseReporter Assay (Promega) was optimized and conductedas per the manufacturerrsquos instructions using 20 120583L of eachreagent for each individual reading at 24 hrs posttransfectionRelative luminescence was measured with a GloMax 2020Luminometer on the Dual Glo setting and the ratio of Renillaprotein (from the pRL plasmid) to Firefly protein (from thepGL3 plasmid) luminescence was graphed

Methylation of theRenilla luciferase reporter plasmidwasconducted using the SssI methylase (Promega M0226S) asthis provided optimal methylation when compared to othermethylases when testing comparative luminescence and alsowith a BstUI restriction digest which cuts DNA at sites ofunmethylated 51015840 CGCG 31015840 (data not shown)

46 Stable Transfection and Differentiation pCDNA3 andTET2-pCDNA3 plasmid constructs were kindly donatedby Professor Olivier Barnard of INSERM Institut GustaveRoussy Villejuif France E14 cells were cultured in gelatincoated plates and transfected using the LipofectamineLTXand PLUS reagents (Invitrogen) with the TET2-pCDNA3plasmid pCDNA3 control plasmid ormock transfected eachin triplicate Incorporation of plasmid DNA was selected

using 300120583gmLminus Geneticin G418 selection antibiotic (Invit-rogen) at 5 days posttransfection for 14 days from whichpoint 100 120583gmLminus G418 was used After the 14-day selectionperiod no mock transfected colonies were apparent

Myeloid differentiation was measured by colony formingcell assay (CFC) conducted using MethoCult Growth FactorH4034 (Stem Cell Technologies) 3 times 3 cm plates of bothTET2-pCDNA3 and pCDNA3 stable transfect cells weregrown with GF H4034 with control plates of the stablytransfected lines grown without GF H4034 At 22 dayscolonies were classified according to the MethoCult GFH4034 protocol and photographed using a light microscope-mounted Canon Powershot G5 with B-52 lens adapter andCarl Zeiss 426126 digital camera adapter

Alkaline phosphatase analysis of stably transfected differ-entiating E14 cells by removal of LIF was conducted usingAlkaline Phosphatase Leukocyte kit (Sigma-Aldrich) Stablytransfected cells were plated onto gelatin coated 10 cm tissueculture plates at 1000 cells per plate in triplicate Media werechanged at 72 hours after plating that did not contain LIFCells were stained 7 days after removal of LIF and each colonygiven a leukocyte alkaline phosphatase activity (LAPA) scoreaccording to the protocol Reference LAPA score colonieswere photographed as above

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are grateful to Dr Helen Stewart for technicalsupport with experiments to Drs John Brewin and GillianHorne for helpful comments on the paper to Dr BernardRamsahoye formass spectrometricmeasurement of theDNAmethylation to Dr Olivier Bernard for provision of the TET2vector and to Professor Ghulam Mufti for kind donation ofmyelodysplasia bone marrow samples

References

[1] F Mohr K Dohner C Buske and V P Rawat ldquoTET genes newplayers in DNA demethylation and important determinants forstemnessrdquo Experimental Hematology vol 39 no 3 pp 272ndash2812011

[2] R L Strausberg E A Feingold L H Grouse et al ldquoGenerationand initial analysis of more than 15000 full-length human andmouse cDNA sequencesrdquo Proceedings of the National Academyof Sciences of the United States of America vol 99 no 26 pp16899ndash16903 2002

[3] M Tahiliani K P Koh Y Shen et al ldquoConversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalianDNAbyMLLpartner TET1rdquo Science vol 324 no 5929 pp 930ndash935 2009

[4] M E Figueroa O Abdel-Wahab C Lu et al ldquoLeukemic IDH1and IDH2 mutations result in a hypermethylation phenotypedisrupt TET2 function and impair hematopoietic differentia-tionrdquo Cancer Cell vol 18 no 6 pp 553ndash567 2010


[5] S Ito A C Dalessio O V Taranova K Hong L C Sowers andY Zhang ldquoRole of tet proteins in 5mC to 5hmC conversion ES-cell self-renewal and inner cell mass specificationrdquo Nature vol466 no 7310 pp 1129ndash1133 2010

[6] M Ko Y Huang A M Jankowska et al ldquoImpaired hydroxyla-tion of 5-methylcytosine inmyeloid cancerswithmutant TET2rdquoNature vol 468 no 7325 pp 839ndash843 2010

[7] W Xu H Yang Y Liu et al ldquoOncometabolite 2-hydroxyglutarate is a competitive inhibitor of 120572-ketoglutarate-dependent dioxygenasesrdquo Cancer Cell vol 19 no 1 pp 17ndash302011

[8] C Loenarz and C J Schofield ldquoOxygenase catalyzed 5-methylcytosine hydroxylationrdquo Chemistry and Biology vol 16no 6 pp 580ndash583 2009

[9] S Ito L Shen Q Dai et al ldquoTet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosinerdquoScience vol 333 no 6047 pp 1300ndash1303 2011

[10] Y He B Li Z Li et al ldquoTet-mediated formation of 5-carboxylcytosine and its excision byTDG inmammalianDNArdquoScience vol 333 no 6047 pp 1303ndash1307 2011

[11] C Chen K Wang and C-K Shen ldquoThe mammalian de novoDNA methyltransferases DNMT3A and DNMT3B are alsoDNA 5-hydroxymethylcytosine dehydroxymethylasesrdquo Journalof Biological Chemistry vol 287 no 40 pp 33116ndash33121 2012

[12] S M C Langemeijer R P Kuiper M Berends et al ldquoAcquiredmutations in TET2 are common in myelodysplastic syn-dromesrdquo Nature Genetics vol 41 no 7 pp 838ndash842 2009

[13] E Hellstrom-Lindberg ldquoSignificance of JAK2 and TET2 muta-tions inmyelodysplastic syndromesrdquo Blood Reviews vol 24 no2 pp 83ndash90 2010

[14] A M Jankowska H Szpurka R V Tiu et al ldquoLoss of heterozy-gosity 4q24 and TET2mutations associated withmyelodysplas-ticmyeloproliferative neoplasmsrdquo Blood vol 113 no 25 pp6403ndash6410 2009

[15] O Abdel-Wahab A Mullally C Hedvat et al ldquoGenetic char-acterization of TET1 TET2 and TET3 alterations in myeloidmalignanciesrdquo Blood vol 114 no 1 pp 144ndash147 2009

[16] O Nibourel O Kosmider M Cheok et al ldquoIncidence andprognostic value of TET2 alterations in de novo acute myeloidleukemia achieving complete remissionrdquo Blood vol 116 no 7pp 1132ndash1135 2010

[17] R Garzon M Garofalo M P Martelli et al ldquoDistinctivemicroRNA signature of acute myeloid leukemia bearing cyto-plasmic mutated nucleophosminrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 105 no10 pp 3945ndash3950 2008

[18] O Kosmider V Gelsi-BoyerM Cheok et al ldquoTET2mutation isan independent favorable prognostic factor in myelodysplasticsyndromes (MDSs)rdquo Blood vol 114 no 15 pp 3285ndash3291 2009

[19] A E Smith A M Mohamedali A Kulasekararaj et alldquoNext-generation sequencing of the TET2 gene in 355 MDSand CMMLpatients reveals low-abundance mutant clones withearly origins but indicates no definite prognostic valuerdquo Bloodvol 116 no 19 pp 3923ndash3932 2010

[20] S K Patra A Patra F Rizzi T C Ghosh and S BettuzzildquoDemethylation of (Cytosine-5-C-methyl) DNA and regulationof transcription in the epigenetic pathways of cancer develop-mentrdquoCancer andMetastasis Reviews vol 27 no 2 pp 315ndash3342008

[21] M M Suzuki and A Bird ldquoDNA methylation landscapesprovocative insights from epigenomicsrdquo Nature Reviews Genet-ics vol 9 no 6 pp 465ndash476 2008

[22] R Lister M Pelizzola R H Dowen et al ldquoHuman DNAmethylomes at base resolution show widespread epigenomicdifferencesrdquo Nature vol 462 no 7271 pp 315ndash322 2009

[23] R S Illingworth and A P Bird ldquoCpG islandsmdashldquoa rough guiderdquordquoFEBS Letters vol 583 no 11 pp 1713ndash1720 2009

[24] C M Bender M L Gonzalgo F A Gonzales C T Nguyen KD Robertson and P A Jones ldquoRoles of cell division and genetranscription in themethylation of CpG islandsrdquoMolecular andCellular Biology vol 19 no 10 pp 6690ndash6698 1999

[25] C A Doege K Inoue T Yamashita et al ldquoEarly-stage epige-neticmodification during somatic cell reprogramming by Parp1and Tet2rdquo Nature vol 488 no 7413 pp 652ndash655 2012

[26] K M Loh and B Lim ldquoEpigenetics actors in the cell repro-gramming dramardquoNature vol 488 no 7413 pp 599ndash600 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


occurring in approximately 12 of acute myeloid leukemia(AML) cases and up to 58 of mixed myeloproliferativeneoplasms (MPN) and myelodysplastic syndromes (MDS)[12ndash14] Therefore it is likely that TET2 plays an importantrole in regulation of hematopoietic differentiation and mayeven serve as a prognostic tool inmyeloidmalignancies AMLis characterized by enhanced proliferation and impaireddifferentiation of myeloid progenitors resulting in clonalpopulations of immature hematologic blasts Furthermorespecific AML subgroups show patterns of dysregulated DNAmethylation patterns which can be correlated with patientsurvival [4] The majority of TET2 mutations recorded inpatients with myeloid malignancy are within regions criticalfor enzymatic activity suggesting they are loss of functionmutations and hence TET2 is seen to be having a tumoursuppressor role [1 3]

The clinical outcome of patients with a mutation in TET2remains contentious In AML patients one study found adecreased survival rate in TET2 mutants when comparedto a wild-type group [15] whereas another study found nosignificant impact of mutation status on clinical outcomebut mutation was strongly associated to a mutation in theNPM1 gene which is also implicated in AML [16 17] InMDSpatients the 5-year survival rate was 769 for TET2mutantsagainst 183 for wild-type indicating amutation as amarkerfor good prognosis [18] Additionally Smith et al couldfind no prognostic significance for TET2 mutation whenpatients were clustered into WHO subtypes scored usingthe International Prognostic Scoring System or grouped intocytogenic status or progression to AML [19]

The isocitrate dehydrogenase enzyme IDH1 and its mito-chondrial homologue IDH2 are responsible for convertingisocitrate to 120572-ketoglutarate in the KrebsTCA cycle Het-erozygous gain of function mutations of IDH1 and IDH2 hasbeen shown to convert isocitrate into 2-hydroxyglutarate (2-HG) which competes with 120572-ketoglutarate to inhibit TETactivity [4] Various similarities exist between TET2 mutantand IDH12 mutant phenotypes including increased 5mC incultured cells and an increase in progenitor cell numbersand stem cells markers [4] These findings provide importantinsights into a possible common mechanism of epigeneticdisruption that may lead to AML or precursor diseases suchas MDS

13 Methylcytosine Hydroxymethylcytosine and Its Effect onPromoter Activity The total 5mC content of the humangenome is 1ndash3 of all cytosine bases [3 20ndash22] occurringalmost exclusively at CpG dinucleotides Clusters of CpGsites occurring at higher than expected frequency withingenomic regions are termed CpG islands (CGIs) which aregenerally hypomethylated within cells Around 65 of CGIslocalise around the point of gene promoter regions includingall constitutively expressed genes [23] Aberrant methylationof CGIs leads to stable transcriptional repression and isthought to be important in silencing tumour suppressorgenes in some conditions such as MDS CGI methylation atpromoter regions upstream of genes has the greatest effect ontranscription via prevention of elongation of the transcript[20] Some evidence suggest that CGI methylation within

exonic regions of genes is permissive of transcription anddoes not block initiation or elongation [24] The recentdiscovery of cytosine hydroxymethylation raises the possi-bility that methylation at CGIs can be reversed via activecytosine demethylation involving TET proteins leading to thereactivation of transcriptionally silenced promoters [9 10]Furthermore new evidence on induced pluripotent stem cells(iPSCs) suggests that the 5hmC base modification not only isan intermediate within cytosine demethylation but acts asan epigenetic mark in itself possibly aiding in recruitmentof factors that promote chromatin remodeling [25 26]

We show here that levels of 5hmC are reduced by TET2knockdown in ES cells and in TET2 mutated MDS bonemarrow samples We investigated the phenotypic effects ofTET2 overexpression on differentiation of murine ES cellsusing stable transfectants Finally using reported genes weshow that transient overexpression of TET2 has no significanteffect on the transcriptional activity ofmethylated promoters

2 Results

21 Differentiation of ES Cells Leads to Decreased 5ℎ119898C Wesought to investigate the levels of 5mC and 5hmC in undif-ferentiated murine ES cells using HPLC mass spectrometryWe found levels of 75 for 5mC and 015 for 5hmC relativeto guanosine bases demonstrating that 5mC is present inES cells at approximately 50-fold greater levels than 5hmC(Figure 1(a))

Next we sought to investigate what happens to levels of5hmCduring induction of differentiationWe compared levelsof 5hmC in undifferentiated ES cells grown in the presence ofleukemia inhibitory factor (LIF) and in differentiated cells 5days post-LIF withdrawal (Figure 1(b)) We found that levelsof 5hmC fall by approximately 50 from 0155 to 0072relative to guanosine bases upon differentiation This resultsuggests that LIF withdrawal induces downregulation of oneor more of the TET family genes leading to a reduction inconversion of 5mC to 5hmC

22 Knockdown of TET2 in ES Cells Causes Reduced Lev-els of 5ℎ119898C Based on this finding we performed siRNAknockdown experiments of the TET2 gene Using two dif-ferent gene-specific siRNA oligos in triplicate knockdownexperiments we measured reductions in 5hmC levels of 5and 20 respectively compared with a scrambled control(Figure 1(c)) These results provide evidence that TET2 likeTET1 catalyses the conversion of 5mC to 5hmC

23 Measurement of 5ℎ119898C Levels in TET2MutatedMDSAMLPatient Marrow Samples Genomic DNA analysis usingmassspectrometry of five patients with myeloid malignancy failedto identify a clear association between the TET2 mutationalstatus and 5hmC levels (Figure 1(d)) Patient samples weregenotyped to identify mutations within the TET2 gene locusany patients found to have a mutation were heterozygouswild-type While this result is based on a small number ofsamples and was not correlated with the size of the malignant


0

1

2

3

4

5

6

7

8D

eoxy

guan

osin

e (

)

5hm C 5

mC


0

002

004

006

008

01

012

014

016

018


Deo

xygu

anos

ine (

)


0

02

04

06

08

1

12

14

16

18


Deo

xygu

anos

ine (

)


0

0005

001

0015

002

0025

Deo

xygu

anos

ine (

)










EV st

able

tran

sfect

s

DAPI DAPI

Tet25hmC

(a)

TET2

stab

le tr

ansfe

cts

DAPI DAPI

5hmC Tet2

(b)







3 Discussion



0

05

1

15

2

25


Inve

rse a

vera

ge L

APA

scor

e per

colo

ny


(a)


(b)







50

55

60

65

70

75

80

85

90


Col

ony

diffe

rent

iatio

n (

)


(a)


(b)








1

10

100

1000

10000


MethylatedpRL +LIF


pRL minusLIF

RLU

Ren

illa

firefl

ytimes1000


1

10

100

1000

10000

Con

trol 1

Con

trol 2

Con

trol 3

unm

ethy

lated

pRL

repo

rter

no T

ET2

cotr

ansfe

ct

empt

y pC

DN

Ave

ctor

pCD

NA-

TET2

05120583

g

pCD

NA-

TET2

1120583

g

pCD

NA-

TET2

2120583

g

RLU

Ren

illa

firefl

ytimes1000




2at 37∘C




0ndash2mins 1 B


7ndash14m mins 1 B














Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3

4

5

6

7

8D

eoxy

guan

osin

e (

)

5hm C 5

mC


0

002

004

006

008

01

012

014

016

018


Deo

xygu

anos

ine (

)


0

02

04

06

08

1

12

14

16

18


Deo

xygu

anos

ine (

)


0

0005

001

0015

002

0025

Deo

xygu

anos

ine (

)










EV st

able

tran

sfect

s

DAPI DAPI

Tet25hmC

(a)

TET2

stab

le tr

ansfe

cts

DAPI DAPI

5hmC Tet2

(b)







3 Discussion



0

05

1

15

2

25


Inve

rse a

vera

ge L

APA

scor

e per

colo

ny


(a)


(b)







50

55

60

65

70

75

80

85

90


Col

ony

diffe

rent

iatio

n (

)


(a)


(b)








1

10

100

1000

10000


MethylatedpRL +LIF


pRL minusLIF

RLU

Ren

illa

firefl

ytimes1000


1

10

100

1000

10000

Con

trol 1

Con

trol 2

Con

trol 3

unm

ethy

lated

pRL

repo

rter

no T

ET2

cotr

ansfe

ct

empt

y pC

DN

Ave

ctor

pCD

NA-

TET2

05120583

g

pCD

NA-

TET2

1120583

g

pCD

NA-

TET2

2120583

g

RLU

Ren

illa

firefl

ytimes1000




2at 37∘C




0ndash2mins 1 B


7ndash14m mins 1 B














Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















EV st

able

tran

sfect

s

DAPI DAPI

Tet25hmC

(a)

TET2

stab

le tr

ansfe

cts

DAPI DAPI

5hmC Tet2

(b)







3 Discussion



0

05

1

15

2

25


Inve

rse a

vera

ge L

APA

scor

e per

colo

ny


(a)


(b)







50

55

60

65

70

75

80

85

90


Col

ony

diffe

rent

iatio

n (

)


(a)


(b)








1

10

100

1000

10000


MethylatedpRL +LIF


pRL minusLIF

RLU

Ren

illa

firefl

ytimes1000


1

10

100

1000

10000

Con

trol 1

Con

trol 2

Con

trol 3

unm

ethy

lated

pRL

repo

rter

no T

ET2

cotr

ansfe

ct

empt

y pC

DN

Ave

ctor

pCD

NA-

TET2

05120583

g

pCD

NA-

TET2

1120583

g

pCD

NA-

TET2

2120583

g

RLU

Ren

illa

firefl

ytimes1000




2at 37∘C




0ndash2mins 1 B


7ndash14m mins 1 B














Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

05

1

15

2

25


Inve

rse a

vera

ge L

APA

scor

e per

colo

ny


(a)


(b)







50

55

60

65

70

75

80

85

90


Col

ony

diffe

rent

iatio

n (

)


(a)


(b)








1

10

100

1000

10000


MethylatedpRL +LIF


pRL minusLIF

RLU

Ren

illa

firefl

ytimes1000


1

10

100

1000

10000

Con

trol 1

Con

trol 2

Con

trol 3

unm

ethy

lated

pRL

repo

rter

no T

ET2

cotr

ansfe

ct

empt

y pC

DN

Ave

ctor

pCD

NA-

TET2

05120583

g

pCD

NA-

TET2

1120583

g

pCD

NA-

TET2

2120583

g

RLU

Ren

illa

firefl

ytimes1000




2at 37∘C




0ndash2mins 1 B


7ndash14m mins 1 B














Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















50

55

60

65

70

75

80

85

90


Col

ony

diffe

rent

iatio

n (

)


(a)


(b)








1

10

100

1000

10000


MethylatedpRL +LIF


pRL minusLIF

RLU

Ren

illa

firefl

ytimes1000


1

10

100

1000

10000

Con

trol 1

Con

trol 2

Con

trol 3

unm

ethy

lated

pRL

repo

rter

no T

ET2

cotr

ansfe

ct

empt

y pC

DN

Ave

ctor

pCD

NA-

TET2

05120583

g

pCD

NA-

TET2

1120583

g

pCD

NA-

TET2

2120583

g

RLU

Ren

illa

firefl

ytimes1000




2at 37∘C




0ndash2mins 1 B


7ndash14m mins 1 B














Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1

10

100

1000

10000


MethylatedpRL +LIF


pRL minusLIF

RLU

Ren

illa

firefl

ytimes1000


1

10

100

1000

10000

Con

trol 1

Con

trol 2

Con

trol 3

unm

ethy

lated

pRL

repo

rter

no T

ET2

cotr

ansfe

ct

empt

y pC

DN

Ave

ctor

pCD

NA-

TET2

05120583

g

pCD

NA-

TET2

1120583

g

pCD

NA-

TET2

2120583

g

RLU

Ren

illa

firefl

ytimes1000




2at 37∘C




0ndash2mins 1 B


7ndash14m mins 1 B














Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article TET2 Inhibits Differentiation of ...downloads.hindawi.com/archive/2014/986571.pdf · HG) which competes with -ketoglutarate to inhibit TET activity [ ]. Various similarities