Dissertation Combined Faculties For The Natural Sciences .
Dissertationsubmitted to theCombined Faculties for the Natural Sciences and for Mathematicsof theRuperto-Carola University of Heidelberg, Germanyfor the degree ofDoctor of Natural SciencesPresented byM. Sc. Laura Wiehleborn in Waren (Müritz), GermanyOral examination: 5th October 2017
TET-dependent DNA methylation patterns in mammaliandevelopment and diseaseReferees:Prof. Dr. Frank LykoProf. Dr. Jan Lohmann
AbstractTET enzymes are relatively novel players in the epigenetic regulation of mammalian DNAmethylation. They participate in DNA demethylation, but their precise roles in differentdevelopmental and disease scenarios are not fully understood. The aim of this work was toinvestigate the biological roles of TET enzymes in lineage-committed normal and cancercells. To this end, murine primary cells with genetic deletion of TET enzymes and humancancer cells with recurrent mutations in the cofactor providing isocitrate dehydrogenases(IDH), provoking competitive inhibition of TET enzymes, were analyzed.By characterizing mouse embryonic fibroblasts adipogenic differentiation defects,inefficient activation of genes relevant to adipogenesis and widespread gene deregulationupon TET1/2-deficiency were discovered. Examination of the genome-wide DNA methylationlandscape demonstrated the hypermethylation of DNA methylation canyons as a anyonswereassociatedwithdevelopmentally important genes and canyon collapse due to hypermethylation coincidedwith developmental gene deregulation, defective induction of adipogenic markers and thehypermethylation of their promoters. Together, these findings uncovered a novel epigeneticregulatory role in the maintenance of DNA methylation canyons for TET1 and TET2 that isessential for epigenetic programming during differentiation.In the second part of this thesis, published array-based DNA methylation profiles of alarge acute myeloid leukemia (AML) patient cohort were used to examine mutant IDH(mIDH) and TET-dependent DNA methylation changes. This confirmed the known associationbetween mIDH and genome-wide hypermethylation. However, similar global methylationchanges were not present in TET2 mutant patients and mIDH carrying patients lackedspecific canyon hypermethylation. Intriguingly, neither overexpression of mIDH, nor treatmentof a leukemia cell line with D-2-hydroxyglutarate, which is a putative TET inhibitor producedby mIDH, recapitulated the mIDH-associated hypermethylation. Instead, comparison withhematopoietic reference methylomes revealed high similarity between mIDH-associated andmyeloid progenitor methylation profiles, suggesting the involvement of differentiation staterather than TET inhibition in the hypermethylation phenotype. These findings implicate apreviously unnoted factor in the epigenomic changes of AML cells with mIDH, which may becritical to understand and therapeutically target mIDH-dependent pathogenesis.
ZusammenfassungTET Enzyme sind relativ neue Akteure in der epigenetischen Regulation von DNAMethylierung in Säugern. Sie wirken an DNA-Demethylierung mit, jedoch sind ihre konkretenRollen in verschiedenen Differenzierungs- und Krankheitsszenarien nicht vollständigaufgeklärt. In der vorliegenden Arbeit sollten die biologischen Funktionen von TET-Enzymenin determinierten normalen sowie Krebszellen erforscht werden. Dazu wurden primäreMauszellen mit genetischer TET-Deletion und humane Krebszellen mit wiederkehrendenMutationen in den Cofaktor-bereitstellenden Isocitrat-Dehydrogenasen (IDH), welche zu einerkompetitiven Enzymhemmung der TET Proteine führen, untersucht.Mittels Charakterisierung von embryonalen Mausfibroblasten wurden Defekte in deradipogenen Differenzierung, der Aktivierung von Adipogenese-relevanten Genen und derGenexpression bei TET1/2-Defizienz entdeckt. Die Untersuchung der genomweiten DNAMethylierung identifizierte die Hypermethylierung von sogenannten DNA-MethylierungsCanyons als wesentliches Merkmal des TET1/2-defizienten Methyloms. Canyons waren mitentwicklungsbiologisch relevanten Genen assoziiert und ihr Zusammenbruch durchHypermethylierung war begleitet von Regulationsdefekten in Entwicklungsgenen, fehlerhafterInduktion von adipogenen Markern und Promoter-Hypermethylierung. Diese Ergebnisseschreiben TET1 und TET2 eine neuartige regulatorische Rolle in der Erhaltung von Canyonszu, die essentiell für die epigenetische Programmierung während der Differenzierung ist.Im zweiten Teil dieser Arbeit wurden publizierte Array-basierte DNA Methylierungsprofileeiner großen akuten myeloischen Leukämie (AML) Patienten-Kohorte verwendet, umMethylierungsveränderungen durch mutante IDH (mIDH) und TET Enzyme zu analysieren.Dies bestätigte die bekannte Assoziation zwischen mIDH und genomweiter DNAHypermethylierung. Allerdings waren ähnliche Veränderungen nicht in TET2 mutantenPatienten vorhanden und Patienten mit mIDH wiesen keine spezifische CanyonHypermethylierung auf. Interessanterweise wurde die mIDH-assoziierte Hypermethylierungauch weder durch die Überexpression von mIDH, noch die Behandlung von Leukämiezellenmit D-2-Hydroxyglutarat, welches ein durch mIDH produzierter, mutmaßlicher TET Inhibitorist, exakt nachgebildet. Stattdessen offenbarte der Vergleich mit hämatopoetischenReferenzmethylomen eine hohe Ähnlichkeit zwischen mIDH-assoziierten und myeloischenProgenitorzell-Methylierungsprofilen, was auf eine Beteiligung des Differenzierungsgradesanstelle der TET Inhibition an der Hypermethylierung hinweist. Diese Ergebnisse involviereneinen bisher unbeachteten Faktor in die epigenomischen Veränderungen von AML-Zellen mitmIDH, welcher entscheidend für das weitere Verständnis und die gezielte Therapie vonmIDH-abhängiger Pathogenese sein könnte.
ContentsContentsList of Abbreviations . IVList of Figures . VIList of Tables . VIII1Introduction . 11.11.1.1DNA methyltransferases . 11.1.2The mammalian DNA methylation landscape . 31.1.3Function of DNA methylation . 41.1.4DNA methylation changes in cancer . 61.2The mammalian DNA demethylation machinery . 71.2.1DNA hydroxymethylation . 81.2.2Ten-eleven translocation enzymes . 101.2.3Biological roles of TET enzymes . 121.3The interaction between IDH und TET enzymes. 151.3.1Molecular functions of IDH and mutated IDH (mIDH) . 151.3.2IDH mutations in cancer . 171.3.3The hematopoietic system and acute myeloid leukemia (AML) . 181.42The mammalian DNA methylation machinery . 1Aims of the thesis . 20Results . 212.1TET function in mouse development . 212.1.1Phenotypic characterization of Tet1/2-deficient MEFs . 212.1.2DNA methylation changes in Tet1/2-deficient MEFs . 252.1.3Gene regulation defects in Tet1/2-deficient MEFs . 292.1.4Dysregulation of canyons and associated genes during adipogenesis . 302.1.5Cooperative role of TET1 and TET2 in canyon maintenance. 322.22.2.1TET function in human hematological disease with IDH mutation . 34DNA methylation changes in IDH mutant AML patients . 34I
Contents32.2.2Generation of IDH mutant AML cell lines . 372.2.3DNA methylation changes in an IDH mutant AML cell line . 412.2.4DNA methylation changes in AML patients with TET mutations . 432.2.5Canyon analysis in IDH mutant AML patients . 442.2.6Effect of D-2-HG on the methylome of an AML cell line . 452.2.7Comparison of IDH mutant AML and normal hematopoietic methylomes . 46Discussion . 493.13.1.1A differentiated model system with impaired DNA demethylation . 493.1.2Hypermethylated canyons are a key feature of the DKO methylome . 503.1.3A role for TET1/2-dependent canyon maintenance in differentiation . 523.1.4A potential role of canyon hypermethylation in cancer . 553.2Genomic hypermethylation is a key feature of AML with mutations in IDH . 563.2.2MIDH-associated methylomes and their relation to TET inhibition . 573.2.3MIDH-associated methylomes in view of hematopoietic differentiation states . 59Summary and Conclusions . 62Materials and Methods . 654.1Materials . 654.2Eukaryotic cells and cell culture . 734.2.1Cell lines and primary cells . 734.2.2Generation of stable cell lines . 744.3Molecular cloning . 754.3.1pLVX-IRES-ZsGreen1 constructs . 764.3.2pHAGE PGK-GFP-IRES-LUC-W constructs . 794.4IIRole of IDH-TET axis in human disease . 553.2.13.34Role of TET enzymes in mouse development . 49Nucleic acid analyses . 824.4.1DNA extraction. 824.4.2Dot blot analyses . 824.4.3RNA extraction. 83
Contents4.4.4Reverse transcription . 844.4.5Quantitative real time PCR (qRT-PCR) . 844.4.6High throughput sequencing. 854.4.7454 targeted amplicon bisulfite sequencing . 864.4.8EPIC Methylation Array . 874.54.5.1Adipogenic differentiation . 884.5.22-HG treatment . 884.5.3D-2-HG measurement . 884.5.4Proliferation assay by Cell Titer Glo . 904.5.5Proliferation assay by assessment of cumulative population doubling . 904.6High throughput data analysis . 914.6.1Whole genome bisulfite sequencing . 914.6.2RNA sequencing . 914.6.3Methylation array. 924.75Cellular assays . 88Statistical analysis . 92Appendix . 935.1Supplemental Figures. 935.2References . 985.3List of Publications . 121III
List of AbbreviationsList of lcytosineAMLacute myeloid leukemiabpbase pairsCGICpG islandChIP(-seq)chromatin immunoprecipitation (sequencing)CIMPCpG island hypermethylator phenotypeCLPcommon lymphoid progenitorCMMLchronic myelomonocytic leukemiaCMPcommon myeloid xynucleotide triphosphateDKOdouble knockoutggravitational acceleration constantGMPgranulocyte macrophage progenitorHGDHhydroxyglutarate dehydrogenaseHSChematopoietic stem cellkbkilo teLSKlineage SCA1 KIT cells (includes HSCs and MPPs)MDSmyelodysplastic syndromeMEFmouse embryonic fibroblastsMEPmegakaryocyte erythrocyte progenitor(m)ESC(mouse) embryonic stem cell(m)IDH(mutant) isocitrate dehydrogenaseminminutesMPPmultipotent progenitorODoptical densityPMDpartially methylated domainqRT-PCRquantitative real time PCRR&Dresearch and developmentIV
List of AbbreviationsRNA-seqRNA sequencingrpmrotations per minutesecsecondsTCGAThe Cancer Genome AtlasTETTen-eleven translocation enzymeTSStranscriptional start siteWGBSwhole genome bisulfite sequencingWTwildtypeAll gene names in introduction and discussion are italic and in capitals independent ofspecies, while protein names are in regular font. Spelling in the results section is according tothe species-specific conventions.V
List of FiguresList of FiguresFigure 1.1: Mammalian DNA (de)methylation machinery. 2Figure 1.2: Schematic presentation of key features of the mammalian methylome. 4Figure 1.3: Mechanisms of DNA methylation and active and passive demethylation. . 10Figure 1.4: Actions of WT IDH enzymes in homeostasis and mutant IDH1/2 in disease. . 16Figure 1.5: Schematic of human hematopoiesis and accompanying DNA methylationchanges. . 19Figure 2.1: Validation of the Tet1/2-deficient MEF model. . 22Figure 2.2: Phenotypic changes in Tet1/2-deficient MEFs. . 23Figure 2.3: Incomplete adipogenesis in Tet1/2-deficient MEFs. . 23Figure 2.4: Inefficient transcription of adipogenic marker genes in Tet1/2-deficient MEFs. . 24Figure 2.5: Tet expression is induced during adipogenesis. . 25Figure 2.6: Tet1/2-deficient MEFs show widespread DNA hypermethylation. . 26Figure 2.7: Tet1/2-deficiency in MEFs results in canyon hypermethylation. . 27Figure 2.8: Canyon borders are maintained by TET1/2. . 28Figure 2.9: Validation of canyon border hypermethylation. . 28Figure 2.10: Tet1/2-deficiency disturbs gene expression in MEFs. . 29Figure 2.11: Validation of gene expression changes in Tet1/2-deficient MEFs. . 30Figure 2.12: Association of gene deregulation and localization in a hypermethylated canyon. 30Figure 2.13: Promoter hypermethylation correlates with defective gene induction duringadipogenesis in Tet1/2-deficient MEFs. 31Figure 2.14: TET1 and TET2 cooperate to maintain canyon borders. . 33Figure 2.15: AML patients with mutations in IDH1/2 display genomic hypermethylation. . 35Figure 2.16. MIDH confers specific methylation patterns to AML cells. 36Figure 2.17: Overexpression of mIDH1 but not WT IDH1 leads to D-2-HG secretion. . 38Figure 2.18: Overexpression of mIDH2 results in robust D-2-HG production. . 39Figure 2.19: MIDH2-expressing HL-60 cells show no overt phenotypic alterations. . 40Figure 2.20: MIDH2-expressing HL-60 cells display gene expression changes. . 41Figure 2.21: MIDH2-expressing HL-60 cells display globally increased DNA methylation. . 42Figure 2.22: DNA methylation changes in AML patients and HL-60 cells with mIDH aredistinct. 43Figure 2.23: Mutations in TET2 do not recapitulate mIDH-associated hypermethylation inAML. . 44VI
List of FiguresFigure 2.24: TET-dependent DNA methylation canyons are not specifically hypermethylatedin mIDH AML. . 45Figure 2.25: D-2-HG does not provoke genomic hypermethylation in vitro. 46Figure 2.26: MIDH-associated hypermethylation in AML resembles methylation patterns ofmyeloid progenitors. . 48Figure 3.1: Model for the proposed actions of TET1, TET2 and DNMT3A on canyons. . 54Figure 3.2: Schematic of the proposed action of mIDH in the myeloid lineage. . 61Figure 4.1: Cloning strategy to introduce IDH genes into different lentiviral backbones. . 75Figure 4.2: Restriction digestion of pLVX-IRES-ZsGreen1. 77Figure 4.3: Contr
investigate the biological roles of TET enzymes in lineage-committed normal and cancer cells. To this end, murine primary cells with genetic deletion of TET enzymes and human cancer cells with recurrent mutations in the cofactor providing isocitrate dehydrogenases (IDH), provoking competitive inhibition of TET enzymes, were analyzed.
May 02, 2018 · D. Program Evaluation ͟The organization has provided a description of the framework for how each program will be evaluated. The framework should include all the elements below: ͟The evaluation methods are cost-effective for the organization ͟Quantitative and qualitative data is being collected (at Basics tier, data collection must have begun)
Silat is a combative art of self-defense and survival rooted from Matay archipelago. It was traced at thé early of Langkasuka Kingdom (2nd century CE) till thé reign of Melaka (Malaysia) Sultanate era (13th century). Silat has now evolved to become part of social culture and tradition with thé appearance of a fine physical and spiritual .
On an exceptional basis, Member States may request UNESCO to provide thé candidates with access to thé platform so they can complète thé form by themselves. Thèse requests must be addressed to esd rize unesco. or by 15 A ril 2021 UNESCO will provide thé nomineewith accessto thé platform via their émail address.
̶The leading indicator of employee engagement is based on the quality of the relationship between employee and supervisor Empower your managers! ̶Help them understand the impact on the organization ̶Share important changes, plan options, tasks, and deadlines ̶Provide key messages and talking points ̶Prepare them to answer employee questions
Dr. Sunita Bharatwal** Dr. Pawan Garga*** Abstract Customer satisfaction is derived from thè functionalities and values, a product or Service can provide. The current study aims to segregate thè dimensions of ordine Service quality and gather insights on its impact on web shopping. The trends of purchases have
Bruksanvisning för bilstereo . Bruksanvisning for bilstereo . Instrukcja obsługi samochodowego odtwarzacza stereo . Operating Instructions for Car Stereo . 610-104 . SV . Bruksanvisning i original
Chính Văn.- Còn đức Thế tôn thì tuệ giác cực kỳ trong sạch 8: hiện hành bất nhị 9, đạt đến vô tướng 10, đứng vào chỗ đứng của các đức Thế tôn 11, thể hiện tính bình đẳng của các Ngài, đến chỗ không còn chướng ngại 12, giáo pháp không thể khuynh đảo, tâm thức không bị cản trở, cái được
10 tips och tricks för att lyckas med ert sap-projekt 20 SAPSANYTT 2/2015 De flesta projektledare känner säkert till Cobb’s paradox. Martin Cobb verkade som CIO för sekretariatet för Treasury Board of Canada 1995 då han ställde frågan
