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0Nducing *Nd Suppressing Phe *5Pern*Pive 5Engphening Of nducing nd suppressing he ern)ive enghening of eomeres mech)nism in cncer ce0s Dissertation submitted by Delia Braun 2018 Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Dipl.-Chem. Delia Maria Braun born in Offenburg, Germany Oral examination: 24.07.2018 nducing nd suppressing he ern)ive enghening of eomeres mech)nism in cncer ce0s Referees: Prof. Dr. Karsten Rippe Prof. Dr. Brian Luke This work was performed from September 2012 to January 2018 under the supervision of Prof. Dr. Karsten Rippe in the Division Chromatin Networks at the DKFZ and the Bioquant Center in Heidelberg, Germany. Declaration I hereby declare that I have written the submitted dissertation “Inducing and suppressing the alternative lengthening of telomeres mechanism in cancer cells” myself and in this process, have used no other sources or materials than those explicitly indicated. I hereby declare that I have not applied to be examined at any other institution, nor have I used the dissertation in this or any other form at any other institution as an examination paper, nor submitted it to any other faculty as a dissertation. ______________________________ ______________________________ (Place, Date) Delia Braun Für Philipp und Lina Tbe of conens Lis of pubicions .................................................. V Summ)r? ............................................................... V! Zusmmenfssung ................................................. X Lis of figures ........................................................ X Lis of .bes ........................................................ X!! Abbrevi).ions ....................................................... XV n.roduc.ion ............................................................. 1 1 Te0omeres nd .e0omere minennce .............................................................................. 1 1.1 Telomere structure and function ........................................................................... 1 1.2 Telomere maintenance mechanisms (TMM) ........................................................ 3 2 A.ern).ive 0enghening of .e0omeres (ALT) ...................................................................... 5 2.1 Molecular and cytological hallmarks of ALT .......................................................... 5 2.2 Other ALT features ............................................................................................... 8 2.3 Databases with telomere maintenance (TM) information ..................................... 9 3 Trge.ing .e0omere minennce ..................................................................................... 10 3.1 Inhibiting telomerase ........................................................................................... 10 3.2 Suppressing the ALT mechanism ....................................................................... 11 4 PML pro.ein in .he ALT p).hw)? ...................................................................................... 12 4.1 Promyelocytic leukemia nuclear bodies (PML-NBs) ........................................... 12 4.2 ALT-associated PML nuclear bodies (APBs) ...................................................... 14 5 Scope of .he .hesis ............................................................................................................. 17 \ Tbe of conens M.eri)0s )nd Me.hods ........................................... 1 1 M.eri)0s .............................................................................................................................. 1 1.1 Buffers ................................................................................................................. 19 1.2 Kits and reagents ................................................................................................ 19 1.3 Instruments ......................................................................................................... 20 1.4 Software .............................................................................................................. 20 2 Coning ................................................................................................................................. 21 3 Ce00 cu0.ure )nd .r)nsfec.ion ............................................................................................ 22 3.1 siRNA transfections ............................................................................................ 24 3.2 Small molecule inhibitors .................................................................................... 25 4 Fuorescence c.iv).ed ce00 sor.ing (FACS) ................................................................... 2 4.1 Analysis of induced apoptosis ............................................................................. 27 4.2 Cell cycle analysis ............................................................................................... 27 5 Wesern b0o. ........................................................................................................................ 28 R C-circe )ss? ..................................................................................................................... 2 7 Fuorescence microscop ................................................................................................ 30 7.1 Fixation and permeabilization ............................................................................. 30 7.2 Telomere PNA FISH ........................................................................................... 30 7.3 Immunofluorescence ........................................................................................... 31 7.4 DAPI staining ...................................................................................................... 31 7.5 Manual and automatic confocal image acquisition ............................................. 31 7.6 Automatic image analysis ................................................................................... 32 8 RNA iso0).ion for RNA-se ............................................................................................... 34 O RNA 0ibr)r? prep)r).ion .................................................................................................... 35 10 RNA-se d).) )n)0?sis ...................................................................................................... 3 10.1 Mapping .............................................................................................................. 36 10.2 Integrative genomics viewer (IGV) ...................................................................... 37 10.3 Differential gene expression and GO pathway analysis ..................................... 37 11 mp0emen.).ion of .he Te#Ne% dbse .......................................................................... 38 \\ Tbe of conen.s Resu.s ................................................................... 41 1 Te#Ne% dbse .................................................................................................................. 41 1.1 Screening studies provide an initial source of TM genes .................................... 41 1.2 General gene annotations are gained from external databases ......................... 43 1.3 Genes are annotated with TM information .......................................................... 44 1.4 TelNet offers different search modes .................................................................. 46 1.5 TelNet is a versatile tool for the identification of TM genes ................................ 48 2 ALT suppression )nd induc.ion ....................................................................................... 51 2.1 Histone deacetylase inhibitor SAHA reduces ALT markers ................................ 51 2.1.1 SAHA reduces ALT markers already at low concentration ................................. 51 2.1.2 SAHA does not specifically induce apoptosis in ALT cells ................................. 53 2.1.3 ALT-positive cells are not hypersensitive to ATR inhibitor (ATRi) ...................... 54 2.1.4 Inflammatory and immune response genes were deregulated upon SAHA ....... 55 2.2 Knockdown of histone chaperone ASF1 induces ALT ........................................ 59 2.2.1 HeLa LT cells differ in several pathways and show low levels of PDK4 ............. 59 2.2.2 ASF1 depletion induces ALT in HeLa LT but not HeLa ST cells ........................ 63 2.2.3 HeLa LT and ST cells respond differentially to ASF1 depletion ......................... 64 2.2.4 HeLa ST cells respond with immune response and virus defense to ASF1 kd .. 65 2.2.5 ALT induction correlates with expression of TGFb and WNT signaling genes ... 67 2.3 HDAC inhibitor SAHA suppresses ALT induction ............................................... 71 2.3.1 SAHA reduces ALT induction capacity of ASF1 depletion in HeLa LT cells ....... 71 2.3.2 Inhibitors of WNT signaling are up- and virus response genes are downregulated upon SAHA-mediated suppression of ALT induction ................. 71 2.4 Yeast survivor formation marginally changes gene expression .......................... 76 3 ALT induc.ion b? recrui.ing PML .o .e0omeres ............................................................... 78 3.1 PML-TRF1 fusion protein leads to artificial APBs at the majority of telomeres ... 79 3.2 PML at telomeres induces telomere clustering ..................................................
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