Transcriptional characterization and functional analysis of long non-coding RNA/protein-coding gene pairs encoded in the human genome Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by Anne-Susann Musahl from Berlin Berlin, 2015 ii The present work was carried out from March 2012 until April 2015 at the Max Planck Institute for Molecular Genetics under the supervision of Dr. Ulf Andersson Ørom. 1st Reviewer: Dr. Ulf Andersson Ørom 2nd Reviewer:Prof. Dr. Markus Wahl Date of Defence: 10.07.2015 iii Für Küsschen und das Entchen. iv v Acknowledgements I would like to thank my PhD advisor Dr. Ulf Ørom for giving me the opportunity to work in his lab and on this exciting project. Ulf provided me with guidance and support but also gave me the freedom to develop and follow own research ideas. I also want to thank Prof. Dr. Markus Wahl for reviewing this PhD thesis. Many thanks goes to my friends and colleagues in the lab: Dubravka Vučićević, Thomas Conrad, Evgenia Ntini, Annita Louloupi, Alexander Kiefer, Julia Liz, Maja Gehre, An- tonia Hilbig and Masha Kenda, you guys provided such a friendly and helpful working environment that enabled me to finish this PhD work. Special thanks goes to Dusa, Thomas and Evgenia for many insightful scientific discussions and their endless technical and moral support to pass all big and small problems that emerged in the last three years. Also, this work would never have been possible without the help of my co-authors at the Max Planck Institute and in Paris and Villejuif (France), therefore I would like to thank Annalisa Marsico, Ruping Sun, Marcus W. Albrecht, Guido Kroemer, Oliver Kepp, Xing Huang and Sylvie Rusakiewicz. Besonders dankbar bin ich meinen Eltern, für ihre dauerhafte Unterstützung und Er- mutigung ohne die meine schulische und universitäre Ausbildung nicht möglich gewesen wäre. Last, but certainly not least I would like to endlessly thank Marcus for always standing at my side and his continuous encouragement. Your love, friendship and support means everything to me. vi Acknowledgements Contents vii Contents Acknowledgements v Abbreviations xiii Abstract xvii Zusammenfassung xix 1 Introduction 1 1.1 Preface ...................................... 1 1.2 Encoding of genetic information in the human genome ........... 1 1.3 The transcription process ........................... 2 1.4 Transcriptional regulatory motifs ....................... 4 1.4.1 Enhancers ................................ 5 1.4.2 Promoters ................................ 6 1.4.2.1 Proximal promoter ...................... 6 1.4.2.2 Core promoter ........................ 6 1.4.2.3 Core promoter classes .................... 8 1.4.3 Silencers ................................. 9 1.4.4 Insulators ................................ 10 1.5 Transcription of the human genome ...................... 10 1.5.1 Bidirectional expression of PCGs ................... 10 1.5.2 Inherent promoter bidirectionality .................. 11 1.5.3 Transcription at enhancers ....................... 12 1.5.4 Similarity of transcription initiation at promoters and enhancers . 13 1.6 ncRNA species encoded in the human genome ................ 13 1.6.1 RNAs functioning in protein synthesis ................ 14 1.6.2 RNAs functioning in RNA processing ................ 14 1.6.3 RNAs functioning in regulation of RNA expression ......... 14 1.6.4 RNAs with diverse functions - long ncRNAs ............. 14 1.7 Functionality of long ncRNAs ......................... 15 1.7.1 Classification of long ncRNAs ..................... 15 viii Contents 1.7.2 Characteristics of long ncRNAs .................... 15 1.7.3 Functions of long ncRNAs ....................... 17 1.7.4 Association of long ncRNAs with PCG promoters .......... 19 1.8 Human tumor suppressors ........................... 21 1.8.1 Functionality of the retinoblastoma protein ............. 22 1.8.2 Functionality of calreticulin ...................... 23 1.9 Aims of the thesis ................................ 25 2 Materials and Methods 27 2.1 Materials .................................... 27 2.1.1 Instrumentation ............................ 27 2.1.2 Consumables .............................. 28 2.1.3 Chemicals ................................ 29 2.1.4 Buffers, solutions and media ...................... 30 2.1.5 Molecular biology kits ......................... 31 2.1.6 Enzymes and proteins ......................... 31 2.1.7 Plasmids ................................. 31 2.1.8 Antibodies ............................... 32 2.1.9 Oligonucleotides ............................ 32 2.1.10 Cell lines ................................ 35 2.1.11 Bacterial strain ............................. 36 2.1.12 Software ................................. 36 2.2 Methods ..................................... 36 2.2.1 Molecular biology methods ...................... 36 2.2.1.1 Isolation of genomic DNA .................. 36 2.2.1.2 Polymerase chain reaction .................. 37 2.2.1.3 Site-directed mutagenesis PCR ............... 37 2.2.1.4 Agarose gel electrophoresis ................. 38 2.2.1.5 DNA gel extraction ..................... 38 2.2.1.6 Determination of nucleic acid concentration and purity . 38 2.2.1.7 A-tailing of PCR products ................. 38 2.2.1.8 TOPO TA cloning ...................... 39 2.2.1.9 Restriction digest ...................... 39 2.2.1.10 Dephosphorylation of vectors ................ 39 2.2.1.11 Ligation of DNA ....................... 39 2.2.1.12 Generation of chemically competent E. coli cells ..... 40 2.2.1.13 Transformation of E. coli cells ............... 40 2.2.1.14 Colony PCR ......................... 40 2.2.1.15 Bacterial culture ....................... 40 2.2.1.16 Plasmid isolation and analytical digest ........... 41 Contents ix 2.2.1.17 Sanger sequencing ...................... 41 2.2.1.18 Diethylpyrocarbonate-treatment of dH2O ......... 41 2.2.1.19 RNA isolation of human cells ................ 41 2.2.1.20 DNase I treatment of RNA ................. 41 2.2.1.21 Complementary DNA synthesis ............... 42 2.2.1.22 Quantitative real-time PCR ................. 42 2.2.1.23 Next generation sequencing ................. 43 2.2.2 Cell culture methods .......................... 43 2.2.2.1 Culture of human cell lines ................. 43 2.2.2.2 Transfection of human cells ................. 44 2.2.2.3 Luciferase assay ....................... 44 2.2.2.4 Preparation of cellular extracts ............... 45 2.2.2.5 Fractionation of cells ..................... 45 2.2.2.6 Generation of stable cell lines using CRISPR ....... 45 2.2.3 Protein biochemical methods ..................... 46 2.2.3.1 Determination of protein concentration .......... 46 2.2.3.2 SDS polyacrylamide gel electrophoresis .......... 46 2.2.3.3 Western blot ......................... 46 2.2.3.4 Immunocytochemistry .................... 47 2.2.3.5 Cell surface immunocytochemistry and flow cytometry .. 47 2.2.3.6 Macrophage uptake assay .................. 47 2.2.4 Computational methods ........................ 48 2.2.4.1 Filtering of ncRNA/PCG pairs ............... 48 2.2.4.2 Conservation analysis of promoters ............. 48 2.2.4.3 Analysis of next generation sequencing data ........ 48 2.2.4.4 Determination of immunofluorescence intensity ...... 49 2.2.5 Statistical data analysis ........................ 49 3 Results 51 3.1 Long ncRNA/PCG pairs encoded in the human genome .......... 52 3.1.1 Association of long ncRNA genes with PCGs ............ 52 3.1.2 Functional categories of PCGs expressed from bidirectional long ncRNA/PCG promoters ........................ 52 3.1.3 Final set of bidirectionally expressed long ncRNA/PCG pairs ... 54 3.1.4 Conservation of bidirectional ncRNA/PCG promoters ....... 54 3.2 Expression of long ncRNA/PCG pairs from bidirectional promoters .... 55 3.2.1 Polymerase II occupancy at bidirectional promoters ......... 55 3.2.2 Capacity of long ncRNA/PCG promoters to initiate transcription bidirectionally .............................. 56 3.2.3 In vivo expression levels of ncRNA/PCG pairs ........... 57 x Contents 3.2.4 Regulation of long ncRNA expression by the exosome complex .. 59 3.3 The bidirectional ncRNA-RB1/RB1 promoter ................ 60 3.3.1 Effect of mutations on ncRNA-RB1/RB1 promoter activity .... 60 3.3.1.1 Mutations within transcription factor binding sites .... 60 3.3.1.2 Mutations within core promoter elements ......... 61 3.4 Transcript characteristics of ncRNA-RB1 ................... 64 3.4.1 Transcript structure .......................... 64 3.4.2 Cellular localization .......................... 64 3.5 Gene regulatory effects of ncRNA-RB1 .................... 65 3.5.1 Cellular depletion of ncRNA-RB1 and RB1 using dsiRNAs .... 65 3.5.2 Effect of ncRNA-RB1 knock-down on RB1 expression levels .... 66 3.5.3 Effect of ncRNA-RB1 and RB1 knock-down on the cellular tran- scriptome ................................ 66 3.5.4 Confirmation of ncRNA target genes by qRT-PCR ......... 68 3.5.5 Double knock-down of ncRNA-RB1 and RB1 ............ 68 3.5.6 Overexpression of ncRNA-RB1 .................... 69 3.5.7 Calreticulin as a ncRNA-RB1 target gene .............. 69 3.5.7.1 Regulation of calreticulin protein levels .......... 69 3.5.7.2 Knock-down of ncRNA-RB1 by antisense oligonucleotides 71 3.5.7.3 Knock-out of ncRNA-RB1 by genome editing using CRISPR 71 3.5.7.4 Knock-down of ncRNA-RB1 in U2OS cells ........ 72 3.6 Mechanistic insights into the regulation of calreticulin by ncRNA-RB1