Institut für Immunologie Direktor: Prof. Dr. Hans-Jürgen Thiesen Transcription Factor Properties of ZNF746 and ZNF777 with Focus on their Domains of Unknown Function 3669 (DUF3669) Dissertation zur Erlangung des akademischen Grades doctor rerum humanarum: “Dr. rer. hum.” der Universitätsmedizin Rostock vorgelegt von M.Sc. Mohannad Al Chiblak, geboren am 08.01.1982, in Daraa, Syrien Rostock, 06.10.20 https://doi.org/10.18453/rosdok_id00002800 Gutachter Prof. Dr. med. Markus Tiedge, Universitätsmedizin Rostock, Institut für Medizinische Biochemie und Molekularbiologie. Prof. Dr. med. Hans-Jürgen Thiesen, Universitätsmedizin Rostock Institut für Immunologie. Prof. Dr. med. Christian Sina, Universitätsklinikum schleswig-holstein Institut für Ernährungsmedizin. Datum der Einreichung: 27.01.2020 Datum der Verteidigung: 06.10.2020 Table of contents 1- Summary -------------------------------------------------------------------------------------------------------------1 2- Introduction ---------------------------------------------------------------------------------------------------------4 2.1- Regulation of gene expression ------------------------------------------------------------------------------4 2.2- C2H2 zinc finger transcription factors ---------------------------------------------------------------------5 2.3- Krüppel associated box (KRAB)–containing zinc finger protein family -----------------------------6 2.3.1- Discovery -------------------------------------------------------------------------------------------------6 2.3.2- Genomic organization and protein domain configuration -----------------------------------------6 2.3.3- Molecular mechanisms of canonical KRAB-mediated transcriptional repression ------------7 2.3.4- The biological function of KRAB-ZNF proteins ------------------------------------------------- 10 2.4- The domain of Unknown function 3669 (DUF3669) -------------------------------------------------- 12 2.5- Zinc finger protein 746 (ZNF746/PARIS) -------------------------------------------------------------- 13 2.5.1- The role of ZNF746/PARIS in Parkinson’s disease ---------------------------------------------- 13 2.5.2- The role of ZNF746/PARIS in cancer -------------------------------------------------------------- 15 2.6- Zinc finger protein 777 (ZNF777) ------------------------------------------------------------------------ 16 2.7. Haploid HAP1 cells – an efficient knockout model to study cellular functions ------------------- 16 3- Aim ----------------------------------------------------------------------------------------------------------------- 18 4- Materials and methods ------------------------------------------------------------------------------------------- 19 4.1- DNA techniques and cloning ------------------------------------------------------------------------------ 19 4.1.1- RT-PCR (Reverse transcription PCR) -------------------------------------------------------------- 19 4.1.2- Specific PCR (DNA amplification) ----------------------------------------------------------------- 19 4.1.3- Purification of PCR products ------------------------------------------------------------------------ 20 4.1.4- Ligation ------------------------------------------------------------------------------------------------- 21 4.1.5- Competent bacteria and Transformation ----------------------------------------------------------- 22 4.1.6- Blue-White Screening --------------------------------------------------------------------------------- 23 4.1.7- Sanger sequencing ------------------------------------------------------------------------------------- 23 4.1.8- Restriction digestion of DNA ------------------------------------------------------------------------ 25 4.1.9- Agarose Gel Electrophoresis ------------------------------------------------------------------------- 26 4.1.10- DNA gel extraction ---------------------------------------------------------------------------------- 27 4.1.11- DNA precipitation ----------------------------------------------------------------------------------- 28 4.1.12- Plasmid Isolation ------------------------------------------------------------------------------------- 28 4.1.13- Cloning Strategies ------------------------------------------------------------------------------------ 32 4.2- Cell Culture -------------------------------------------------------------------------------------------------- 36 4.2.1- Cell lines and media ----------------------------------------------------------------------------------- 36 4.2.2- Freezing and Thawing human Cell lines ----------------------------------------------------------- 36 4.2.3- Cell passaging and seeding --------------------------------------------------------------------------- 36 4.2.4- Transient transfection --------------------------------------------------------------------------------- 37 4.2.5- Generation of stable cell lines ----------------------------------------------------------------------- 37 4.3- Production of recombinant proteins in E. coli ---------------------------------------------------------- 39 4.4- Immunofluorescence --------------------------------------------------------------------------------------- 40 4.5- Immunoprecipitation and Pulldown Assays ------------------------------------------------------------ 41 4.5.1- Lysis of eukaryotic cells ------------------------------------------------------------------------------ 41 I 4.5.2- Immunopreciptation and pulldown assays in eukaryotic cells ---------------------------------- 42 4.5.3- Pulldown assay with NiNTA agarose beads ------------------------------------------------------- 42 4.6- SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) ------------------------ 43 4.7- Western blotting --------------------------------------------------------------------------------------------- 44 4.8- Dual-luciferase reporter assay----------------------------------------------------------------------------- 46 4.9- Compilation of DUF3669-containing KRAB-ZNFs -------------------------------------------------- 47 5- Results-------------------------------------------------------------------------------------------------------------- 49 5.1- Six human KRAB-ZNFs have an extremely evolutionarily conserved DUF3669 and specific to amniotes ------------------------------------------------------------------------------------------------------------ 49 5.2- Protein constructs and expression verification --------------------------------------------------------- 52 5.3- Contribution of the protein domain setup of ZNF746 to its transcriptional repression activities ----------------------------------------------------------------------------------------------------------------------- 57 5.4- Impact of the ZNF777 domain organization on transcriptional repression ------------------------ 59 5.5- Evaluation of the dependency of ZNF746 and ZNF777 repressor potential on TRIM28 and SETDB1 ------------------------------------------------------------------------------------------------------------ 60 5.6- Investigation of the interaction between ZNF746 or-/ ZNF777 protein segments and endogenous TRIM28. -------------------------------------------------------------------------------------------- 63 5.7- Lack of a conserved glutamic acid residue in ZNF746 KRAB-A explains the weak repressor activity and insufficient interaction with TRIM28.---------------------------------------------------------- 65 5.8- Lack of transferability of the DUF3669 effect on repression to a canonical KRAB domain --- 68 5.9- SUMOylation at K189 in ZNF746a is critical for its transcriptional repression activities ------ 69 5.10- Investigation of DUF3669 Homo- and Hetero-oligomerization ----------------------------------- 69 6- Discussion --------------------------------------------------------------------------------------------------------- 73 7- Conclusion and outlook ----------------------------------------------------------------------------------------- 81 8- Acronyms and Abbreviations ----------------------------------------------------------------------------------- 82 9- References --------------------------------------------------------------------------------------------------------- 83 10- Supplements ----------------------------------------------------------------------------------------------------- 91 11- Acknowledgment --------------------------------------------------------------------------------------------- 105 12- Declaration of Originality ----------------------------------------------------------------------------------- 106 II Summary 1- Summary Krüppel-associated box-containing zinc finger proteins (KRAB-ZNFs) represent a large family of transcription regulators in tetrapods. Typically, KRAB-ZNF proteins consist of an amino-terminal protein interaction domain, designated KRAB, and a carboxy-terminal tandem array of DNA-binding C2H2 zinc finger motifs as initially determined in KOX1/ZNF10. In the canonical model of function, zinc finger motifs specifically bind to genomic target sites while the KRAB domain interacts with TRIM28 that in turn recruits chromatin-modifying protein complexes including enzymes such as histone methyltransferase SETDB1 and histone deacetylase HDAC1. The KRAB/TRIM28-mediated interaction initiates a local spreading of repressive chromatin structures promoting epigenetic silencing. A subset of KRAB-ZNFs has been described to contain N-terminally an additional conserved protein domain designated “domain of unknown function 3669” (DUF3669).