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Functional Characterization and Physiological Significance of SETD3 This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore. Functional characterization and physiological significance of SETD3 Chia, Shyh Jenn 2017 Chia, S. J. (2017). Functional characterization and physiological significance of SETD3. Doctoral thesis, Nanyang Technological University, Singapore. http://hdl.handle.net/10356/72760 https://doi.org/10.32657/10356/72760 Downloaded on 24 Sep 2021 15:05:18 SGT Chia Shyh Jenn G1500804C Functional characterization and physiological significance of SETD3 Chia Shyh Jenn School of Biological Sciences 2017 i Chia Shyh Jenn G1500804C Acknowledgements First and foremost, I wish to express my sincere gratitude to Assoc/prof Su I-Hsin, wise and knowledgeable supervisor for her precious guidance, criticisms and trust in all of my endeavors. I would like to thank my TAC committee members, Dr. Francesc Xavier Roca Castella and Dr. Zhang Li Feng, for their scientific suggestions and criticisms on my experiments. I would also like to gratefully acknowledge my earnest colleagues Dr Nandini Prabhakar, Dr Wong Jong Fu, Dr Loh Jia Tong, Lim Jun Feng and Maegan Bunjamin for their useful advice and assistance on my experiments. In addition, I also thank my attachment student Yeo Kian Hua for his contribution to this project. I would also like to thank the scientists from Transgenic Mouse Model Core, National Taiwan University for the generation of SETD3 conditional knockout mouse model. i Chia Shyh Jenn G1500804C Table of Contents Acknowledgements ........................................................................................................... i Table of Contents ............................................................................................................. ii List of Figures ................................................................................................................. iv List of Tables .................................................................................................................... v Abbreviations .................................................................................................................. vi Abstract .......................................................................................................................... vii 1. Introduction.................................................................................................................. 1 1.1 Protein Phosphorylation ................................................................................. 1 1.2 Protein Acetylation ......................................................................................... 2 1.3 Protein Glycosylation ..................................................................................... 2 1.4 Protein Methylation ........................................................................................ 3 1.5 Lysine Methyltransferase - SET Domain Containing Protein ...................... 3 1.6 Lysine Methylation on Histone Protein ......................................................... 4 1.7 Lysine Methylation on Non-Histone Proteins ................................................ 6 1.8 Readers of Lysine Methylation ...................................................................... 8 1.9 Self-Methylation of Protein Methyltransferase ............................................. 9 1.10 SETD3 Lysine Methyltransferase .............................................................. 10 1.11 Research Objectives .................................................................................... 13 2. Materials and Methods .............................................................................................. 14 2.1 In Vitro Methylation Assays......................................................................... 14 2.2 Polymerization of Purified G-actin .............................................................. 14 2.3 Recombinant Protein Expression and Purification ..................................... 15 2.4 Actin Lysine Site Directed Mutagenesis ...................................................... 15 2.5 Immunoprecipitation of 3x FLAG-Actin ..................................................... 16 2.6 B Cell Purification and In Vitro Stimulation............................................... 16 2.7 Bone Marrow Derived Dendritic Cell (BMDC) Culture and In Vitro Stimulation.......................................................................................................... 17 2.8 Bone Marrow Derived Macrophage (BMDM) Culture and In Vitro Stimulation.......................................................................................................... 18 ii Chia Shyh Jenn G1500804C 2.9 SETD3-GFP Pull-Down ................................................................................ 18 3. Results ........................................................................................................................ 19 3.1 Actin is the potential cytosolic substrate of SETD3 ..................................... 19 3.2 SETD3 is a Self-Methylating Lysine Methyltransferase ............................. 22 3.3 SETD3 Could Methylate Actin at Multiple Lysine Residues ...................... 24 3.4 SETD3 Conditional Knockout Mouse Model .............................................. 27 3.5 SETD3 Possibly Regulates Lymphocyte and Granulocyte Development ... 29 3.6 SETD3 Expression Increased in B Cells upon Activation ........................... 32 4. Discussion ................................................................................................................... 35 4.1 Self Methylation of SETD3 ........................................................................... 35 4.2 Posttranslational Modifications (PTMs) on Actin ....................................... 35 4.3 Actin Cytoskeleton Events in Hematopoietic Cells...................................... 37 5. Conclusion .................................................................................................................. 39 6. Future Direction ......................................................................................................... 40 7. References................................................................................................................... 43 iii Chia Shyh Jenn G1500804C List of Figures FIGURE 1.10. 1 CYTOPLASMIC LOCALIZATION OF SETD3 IN BOSC23 CELLS ............... 12 FIGURE 1.10. 2 MRNA EXPRESSION LEVEL OF SETD3 IN CHARACTERIZED IMMUNOLOGICAL CELL LINEAGE ............................................................................. 12 FIGURE 3.1. 1 SETD3 METHYLATE PROTEIN AT THE SIZE AROUND 37-45 KDA ............... 20 FIGURE 3.1. 2 SETD3 METHYLATES BOTH MONOMERIC AND POLYMERIC FORM OF ACTIN IN VITRO ................................................................................................................... 21 FIGURE 3.2. 1 THE METHYLATION OF SETD3 MIGHT OCCUR IN CIS................................ 22 FIGURE 3.2. 2 THE METHYLATION ON SETD3 IS INHIBITED IN THE PRESENCE OF ITS NUCLEAR SUBSTRATE ............................................................................................... 23 FIGURE 3.3. 1 SETD3 COULD METHYLATE ACTIN AT MULTIPLE LYSINE RESIDUES ........ 26 FIGURE 3.4. 1 GENERATION OF CONDITIONAL SETD3 KNOCKOUT MICE ........................ 28 FIGURE 3.5. 1 SETD3 COULD HAVE REGULATORY EFFECT ON B CELL DEVELOPMENT AND NEUTROPHIL PRODUCTION ....................................................................................... 30 FIGURE 3.5. 2 SETD3 DOES NOT REGULATE THE ACTIVATION PROCESSES OF BONE MARROW DERIVED DENDRITIC CELLS (BMDC) AND MACROPHAGES (BMDM) ....... 31 FIGURE 3.5. 3 SETD3 COULD HAVE REGULATORY ROLE IN T CELL DEVELOPMENT ........ 32 FIGURE 3.6. 1 B CELLS UPREGULATED SETD3 EXPRESSION UPON STIMULATION ........... 34 iv Chia Shyh Jenn G1500804C List of Tables TABLE 1.7. 1 SUMMARY OF THE NON-HISTONE SUBSTRATE OF LYSINE METHYLTRANSFERASE, METHYLATION SITE AND THE EFFECT OF METHYLATION ...... 8 TABLE 2.4. 1 PRIMERS FOR ACTIN LYSINE SITE DIRECTED MUTAGENESIS ....................... 15 TABLE 2.4. 2 PCR CYCLE FOR SITE DIRECTED MUTAGENESIS OF 3XFLAG-ACTIN.......... 16 v Chia Shyh Jenn G1500804C Abbreviations Rubisco LSMT Rubisco large subunit methyltransferase LPS Lipopolysaccharides PTMs Post translational modifications H3 Histone 3 H4 Histone 4 HDAC Histone deacetylases MBP Maltose binding protein GTPase Guanosine triphosphate hydrolase enzymes KMT Lysine-specific methyltransferase PRMT Protein arginine methyltransferase SAM S-adenosylmethionine PRC2 Polycomb repressive complex 2 Me0 unmethylated Me1 monomethylated Me2 dimethylated Me3 trimethylated EZH2 Enhancer of zeste homolog 2 SETD3 SET domain containing 3 CrispR Clustered regularly interspaced short palindromic repeats SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis GFP Green fluorescent protein WT Wild type MT Mutant kDa Kilodalton PCR Polymerase chain reaction BMDM Bone marrow derived macrophages BMDC Bone marrow derived dendritic cells KO Knockout vi Chia Shyh Jenn G1500804C Abstract Lysine methyltransferase family plays important roles in various cellular processes, for instance transcriptional regulation, embryonic development, cell proliferation, migration and more. My project aims to characterize a novel SET-domain containing lysine methyltransferase SETD3 and understand the physiological significance of this protein. SET-domain containing protein SETD3 methylates Lys-4 and Lys-36 of histone 3, which is linked to the transcriptional
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    PRODUCT INFORMATION SETD3 (human, recombinant) Item No. 27355 Overview and Properties Synonyms: Actin Histidine Methyltransferase, Actin Histidine N-Methyltransferase, C14orf154, Chromosome 14 Open Reading Frame 154, HSETD3, SET Doman-Containing 3, SET Domain-containing Protein 3 Source: Active recombinant N-terminal His-tagged SETD3 expressed in E. coli Amino Acids: 2-594 (full length) Uniprot No.: Q86TU7 Molecular Weight: 69.2 kDa Storage: -80°C (as supplied) Stability: ≥1 year Purity: batch specific (≥80% estimated by SDS-PAGE) Supplied in: 50 mM HEPES, pH 8.0, with 150 mM sodium chloride and 10% glycerol Protein Concentration: batch specific mg/ml Activity: batch specific U/ml Specific Activity: batch specific U/mg Unit Definition: nmol/min/mg. One unit is defined as the amount of enzyme required to transfer one methyl group to actin peptide per minute using 8 µM actin peptide (LKYPIEHGIVTNWDDMEKIW-amide) at 37°C in Cayman’s Methyltransferase Colorimetric Assay Kit (Item No. 700140). Information represents the product specifications. Batch specific analytical results are provided on each certificate of analysis. Images SETD3 Acǎvity 1 2 3 250 kDa · · · · · · · 150 kDa · · · · · · · 100 kDa · · · · · · · 75 kDa · · · · · · · 50 kDa · · · · · · · 37 kDa · · · · · · · 25 kDa · · · · · · · 20 kDa · · · · · · · 15 kDa · · · · · · · Figure 2: Acǎvity Assay. SETD3 acǎvity was Lane 1: MW Markers determined using Cayman’s Methyltransferase Lane 2: SETD3 (2 µg) Colorimetric Assay Kit (Item No. 700140). Lane 3: SETD3 (4 µg) Figure 1: SDS-PAGE Analysis of SETD3 Representaìve gel image shown; actual purity may vary between each batch. WARNING CAYMAN CHEMICAL THIS PRODUCT IS FOR RESEARCH ONLY - NOT FOR HUMAN OR VETERINARY DIAGNOSTIC OR THERAPEUTIC USE.
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