Heterodimeric DNA methyltransferases as a platform for creating designer zinc finger methyltransferases for targeted DNA methylation in cells

The ability to target methylation to specific genomic sites would further the study of DNA methylation’s biological role and potentially offer a tool for silencing gene expression and for treating diseases involving abnormal hypomethylation. The end-to-end fusion of DNA methyltransferases to zinc fingers has been shown to bias methylation to desired regions. However, the strategy is inherently limited because the methyltransferase domain remains active regardless of whether the zinc finger domain is bound at its cognate site and can methylate non-target sites. We demonstrate an alternative strategy in which fragments of a DNA methyltransferase, compromised in their ability to methylate DNA, are fused to two zinc fingers designed to bind 9 bp sites flanking a methylation target site. Using the naturally heterodimeric DNA methyltransferase M.EcoHK31I, which methylates the inner cytosine of 5′-YGGCCR-3′, we demonstrate that this strategy can yield a methyltransferase capable of significant levels of methylation at the target site with undetectable levels of methylation at non-target sites in Escherichia coli. However, some non-target methylation could be detected at higher expression levels of the zinc finger methyltransferase indicating that further improvements will be necessary to attain the desired exclusive target specificity

Cloning and characterization of EcoHK31I restriction and modification system from escherichia coli HK31.

by Lee Kai Fai, Calvin.Thesis (Ph.D.)--Chinese University of Hong Kong, 1995.Includes bibliographical references (leaves 159-167).ACKNOWLEDGMENTS --- p.iABSTRACT --- p.iiCONTENTS --- p.ivABBREVIATIONS --- p.xiChapter CHAPTER ONE --- General Introduction --- p.1Chapter 1.1 --- The Phenomenon of Host-controlled Restriction --- p.1Chapter 1.2 --- Classification of Restriction and Modification Systems --- p.2Chapter 1.2.1 --- Type I Restriction-Modification Systems --- p.2Chapter 1.2.2 --- Type II Restriction-Modification Systems --- p.3Chapter 1.2.3 --- Type III Restriction-Modification Systems --- p.4Chapter 1.2.4 --- Type IV Restriction-Modification Systems --- p.5Chapter 1.3 --- Occurrence of Restriction-Modification Systems --- p.6Chapter 1.4 --- Effect of Methylation --- p.7Chapter 1.5 --- Alternation of Recognition Specificities --- p.7Chapter 1.5.1 --- Cross Protection by DNA Methyltransferase --- p.8Chapter 1.5.2 --- A-Assisted Restriction Endonuclease (RARE) Cleavage --- p.9Chapter 1.5.3 --- Site-specific Cleavage mediated by Triple-helix formation --- p.9Chapter 1.5.4 --- Site-specific Cleavage of Duplex DNA with a λ repressor- Staphylococcal Nuclease Hybrid --- p.10Chapter 1.5.5 --- Achilles' heel Cleavage --- p.10Chapter 1.5.6 --- Chimeric Restriction Endonuclease --- p.11Chapter 1.6 --- Cloning of Restriction and Modification Systems --- p.11Chapter 1.6.1 --- Selection based on Modification --- p.11Chapter 1.6.2 --- Other Cloning Strategies --- p.12Chapter 1.6.2.1 --- Sub-Cloning of Plasmids --- p.12Chapter 1.6.2.2 --- Selection based on Restriction --- p.13Chapter 1.6.2.3 --- Multi-step Cloning --- p.13Chapter 1.6.2.4 --- Cloning in AP1-200 and AP1-200-9 strain --- p.13Chapter 1.6.2.5 --- Direct Cloning of Restriction gene by 'endo-blue' method --- p.14Chapter 1.7 --- Genetic Location of Restriction-Modification Systems --- p.14Chapter 1.8 --- Sequences of Restriction-Modification Systems --- p.15Chapter 1.9 --- Catalytic Properties of Type II Restriction-Modification Systems --- p.17Chapter 1.10 --- Crystallography of Type II Restriction and Modification Enzymes --- p.19Chapter 1.11 --- Evolution of Type II Restriction and Modification Enzymes --- p.22Chapter 1.12 --- Aim of Study --- p.23Chapter CHAPTER TWO --- Materials and Methods --- p.24Chapter 2.1 --- Bacterial Strains --- p.24Chapter 2.2 --- General Techniques --- p.25Chapter 2.2.1 --- Phenol/Chloroform Extraction --- p.25Chapter 2.2.2 --- Ethanol Precipitation --- p.25Chapter 2.2.3 --- Spectrophotometry --- p.25Chapter 2.2.4 --- Restriction digestion of DNA --- p.26Chapter 2.2.5 --- Agarose Gel Electrophoresis of DNA --- p.26Chapter 2.2.6 --- Recovery of DNA fragment from Agarose gel --- p.26Chapter 2.2.7 --- Minipreparation of Plasmid --- p.27Chapter 2.2.8 --- Large-Scale Preparation of Plasmid DNA --- p.28Chapter 2.2.8A --- By Equilibrium Centrifugation in Cesium Chloride- Ethidium Bromide Gradient --- p.28Chapter 2.2.8B --- By Using Qiagen-tip 100 Cartridge --- p.29Chapter 2.2.9 --- Preparation of Competent Cells --- p.30Chapter 2.2.10 --- Transformation of Competent Cells --- p.31Chapter 2.2.11 --- Screening of Recombinant Plasmids --- p.32Chapter 2.2.11A --- Using Selective media --- p.32Chapter 2.2.11B --- Rapid Alkaline Lysis Method --- p.32Chapter 2.2.12 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.33Chapter 2.2.13 --- Size Exclusion Chromatography --- p.34Chapter 2.2.14 --- Electroblotting of Protein on Polyvinylidene Difluoride (PVDF) membrane --- p.35Chapter 2.2.15 --- Isoelectric Focusing (EEF) --- p.36Chapter 2.2.16 --- Protein Assay --- p.37Chapter 2.3 --- DNA Sequencing --- p.37Chapter 2.3.1 --- Isolation of a template DNA --- p.38Chapter 2.3.2 --- DNA Denaturation and Annealing Reaction --- p.38Chapter 2.3.3 --- Labeling and Termination Reaction --- p.38Chapter 2.3.4 --- DNA Sequencing Electrophoresis --- p.39Chapter 2.3.5 --- Autoradiography --- p.40Chapter CHAPTER THREE --- Purification and Characterization of Restriction Endonuclease from Escherichia coli HK31 --- p.41Chapter 3.1 --- Introduction --- p.41Chapter 3.2 --- Materials and Methods --- p.42Chapter 3.2.1 --- Preparation of Crude enzyme Extract --- p.42Chapter 3.2.2 --- Purification of R.EcoHK31I --- p.42Chapter 3.2.3 --- Characterization of Restriction endonuclease --- p.43Chapter 3.2.3.1 --- Enzyme Activity assay --- p.43Chapter 3.2.3.2 --- "Optimal pH, Temperature, Metal Ion and Salt concentration of R.EcoHK31I" --- p.43Chapter 3.2.3.3 --- Assay for the Purity of R.EcoHK31I --- p.43Chapter 3.2.3.4 --- Determination of Recognition Specificity --- p.44Chapter 3.2.3.5 --- Determination of the Cleavage Specificity --- p.44Chapter 3.3 --- Results and Discussion --- p.45Chapter 3.3.1 --- Purification ofR.EcoHK31I from Escherichia coli HK31 --- p.45Chapter 3.3.2 --- "Optimal pH,Temperature, Metal ions and Salt concentration of R.EcoHK31I" --- p.46Chapter 3.3.3 --- Unit Definition --- p.51Chapter 3.3.4 --- Purity of the R.EcoHK31I --- p.51Chapter 3.3.5 --- Recognition Site of the R.EcoHK31I --- p.51Chapter 3.3.6 --- Sensitivity of the R.EcoHK31I to dcm Methylation --- p.52Chapter 3.3.7 --- Cleavage Specificity of R.EcoHK31I --- p.52Chapter CHAPTER FOUR --- Cloning of EcoEK31I Restriction and Modification (R-M) System from Escherichia coli HK31 --- p.57Chapter 4.1 --- Introduction --- p.57Chapter 4.2 --- Materials and Methods --- p.58Chapter 4.2.1 --- Extraction of genomic DNA from E. coli HK31 --- p.58Chapter 4.2.2 --- Extraction of Extra-Chromosomal DNA from E. coli HK31 --- p.59Chapter 4.2.3 --- Restriction Digestion of the Total DNA --- p.59Chapter 4.2.4 --- Preparation of Linearized and Dephosphorylated Vector --- p.60Chapter 4.2.5 --- Fill-in Reaction --- p.60Chapter 4.2.6 --- Ligation between Vector and Digested Chromosomal DNA --- p.61Chapter 4.2.7 --- Selection of Clones Harboring Methyltransferase gene --- p.61Chapter 4.2.8 --- Screening of the Survival Clones --- p.62Chapter 4.3 --- Results --- p.62Chapter 4.3.1 --- Construction of Genomic Libraries --- p.62Chapter 4.3.2 --- Selection of the Methyltransferase Gene --- p.66Chapter 4.3.3 --- In vitro Detection of R.EcoHK31I activity --- p.67Chapter 4.3.4 --- Functional Localization of EcoHK31I --- p.67Chapter 4.3.5 --- Subcloning of the Complete EcoHK31I R-M System --- p.72Chapter 4.4 --- Discussion --- p.72Chapter 4.4.1 --- Construction of Genomic Libraries --- p.72Chapter 4.4.2 --- Cloning of EcoHK31I Restriction and Modification System --- p.75Chapter 4.4.2.1 --- Selecting Endonuclease --- p.75Chapter 4.4.2.2 --- Detection of Restriction Endonuclease Activity --- p.76Chapter 4.4.3 --- Functional Localization of the R-M System --- p.76Chapter CHAPTER FIVE --- The Nucleotide Sequences of the EcoHK31I R-M System --- p.78Chapter 5.1 --- Introduction --- p.78Chapter 5.2 --- Materials and Methods --- p.79Chapter 5.2.1 --- Sequencing Strategies --- p.79Chapter 5.2.2 --- DNA Sequencing --- p.80Chapter 5.2.3 --- Sequence Analysis --- p.80Chapter 5.3 --- Results and Discussion --- p.80Chapter 5.3.1 --- Nucleotide Sequences and Deduced Amino Acid sequences --- p.80Chapter 5.3.2 --- Comparison of Amino Acid Sequences --- p.85Chapter CHAPTER SIX --- Purification and Characterization of EcoHK31I Methyltransferase from E. coli K802 [pEcoHK31E] --- p.91Chapter 6.1 --- Introduction --- p.91Chapter 6.2 --- Materials and Methods --- p.92Chapter 6.2.1 --- Preparation of Crude enzyme Extract --- p.92Chapter 6.2.2 --- Purification of M.EcoHK31I --- p.92Chapter 6.2.3 --- Characterization of EcoHK31I Methyltransferase --- p.93Chapter 6.2.3.1 --- Enzyme Activity assay --- p.93Chapter 6.2.3.2 --- Determination of Methylation specificity --- p.93Chapter 6.2.3.3 --- Determination of Molecular weight of M.EcoHK31I --- p.94Chapter 6.2.3.4 --- Determination ofM.EcoHK31I Kinetics --- p.94Chapter 6.3 --- Results and Discussion --- p.96Chapter 6.3.1 --- Purification of EcoHK31I Methyltransferase --- p.96Chapter 6.3.2 --- M.EcoHK31I Modification Specificity --- p.99Chapter 6.3.3 --- "Determination of Molecular Weight ofM,EcoHK31I" --- p.99Chapter 6.3.4 --- Catalytic Properties of EcoHK31I Methyltransferase --- p.103Chapter 6.3.5 --- A Novel m5C-MTase M.EcoHK31I --- p.103Chapter CHAPTER SEVEN --- Over-expression and Characterization of EcoHK31I Restriction and Modification Enzymes --- p.106Chapter 7.1 --- Introduction --- p.106Chapter 7.1.1 --- Expression Vector pTrc series --- p.107Chapter 7.1.2 --- Expression Vector pET series --- p.107Chapter 7.2 --- Materials and Methods --- p.109Chapter 7.2.1 --- General technique --- p.109Chapter 7.2.2 --- Polymerase Chain Reaction --- p.110Chapter 7.2.3 --- Construction of plysSM13 --- p.110Chapter 7.2.4 --- Construction of pTrc99A-R36 --- p.110Chapter 7.2.5 --- Construction of pET3a-M38 --- p.111Chapter 7.2.6 --- Construction of pET3a-C23 --- p.111Chapter 7.2.7 --- Expression of Recombinant Proteins in E. coli hosts --- p.115Chapter 7.2.8 --- Purification of Recombinant R.EcoHK31I --- p.115Chapter 7.2.9 --- Determination of Molecular Weight of Recombinant R. EcoHK31I --- p.115Chapter 7.2.10 --- Polyclonal Antibodies against R.EcoHK31I --- p.116Chapter 7.2.11 --- Western Blotting --- p.116Chapter 7.2.12 --- Purification of Recombinant M.EcoHK31I polypeptide α --- p.117Chapter 7.2.13 --- Purification of Recombinant M.EcoHK31I polypeptide β --- p.118Chapter 7.2.14 --- In vitro Complementation Methylation Activity --- p.118Chapter 7.2.15 --- Incorporation of [3H]-AdoMet to non-methylated Lambda DNA --- p.119Chapter 7.3 --- Results and Discussion --- p.119Chapter 7.3.1 --- Expression of Recombinant R. EcoHK31I --- p.119Chapter 7.3.2 --- Purification and Characterization of Recombinant R.EcoHK31I --- p.120Chapter 7.3.2.1 --- Purification of Recombinant R.EcoHK31I --- p.120Chapter 7.3.2.2 --- Characterization of Recombinant R.EcoHK31I --- p.122Chapter 7.3.2.2.1 --- Molecular Weight and Isoelectric point of the Recombinant R.EcoHK31I --- p.122Chapter 7.3.2.2.2 --- Antibodies to Recombinant R.EcoHK31I --- p.125Chapter 7.3.3 --- Expression and Purification of M.EcoHK31Ipolypeptide α --- p.127Chapter 7.3.4 --- Expression and Purification of M.EcoHK31I polypeptide β --- p.127Chapter 7.3.5 --- Characterization of M.EcoHK31I polypeptides a and β --- p.129Chapter 7.3.5.1 --- Molecular Weight Determination --- p.129Chapter 7.3.5.2 --- Isoelectric Point Determination --- p.132Chapter 7.3.5.3 --- In vivo and in vitro Methylation Activity --- p.132Chapter CHAPTER EIGHT --- Generation and Activity Assay of Q193G Mutein of M.EcoHK31I Polypeptide a --- p.138Chapter 8.1 --- Introduction --- p.138Chapter 8.2 --- Materials and Methods --- p.139Chapter 8.2.1 --- Construction of pET3a-M38 (Q193G) --- p.139Chapter 8.2.2 --- Expression and Purification of Q193G protein --- p.140Chapter 8.2.3 --- In vivo and in vitro methylation activity of Q193G Mutein --- p.140Chapter 8.3 --- Results and Discussion --- p.145Chapter 8.3.1 --- "Construction, Expression and Purification of Q193G Mutein" --- p.145Chapter 8.3.2 --- Determination of Molecular Weight and Isoelectric point of Q193G --- p.145Chapter 8.3.3 --- In vivo and in vitro methylation activity of Q193G Mutein --- p.145Chapter 8.3.4 --- Recognition Specificity of Q193G Mutein --- p.147Chapter CHAPTER NINE --- General Discussion --- p.151REFERENCES --- p.159APPENDIX A --- p.16

Targeting bifurcated methyltransferases towards user-defined DNA sequences

There would be great value in targeting methylation toward user-defined DNA sequences. Directing methylation toward single CpG sites within a genome would provide a means to examine the effects of single epigenetic alterations on cellular phenotype. The spread, erasure, or maintenance of such modifications could be examined in different cellular contexts and at different genomic loci. Further, as aberrant methylation patterns cause or are implicated in many disease states, targeted methylation might be used as a therapeutic. Many groups have attempted to target methylation toward user-defined sites by fusing a methyltransferase enzyme to a sequence specific DNA binding domain. This strategy biases the methyltransferase toward specific DNA sequences, but the methyltransferase enzyme is active in the absence of the sequence specific DNA binding event. A better strategy would involve linking the DNA binding event of sequence specific proteins to the activity of the methyltransferase enzyme. The contents of this thesis describe work on an assisted protein assembly strategy for targeting methylation to single CpG sites within a genome. This strategy utilizes naturally or unnaturally bifurcated methyltransferases fused to zinc fingers to affect reassembly over a desired site. The bifurcated methyltransferases are engineered to have reduced affinity for each other and/or for DNA, preventing unassisted enzymatic reassembly at non-targeted CpG sites. Zinc finger binding to sequences flanking an internal CpG site increase the local concentration of these assembly-deficient, bifurcated methyltransferases, enabling enzymatic reassembly and methylation only over the targeted CpG site. In Chapter 2, we demonstrate the successful implementation of this strategy for two prokaryotic methyltransferases, M.HhaI and M.SssI. Further, we elucidate design parameters important for constructing active, targeted, bifurcated methyltransferases. In Chapter 3, we describe a novel directed-evolution strategy to quickly identify optimized zinc finger-fused bifurcated methyltransferases. Importantly, we also demonstrate that substitution of bifurcated methyltransferase fragments with new zinc fingers predictably targets methylation toward new zinc finger cognate sequences. Finally, in Chapter 4, we describe successful preliminary studies in human cell lines. We demonstrate the eukaryotic expression of both fragments, targeting specific sites in a mammalian expression vector and methyltransferase activity on chromosomal DNA. Advisor: Professor Marc Ostermeier Readers: Professor Sarah Woodson Professor Jeffrey J. Gra