The Expression Of DNA Targeting Methyltranferase And The Study Of Its Function

DNA methylation, modifications of histone, DNA remodeling, etc, which increases the inheritable information of DNA without the changes of the primary property of base pairing and encoding information, are called epigenetic modification. In the advanced eukaryotic organisms, DNA methylation plays a critical role in the development and differentiation, temporal and spatial regulation of gene expression as well as the maintenance of genomic stability.DNA methyltransferase can be divided into two types which are responsible for de novo methylation and maintenance methylation, respectively; in mammalian cells, the identified primary de novo methyltransferase are Dnmt 3a and Dnmt 3b and the maintenance methyltransferase is Dnmt1. The main function of the de novo methyltransferase is to introduce new methylated DNA sites and it does not have selectivity to the substrate DNA; the maintenance methyltransferase Dnmt1 has selectivity towards its substrate DNA and it preferentially recognizes and catalyses the double-strand DNA with one methylated strand (named hemimethlated double-strand DNA) so that its main function is to copy the methylation pattern of the parent chain into the nascent chain during DNA replication which is also the molecular basis for the inheritance of DNA methylation. The de novo methylation guarantees the dynamic state and the adaptability to the environment. A great deal of studies indicated that DNA methylation is closely related with gene silencing. Generally, the methylation of CG islands in the promoter region of a gene results the close of the gene expression or so-called gene silencing. The property of gene silencing induced by DNA methylation suggested that the selective close of gene expression may be acquired through specific DNA methylation which is to specifically introduce the modification of DNA methylation into the CG islands of the promoter or the expression regulating region of the target gene.The rapid development of the designation and library screening technology of artificial zinc finger proteins in recent years turns it into reality to acquire the engineered DNA binding protein (zinc finger protein) targeting any specific gene sequence. In one study, the engineered zinc finger protein, which binds VP16 site in the promoter IE175 of HSV, was fused with DNA methyltransferase, and recombinant protein induced the hypermethylation of the promoter and the gene silencing as well as the significant inhibition of HSV virus in COS cells. To increase the general applicability of targeted DNA methylation technology, we will exploit the targeted DNA methylation specified by artificial zinc finger protein and this will also be a creative expanding of the application of zinc finger protein technology. To solve above-mentioned problems, we will construct and expresse recombinant DNA methyltransferase specified by artificial zinc finger protein, exert ex vivo analysis of its functional parameters such as its specificity and efficacy of targeted DNA methylation to provide necessary basis for the intracellular targeted DNA methylation; then, take yeast as the model to observe and analyze the efficacy and specificity to the target gene of the targeted DNA methyltransferase at yeast chromatic level through oriented integration of the target gene sequence by homologous recombination into the yeast genome.Based on above statements, the aim of our experiment is to express and purify targeted DNA methylatransferase in prokaryotic system and Pichia yeast and conduct biochemistry analysis of its function: affinity features to the target substrate and non-target substrate, the maintenance of the activity of methyltransferase, the efficacy of DNA methylation to target substrate and non-target substrate as well as the valid range of targeted DNA methylation; meanwhile, the yeast genome does not contain the sequence which recognized and binded by the zinc finger protein here we used and we introduced the DNA recognized by the zinc finger protein into the fixed-point of yeast genome to design artificial target gene (the near sequence of binding site). Use the bisulfate sequencing method to analyze the methylation caused by the targeted DNA methyltransferase at yeast chromatin level to provide some basis for the following studies.The study was carried out as the following proposal:1 . Cloning of targeted DNA methyltransferase. Took pcDNA4-B1-3a-Myc-6His plasmid kept by our laboratory as the template, amplified the DNA by PCR, recovered and purified the PCR product and inserted it into pMD 19-T vector and sequenced. Sequences thus obtained were validated to GenBank database using BLAST program.2. The prokaryotic expression of targeted DNA methyltransferase B1-3a. The validated gene fragment was inserted into pET32a vector. Protein expression was induced by IPTG in Bl 21. The expressed protein was insoluble.3. The expression of targeted DNA methyltransferase in Pichia yeast. Took pcDNA4-B1-3a-Myc-6His plasmid kept by our laboratory as the template, amplified the DNA by PCR, recovered and purified the PCR product and inserted it into the yeast expression vector pPIC3.5k and sequenced. Sequences thus obtained were validated to GenBank database using BLAST program. The expressed fusion protein B1-3a-Myc-6His was mainly insoluble. The expression fusion protein in supernatant of yeast split product was purified by Ni-NTA agarose beads affinity chromatography. The obtained protein was analyzed by peptide mass fingerprint.4. Inserted the DNA recognizable to zinc finger protein into the fixed-point of the yeast genome to construct artificial target gene. Took pcDNA4-B1-3a-Myc-6His plasmid kept by our laboratory as the template and designed 3' end primers to link the B1-3a binding sequence to the down stream of B1-3a-Myc-6His through twice PCRs, recovered and purified the PCR product and inserted it into the yeast expression vector pPIC3.5k and sequenced. Sequences thus obtained were validated to GenBank database using BLAST program. Integrated the targeted gene sequence into the fixed-point of the yeast genome through homologous recombination.5. The analysis of the methylation function of targeted DNA methyltransferase at yeast chromatin level. Extract the induced and non-induced yeast genomes containing B1-3a-Myc-6His-BS sequence, treated the genomic DNA by bisulfite and took them as the templates for PCR, recovered and purified the PCR products and inserted them into pMD 19-T vectors and sequenced. Sequences thus obtained were validated to GenBank database using BLAST program.