טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentFisher Ohad
SubjectThe Role of DNA Methylation in the Parasite
Entamoeba histolytica
DepartmentDepartment of Medicine
Supervisor Professor Serge Ankri
Full Thesis textFull thesis text - English Version


Abstract

DNA methylation is an epigenetic modification, occurring in a wide range of organisms by which enzymes known as DNA methyltransferases catalyze the transfer of a methyl group that is provided by the co-factor S-adenosylmethionine (SAM) to one of the four bases, in general cytosine.

The DNA methylation status of the protozoan parasite Entamoeba histolytica was unknown. We used an antibody against 5mC to detect this modified base in the parasite’s DNA. The gene that encodes a DNA methyltransferase (Ehmeth) was cloned and its product was characterized. Ehmeth belongs to the DNMT2 protein family. This highly conserved protein family is widely distributed and its biological role was unknown.

Ehmeth was isolated from the nuclear matrix of trophozoites. Its inhibition by 5-Azacytidine, a potent DNA methyltransferase inhibitor, led to demethylation of the parasite’s DNA and a reduced virulence. Over-expression of Ehmeth in the parasite led to a pleiotropic phenotype which included the accumulation of multinucleated cells, up-regulation of heat shock protein 70 (HSP70) expression and resistance to oxidative stress. The presence of m5C in the promotor of the HSP70 gene was demonstrated by bisulfite analysis although no difference in the DNA methylation pattern was found between control and Ehmeth over-express trophozoites. This pleiotropic phenotype suggests that Ehmeth plays an important role in the control of key cellular processes in the parasite.

 To identify methylated regions in the parasite's DNA, we developed a novel affinity column with anti-5mC antibodies as ligand. rDNA was isolated by this method and the presence of m5C was confirmed by bisulfite analysis.

 The presence of methylated cytosines was also detected in the 3'-end of an EhMRS2 element containing a scaffold/matrix attachment regions (S/MAR) bipartite recognition sequence. S/MAR is a eukaryotic consensus repetitive sequence known to take part in anchoring the chromatin to the nuclear matrix.

The same affinity method was used to identify methylated DNA sequences in Drosophila melanogaster. The fly, like the amoeba encodes a single DNA methyltransferase gene from the DNMT2 family. The sequences isolated by this method consist of repetitive elements including satellite repeats and retro-transposons. 

The methylated DNA sequences identified in this work point out the role of the DNMT2 protein as a DNA methyltransferase responsible for methylating  repetitive elements in the DNA of organisms encoding a single DNA methyltransferase gene from the DNMT2 family.