טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentHarony Hala
SubjectDNA Methylation and Targeting of LINE Retrotransposons in
Entamoeba histolytica and Entamoeba invadens
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 and is involved in developmental processes as well as in inactivation of transposable elements. This modification is catalyzed by enzymes known as DNA methyltransferases (MTases) which use S-adenosyl methionine as a methyl donor. Previous work in the human parasite Entamoeba histolytica had demonstrated the presence of methylated cytosines in the parasite’s genome and characterized the only m5C-DNA MTase expressed in this parasite; Ehmeth. In this work, we established the presence of methylated cytosines in the reptile’s parasite Entamoeba invadens and determined that Eimeth, similarly to Ehmeth, is related to the DNMT2 family. We also showed that the levels of m5C and Eimeth expression in E.invadens fluctuate among the different stages of the parasite life cycle, which suggested that DNA methylation could be developmentally regulated in this parasite. Moreover, we have isolated by affinity chromatography, using anti-m5C antibody, a DNA encoding reverse transcriptase of LINE retrotransposon (RT LINE) from both parasites.  RT LINE transcripts were detected in E.histolytica but were absent in E.invadens. Treating both parasites with the DNA MTase inhibitor; 5-azacytidine (5-azaC) resulted in a slight increase on the transcriptional levels of E.histolytica RT LINEs but had no effect on E.invadens RT LINEs transcription. The methylation status of genomic copies of E.invadens RT LINEs was confirmed by bisulfite analysis. In contrast, we were unable to isolate methylated RT LINEs from E.histolytica. Interestingly, many of the genomic copies of the E.histolytica RT LINEs, analyzed in this study diverge from the RT LINE isolated by m5C affinity chromatography by a number of mutations that includes conversion of C to T and G to A. These mutations are reminiscent of the conversion of C to T (and G to A on the complementary DNA strand) that occurred during primate evolution in Alu elements following accelerated deamination of methylated cytosines. E.invadens and E.histolytica RT LINEs, isolated by the affinity chromatography, were both cloned in a pEhAct Neo vector, amplified in E.coli methylation deficient strain, and transfected to E.histolytica. Bisulfite analysis on the vectors purified from transfected amoeba showed the presence of m5C in E.invadens RT LINE replicated in E.histolytica, but not in E.histolytica RT LINE or in the neomycine phosphotransferase (Neo) gene, which is also carried by the pEhAct Neo vector. Taken together, these results suggest the existence of a specific mechanism based on DNA methylation that controls retrotransposons in these parasites.