טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentKatz Sophia
SubjectThe E.histolytica Methylated LINE Binding Protein(EhMLBP)
Serves as a Bridge between Environmental Stress
Response and Epigenetic Regulation
DepartmentDepartment of Medicine
Supervisor Professor Serge Ankri
Full Thesis textFull thesis text - English Version


Abstract

Adaptation to environmental stress is a key process that allows the unicellular parasite Entamoeba histolytica to survive in its human host. EhMLBP (E. histolytica Methylated LINE Binding Protein) was previously characterized as an essential protein for the growth and the virulence of the parasite. EhMLBP binds to methylated repetitive DNA, and along with Ehmeth (E. histolytica DNMT2 homologue) constitute the core proteins of the parasite’s epigenetic machinery. Here, we show that EhMLBP and heat shock proteins have common properties; EhMLBP contains a heat shock domain. Its expression is induced by heat shock via the binding of heat shock transcription factor to heat shock element binding sites present in EhMLBP 5’ non-coding region. Overexpression of EhMLBP protects trophozoites against heat shock and reduces protein aggregation. Furthermore, EhMLBP binds polyubiquitinated proteins upon heat shock.  These properties are lost in trophozoites that overexpress a mutated form of EhMLBP, which is devoid of its heats shock domain. Following heat shock, the perinuclear localization of EhMLBP in control trophozoites is replaced by an even distribution within the nucleus, alongside with its appearance in cytoplasmic vesicles. These vesicles are probably stress granules based on the prevention of their formation by cycloheximide (a drug that prevents the formation of stress granules). In addition, EhMLBP and ubiquitin (which was reported to be present in stress granules) co-localized in these vesicules. Stress granules are a site of interaction between RNA and RNA binding proteins. EhMLBP was found to bind short interspersed element (SINE) RNA thus supporting its role as a stress granule protein.  The properties of EhMLBP as a DNA binding protein under heat shock stress were also investigated. Following heat shock, EhMLBP displays enhanced recruitment to the reverse transcriptase of long interspersed nucleotide element (LINE) DNA which is known to be up-regulated in response to heat shock. Moreover, constitutive over-expression of EhMLBP leads to an enhanced transcription of RT LINE and also affects SINE transcription. Interestingly, the cross talk between RT LINE and EhMLBP is reciprocal as over expression or down regulation of RT LINE transcription directly affects the expression level of EhMLBP. We also demonstrate that over expression of RT LINE RNA induces the localization of EhMLBP to cytoplasmic vesicles that resembles stress granules. The biological meaning of the cross talk between EhMLBP and RT LINE requires further investigation. To the best of our knowledge, this is the first report of a methyl DNA binding protein that plays a protective role against heat shock.