title: Sensing DNA methylation in the protozoan parasite Entamoeba histolytica.
abstract: In the protozoan parasite Entamoeba histolytica, 5-methylcytosine (m5C) was found predominantly in repetitive elements. Its formation is catalysed by Ehmeth, a DNA methyltransferase that belongs to the Dnmt2 subfamily. Here we describe a 32 kDa nuclear protein that binds in vitro with higher affinity to the methylated form of a DNA encoding a reverse transcriptase of an autonomous non-long-terminal repeat retrotransposon (RT LINE) compared with the non-methylated RT LINE. This protein, named E. histolytica-methylated LINE binding protein (EhMLBP), was purified from E. histolytica nuclear lysate, identified by mass spectrometry, and its corresponding gene was cloned. EhMLBP corresponds to a gene of unknown function that shares strong homology with putative proteins present in Entamoeba dispar and Entamoeba invadens. In contrast, the homology dropped dramatically when non-Entamoebidae sequences were considered and only a weak sequence identity was found with Trypanosoma and several prokaryotic histone H1. Recombinant EhMLBP showed the same binding preference for methylated RT LINE as the endogenous EhMLBP. Deletion mapping analysis localized the DNA binding region at the C-terminal part of the protein. This region is sufficient to assure the binding to methylated RT LINE with high affinity. Western blot and immunofluorescence microscopy, using an antibody raised against EhMLBP, showed that it has a nuclear localization. Chromatin immunoprecipitation (ChIP) confirmed that EhMLBP interacts with RT LINE in vivo. Finally, we showed that EhMLBP can also bind rDNA episome, a DNA that is methylated in the parasite. This suggests that EhMLBP may serve as a sensor of methylated repetitive DNA. This is the first report of a DNA-methylated binding activity in protozoa.
authors.affiliation: Department of Molecular Microbiology, The Bruce Rappaport Faculty of Medicine, Technion, Israel.
authors.forename: Tal
authors.initials: T
authors.lastname: Lavi
authors.forename: Elada
authors.initials: E
authors.lastname: Isakov
authors.forename: Hala
authors.initials: H
authors.lastname: Harony
authors.forename: Ohad
authors.initials: O
authors.lastname: Fisher
authors.forename: Rama
authors.initials: R
authors.lastname: Siman-Tov
authors.forename: Serge
authors.initials: S
authors.lastname: Ankri
mesh_terms: D000818:Animals; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D019175:DNA Methylation; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004748:Entamoeba histolytica / Q000235:genetics* / Q000502:physiology
chemical_list: D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D004248:DNA (Cytosine-5-)-Methyltransferases