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Sensing DNA methylation in the protozoan parasite Entamoeba histolytica
Author(s) -
Lavi Tal,
Isakov Elada,
Harony Hala,
Fisher Ohad,
SimanTov Rama,
Ankri Serge
Publication year - 2006
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2006.05464.x
Subject(s) - biology , entamoeba histolytica , microbiology and biotechnology , dna methylation , chromatin immunoprecipitation , methylation , histone , dna , gene , biochemistry , genetics , promoter , gene expression
Summary 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.