Sensing DNA methylation in the protozoan parasite Entamoeba histolytica

Abstract

SummaryIn 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 nonEntamoebidae 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.

Nucleus-localized Antisense Small RNAs with 5′-Polyphosphate Termini Regulate Long Term Transcriptional Gene Silencing in Entamoeba histolytica G3 Strain

“…In E. histolytica, 5-methylcytosine modifications have been confirmed, an active DNA methyltransferase has been reported (44), and an E. histolytica methylated LINE binding protein that binds preferentially to methylated DNA has been characterized (45). In addition, histone acetylation and deacetylation are operational in E. histolytica (46).…”

Toll-Like Receptor 9-Dependent Macrophage Activation byEntamoeba histolyticaDNA

“…In contrast to bacterial DNA, E. histolytica DNA has an AT-rich (75.3%) genome and CG dinucleotides are underrepresented (22). It has been reported that E. histolytica has a DNA methyltransferase of the DNMT2 family which targets rRNA (29). Methylation seems to occur mostly at AT dinucleotides and not at the common CpG site as in vertebrate systems (5).…”

The Entamoeba histolytica Dnmt2 Homolog (Ehmeth) Confers Resistance to Nitrosative Stress

“…For Western blotting, nuclear fractions of pJST4-Ehmeth-or pcontrol-transformed E. histolytica trophozoites were prepared using a previously described protocol (24). Proteins were resolved on 12% SDS-polyacrylamide gel electrophoresis gels and then transferred to nitrocellulose membranes.…”