Entamoeba histolytica is a protozoan parasite and a major cause of dysentery and diarrheal disease in developing countries. Disease transmission from one host to another occurs via cysts which can survive in environmental extremes and are transmitted through contaminated food and water. Recent studies in our lab identified a novel transcription factor, Encystation Regulatory Motif-Binding Protein (ERM-BP), which is responsive to NAD + and has an important role in encystation. The key residues important for ERM-BP function were demonstrated in vitro using recombinant protein. In this study we demonstrate the in vivo functional consequences of mutations in key domains and their impact on Entamoeba encystation. Our results show that mutations in the DNA binding domain (ERM-BP-DBM) and in the nicotinamidase domain (ERM-BP-C198A) lead to protein mis-localization in both trophozoites and cysts and significantly reduce encystation efficiency. Additionally, we showed that silencing of ERM-BP significantly decreased the size and number of multi-nucleated giant cells (MGC) that form during encystation, indicating that ERM-BP functions upstream of the cellular aggregation that precedes stage conversion. Dissection of epistatic interactions between ERM-BP and a second encystation-related transcription factor, NF-Y revealed that ERM-BP is upstream of NF-Y in controlling the developmental cascade and appears to be one of the earliest regulators of development identified to date in Entamoeba. We also demonstrated that ERM-BP is upregulated during heat stress in Entamoeba, another condition which increases intracellular NAD + levels and that overexpression of ERM-BP makes E. histolytica and E. invadens parasites more resistant to heat stress. Overexpression of ERM-BP in E. histolytica also induced the formation of cyst-like quadrinucleated cells and formation of MGCs. Overall, our work has identified an important role of ERM-BP in Entamoeba stress response and links an NAD +-responsive transcription factor to both development and heat shock response. Characterization of stress and developmental cascades are important avenues to investigate for Entamoeba, an important human parasitic pathogen.

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BackgroundIn human hosts, Entamoeba histolytica cysts can develop into trophozoites, suggesting that the life cycle of this parasite are regulated by changes in gene expression. To date, some evidence has suggested that epigenetic mechanisms such as DNA methylation and histone modification are involved in the regulation of gene expression in Entamoeba. Some post–translational modifications (PTMs) at the N-terminus of E. histolytica’s histones have been reported experimentally, including tri-methylation in the lysine 4 of histone H3 (H3K4me3) and dimethylation in the lysine 27 of histone H3 (H3K27me2), dimethylation of arginine 3 (H4R3me2) and the indirect acetylation of histone H4 in the N-terminal region. However, it is not known which residues of histone H4 are subject to acetylation and/or methylation or where in the nucleus these epigenetic marks are located.MethodsHistones from trophozoites of E. histolytica were obtained and analyzed by LC-MS/MS. WB assays were performed using antibodies against epigenetic marks (acetylated lysines and methylated arginines). Immunofluorescence assays (IFA) were carried out to determine the distribution of PTMs and the localization of DNA methylation as a heterochromatin marker. Nuclear bodies such as the nucleolus were identified by using antibodies against fibrillarin and nucleolin and speckles by using anti-PRP6 antibody.ResultsSome new PTMs in histone H4 of E. histolytica, such as the acetylation of lysines 5, 8, 12 and 16 and the monomethylation of arginine 3, were identified by WB. IFA demonstrated that some marks are associated with transcriptional activity (such as acetylation and/or methylation) and that these marks are distributed throughout the E. histolytica nucleus. Staining with antibodies against anti-pan-acetylated lysine H4 histone and 5-methyl cytosine showed that the activation and transcriptional repression marks converge. Additionally, two nuclear bodies, the nucleolus and speckles, were identified in this parasite.ConclusionsThis study provides the first evidence that the nucleus of E. histolytica is not compartmentalized and contains two nuclear bodies, the nucleolus and speckles, the latter of which was not identified previously. The challenge is now to understand how these epigenetic marks and nuclear bodies work together to regulate gene expression in E. histolytica.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1298-7) contains supplementary material, which is available to authorized users.

Post‐translational modifications of histone H3 N‐terminal tails are key epigenetic regulators of virulence gene expression and sexual commitment in the human malaria parasite Plasmodium falciparum . Here, we identify proteolytic clipping of the N‐terminal tail of nucleosome‐associated histone H3 at amino acid position 21 as a new chromatin modification. A cathepsin C‐like proteolytic clipping activity is observed in nuclear parasite extracts. Notably, an ectopically expressed version of clipped histone H3, PfH3p‐ HA , is targeted to the nucleus and integrates into mononucleosomes. Furthermore, chromatin immunoprecipitation and next‐generation sequencing analysis identified PfH3p‐ HA as being highly enriched in the upstream region of six genes that play a key role in DNA replication and repair: In these genes, PfH3p‐ HA demarcates a specific 1.5 kb chromatin island adjacent to the open reading frame. Our results indicate that, in P. falciparum , the process of histone clipping may precede chromatin integration hinting at preferential targeting of pre‐assembled PfH3p‐containing nucleosomes to specific genomic regions. The discovery of a protease‐directed mode of chromatin organization in P. falciparum opens up new avenues to develop new anti‐malarials.