Background: The mechanism(s) of G3-based TGS in E. histolytica is largely unknown. Results: 5Ј-polyphosphate antisense sRNAs are identified; mechanistic insights linking these sRNAs with TGS are provided by IFA, FISH, IP, and ChIP assays. Conclusion: TGS in E. histolytica G3 strain is mediated by an siRNA pathway, which utilizes antisense 5Ј-polyphosphate sRNAs. Significance: This is the first demonstration of (a) 5Ј-polyphosphate antisense sRNAs mediating TGS and (b) RNAi-mediated TGS in protozoan parasites.

ABSTRACTWe report the characterization of a bacterial-type oxygen reductase abundant in the cytoplasm of the anaerobic protozoan parasiteEntamoeba histolytica. Upon host infection,E. histolyticais confronted with various oxygen tensions in the host intestine, as well as increased reactive oxygen and nitrogen species at the site of local tissue inflammation. Resistance to oxygen-derived stress thus plays an important role in the pathogenic potential ofE. histolytica. The genome ofE. histolyticahas four genes that encode flavodiiron proteins, which are bacterial-type oxygen or nitric oxide reductases and were likely acquired by lateral gene transfer from prokaryotes. TheEhFdp1gene has higher expression in virulent than in nonvirulentEntamoebastrains and species, hinting that the response to oxidative stress may be one correlate of virulence potential. We demonstrate that EhFdp1 is abundantly expressed in the cytoplasm ofE. histolyticaand that the protein levels are markedly increased (up to ∼5-fold) upon oxygen exposure. Additionally, we produced fully functional recombinant EhFdp1 and demonstrated that this enzyme is a specific and robust oxygen reductase but has poor nitric oxide reductase activity. This observation represents a new mechanism of oxygen resistance in the anaerobic protozoan pathogenE. histolytica.

Despite Bisphenol-A (BPA) being subject to extensive study, a thorough understanding of molecular mechanism remains elusive. Here we show that using weighted gene correlation network analysis (WGCNA), which takes advantage of a graph theoretical approach to understanding correlations amongst genes and grouping genes into modules that typically have co-ordinated biological functions and regulatory mechanisms, that despite some commonality in altered genes, there is minimal overlap between BPA and estrogen in terms of network topology. We confirmed previous findings that ZNF217 and TFAP2C are involved in the estrogen pathway, and are implicated in BPA as well, although for BPA they appear to be active in the absence of canonical estrogen-receptor driven gene expression. Furthermore, our study suggested that PADI4 and RACK7/ZMYNDB8 may be involved in the overlap in gene expression between estradiol and BPA. Lastly, we demonstrated that even at low doses there are unique transcription factors that appear to be driving the biology of BPA, such as SREBF1. Overall, our data is consistent with other reports that BPA leads to subtle gene changes rather than profound aberrations of a conserved estrogen signaling (or other) pathways.

The RNA interference (RNAi) pathway regulates gene expression in many eukaryotic organisms. Argonaute (Ago) proteins, together with bound small RNAs (sRNAs), are key effectors that mediate gene silencing function. However, there is limited knowledge of Ago proteins and their functions in nonmodel systems. In the protozoan parasite Entamoeba histolytica, RNAi is a robust means for stable gene silencing mediated via large populations of antisense sRNAs. Here, we report functional characterization of three Ago proteins in E. histolytica (EhAgo2-1, EhAgo2-2, and EhAgo2-3). Our data show that each EhAgo protein has a distinct subcellular localization and binds 27-nucleotide (nt) sRNAs and that the localization of EhAgo proteins is altered in response to stress conditions. Via mutagenesis analyses, we demonstrated that the Ago PAZ (Piwi/Argonaute/Zwille) domain in all three EhAgos is essential for sRNA binding. With mutation of the PAZ domain in EhAgo2-2, there was no effect on the nuclear localization of the protein but a strong phenotype and a growth defect. We further show that EhAgo2-2 contains an unusual repetitive DR-rich (aspartic acid, arginine-rich) motif region which functions as a nuclear localization signal (NLS) and is both necessary and sufficient to mediate nuclear localization. Overall, our data delineate the localization and sRNA binding features of the three E. histolytica Ago proteins and demonstrate that the PAZ domain is necessary for sRNA binding. The repetitive DR-rich motif region in EhAgo2-2 has not previously been defined in other systems, which adds to the novel observations that can be made when studies of the RNAi pathway are extended to nonmodel systems. IMPORTANCE The protozoan parasite Entamoeba histolytica, which causes amebiasis and affects over 50 million people worldwide, contains an important RNAi pathway for gene silencing. Gene silencing via the RNAi pathway is mediated by the Argonaute (Ago) proteins. However, we lack knowledge on Ago function(s) in this nonmodel system. In this paper, we discovered that three E. histolytica Ago proteins (EhAgo2-1, EhAgo2-2, and EhAgo2-3) all bind 27-nt small RNAs and have distinct subcellular localizations, which change in response to stress conditions. The EhAgos bind small RNA populations via their PAZ domains. An unusual repetitive DR-rich motif region is identified in EhAgo2-2 that functions as a nuclear localization signal. Our results show for the first time an active nuclear transport process of the EhAgo2-2 RNA-induced silencing complex (RISC) in this parasite. These data add to the novel observations that can be made when studies of the RNAi pathway are extended to nonmodel systems.

Cancer is a comparatively well-studied disease, yet despite decades of intense focus, we demonstrate here using data from The Cancer Genome Atlas that a substantial number of genes implicated in cancer are relatively poorly studied. Those genes will likely be missed by any data analysis pipeline, such as enrichment analysis, that depends exclusively on annotations for understanding biological function. There is no indication that the amount of research - indicated by number of publications - is correlated with any objective metric of gene significance. Moreover, these genes are not missing at random but reflect that our information about genes is gathered in a biased manner: poorly studied genes are more likely to be primate-specific and less likely to have a Mendelian inheritance pattern, and they tend to cluster in some biological processes and not others. While this likely reflects both technological limitations as well as the fact that well-known genes tend to gather more interest from the research community, in the absence of a concerted effort to study genes in an unbiased way, many genes (and biological processes) will remain opaque.

Failure to adequately characterize cell lines, and understand the differences between in vitro and in vivo biology, can have serious consequences on the translatability of in vitro scientific studies to human clinical trials. This project focuses on the Michigan Cancer Foundation-7 (MCF-7) cells, a human breast adenocarcinoma cell line that is commonly used for in vitro cancer research, with over 42,000 publications in PubMed. In this study, we explore the key similarities and differences in gene expression networks of MCF-7 cell lines compared to human breast cancer tissues. We used two MCF-7 data sets, one data set collected by ARCHS4 including 1032 samples and one data set from Gene Expression Omnibus GSE50705 with 88 estradiol-treated MCF-7 samples. The human breast invasive ductal carcinoma (BRCA) data set came from The Cancer Genome Atlas, including 1212 breast tissue samples. Weighted Gene Correlation Network Analysis (WGCNA) and functional annotations of the data showed that MCF-7 cells and human breast tissues have only minimal similarity in biological processes, although some fundamental functions, such as cell cycle, are conserved. Scaled connectivity—a network topology metric—also showed drastic differences in the behavior of genes between MCF-7 and BRCA data sets. Finally, we used canSAR to compute ligand-based druggability scores of genes in the data sets, and our results suggested that using MCF-7 to study breast cancer may lead to missing important gene targets. Our comparison of the networks of MCF-7 and human breast cancer highlights the nuances of using MCF-7 to study human breast cancer and can contribute to better experimental design and result interpretation of study involving this cell line.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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