Identification of a polypeptide containing Tudor and staphyloccocal nuclease-like domains as the sequence-specific binding protein to the upstream regulatory element 1 of Entamoeba histolytica

Calixto-Gálvez, Mercedes; Romero-Díaz, Mónica; García-Muñoz, Alejandro; Salas‐Casas, Andrés; Pais-Morales, Jonnatan; Galván, Iván J.; Orozco, Esther; Rodríguez, Mario A.

doi:10.1016/j.ijpara.2011.02.002

Cited by 9 publications

(15 citation statements)

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“…For example, the Toxoplasma gondii PRMT1 methylates H4R3, but it also catalyzes the methylation of the argonaute ortholog to regulate the recruitment of a Tudor staphylococcal nuclease (TSN), a more potent second slicer to perform the parasitic RNA silencing [ 43 ]. Interestingly, an E. histolytica TSN was identified as the transcription factor that binds to the cis -regulatory sequence URE1 (EhURE1-BP) [ 44 ], suggesting that probably the methylation of non-histone substrates could affect the transcriptional activity of EhURE1-BP. Future investigations to search the substrates of EhPRMT1 proteins may reveal novel pathways in which these enzymes play regulatory roles in this parasite.…”

Section: Discussionmentioning

confidence: 99%

Entamoeba histolytica: protein arginine transferase 1a methylates arginine residues and potentially modify the H4 histone

et al. 2015

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BackgroundIn eukaryotes, histone arginine methylation associates with both active and repressed chromatin states depending on the residues involved and the status of methylation. Even when the amino-terminus of Entamoeba histolytica histones diverge from metazoan sequences, these regions contain arginine residues that are potential targets for methylation. However, histone arginine methylation as well as the activity of arginine methyltransferases (PRMTs) has not been studied in this parasite. The aim of this work was to examine the dimethylation of arginine 3 of H4 histone (H4R3me2) and to identify the parasite PRMT that could be responsible for this modification (EhPRMT1).MethodsTo examine the presence of H4R3me2 in E histolytica, we performed Western blot and immunofluorescence assays on trophozoites using an antibody against this epigenetic mark. To recognize the PRMT1 enzyme of this parasite that possibly perform that modification, we first performed a phylogenetic analysis of E. histolytica and human PRMTs. RT-PCR assays were carried out to analyze the expression of the putative PRMT1 genes. One of these genes was cloned and expressed in Escherichia coli. The recombinant protein was tested by its recognition by an antibody against human PRMT1 and in its ability to form homodimers and to methylate commercial histones.ResultsThe arginine 3 of human H4, which is subjected to post translational methylation, was aligned with the arginine 8 of E. histolytica H4, suggesting that this residue could be methylated. The recognition of an 18 kDa nuclear protein of E. histolytica by an antibody against H4R3me2 confirmed this assumption. We found that this parasite expresses three phylogenetic and structural proteins related to PRMT1. Antibodies against the human PRMT1 detected E. histolytica proteins in cytoplasm and nuclei and recognized a recombinant PRMT1 of this parasite. The recombinant protein was able to form homodimers and homotetramers and displayed methyltransferase activity on arginine 3 of chicken H4.ConclusionAll these results suggest that E. histolytica contains as a minimum one structural and functional protein ortholog to PRMT1, enzyme that potentially dimethylates H4R8. This modification may play an important role in the gene expression regulation of this microorganism.

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Section: Discussionmentioning

confidence: 99%

Entamoeba histolytica: protein arginine transferase 1a methylates arginine residues and potentially modify the H4 histone

et al. 2015

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“…Sections were obtained and mounted on formvar-covered nickel grids. Later, they were incubated with rabbit polyclonal antibodies α-EhTSN (Calixto-Gálvez et al, 2011 ; 1:50 dilution). Goat anti-rabbit IgG conjugated to 30 nm gold particles (Ted Pella Inc.) were used as secondary antibodies (1:60 dilution).…”

Section: Methodsmentioning

confidence: 99%

“…Five amoebic-specific upstream regulatory elements, named URE1 to URE5, are involved in the transcription regulation of several genes of this parasite (Gilchrist et al, 1998 , 2010 ; Romero-Díaz et al, 2007 ). The transcription factor that binds to URE1 belongs to the TSN family (EhTSN; former EhURE1BP), because it contains two canonical SNase motifs in its N-terminus, two divergent SNase motifs in the middle, and a Tudor-SNase domain in its C-terminus (Calixto-Gálvez et al, 2011 ). Western blot assays on nuclear and cytoplasmic fractions as well as immunofluorescence analyzes performed with antibodies against EhTSN showed that the native protein is situated in nucleus and cytoplasm of trophozoites, suggesting that this protein may display distinct roles, depending on its cellular location (Calixto-Gálvez et al, 2011 ).…”

Section: Introductionmentioning

confidence: 99%

The Tudor Staphylococcal Nuclease Protein of Entamoeba histolytica Participates in Transcription Regulation and Stress Response

Cázares-Apátiga

Medina-Gómez

Chávez‐Munguía

et al. 2017

Front. Cell. Infect. Microbiol.

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Entamoeba histolytica is the protozoa parasite responsible of human amoebiasis, disease that causes from 40,000 to 100,000 deaths annually worldwide. However, few are known about the expression regulation of molecules involved in its pathogenicity. Transcription of some virulence-related genes is positively controlled by the cis-regulatory element named URE1. Previously we identified the transcription factor that binds to URE1, which displayed a nuclear and cytoplasmic localization. This protein belongs to the Tudor Staphyococcal nuclease (TSN) family, which in other systems participates in virtually all pathways of gene expression, suggesting that this amoebic transcription factor (EhTSN; former EhURE1BP) could also play multiple functions in E. histolytica. The aim of this study was to identify the possible cellular events where EhTSN is involved. Here, we found that EhTSN in nucleus is located in euchromatin and close to, but not into, heterochromatin. We also showed the association of EhTSN with proteins involved in transcription and that the knockdown of EhTSN provokes a diminishing in the mRNA level of the EhRabB gene, which in its promoter region contains the URE1 motif, confirming that EhTSN participates in transcription regulation. In cytoplasm, this protein was found linked to the membrane of small vesicles and to plasma membrane. Through pull-down assays and mass spectrometry we identity thirty two candidate proteins to interact with EhTSN. These proteins participate in transcription, metabolism, signaling, and stress response, among other cellular processes. Interaction of EhTSN with some candidate proteins involved in metabolism, and signaling was validated by co-immunoprecipitation or co-localization. Finally we showed the co-localization of EhTSN and HSP70 in putative stress granules during heat shock and that the knockdown of EhTSN increases the cell death during heat shock treatment, reinforcing the hypothesis that EhTSN has a role during stress response. All data support the proposal that EhTSN is a multifunctional protein of E. histolytica.

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“…The Entamoeba histolytica genome possesses 8201 protein-coding genes, including virulence genes and canonical transcription factors related to the basal transcription machinery [ 2 , 3 ]. The characterization of some gene promoters, such as those of the hgl5 , fdx , Ehpgp1, Ehpgp5 and actin genes [ 4 – 8 ] has allowed the identification of key elements in promoters, including the TATA-box (GTATTTAAAG/C), Inr (AAAAATTCA), GAAC (AATGAACT) and GAAC-like (GAACTACAAA) core promoter elements, as well as other motifs, including the H 2 O 2 -regulatory motif (HRM) [ 9 ], URE-1 [ 10 , 11 ], URE-3 [ 12 ], URE-4 [ 13 ], CCAAT-box [ 14 ], and CCCCCC motif [ 15 ]. The unusual core promoter element GAAC-box is described as a key player in driving the transcription start site of the hgl5 gene promoter lacking both the TATA and Inr-boxes [ 4 ].…”

Section: Introductionmentioning

confidence: 99%

The Entamoeba histolytica TBP and TRF1 transcription factors are GAAC-box binding proteins, which display differential gene expression under different stress stimuli and during the interaction with mammalian cells

Narayanasamy

Castañón-Sánchez

Luna-Arias³

et al. 2018

Parasites Vectors

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BackgroundEntamoeba histolytica is the protozoan parasite responsible for human amebiasis. It causes up to 100,000 deaths worldwide each year. This parasite has two closely related basal transcription factors, the TATA-box binding protein (EhTBP) and the TBP-related factor 1 (EhTRF1). TBP binds to the canonical TATTTAAA-box, as well as to different TATA variants. TRF1 also binds to the TATTTAAA-box. However, their binding capacity to diverse core promoter elements, including the GAAC-element, and their role in gene regulation in this parasite remains unknown.MethodsEMSA experiments were performed to determine the binding capacity of recombinant TBP and TRF1 to TATA variants, GAAC and GAAC-like boxes. For the functional analysis under different stress stimuli (e.g. growth curve, serum depletion, heat-shock, and UV-irradiation) and during the interaction with mammalian cells (erythrocytes, MDCK cell monolayers, and hepatocytes of hamsters), RT-qPCR, and gene knockdown were performed.ResultsBoth transcription factors bound to the different TATA variants tested, as well as to the GAAC-boxes, suggesting that they are GAAC-box-binding proteins. The KD values determined for TBP and TRF1 for the different TATA variants and GAAC-box were in the range of 10-12 M to 10-11 M. During the death phase of growth or in serum depletion, Ehtbp mRNA levels significantly increased, whereas the mRNA level of Ehtrf1 did not change under these conditions. Ehtrf1 gene expression was negatively regulated by UV-irradiation and heat-shock stress, with no changes in Ehtbp gene expression. Moreover, Ehtrf1 gene also showed a negative regulation during erythrophagocytosis, liver abscess formation, and a transient expression level increase at the initial phase of MDCK cell destruction. Finally, the Ehtbp gene knockdown displayed a drastic decrease in the efficiency of erythrophagocytosis in G3 trophozoites.ConclusionsTo our knowledge, this study reveals that these basal transcription factors are able to bind multiple core promoter elements. However, their immediate change in gene expression level in response to different stimuli, as well as during the interaction with mammalian cells, and the diminishing of erythrophagocytosis by silencing the Ehtbp gene indicate the different physiological roles of these transcription factors in E. histolytica.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-2698-7) contains supplementary material, which is available to authorized users.

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Identification of a polypeptide containing Tudor and staphyloccocal nuclease-like domains as the sequence-specific binding protein to the upstream regulatory element 1 of Entamoeba histolytica

Cited by 9 publications

References 47 publications

Entamoeba histolytica: protein arginine transferase 1a methylates arginine residues and potentially modify the H4 histone

Entamoeba histolytica: protein arginine transferase 1a methylates arginine residues and potentially modify the H4 histone

The Tudor Staphylococcal Nuclease Protein of Entamoeba histolytica Participates in Transcription Regulation and Stress Response

The Entamoeba histolytica TBP and TRF1 transcription factors are GAAC-box binding proteins, which display differential gene expression under different stress stimuli and during the interaction with mammalian cells

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