Identification of the gene encoding the TATA box-binding protein-associated factor 1 (TAF1) and its putative role in the heat shock response in the protozoan parasite Entamoeba histolytica
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Transcription factor IID (TFIID) is a cornerstone in the transcription initiation in eukaryotes. It is composed of TBP and approximately 14 different subunits named TBP-associated factors (TAFs). TFIID has a key role in transcription of many genes involved in cell proliferation, cell growth, cell cycle, cell cycle checkpoint, and various other processes as well. Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis, represents a major global health concern. Our research group has previously reported the genes coding the TATA box-binding protein (EhTBP) and TBP-related factor 1 (EhTRF1), which displayed different mRNA levels in trophozoites under different stress conditions. In this work, we identified the TBP-associated factor 1 (Ehtaf1) gene in the E. histolytica genome, which possess a well-conserved DUF domain and a Bromo domain located in the middle and C-terminus of the protein, respectively. The EhTAF1-DUF domain tertiary structure is similar to the corresponding HsTAF1 DUF domain. RT-qPCR experiments with RNA isolated from trophozoites harvested at different time points of the growth curve and under different stress conditions revealed that the Ehtaf1 gene was found slightly upregulated in the death phase of growth curve, but under heat shock stress, it was found upregulated 10 times, suggesting that Ehtaf1 might have an important role in the heat shock stress response. We also found that EhTAF1 is expressed in the nucleus and cytoplasm at 37 °C, but under heat shock stress, it is overexpressed in both the nucleus and cytoplasm, and partially colocalized with EhHSP70 in cytoplasm.
KeywordsEntamoeba histolytica TFllD TAF1 Heat shock stress Cloning
The authors are grateful to Dr. Javier Cázares-Apátiga for his technical support in confocal microscopy experiments, Emmanuel Ríos-Castro MSc. for his technical support in MS analysis at the Unidad de Genómica, Proteómica y Metabolómica (UGPM), LaNSE, Cinvestav-IPN, and Cesar Isaac Bazán-Méndez for spelling and grammar review of manuscript.
BA-B, performed, designed the experiments, discussed the results, and wrote the manuscript; RKN, designed the experiments; GG-R, helped in some experiments with E. histolytica trophozoites, MLL-B, participated in the generation of antibodies and Western blots; AEL-G, performed the electron microscopy experiments; BCHM, designed and performed the electron microscopy experiments; CAC-S, discussed experiments and results; EO, supplied the E. histolytica strain and laboratory facilities for the development of some experiments; JPL-A, conducted experiments, discussed experiments and results, wrote and revised the manuscript.
We are grateful to the National Council of Science and Technology of Mexico (CONACyT) for the support to this work with the Grant number CB-2009-132020-B to Dr. Juan Pedro Luna-Arias, and for the PhD fellowship to Bartolo Avendaño Borromeo (CVU 229747).
Compliance with ethical standards
Cinvestav fulfills the standard of the Mexican Official Norm (NOM-062-ZOO-1999) “Technical Specifications for the Care and Use of Laboratory Animals” based on the Guide for the Care and Use of Laboratory Animals “The Guide,” 2011, NRC, USA with the Federal Register Number BOO.02.03.02.01.908. The Institutional Animal Care and Use Committee (IACUC/ethics committee) from Cinvestav, as the regulatory office for the approval of research protocols involving the use of laboratory animals and in fulfillment of the Mexican Official Norm, has reviewed and approved all animal experiments.
Conflict of interest
The authors declare that they have no conflict of interest.
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