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Characterization of a Trypanosoma cruzi polypyrimidine tract binding protein interacting with single-stranded forming DNA
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. (Lena Åslund)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. (Lena Åslund)
Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús, CONICET-UNSAM, (1650) San Martin, Provincia de Buenos Aires, Argentina. (Alberto C. C. Frasch)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. (Lena Åslund)
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(English)Manuscript (Other academic)
Abstract [en]

 Although crucial to their biology, regulation of gene expression in trypanosomatid parasites is not fully understood. Our knowledge is limited to the fact that transcript levels are mainly determined post-transcriptionally. However, where and how transcription is initiated is largely unknown and few DNA and RNA binding proteins have been characterized. Therefore, identification of regulatory sequences and counteracting proteins will considerably improve our understanding of the parasite biology. In this work, we have characterized a novel protein in the protozoan parasite Trypanosoma cruzi named T. cruzi polypyrimidine tract binding protein (TcPTB) and investigated its potential role in transcription initiation. TcPTB shares structural and functional properties with mammalian PTB, a protein with multiple roles in mRNA metabolism and transcription initiation. We show that TcPTB is expressed in all parasite stages and is predominantly located in the nucleus. It binds in vitro to polypyrimidine-rich ssDNA including cis-regulatory elements implicated in transcription initiation in mammals and in the related parasite Leishmania major. Chromatin immunoprecipitation analysis associates TcPTB to the RNA pol II promoter of the spliced leader gene. Finally, we also show that T. cruzi polypyrimidine/polypurine sequences form triple helix, a condition necessary for the binding of mammalian PTB. These results suggest that TcPTB has a similar multifunctional role in gene regulation as in mammals.

Keyword [en]
Trypanosoma cruzi, polypyrimidine-binding protein, CL Brener, TcPTB
URN: urn:nbn:se:uu:diva-100258OAI: oai:DiVA.org:uu-100258DiVA: diva2:210552
Available from: 2009-04-02 Created: 2009-03-29 Last updated: 2010-01-14
In thesis
1. Gene Regulation and Epigenetic Mechanisms in the Parasite Trypanosoma cruzi
Open this publication in new window or tab >>Gene Regulation and Epigenetic Mechanisms in the Parasite Trypanosoma cruzi
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Trypanosomes are unicellular protozoan parasites responsible for several human diseases that affect millions of people and cause thousands of casualties every year. They also represent a primitive eukaryotic model system harboring unique processes and basic regulatory mechanisms such as RNA-editing, polycistronic transcription and trans-splicing, first described in these organisms. Unlike most eukaryotes where levels of gene expression are controlled at initiation of transcription, trypanosomes use post-transcriptional events as the main regulators. This thesis explores the epigenetic mechanisms involved in gene expression control in trypanosomes, providing the first evidences for a functional “histone-code” as well as the existence and location of DNA methylation in Trypanosoma cruzi.

Chromatin immunoprecipitation (ChIP) was used for the profiling of acetylated and methylated histones in T. cruzi, showing that the modified histones were exclusively localized at bidirectional transcription start sites. In addition, promoters from highly transcribed genes were found depleted of nucleosomes, while DNA regions expected to be silent were not enriched in the investigated modified histones. Furthermore, we showed that the histone patterns were developmentally regulated.

The first in depth characterization of the DNA methylation patterns in T. cruzi was presented in this work. We detected m5C in regions of transcriptional initiation and termination, retrotransposons, pseudogenes and the kinetoplast minicircle. We also showed that the amount of methylation changes during development, with an increase in non-replicative forms.

We also characterized the DNA-interacting properties of a T. cruzi polypyrimidine-tract binding protein (TcPTB), and its potential role as a transcription factor. TcPTB was found to interact with single-stranded DNA present in promoters bound by its mammalian homologue as well as to the region of transcriptional initiation in Leishmania major. We also demonstrated that T. cruzi polypyrimidine stretches were able to confer ssDNA conformations.

Overall, these results provide new insights into the biology of ancient pathogenic parasites, which might be exploited for drug development.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 55 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 446
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
urn:nbn:se:uu:diva-100265 (URN)978-91-554-7490-4 (ISBN)
Public defence
2009-05-14, Rudbeck Hall, Rudbeck Laboratory, Dag Hammarskjölds väg 20, Uppsala, 14:15 (English)
Available from: 2009-04-23 Created: 2009-03-29 Last updated: 2010-02-22Bibliographically approved

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