Trypanosoma cruzi is a single cell protozoan parasite that causes Chagas'disease, characterized by cardiac or gastrointestinal complications in chronically infected patients. Related trypanosomatid pathogens, including Trypanosoma brucei (the causative agent of African sleeping sickness) and numerous Leishmania species (which cause a diverse spectrum of visceral and cutaneous disease), cause even more morbidity and mortality worldwide. Each of these parasites undergoes a complex developmental cycle, alternating between insect and several possible vertebrate hosts including humans. The regulation of gene expression is essential for parasite development and survival in their sequential host environments. Exactly how trypanosomatid gene expression is regulated is currently not well understood, but the relative contribution of transcriptional control was thought to be absent. We have demonstrated a role for a novel modified DNA base, represented by glycosylation of thymine residues (called base J), in the regulation of RNA polymerase II (Pol II) transcription in trypanosomatids. Loss of J at transcription start sites leads to changes in Pol II occupancy and gene expression in T. cruzi. Loss of J at transcription termination sites leads to defects in Pol II termination in Leishmania. This project seeks to determine the mechanism of J regulation of Pol II transcription initiation, elongation and termination and the biological relevance in regulating gene expression in trypanosomatids.
Aim 1 will establish the role of J in regulating Pol II transcription initiation by globally and quantitatively assessing the effect of J on rate of Pol II transcription.
Aim 2 will utilize L. major to elucidate the role of J in Pol II termination and consequence of read-through transcription and colliding Pol II elongating complexes.
Aim 3 will determine whether suppression of transcription initiation and transcriptional read-through act through similar mechanisms;involving the creation of repressive chromatin or modulating Pol II elongation kinetics. The proposed work will provide a fundamental understanding trypanosomatid gene expression, yielding a comprehensive view of the role of transcriptional control in trypanosomatids and clues to it mechanisms, as well as new information the differences in transcriptional control among the various species. These studies may prove useful in identifying novel approaches to prevention, treatment and diagnosis of the debilitating and deadly diseases caused by these parasites.
Parasitic protozoa are a major cause of global infectious disease and therefore represent a serious threat to public health. One such protozoan, Trypanosoma cruzi, is the causative agent of Chagas'disease in humans, where parasite-associated inflammation and tissue damage in the heart and gastrointestinal tract lead to progressive organ failure. We are studying a unique modification of DNA that is involved in regulating parasite gene expression and virulence. A detailed understanding of the biology of this modified base will lead us to new parasite specific interventions to treat and prevent disease. We expect that our findings will be relevant to the mission of the NIH and be broadly interesting to researchers studying molecular mechanisms of gene expression.
|Reynolds, David; Hofmeister, Brigitte T; Cliffe, Laura et al. (2016) Histone H3 Variant Regulates RNA Polymerase II Transcription Termination and Dual Strand Transcription of siRNA Loci in Trypanosoma brucei. PLoS Genet 12:e1005758|
|Reynolds, David L; Hofmeister, Brigitte T; Cliffe, Laura et al. (2016) Base J represses genes at the end of polycistronic gene clusters in Leishmania major by promoting RNAP II termination. Mol Microbiol 101:559-74|
|Bullard, Whitney; Cliffe, Laura; Wang, Pengcheng et al. (2015) Base J glucosyltransferase does not regulate the sequence specificity of J synthesis in trypanosomatid telomeric DNA. Mol Biochem Parasitol 204:77-80|
|Reynolds, David; Cliffe, Laura; FÃ¶rstner, Konrad U et al. (2014) Regulation of transcription termination by glucosylated hydroxymethyluracil, base J, in Leishmania major and Trypanosoma brucei. Nucleic Acids Res 42:9717-29|