Unlike other eukaryotes, the protein-coding genes of Trypanosoma cruzi are arranged in large polycistronic gene clusters transcribed by polymerase II (Pol II). Thus, it is thought that trypanosomes rely solely on posttranscriptional processes to regulate gene expression. Here, we show that the glucosylated thymine DNA base (␤-D-glucosyl-hydroxymethyluracil or base J) is present within sequences flanking the polycistronic units (PTUs) in T. cruzi. The loss of base J at sites of transcription initiation, via deletion of the two enzymes that regulate base J synthesis (JBP1 and JBP2), correlates with an increased rate of Pol II transcription and subsequent genome-wide increase in gene expression. The affected genes include virulence genes, and the resulting parasites are defective in host cell invasion and egress. These studies indicate that base J is an epigenetic factor regulating Pol II transcription initiation in kinetoplastids and provides the first biological role of the only hypermodified DNA base in eukaryotes.Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, is the major cause of cardiac disease in South and Central America (1). The parasite has a complex life cycle with two hosts and four developmental stages. Epimastigotes develop in the hindgut of the triatomine insect vector and differentiate into metacyclic forms. Infective metacyclic forms enter the vertebrate host, invade the host cell, and differentiate to form amastigotes. Trypomastigotes released from the infected cell are able to reinvade a wide variety of host cells. Success of the parasite throughout the life cycle is ensured by the regulated expression of surface proteins such as mucin and trans-sialidase, which allow differential adherence and evasion of the host immune responses (2). Members of the surface glycoprotein gene family colocalize with a novel hypermodified DNA base, ␤-D-glucosyl-hydroxymethyluracil or base J, suggesting an epigenetic mechanism of regulating T. cruzi pathogenesis (3).In base J, the thymine base exhibits O-linked glucosylation in telomeric DNA of all kinetoplastid flagellates and some closely related unicellular flagellates, but base J is not present in the genomes of other protozoa or metazoa (3, 4). Base J was initially discovered on the basis of its distinct presence within the 19 silent telomeric variant surface glycoprotein (VSG) expression sites (ES) of Trypanosoma brucei but absence from the single transcribed ES, suggesting its role in the regulation of telomeric VSG gene expression (3, 35). Recent genomewide analysis revealed that base J is also present throughout the T. brucei genome, enriched at regions flanking polymerase II (Pol II) polycistronic transcription units (PTUs) (9). PTUs are large gene clusters that are cotranscribed by Pol II to yield polycistronic pre-mRNAs that are then processed into mature mRNAs by trans-splicing and polyadenylation (7). The localization of base J at PTU-flanking regions suggests a role for the modified base in regulating Pol II transcription initi...

Cryptosporidiosis is a rapidly emerging disease in the tropics. This is the first report of Cryptosporidium and other protozoan infections (Entamoeba spp., Iodamoeba, Chilomastix, and Balantidium spp.) in wild primates that inhabit the natural forest of Sri Lanka. It is unclear if non-human primates serve as a reservoir for these parasites under certain conditions. A cross-sectional coprologic survey among 125 monkeys (89 toque macaques, 21 gray langurs, and 15 purple-faced langurs) indicated that Cryptosporidium was detected in all three primate species and was most common among monkeys using areas and water that had been heavily soiled by human feces and livestock. Most macaques (96%) shedding Cryptosporidium oocysts were co-infected with other protozoans and important anthropozoonotic gastrointestinal parasites (e.g., Enterobius and Strongyloides). The transmission of these parasites among primates in the wild may have important implications for public health as well as wildlife conservation management.

Very little is understood regarding how transcription is initiated/regulated in the early-diverging eukaryote Trypanosoma cruzi. Unusually for a eukaryote, genes transcribed by RNA polymerase (Pol) II in T. cruzi are arranged in polycistronic transcription units (PTUs). On the basis of this gene organization, it was previously thought that trypanosomes rely solely on posttranscriptional processes to regulate gene expression. We recently localized a novel glucosylated thymine DNA base, called base J, to potential promoter regions of PTUs throughout the trypanosome genome. Loss of base J, following the deletion of JBP1, a thymidine hydroxylase involved with synthesis, led to a global increase in the Pol II transcription rate and gene expression. In order to determine the mechanism by which base J regulates transcription, we have characterized changes in chromatin structure and Pol II recruitment to promoter regions following the loss of base J. The loss of base J coincides with a decrease in nucleosome abundance, increased histone H3/H4 acetylation, and increased Pol II occupancy at promoter regions, including the well-characterized spliced leader RNA gene promoter. These studies present the first direct evidence for epigenetic regulation of Pol II transcription initiation via DNA modification and chromatin structure in kinetoplastids as well as provide a mechanism for regulation of trypanosome gene expression via the novel hypermodified base J.Trypanosoma cruzi is a major human parasitic pathogen with a complex life cycle, alternating between an insect vector and mammalian hosts (42). In both, T. cruzi undergoes differential morphological and functional changes that require rapid and selective changes in gene expression profile. Unlike other eukaryotes, the genes in T. cruzi are arranged in large polycistronic bidirectional gene clusters (34). These polycistronic transcription units (PTUs) are transcribed by RNA polymerase (Pol) II to yield polycistronic pre-mRNAs that are then processed into mature mRNAs by trans-splicing and polyadenylation. Given this genome arrangement and the apparent lack of a functional relationship of genes within a PTU, the regulation of trypanosome gene expression was thought to occur primarily via differential mRNA decay or other posttranscriptional mechanisms (5). Until very recently, there was no evidence for regulation of gene expression at the level of Pol II transcription in kinetoplastids (11). Furthermore, very little is understood about the DNA sequences and proteins involved in transcription initiation and termination in kinetoplastids. With the exception of the spliced-leader RNA promoter, Pol II promoters and associated factors have not been identified or functionally characterized. While genome localization studies suggest that histone modifications and histone variants are involved in transcription initiation and termination in trypanosomes, direct evidence from functional studies is lacking (34, 39). Thus, how Pol II transcription is initiated/regulated, including the rol...

Background: Base J regulates Pol II transcription. Results: JBP1 and -2 stimulate the first step of base J synthesis: hydroxylation of thymidine. Conclusion: JBP are Fe 2ϩ /2-OG-dependent dioxygenases sensitive to physiologically relevant O 2 tensions. Significance: These results predict that JBPs can act as oxygen sensors regulating trypanosome gene expression and adaption to different host niches.

Synthesis of the modified thymine base, β-D-glucosyl-hydroxymethyluracil or J, within telomeric DNA of Trypanosoma brucei correlates with the bloodstream-form specific epigenetic silencing of telomeric variant surface glycoprotein genes involved in antigenic variation. In order to analyze the function of base J in the regulation of antigenic variation, we are characterizing the regulatory mechanism of J biosynthesis. We have recently proposed a model in which chromatin remodeling by a SWI2/SNF2-like protein (JBP2) regulates the developmental and de-novo site-specific localization of J synthesis within bloodstream-form trypanosome DNA. Consistent with this model, we now show that JBP2 (−/−) bloodstream-form trypanosomes contain 5-fold less base J and are unable to stimulate de-novo J synthesis in newly generated telomeric arrays.