Localization of Epigenetic Markers in Leishmania Chromatin.

Eukaryotes use histone variants and post-translation modifications (PTMs), as well as DNA base modifications, to regulate DNA replication/repair, chromosome condensation, and gene expression. Despite the unusual organization of their protein-coding genes into large polycistronic transcription units (PTUs), trypanosomatid parasites also employ a "histone code" to control these processes, but the details of this epigenetic code are poorly understood. Here, we present the results of experiments designed to elucidate the distribution of histone variants and PTMs over the chromatin landscape of Leishmania tarentolae. These experiments show that two histone variants (H2A.Z and H2B.V) and three histone H3 PTMs (H3K4me3, H3K16ac, and H3K76me3) are enriched at transcription start sites (TSSs); while a histone variant (H3.V) and the trypanosomatid-specific hyper-modified DNA base J are located at transcription termination sites (TTSs). Reduced nucleosome density was observed at all TTSs and TSSs for RNA genes transcribed by RNA polymerases I (RNAPI) or RNAPIII; as well as (to a lesser extent) at TSSs for the PTUs transcribed by RNAPII. Several PTMs (H3K4me3, H3K16ac H3K20me2 and H3K36me3) and base J were enriched at centromeres, while H3K50ac was specifically associated with the periphery of these centromeric sequences. These findings significantly expand our knowledge of the epigenetic markers associated with transcription, DNA replication and/or chromosome segregation in these early diverging eukaryotes and will hopefully lay the groundwork for future studies to elucidate how they control these fundamental processes.

The transcriptome of Balamuthia mandrillaris trophozoites for structure-guided drug design.

Balamuthia mandrillaris, a pathogenic free-living amoeba, causes cutaneous skin lesions as well as granulomatous amoebic encephalitis, a 'brain-eating' disease. As with the other known pathogenic free-living amoebas (Naegleria fowleri and Acanthamoeba species), drug discovery efforts to combat Balamuthia infections of the central nervous system are sparse; few targets have been validated or characterized at the molecular level, and little is known about the biochemical pathways necessary for parasite survival. Current treatments of encephalitis due to B. mandrillaris lack efficacy, leading to case fatality rates above 90%. Using our recently published methodology to discover potential drugs against pathogenic amoebas, we screened a collection of 85 compounds with known antiparasitic activity and identified 59 compounds that impacted the growth of Balamuthia trophozoites at concentrations below 220 µM. Since there is no fully annotated genome or proteome of B. mandrillaris, we sequenced and assembled its transcriptome from a high-throughput RNA-sequencing (RNA-Seq) experiment and located the coding sequences of the genes potentially targeted by the growth inhibitors from our compound screens. We determined the sequence of 17 of these target genes and obtained expression clones for 15 that we validated by direct sequencing. These will be used in the future in combination with the identified hits in structure guided drug discovery campaigns to develop new approaches for the treatment of Balamuthia infections.

Chromatin-Associated Protein Complexes Link DNA Base J and Transcription Termination in Leishmania.

Unlike most other eukaryotes, Leishmania and other trypanosomatid protozoa have largely eschewed transcriptional control of gene expression, relying instead on posttranscriptional regulation of mRNAs derived from polycistronic transcription units (PTUs). In these parasites, a novel modified nucleotide base (ß-d-glucopyranosyloxymethyluracil) known as J plays a critical role in ensuring that transcription termination occurs only at the end of each PTU, rather than at the polyadenylation sites of individual genes. To further understand the biology of J-associated processes, we used tandem affinity purification (TAP) tagging and mass spectrometry to reveal proteins that interact with the glucosyltransferase performing the final step in J synthesis. These studies identified four proteins reminiscent of subunits in the PTW/PP1 complex that controls transcription termination in higher eukaryotes. Moreover, bioinformatic analyses identified the DNA-binding subunit of Leishmania PTW/PP1 as a novel J-binding protein (JBP3), which is also part of another complex containing proteins with domains suggestive of a role in chromatin modification/remodeling. Additionally, JBP3 associates (albeit transiently and/or indirectly) with the trypanosomatid equivalent of the PAF1 complex involved in the regulation of transcription in other eukaryotes. The downregulation of JBP3 expression levels in Leishmania resulted in a substantial increase in transcriptional readthrough at the 3' end of most PTUs. We propose that JBP3 recruits one or more of these complexes to the J-containing regions at the end of PTUs, where they halt the progression of the RNA polymerase. This decoupling of transcription termination from the splicing of individual genes enables the parasites' unique reliance on polycistronic transcription and posttranscriptional regulation of gene expression.IMPORTANCELeishmania parasites cause a variety of serious human diseases, with no effective vaccine and emerging resistance to current drug therapy. We have previously shown that a novel DNA base called J is critical for transcription termination at the ends of the polycistronic gene clusters that are a hallmark of Leishmania and related trypanosomatids. Here, we describe a new J-binding protein (JBP3) associated with three different protein complexes that are reminiscent of those involved in the control of transcription in other eukaryotes. However, the parasite complexes have been reprogrammed to regulate transcription and gene expression in trypanosomatids differently than in the mammalian hosts, providing new opportunities to develop novel chemotherapeutic treatments against these important pathogens.

Dynamic colocalization of 2 simultaneously active VSG expression sites within a single expression-site body in Trypanosoma brucei.

Monoallelic exclusion ensures that the African trypanosome Trypanosoma brucei exclusively expresses only 1 of thousands of different variant surface glycoprotein (VSG) coat genes. The active VSG is transcribed from 1 of 15 polycistronic bloodstream-form VSG expression sites (ESs), which are controlled in a mutually exclusive fashion. Unusually, T. brucei uses RNA polymerase I (Pol I) to transcribe the active ES, which is unprecedented among eukaryotes. This active ES is located within a unique extranucleolar Pol I body called the expression-site body (ESB). A stringent restriction mechanism prevents T. brucei from expressing multiple ESs at the same time, although how this is mediated is unclear. By using drug-selection pressure, we generated VSG double-expresser T. brucei lines, which have disrupted monoallelic exclusion, and simultaneously express 2 ESs in a dynamic fashion. The 2 unstably active ESs appear epigenetically similar to fully active ESs as determined by using chromatin immunoprecipitation for multiple epigenetic marks (histones H3 and H1, TDP1, and DNA base J). We find that the double-expresser cells, similar to wild-type single-expresser cells, predominantly contain 1 subnuclear ESB, as determined using Pol I or the ESB marker VEX1. Strikingly, simultaneous transcription of the 2 dynamically transcribed ESs is normally observed only when the 2 ESs are both located within this single ESB. This colocalization is reversible in the absence of drug selection. This discovery that simultaneously active ESs dynamically share a single ESB demonstrates the importance of this unique subnuclear body in restricting the monoallelic expression of VSG.