Project description:Protein-coding genes in kinetoplastid protists are transcribed from polycistronic arrays, yielding RNA precursors which are processed to form mature transcripts bearing a 5M-bM-^@M-^Y spliced leader (SL) and 3M-bM-^@M-^Y poly(A) tract. Regions of transcription initiation and termination lack known eukaryotic promoter and terminator elements, and current data suggest that transcription is instead regulated predominantly through epigenetic mechanisms. Several epigenetic marks, including histone modifications, histone variants, and an atypical DNA modification known as base J have been localized to regions of transcription initiation or termination in Trypanosoma brucei, Trypanosoma cruzi, and/or Leishmania major. Despite this conservation, the phenotypes of base J mutants vary significantly across trypanosomatids, suggesting that the specific epigenetic networks governing transcription initiation and termination have diverged significantly during evolution. In this light, we sought to characterize and compare the roles of the histone variants H2A.Z, H2B.V, and H3.V in L. major. As in T. brucei, the histone variants H2A.Z and H2B.V were shown to be essential in L. major using a powerful quantitative plasmid segregation-based test. In contrast and again similar to T. brucei, H3.V is not essential in Leishmania as H3.V-null lines grew normally, resembled WT, and remained infectious. Using SL-primed RNA-seq, we found that H3.V-null parasites have steady-state transcript levels comparable to WT parasites and display no defects in the efficiency of transcription termination at convergent strand switch regions (SSRs). Our results show a conservation of histone variant phenotypes between L. major and T. brucei, in contrast to the phenotypes associated with the epigenetic DNA base J modification. Total RNA from Four LmjF samples were analyzed using RNA-Seq. One of them is wildtype parasites, one is single knockout for H3V gene and two independent double knockouts for H3V gene.
Project description:Protein-coding genes in kinetoplastid protists are transcribed from polycistronic arrays, yielding RNA precursors which are processed to form mature transcripts bearing a 5’ spliced leader (SL) and 3’ poly(A) tract. Regions of transcription initiation and termination lack known eukaryotic promoter and terminator elements, and current data suggest that transcription is instead regulated predominantly through epigenetic mechanisms. Several epigenetic marks, including histone modifications, histone variants, and an atypical DNA modification known as base J have been localized to regions of transcription initiation or termination in Trypanosoma brucei, Trypanosoma cruzi, and/or Leishmania major. Despite this conservation, the phenotypes of base J mutants vary significantly across trypanosomatids, suggesting that the specific epigenetic networks governing transcription initiation and termination have diverged significantly during evolution. In this light, we sought to characterize and compare the roles of the histone variants H2A.Z, H2B.V, and H3.V in L. major. As in T. brucei, the histone variants H2A.Z and H2B.V were shown to be essential in L. major using a powerful quantitative plasmid segregation-based test. In contrast and again similar to T. brucei, H3.V is not essential in Leishmania as H3.V-null lines grew normally, resembled WT, and remained infectious. Using SL-primed RNA-seq, we found that H3.V-null parasites have steady-state transcript levels comparable to WT parasites and display no defects in the efficiency of transcription termination at convergent strand switch regions (SSRs). Our results show a conservation of histone variant phenotypes between L. major and T. brucei, in contrast to the phenotypes associated with the epigenetic DNA base J modification.
Project description:Genome sequencing of species of the kinetoplastid parasite, Leishmania, that give rise to a range of disease phenotypes in the host has revealed highly conserved gene content and synteny across the genus. Only a small number of genes are differentially distributed between the three species studied to date, L. major, L. infantum and L. braziliensis. Here, we focus on RNA expression in the disease-promoting intracellular amastigotes and use customised oligonucleotide microarrays to confirm that all of these differentially-distributed genes are expressed in this critical stage of the parasite life cycle, with only a few regulated between species.
Project description:Base J and H3.V promote RNA Polymerase (RNAP) II termination within polycistronic gene clusters in the kinetoplastid species Trypanosoma brucei. Although base J has been shown to promote RNAP II termination in the related kinetoplastid species Leishmania major and Leishmania tarentolae, the role of H3.V was unclear. The effect of acute J loss on mRNA transcript abundance was also unknown. We find here that H3.V does not promote transcription termination in Leishmania major, but loss of H3.V does reduce J levels. The J loss in H3.V knockout cells is not enough to result in a termination defect, which we show is due to a threshold level of J that is sufficient to promote termination. Loss of J beyond that threshold results in termination defects. Further, the decreased J in H3.V knockout cells allowed greater reduction of J by dimethyloxalylglycine (DMOG), which inhibits J synthesis, compared to wild type cells treated with DMOG, and resulted in stronger defects in RNAP II termination and cell growth. By mRNA-seq we see largely upregulation of genes near the ends of gene clusters following J loss, indicating that J represses genes near termination sites. These findings reveal a conserved role of J in promoting termination prior to the end of polycistronic gene clusters in kinetoplastid parasites and suggest that the essential nature of J is related to its role in repressing genes by promoting termination. The role of base J and H3.V in promoting RNA Polymerase II transcription termination was assessed by small RNA-seq, mRNA-seq, and strand-specific RT-PCR. Wild type cells were compared to H3.V knockout cells and to WT and H3.V knockout cells treated with dimethyloxalylglycine (DMOG) to reduce base J.
Project description:The genomic DNAs of strains JPCM5 and 263 of L. infantum, strains LV39 and Friedlin of L. major and strains Parrot-TarII and S125 of L. tarentolae were used in comparative genomic hybridizations to reveal the intra-species and inter-species gene content, and to validate L. tarentolae Parrot-TarII genome sequencing results. Leishmania (Sauroleishmania) tarentolae was first isolated in the lizard Tarentola mauritanica. This species is not known to be pathogenic to humans but is often used as a model organism for molecular analyses or protein overproduction. The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved by high-throughput sequencing technologies. The L. tarentolae genome was first assembled de novo and then aligned against the reference L. major Friedlin genome to facilitate contig positioning and annotation, providing a 23-fold coverage of the genome. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described, and it provides an opportunity for comparison with the completed genomes of the pathogenic Leishmania species. A high synteny was observed in de novo assembled contigs between all sequenced Leishmania species. A number of limited chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic with L. major. Globally, over 90% of the L. tarentolae gene content was shared with the other Leishmania species. There were 250 L. major genes absent from L. tarentolae, and interestingly these missing genes were primarily expressed in the intracellular amastigote stage of the pathogenic parasites. This implies that L. tarentolae may have impaired ability to survive as an intracellular parasite. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the leishmanolysin (GP63) and a gene related to the promastigote surface antigen (PSA31C). Overall, L. tarentolae appears to have a gene content more adapted to the insect stage rather than the mammalian one. This may partly explain its inability to replicate within mammalian macrophages and its suspected preferred life style as promastigote in the lizards.
Project description:Base J and H3.V promote RNA Polymerase (RNAP) II termination within polycistronic gene clusters in the kinetoplastid species Trypanosoma brucei. Although base J has been shown to promote RNAP II termination in the related kinetoplastid species Leishmania major and Leishmania tarentolae, the role of H3.V was unclear. The effect of acute J loss on mRNA transcript abundance was also unknown. We find here that H3.V does not promote transcription termination in Leishmania major, but loss of H3.V does reduce J levels. The J loss in H3.V knockout cells is not enough to result in a termination defect, which we show is due to a threshold level of J that is sufficient to promote termination. Loss of J beyond that threshold results in termination defects. Further, the decreased J in H3.V knockout cells allowed greater reduction of J by dimethyloxalylglycine (DMOG), which inhibits J synthesis, compared to wild type cells treated with DMOG, and resulted in stronger defects in RNAP II termination and cell growth. By mRNA-seq we see largely upregulation of genes near the ends of gene clusters following J loss, indicating that J represses genes near termination sites. These findings reveal a conserved role of J in promoting termination prior to the end of polycistronic gene clusters in kinetoplastid parasites and suggest that the essential nature of J is related to its role in repressing genes by promoting termination.
Project description:The genomic DNAs of strains JPCM5 and 263 of L. infantum, strains LV39 and Friedlin of L. major and strains Parrot-TarII and S125 of L. tarentolae were used in comparative genomic hybridizations to reveal the intra-species and inter-species gene content, and to validate L. tarentolae Parrot-TarII genome sequencing results. Leishmania (Sauroleishmania) tarentolae was first isolated in the lizard Tarentola mauritanica. This species is not known to be pathogenic to humans but is often used as a model organism for molecular analyses or protein overproduction. The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved by high-throughput sequencing technologies. The L. tarentolae genome was first assembled de novo and then aligned against the reference L. major Friedlin genome to facilitate contig positioning and annotation, providing a 23-fold coverage of the genome. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described, and it provides an opportunity for comparison with the completed genomes of the pathogenic Leishmania species. A high synteny was observed in de novo assembled contigs between all sequenced Leishmania species. A number of limited chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic with L. major. Globally, over 90% of the L. tarentolae gene content was shared with the other Leishmania species. There were 250 L. major genes absent from L. tarentolae, and interestingly these missing genes were primarily expressed in the intracellular amastigote stage of the pathogenic parasites. This implies that L. tarentolae may have impaired ability to survive as an intracellular parasite. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the leishmanolysin (GP63) and a gene related to the promastigote surface antigen (PSA31C). Overall, L. tarentolae appears to have a gene content more adapted to the insect stage rather than the mammalian one. This may partly explain its inability to replicate within mammalian macrophages and its suspected preferred life style as promastigote in the lizards. Six strains of three Leishmania species were hybridizated to 12 microarrays, each with four biological replicates (independent cultures). Supplementary file: Represents final results obtained after statistical analysis of all replicates.
Project description:Base J and H3.V promote RNA Polymerase (RNAP) II termination within polycistronic gene clusters in the kinetoplastid species Trypanosoma brucei. Although base J has been shown to promote RNAP II termination in the related kinetoplastid species Leishmania major and Leishmania tarentolae, the role of H3.V was unclear. The effect of acute J loss on mRNA transcript abundance was also unknown. We find here that H3.V does not promote transcription termination in Leishmania major, but loss of H3.V does reduce J levels. The J loss in H3.V knockout cells is not enough to result in a termination defect, which we show is due to a threshold level of J that is sufficient to promote termination. Loss of J beyond that threshold results in termination defects. Further, the decreased J in H3.V knockout cells allowed greater reduction of J by dimethyloxalylglycine (DMOG), which inhibits J synthesis, compared to wild type cells treated with DMOG, and resulted in stronger defects in RNAP II termination and cell growth. By mRNA-seq we see largely upregulation of genes near the ends of gene clusters following J loss, indicating that J represses genes near termination sites. These findings reveal a conserved role of J in promoting termination prior to the end of polycistronic gene clusters in kinetoplastid parasites and suggest that the essential nature of J is related to its role in repressing genes by promoting termination.