Leishmania major
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ABSTRACT:
PROVIDER: PRJNA311271 | ENA |
REPOSITORIES: ENA
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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.
2016-07-20 | E-GEOD-77632 | biostudies-arrayexpress
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.
2016-07-20 | GSE77632 | GEO
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.
2016-09-26 | GSE77713 | GEO
Project description:Base J represses genes at the end of polycistronic gene clusters in Leishmania major by promoting RNAP II termination [RNA-seq]
| PRJNA311056 | ENA
Project description:Base J, β-D-glucosyl-hydroxymethyluracil, is an epigenetic modification of thymine in the nuclear DNA of flagellated protozoa of the order Kinetoplastida. J is enriched at sites involved in RNA Polymerase (RNAP) II initiation and termination. We have previously demonstrated a role of J in regulating RNAP II initiation in Trypanosoma cruzi. Reduction of J in Leishmania tarentolae via growth in BrdU resulted in cell death and indicated a role of J in the regulation of RNAP II termination. To further explore J function in RNAP II termination among kinetoplastids and avoid indirect effects associated with BrdU toxicity and genetic deletions, we inhibited J synthesis in L. major and T. brucei using DMOG. Reduction of J in L. major resulted in genome-wide defects in transcription termination and the generation of antisense RNAs, without cell death. In contrast, loss of J in T. brucei did not lead to genome-wide termination defects; however, the loss of J at specific sites within polycistronic gene clusters led to altered transcription elongation and increased expression of downstream genes. Thus, J regulation of transcription termination genome-wide is restricted to Leishmania spp., while in T. brucei it regulates RNAP II elongation and gene expression at specific genomic loci. We studied the effect of loss of base J on transcription using WT and WT cells grown in DMOG-containing medium. We used 2 RNA-seq libraries containing processed RNA products and 4 small RNA libraries representing the entire transcriptome.
2015-05-18 | E-GEOD-57621 | biostudies-arrayexpress
Project description:Base J, β-D-glucosyl-hydroxymethyluracil, is an epigenetic modification of thymine in the nuclear DNA of flagellated protozoa of the order Kinetoplastida. J is enriched at sites involved in RNA Polymerase (RNAP) II initiation and termination. We have previously demonstrated a role of J in regulating RNAP II initiation in Trypanosoma cruzi. Reduction of J in Leishmania tarentolae via growth in BrdU resulted in cell death and indicated a role of J in the regulation of RNAP II termination. To further explore J function in RNAP II termination among kinetoplastids and avoid indirect effects associated with BrdU toxicity and genetic deletions, we inhibited J synthesis in L. major and T. brucei using DMOG. Reduction of J in L. major resulted in genome-wide defects in transcription termination and the generation of antisense RNAs, without cell death. In contrast, loss of J in T. brucei did not lead to genome-wide termination defects; however, the loss of J at specific sites within polycistronic gene clusters led to altered transcription elongation and increased expression of downstream genes. Thus, J regulation of transcription termination genome-wide is restricted to Leishmania spp., while in T. brucei it regulates RNAP II elongation and gene expression at specific genomic loci.
2015-05-18 | GSE57621 | GEO
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.
2013-10-26 | E-GEOD-51737 | biostudies-arrayexpress
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.
2013-10-26 | GSE51737 | GEO
Project description:Transcriptional profiling of Leishmania major parasites overexpressing LmSir2rp3 protein compared with wild-type cell line transfected with empty vector in normal growth conditions. Goal of this experiment was to evaluate the possible effect of LmSir2rp3 in the control of gene expression in this protozoan parasite that has a polycistronic transcription.
2012-09-10 | GSE38731 | GEO
Project description:Base J is a modified DNA base that is enriched at RNA polymerase II termination sites. We previously identified a complex in Leishmania major and T. brucei composed of PNUTS, PP1, Wdr82 and a J-binding protein (JBP3). We show that deletion of PP1 in L. major leads to readthrough transcription and expression of downstream genes.
2022-10-01 | GSE200788 | GEO