Unknown,Transcriptomics,Genomics,Proteomics

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Base J represses genes at the end of polycistronic gene clusters in Leishmania major by promoting RNAP II termination

ABSTRACT: 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.

ORGANISM(S): Leishmania major

SUBMITTER: Robert Schmitz

PROVIDER: E-GEOD-77632 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Base J represses genes at the end of polycistronic gene clusters in Leishmania major by promoting RNAP II termination.

Reynolds David L DL Hofmeister Brigitte T BT Cliffe Laura L Siegel T Nicolai TN Anderson Britta A BA Beverley Stephen M SM Schmitz Robert J RJ Sabatini Robert R

Molecular microbiology 20160601 4

The genomes of kinetoplastids are organized into polycistronic gene clusters that are flanked by the modified DNA base J. Previous work has established a role of base J in promoting RNA polymerase II termination in Leishmania spp. where the loss of J leads to termination defects and transcription into adjacent gene clusters. It remains unclear whether these termination defects affect gene expression and whether read through transcription is detrimental to cell growth, thus explaining the essenti ...[more]

PMID: 27125778

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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.

Dataset Information

Base J represses genes at the end of polycistronic gene clusters in Leishmania major by promoting RNAP II termination

Publications

Base J represses genes at the end of polycistronic gene clusters in Leishmania major by promoting RNAP II termination.

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