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RNA polymerase backtracking results in the accumulation of fission yeast condensin at active genes.

ABSTRACT: The mechanisms leading to the accumulation of the SMC complexes condensins around specific transcription units remain unclear. Observations made in bacteria suggested that RNA polymerases (RNAPs) constitute an obstacle to SMC translocation, particularly when RNAP and SMC travel in opposite directions. Here we show in fission yeast that gene termini harbour intrinsic condensin-accumulating features whatever the orientation of transcription, which we attribute to the frequent backtracking of RNAP at gene ends. Consistent with this, to relocate backtracked RNAP2 from gene termini to gene bodies was sufficient to cancel the accumulation of condensin at gene ends and to redistribute it evenly within transcription units, indicating that RNAP backtracking may play a key role in positioning condensin. Formalization of this hypothesis in a mathematical model suggests that the inclusion of a sub-population of RNAP with longer dwell-times is essential to fully recapitulate the distribution profiles of condensin around active genes. Taken together, our data strengthen the idea that dense arrays of proteins tightly bound to DNA alter the distribution of condensin on chromosomes.

SUBMITTER: Rivosecchi J

PROVIDER: S-EPMC8046420 | biostudies-literature |

REPOSITORIES: biostudies-literature

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RNA pol II transcript abundance controls condensin accumulation at mitotically up-regulated and heat-shock-inducible genes in fission yeast.

Project description:Condensin plays fundamental roles in chromosome dynamics. In this study, we determined the binding sites of condensin on fission yeast (Schizosaccharomyces pombe) chromosomes at the level of nucleotide sequences using chromatin immunoprecipitation (ChIP) and ChIP sequencing (ChIP-seq). We found that condensin binds to RNA polymerase I-, II- and III-transcribed genes during both mitosis and interphase, and we focused on pol II constitutive and inducible genes. Accumulation sites for condensin are distinct from those of cohesin and DNA topoisomerase II. Using cell cycle stage and heat-shock-inducible genes, we show that pol II-mediated transcripts cause condensin accumulation. First, condensin's enrichment on mitotically activated genes was abolished by deleting the sep1(+) gene that encodes an M-phase-specific forkhead transcription factor. Second, by raising the temperature, condensin accumulation was rapidly induced at heat-shock protein genes in interphase and even during mid-mitosis. In interphase, condensin accumulates preferentially during the postreplicative phase. Pol II-mediated transcription was neither repressed nor activated by condensin, as levels of transcripts per se did not change when mutant condensin failed to associate with chromosomal DNA. However, massive chromosome missegregation occurred, suggesting that abundant pol II transcription may require active condensin before proper chromosome segregation.

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Dissection of the essential steps for condensin accumulation at kinetochores and rDNAs during fission yeast mitosis.

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Deciphering the RNA polymerase II CTD code in fission yeast.

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RNA pol II transcript abundance controls condensin accumulation at mitotically upregulated and heat shock-inducible genes in fission yeast

Project description:Condensin plays fundamental roles in chromosome dynamics. In this study, we determined the binding sites of condensin on fission yeast (Schizosaccharomyces pombe) chromosomes at the level of nucleotide sequences using chromatin immunoprecipitation (ChIP) and ChIP-sequencing (ChIP-seq). We found that condensin binds to RNA polymerase I-, II- and III-transcribed genes during both mitosis and interphase, and we focused on pol II constitutive and inducible genes. Accumulation sites for condensin are distinct from those of cohesin and DNA topoisomerase II. Using cell cycle stage- and heat shock-inducible genes, we demonstrate that pol II-mediated transcripts cause condensin accumulation. First, condensin’s enrichment on mitotically activated genes was abolished by deleting the sep1+ gene that encodes an M-phase-specific forkhead transcription factor. Second, by raising the temperature, condensin accumulation was rapidly induced at heat shock protein genes in interphase and even during mid-mitosis. In interphase, condensin accumulates preferentially during the post-replicative phase. pol II-mediated transcription was neither repressed nor activated by condensin, as levels of transcripts per se did not change when mutant condensin failed to associate with chromosomal DNA. However, massive chromosome missegregation occurred, suggesting that abundant pol II transcription may require active condensin prior to proper chromosome segregation.

2015-04-15 | GSE65956 | GEO

Pathogenic bacteria experience pervasive RNA polymerase backtracking during infection.

Project description:ImportanceEukaryotic hosts have defense mechanisms that may disrupt molecular transactions along the pathogen's chromosome through excessive DNA damage. Given that DNA damage stalls RNA polymerase (RNAP) thereby increasing mutagenesis, investigating how host defense mechanisms impact the movement of the transcription machinery on the pathogen chromosome is crucial. Using a new methodology we developed, we elucidated the dynamics of RNAP movement and association with the chromosome in the pathogenic bacterium Salmonella enterica during infection. We found that dynamics of RNAP movement on the chromosome change significantly during infection genome-wide, including at regions that encode for key virulence genes. In particular, we found that there is pervasive RNAP backtracking on the bacterial chromosome during infections and that anti-backtracking factors are critical for pathogenesis. Altogether, our results suggest that, interestingly, the host environment can promote the development of antimicrobial resistance and hypervirulence as stalled RNAPs can accelerate evolution through increased mutagenesis.

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Pathogenic bacteria experience pervasive RNA polymerase backtracking during infection.

Project description:Pathogenic bacteria and their eukaryotic hosts are in a constant arms race. Hosts have numerous defense mechanisms at their disposal that not only challenge the bacterial invaders, but have the potential to disrupt molecular transactions along the bacterial chromosome. However, it is unclear how the host impacts association of proteins with the bacterial chromosome at the molecular level during infection. This is partially due to the lack of a method that could detect these events in pathogens while they are within host cells. We developed and optimized a system capable of mapping and measuring levels of bacterial proteins associated with the chromosome while they are actively infecting the host (referred to as PIC-seq). Here, we focused on the dynamics of RNA polymerase (RNAP) movement and association with the chromosome in the pathogenic bacterium Salmonella enterica as a model system during infection. Using PIC-seq, we found that RNAP association patterns with the chromosome change during infection genome-wide, including at regions that encode for key virulence genes. Importantly, we found that infection of a host significantly increases RNAP backtracking on the bacterial chromosome. RNAP backtracking is the most common form of disruption to RNAP progress on the chromosome. Interestingly, we found that the resolution of backtracked RNAPs via the anti-backtracking factors GreA and GreB is critical for pathogenesis, revealing a new class of virulence genes. Altogether, our results strongly suggest that infection of a host significantly impacts transcription by disrupting RNAP movement on the chromosome within the bacterial pathogen. The increased backtracking events have important implications not only for efficient transcription, but also for mutation rates as stalled RNAPs increase the levels of mutagenesis.

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Real-Time Observation of Backtracking by Bacterial RNA Polymerase.

Project description:RNA polymerase (RNAP) backtracking is a backward sliding of the enzyme along DNA and RNA. It plays important roles in many essential processes in bacteria and in eukaryotes. We describe here a fluorescence-based approach that allows a real-time observation of bacterial RNAP backtracking. A Cy3 fluorescence probe, when incorporated into a specific site in the nontemplate strand near the site of backtracking, allows RNAP movements to be monitored near the probe because of a robust enhancement of fluorescence caused by protein proximity. Using this approach, we showed that binding of NTP to the active site prior to phosphodiester bond formation inhibited backtracking, consistent with the coupling of NTP binding to translocation. The extent and the kinetics of backtracking did not show a simple correlation with the instability of the DNA-RNA hybrid, indicating a more complex dependence of backtracking on DNA template sequence. Experiments with transcription through an abasic site in DNA template or neutravidin bound to biotinylated template strand base illustrated an important role of backtracking in defining how RNAP reacts to such obstacles in the DNA template. The described approach will be a useful tool in deciphering the mechanism of backtracking and in studying factors that affect the backtracking.

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Condensin-mediated remodeling of the mitotic chromatin landscape in fission yeast.

Project description:The eukaryotic genome consists of DNA molecules far longer than the cells that contain them. They reach their greatest compaction during chromosome condensation in mitosis. This process is aided by condensin, a structural maintenance of chromosomes (SMC) family member. The spatial organization of mitotic chromosomes and how condensin shapes chromatin architecture are not yet fully understood. Here we use chromosome conformation capture (Hi-C) to study mitotic chromosome condensation in the fission yeast Schizosaccharomyces pombe. This showed that the interphase landscape characterized by small chromatin domains is replaced by fewer but larger domains in mitosis. Condensin achieves this by setting up longer-range, intrachromosomal DNA interactions, which compact and individualize chromosomes. At the same time, local chromatin contacts are constrained by condensin, with profound implications for local chromatin function during mitosis. Our results highlight condensin as a major determinant that changes the chromatin landscape as cells prepare their genomes for cell division.

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A genetic screen for functional partners of condensin in fission yeast.

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Recruitment of Xrn1 to stress-induced genes allows efficient transcription by controlling RNA polymerase II backtracking.

Project description:A new paradigm has emerged proposing that the crosstalk between nuclear transcription and cytoplasmic mRNA stability keeps robust mRNA levels in cells under steady-state conditions. A key piece in this crosstalk is the highly conserved 5'-3' RNA exonuclease Xrn1, which degrades most cytoplasmic mRNAs but also associates with nuclear chromatin to activate transcription by not well-understood mechanisms. Here, we investigated the role of Xrn1 in the transcriptional response of Saccharomyces cerevisiae cells to osmotic stress. We show that a lack of Xrn1 results in much lower transcriptional induction of the upregulated genes but in similar high levels of their transcripts because of parallel mRNA stabilization. Unexpectedly, lower transcription in xrn1 occurs with a higher accumulation of RNA polymerase II (RNAPII) at stress-inducible genes, suggesting that this polymerase remains inactive backtracked. Xrn1 seems to be directly implicated in the formation of a competent elongation complex because Xrn1 is recruited to the osmotic stress-upregulated genes in parallel with the RNAPII complex, and both are dependent on the mitogen-activated protein kinase Hog1. Our findings extend the role of Xrn1 in preventing the accumulation of inactive RNAPII at highly induced genes to other situations of rapid and strong transcriptional upregulation.

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