Leo1 is essential for dynamic regulation of heterochromatin and gene expression during cellular quiescence [ChIP microarray]
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PROVIDER: PRJNA476876 | ENA |
REPOSITORIES: ENA
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Project description:Cellular quiescence is a reversible differentiation state when cells are changing the gene expression programme to reduce metabolic functions and adapt to a new cellular environment. The epigenetic changes that accompany these alterations are not so well understood. Here we investigate the role of Leo1, a subunit of the conserved Paf1 (RNA polymerase-associated factor 1) complex, in the quiescence process using fission yeast as a model organism. Fission yeast cells enter the G0 phase of the cell cycle when exposed to nitrogen starvation and the heterochromatin regions become very dynamic. The reduction of heterochromatin in early G0 correlates with the reduced activity of target of rapamycin, TORC2, signalling. Cells lacking the Leo1 show reduced survival in G0. In these cells heterochromatin regions including subtelomeres are stabilized and many genes, including membrane transport genes, fail to be expressed. Our results suggest that Leo1 is essential for dynamic regulation of heterochromatin and gene expression during cellular quiescence.
2019-06-30 | GSE116038 | GEO
Project description:Cellular quiescence is a reversible differentiation state when cells are changing the gene expression programme to reduce metabolic functions and adapt to a new cellular environment. The epigenetic changes that accompany these alterations are not so well understood. Here we investigate the role of Leo1, a subunit of the conserved Paf1 (RNA polymerase-associated factor 1) complex, in the quiescence process using fission yeast as a model organism. Fission yeast cells enter the G0 phase of the cell cycle when exposed to nitrogen starvation and the heterochromatin regions become very dynamic. The reduction of heterochromatin in early G0 correlates with the reduced activity of target of rapamycin, TORC2, signalling. Cells lacking the Leo1 show reduced survival in G0. In these cells heterochromatin regions including subtelomeres are stabilized and many genes, including membrane transport genes, fail to be expressed. Our results suggest that Leo1 is essential for dynamic regulation of heterochromatin and gene expression during cellular quiescence.
2019-06-30 | GSE116657 | GEO
Project description:Leo1 is essential for dynamic regulation of heterochromatin and gene expression during cellular quiescence [RNA-Seq]
| PRJNA479830 | ENA
Project description:Domains of heterochromatin play important roles in the maintenance and regulation of eukaryotic genomes. However, the repressive nature of heterochromatin combined with its propensity to self-propagate necessitates the existence of robust mechanisms that limit heterochromatin spreading and thereby avoid silencing of expressed genes. A number of specific sequence elements have been found to serve as barriers to heterochromatin spreading; however, the mechanisms by which spreading is curtailed are generally not well understood. Here we uncover a role for PAF complex component Leo1 in regulating heterochromatin cis-spreading. A genetic screen revealed that loss of Leo1 results in spreading of heterochromatin across a centromeric (IRC) boundary element in fission yeast. Similar heterochromatin spreading was seen upon deletion of other components of the PAF complex, but not other factors involved in transcription-coupled chromatin modification, indicating a specific role for the PAF complex in heterochromatin regulation. Loss of Leo1 is associated with reduced levels of H4K16 acetylation at the boundary, while tethering of the H4K16 acetyltransferase Mst1 to boundary chromatin suppresses heterochromatin spreading in leo1? cells, suggesting that Leo1 antagonises heterochromatin spreading by facilitating H4K16 acetylation. Interestingly, Leo1 also regulates heterochromatin spreading independently of boundaries, and loss of Leo1 causes redistribution of heterochromatin, in particular resulting in substantial expansion of telomeric heterochromatin domains. The PAF complex is known to be an important regulator of transcription-related chromatin modifications; our findings reveal a previously undescribed role for this complex in global regulation of heterochromatin spreading in cis. 8 samples: input (whole cell extract) and IP from H3K9me2 ChIP in wild-type and leo1? cells, in duplicate
2015-05-22 | E-GEOD-61688 | biostudies-arrayexpress
Project description:Domains of heterochromatin play important roles in the maintenance and regulation of eukaryotic genomes. However, the repressive nature of heterochromatin combined with its propensity to self-propagate necessitates the existence of robust mechanisms that limit heterochromatin spreading and thereby avoid silencing of expressed genes. A number of specific sequence elements have been found to serve as barriers to heterochromatin spreading; however, the mechanisms by which spreading is curtailed are generally not well understood. Here we uncover a role for PAF complex component Leo1 in regulating heterochromatin cis-spreading. A genetic screen revealed that loss of Leo1 results in spreading of heterochromatin across a centromeric (IRC) boundary element in fission yeast. Similar heterochromatin spreading was seen upon deletion of other components of the PAF complex, but not other factors involved in transcription-coupled chromatin modification, indicating a specific role for the PAF complex in heterochromatin regulation. Loss of Leo1 is associated with reduced levels of H4K16 acetylation at the boundary, while tethering of the H4K16 acetyltransferase Mst1 to boundary chromatin suppresses heterochromatin spreading in leo1Δ cells, suggesting that Leo1 antagonises heterochromatin spreading by facilitating H4K16 acetylation. Interestingly, Leo1 also regulates heterochromatin spreading independently of boundaries, and loss of Leo1 causes redistribution of heterochromatin, in particular resulting in substantial expansion of telomeric heterochromatin domains. The PAF complex is known to be an important regulator of transcription-related chromatin modifications; our findings reveal a previously undescribed role for this complex in global regulation of heterochromatin spreading in cis.
2015-05-22 | GSE61688 | GEO
Project description:BackgroundCellular quiescence is a reversible differentiation state during which cells modify their gene expression program to inhibit metabolic functions and adapt to a new cellular environment. The epigenetic changes accompanying these alterations are not well understood. We used fission yeast cells as a model to study the regulation of quiescence. When these cells are starved for nitrogen, the cell cycle is arrested in G1, and the cells enter quiescence (G0). A gene regulatory program is initiated, including downregulation of thousands of genes-for example, those related to cell proliferation-and upregulation of specific genes-for example, autophagy genes-needed to adapt to the physiological challenge. These changes in gene expression are accompanied by a marked alteration of nuclear organization and chromatin structure.ResultsHere, we investigated the role of Leo1, a subunit of the conserved RNA polymerase-associated factor 1 (Paf1) complex, in the quiescence process using fission yeast as the model organism. Heterochromatic regions became very dynamic in fission yeast in G0 during nitrogen starvation. The reduction of heterochromatin in early G0 was correlated with reduced target of rapamycin complex 2 (TORC2) signaling. We demonstrated that cells lacking Leo1 show reduced survival in G0. In these cells, heterochromatic regions, including subtelomeres, were stabilized, and the expression of many genes, including membrane transport genes, was abrogated. TOR inhibition mimics the effect of nitrogen starvation, leading to the expression of subtelomeric genes, and this effect was suppressed by genetic deletion of leo1.ConclusionsWe identified a protein, Leo1, necessary for survival during quiescence. Leo1 is part of a conserved protein complex, Paf1C, linked to RNA polymerase II. We showed that Leo1, acting downstream of TOR, is crucial for the dynamic reorganization of chromosomes and the regulation of gene expression during cellular quiescence. Genes encoding membrane transporters are not expressed in quiescent leo1 mutant cells, and cells die after 2 weeks of nitrogen starvation. Taken together, our results suggest that Leo1 is essential for the dynamic regulation of heterochromatin and gene expression during cellular quiescence.
| S-EPMC6636030 | biostudies-literature
Project description:Abstract: (1) Background: The LEO1 (Left open reading frame 1) protein is a conserved subunit of the PAF1C complex (RNA polymerase II associated factor 1 complex). PAF1C has well-established mechanistic functions in elongation of transcription and RNA processing. We have previously shown, in fission yeast, that LEO1 is controlling histone H3K9 methylation levels by affecting the turnover of histone H3 in chromatin, and that it is essential for proper regulation of gene expression during cellular quiescence. Human fibroblasts enter a reversible quiescence state upon serum deprivation in the growth media. Here we investigate the function of LEO1 in human fibroblasts. (2) Methods: We knocked out the LEO1 gene using CRISPR/Cas9 methodology in human fibroblasts and verified that the LEO1 protein was undetectable by Western blot. We characterized the phenotype of the DLEO1 knockout cells with FACS analysis and cell growth assays. We used RNA-sequencing using spike-in controls to measure gene expression and spike-in controlled ChIP-sequencing experiments to measure the histone modification H3K9me2 genome-wide. (3) Results: Gene expression levels are altered in quiescent cells, however factors controlling chromatin and gene expression changes in quiescent human cells are largely unknown. The DLEO1 knockout fibroblasts are viable but have reduced metabolic activity compared to wild type cells. DLEO1 cells showed a slower entry into quiescence and a different morphology compared to wild type cells. Gene expression was generally reduced in quiescent wild type cells. The downregulated genes included genes involved in cell proliferation. A small number of genes were upregulated in quiescent wild type cells including several genes involved in ERK1/ERK2 and Wnt signaling. In quiescent DLEO1 cells many genes were mis-regulated compared to wild type cells. This included genes involved in Calcium ion transport and cell morphogenesis. Finally, spike-in controlled ChIP-sequencing experiments demonstrated that the histone modification H3K9me2 levels are globally increased in quiescent DLEO1 cells. (4) Conclusions: Thus, LEO1 is important for proper entry into cellular quiescence, control H3K9me2 levels and gene expression in human fibroblasts.
2023-11-29 | GSE247831 | GEO
Project description:Abstract: (1) Background: The LEO1 (Left open reading frame 1) protein is a conserved subunit of the PAF1C complex (RNA polymerase II associated factor 1 complex). PAF1C has well-established mechanistic functions in elongation of transcription and RNA processing. We have previously shown, in fission yeast, that LEO1 is controlling histone H3K9 methylation levels by affecting the turnover of histone H3 in chromatin, and that it is essential for proper regulation of gene expression during cellular quiescence. Human fibroblasts enter a reversible quiescence state upon serum deprivation in the growth media. Here we investigate the function of LEO1 in human fibroblasts. (2) Methods: We knocked out the LEO1 gene using CRISPR/Cas9 methodology in human fibroblasts and verified that the LEO1 protein was undetectable by Western blot. We characterized the phenotype of the DLEO1 knockout cells with FACS analysis and cell growth assays. We used RNA-sequencing using spike-in controls to measure gene expression and spike-in controlled ChIP-sequencing experiments to measure the histone modification H3K9me2 genome-wide. (3) Results: Gene expression levels are altered in quiescent cells, however factors controlling chromatin and gene expression changes in quiescent human cells are largely unknown. The DLEO1 knockout fibroblasts are viable but have reduced metabolic activity compared to wild type cells. DLEO1 cells showed a slower entry into quiescence and a different morphology compared to wild type cells. Gene expression was generally reduced in quiescent wild type cells. The downregulated genes included genes involved in cell proliferation. A small number of genes were upregulated in quiescent wild type cells including several genes involved in ERK1/ERK2 and Wnt signaling. In quiescent DLEO1 cells many genes were mis-regulated compared to wild type cells. This included genes involved in Calcium ion transport and cell morphogenesis. Finally, spike-in controlled ChIP-sequencing experiments demonstrated that the histone modification H3K9me2 levels are globally increased in quiescent DLEO1 cells. (4) Conclusions: Thus, LEO1 is important for proper entry into cellular quiescence, control H3K9me2 levels and gene expression in human fibroblasts.
2023-11-29 | GSE247829 | GEO
Project description:Maintenance of open and repressed chromatin states is crucial for regulation of gene expression. To study the genes involved in maintaining chromatin states we generated a random mutant library using the Hermes transposon mutagenesis system in fission yeast Schizosacchromyces pombe. The silencing of reporter genes inserted in the euchromatic region adjacent to the heterochromatic mating type locus was monitored. We identified Leo1-Paf1, a subcomplex of the RNA Polymerase II Associated Factor 1 Complex (Paf1C), required to prevent spreading of heterochromatin into euchromatin. Through high-resolution genome-wide ChIP (ChIP-exo) we mapped the heterochromatin mark H3K9me2 in leo1∆ cells. Loss of Leo1-Paf1 led to increased heterochromatin stability at several facultative heterochromatin loci. The RNAi machinery is the major pathway for heterochromatin formation in S. pombe. However, small RNA sequencing showed that heterochromatin assembly in leo1∆ cells was RNAi-independent. By examining histone turnover rate in leo1∆ cells, we showed that deletion of Leo1 decreased nucleosome turnover, which led to heterochromatin spreading. Our data revealed that Leo1-Paf1 promotes chromatin state fluctuations by enhancing histone turnover.
2015-10-18 | GSE66940 | GEO
Project description:Maintenance of open and repressed chromatin states is crucial for regulation of gene expression. To study the genes involved in maintaining chromatin states we generated a random mutant library using the Hermes transposon mutagenesis system in fission yeast Schizosacchromyces pombe. The silencing of reporter genes inserted in the euchromatic region adjacent to the heterochromatic mating type locus was monitored. We identified Leo1-Paf1, a subcomplex of the RNA Polymerase II Associated Factor 1 Complex (Paf1C), required to prevent spreading of heterochromatin into euchromatin. Through high-resolution genome-wide ChIP (ChIP-exo) we mapped the heterochromatin mark H3K9me2 in leo1∆ cells. Loss of Leo1-Paf1 led to increased heterochromatin stability at several facultative heterochromatin loci. The RNAi machinery is the major pathway for heterochromatin formation in S. pombe. However, small RNA sequencing showed that heterochromatin assembly in leo1∆ cells was RNAi-independent. By examining histone turnover rate in leo1∆ cells, we showed that deletion of Leo1 decreased nucleosome turnover, which led to heterochromatin spreading. Our data revealed that Leo1-Paf1 promotes chromatin state fluctuations by enhancing histone turnover.
2015-10-18 | GSE66939 | GEO