ABSTRACT: Chromatin immunoprecipitation and RNA-seq analysis in purified quiescent yeast cells to identify the genomic RNA polymerase II and histone H3 methylation occupancy profiles and the transcriptome
Project description:A defining characteristic of quiescent cells is their low level of gene activity compared to growing cells. Using a yeast model for cellular quiescence, we compared the genome-wide profiles of multiple histone modifications between growing and quiescent cells, and correlated these profiles with the presence of RNA polymerase II and its transcripts. Quiescent cells retained several forms of histone methylation normally associated with transcriptionally active chromatin and had many transcripts in common with growing cells. Quiescent cells also contained high levels of RNA polymerase II, but only low levels of the canonical initiating and elongating forms of the polymerase. The data suggest that the transcript and histone methylation marks in quiescent cells were either inherited from growing cells or established early during the development of quiescence and then retained in this non-growing cell population. This might ensure that quiescent cells can rapidly adapt to a changing environment to resume growth. RNA-seq analysis was performed in yeast Log-phase cells and purified Quiescent yeast cells and the transcriptomes in each were compared. The RNA data was correlated with genomic RNA polymerase II and histone H3 methylation occupancy profiles in the log and quiescent cells.
Project description:Chromatin immunoprecipitation and RNA-seq analysis in purified quiescent yeast cells to identify the genomic RNA polymerase II and histone H3 methylation occupancy profiles and the quiescent cell transcriptome.
Project description:A defining characteristic of quiescent cells is their low level of gene activity compared to growing cells. Using a yeast model for cellular quiescence, we compared the genome-wide profiles of multiple histone modifications between growing and quiescent cells, and correlated these profiles with the presence of RNA polymerase II and its transcripts. Quiescent cells retained several forms of histone methylation normally associated with transcriptionally active chromatin and had many transcripts in common with growing cells. Quiescent cells also contained high levels of RNA polymerase II, but only low levels of the canonical initiating and elongating forms of the polymerase. The data suggest that the transcript and histone methylation marks in quiescent cells were either inherited from growing cells or established early during the development of quiescence and then retained in this non-growing cell population. This might ensure that quiescent cells can rapidly adapt to a changing environment to resume growth. Immunoprecipitation experiments were carried out for Pol II, H3K36me3, H3K4me3, H3K79me3 and H3 separately using both Log-phase and Quiescent cells. Each ChIP-chip assay was conducted using a control (IN) and in all instances, at least one replicate experiment was performed.
Project description:Occupancy profiling of lysine 9 dimethylated histone H3 in fission yeast. Occupancy profiling of Red1, Mtl1, and Mmi1 proteins in fission yeast. Agilent 60mer array was used to analyze DNA recovered by immunoprecipitation of lysine 9 dimethylated histone H3, Red1, Mtl1, and Mmi1 proteins from asynchronous culture of fission yeast.
Project description:(1) Background: Quiescent cells have stopped dividing and show strongly reduced levels of gene expression during dormancy. In response to appropriate signals the cells can wake up and start growing again. Many histone modifications are regulated in quiescence, but their exact functions remain to be determined. (2) Methods: Here we map the different histone modifications: H3K4me3, H3K9ac, H3K9me2, H3K9me3, and the histone variant H2A.Z, comparing vegetative and quiescent fission yeast (S. pombe) cells. We also map histone H3 as a control and RNA polymerase II (phosphorylated at S2 and S5) to enable comparisons of their occupancies within genes. We use ChIP-seq methodology and several different bioinformatics tools. (3) Results: The histone modification mapping data show that H3K4me3 changes stand out as being the most significant. Changes in occupancy of histone variant H2A.Z was also significant consistent with earlier studies. Regarding gene expression changes in quiescence, we found that changes in mRNA levels were associated with changes in occupancy of RNA polymerase II (S2 and S5). Analysis of quiescence genes showed that increased H3K4me3 levels and RNA polymerase II occupancy was super-significant at a small set of core quiescence genes which are continuously upregulated during dormancy. We demonstrate that several of these genes were require Set1C/COMPASS activity for their strong induction during quiescence. (4) Conclusions: Our results imply that regulation of gene expression in quiescent cells involves epigenome changes with a key role for H3K4me3 in regulation of RNA polymerase II activity, and that different gene activation mechanisms are controlling early and core quiescence genes. Thus, our data give further insights into important epigenome changes in quiescence using fission yeast as experimental model.
Project description:Occupancy profiling of lysine 9 dimethylated histone H3 in fission yeast. Agilent 60mer array was used to analyze DNA recovered by immunoprecipitation of lysine 9 dimethylated histone H3 from asynchronous culture of fission yeast.
Project description:Expression profile of mutant cells compared to wild-type cells: Expression profile of Schizosaccharomyces pombe genome in ∆pht1, ∆ago1, ∆clr4, rik1, ∆pht1rik1, ∆pht1∆ago1 and ∆pht1∆clr4 cells. Expression profile of Schizosaccharomyces pombe genome in ∆rrp6, ∆cid14, ∆swi6, ∆swr1, ∆set1, ∆pht1∆swi6 and ∆pht1∆set1 cells. Expression profile of Schizosaccharomyces pombe genome in ∆alp13, ∆cph1, ∆set2, clr6-1 and ∆cph1∆pht1 cells. Occupancy profiling: Occupancy profiling of RNA polymerase II, histone variant H2A.Z and ClrC subunit Rik1 in fission yeast Schizosaccharomyces pombe Occupancy profiling of histone variant H2A.Z in ∆msc1 cells. Agilent 60mer oligonucleotide custom array containing probes spanning large portion of chromosome 2 at 50bp resolution was used to profile expression levels in mutant cells and to compare them to levels in wild-type cells. Agilent 60mer array was used to analyze DNA recovered by immunoprecipitation of RNA polymerase II, H2A.Z or Rik1 from asynchronous culture of fission yeast. Agilent 60mer array was used to analyze DNA recovered by immunoprecipitation of histone H2A.Z from asynchronous culture of fission yeast.
Project description:Occupancy profiling of lysine 9 dimethylated histone H3 in fission yeast. Occupancy profiling of Red1, Mtl1, and Mmi1 proteins in fission yeast.
Project description:Occupancy profiling of lysine 9 dimethylated histone H3 in fission yeast. Occupancy profiling of Psc3, Rec8, Rad21 and Rec11 proteins in fission yeast.
