Project description:Histones of heterochromatin are deacetylated in yeast and methylated in more complex eukaryotes to regulate heterochromatin structure and gene silencing. Here, we report that histone H2A phosphorylated at serine 129 (γH2A) in Saccharomyces cerevisiae is a conceptually new type of heterochromatin modification that functions downstream of silent chromatin assembly. We show that γH2A is enriched throughout yeast telomeric and silent mating locus (HM) heterochromatin where γH2A results from the action of kinases Tel1 and Mec1. Interestingly, mutation of γH2A has no apparent effect on the binding of Sir (silent information regulator) complex or on gene silencing. In contrast, deletion of SIR3 abolishes the formation of γH2A at heterochromatin. To address the function of γH2A, we used a ∆rif1 mutant strain in which telomeres are excessively elongated to show that γH2A is required for the optimal recruitment of Cdc13, a regulator of telomere elongation, and for telomere elongation itself. Thus, a histone modification that parallels Sir3 protein binding is shown here to be dispensable for the formation of a silent structure but is important for a crucial heterochromatin-specific downstream function in telomere homeostasis.
Project description:Telomere chromatin structure is pivotal for maintaining genome stability by regulating the binding of telomere-associated proteins and inhibition of a DNA damage response. In yeast, the silent information regulator (Sir) proteins bind to terminal telomeric repeats and to subtelomeric X-elements resulting in histone deacetylation and transcriptional silencing. Herein, we show that sir2 mutant strains display a very specific loss of a nucleosome residing in the X-element. Most yeast telomeres contain an X-element and the nucleosome occupancy defect in sir2 mutants is remarkably consistent between different telomeres.
Project description:We report the application of single-molecule-based sequencing technology for high-throughput profiling of histone modifications in yeast. By obtaining bases of sequence from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of saccharomuces cerevisiae.We find that H3T11 phosphorylationlysine is widely distributed in gene promoter region and chromosome telomere region
Project description:Occupancy profiling of Shelterin components in fission yeast. Facultative heterochromatin regulates gene expression, but its assembly is poorly understood. Previously, we identified facultative heterochromatin islands in the fission yeast genome and found that RNA elimination machinery promotes island assembly at meiotic genes. Here, we report that Taz1, a component of the telomere protection complex Shelterin, is required to assemble heterochromatin islands at regions corresponding to late replication origins that are sites of double-strand break formation during meiosis. The loss of Taz1 and other Shelterin subunits, including Ccq1 that interacts with Clr4/Suv39h, abolishes heterochromatin at late origins and causes defective silencing of associated genes. Moreover, the late origin regulator Rif1 affects heterochromatin at Taz1-dependent islands and subtelomeric regions. We uncover a connection between heterochromatin and replication control, and show that heterochromatin factors affect timing of replication. These analyses implicate Shelterin in facultative heterochromatin assembly at late origins, which has important implications for the maintenance of genome stability and gene regulation.
Project description:Occupancy profiling of Rec12 during fission yeast meiosis. Facultative heterochromatin regulates gene expression, but its assembly is poorly understood. Previously, we identified facultative heterochromatin islands in the fission yeast genome and found that RNA elimination machinery promotes island assembly at meiotic genes. Here, we report that Taz1, a component of the telomere protection complex Shelterin, is required to assemble heterochromatin islands at regions corresponding to late replication origins that are sites of double-strand break formation during meiosis. The loss of Taz1 and other Shelterin subunits, including Ccq1 that interacts with Clr4/Suv39h, abolishes heterochromatin at late origins and causes defective silencing of associated genes. Moreover, the late origin regulator Rif1 affects heterochromatin at Taz1-dependent islands and subtelomeric regions. We uncover a connection between heterochromatin and replication control, and show that heterochromatin factors affect timing of replication. These analyses implicate Shelterin in facultative heterochromatin assembly at late origins, which has important implications for the maintenance of genome stability and gene regulation.
Project description:BrdU profiling of replication activity in hydroxyurea treated synchronous culture of fission yeast. Facultative heterochromatin regulates gene expression, but its assembly is poorly understood. Previously, we identified facultative heterochromatin islands in the fission yeast genome and found that RNA elimination machinery promotes island assembly at meiotic genes. Here, we report that Taz1, a component of the telomere protection complex Shelterin, is required to assemble heterochromatin islands at regions corresponding to late replication origins that are sites of double-strand break formation during meiosis. The loss of Taz1 and other Shelterin subunits, including Ccq1 that interacts with Clr4/Suv39h, abolishes heterochromatin at late origins and causes defective silencing of associated genes. Moreover, the late origin regulator Rif1 affects heterochromatin at Taz1-dependent islands and subtelomeric regions. We uncover a connection between heterochromatin and replication control, and show that heterochromatin factors affect timing of replication. These analyses implicate Shelterin in facultative heterochromatin assembly at late origins, which has important implications for the maintenance of genome stability and gene regulation.
Project description:Occupancy profiling of Rec12 during fission yeast meiosis. Facultative heterochromatin regulates gene expression, but its assembly is poorly understood. Previously, we identified facultative heterochromatin islands in the fission yeast genome and found that RNA elimination machinery promotes island assembly at meiotic genes. Here, we report that Taz1, a component of the telomere protection complex Shelterin, is required to assemble heterochromatin islands at regions corresponding to late replication origins that are sites of double-strand break formation during meiosis. The loss of Taz1 and other Shelterin subunits, including Ccq1 that interacts with Clr4/Suv39h, abolishes heterochromatin at late origins and causes defective silencing of associated genes. Moreover, the late origin regulator Rif1 affects heterochromatin at Taz1-dependent islands and subtelomeric regions. We uncover a connection between heterochromatin and replication control, and show that heterochromatin factors affect timing of replication. These analyses implicate Shelterin in facultative heterochromatin assembly at late origins, which has important implications for the maintenance of genome stability and gene regulation. Whole cell extract DNA and DNA recovered by Rec12 ChIP from fission yeast undergoing synchronous meiosis were random-prime PCR amplified and labeled with Cy3 (whole cell extract) or Cy5 (IP DNA) and analyzed using custom 60mer Agilent array that tiles Schizosaccharomyces pombe genome in 300bp intervals alternately on both strands.
Project description:Occupancy profiling of Shelterin components in fission yeast. Facultative heterochromatin regulates gene expression, but its assembly is poorly understood. Previously, we identified facultative heterochromatin islands in the fission yeast genome and found that RNA elimination machinery promotes island assembly at meiotic genes. Here, we report that Taz1, a component of the telomere protection complex Shelterin, is required to assemble heterochromatin islands at regions corresponding to late replication origins that are sites of double-strand break formation during meiosis. The loss of Taz1 and other Shelterin subunits, including Ccq1 that interacts with Clr4/Suv39h, abolishes heterochromatin at late origins and causes defective silencing of associated genes. Moreover, the late origin regulator Rif1 affects heterochromatin at Taz1-dependent islands and subtelomeric regions. We uncover a connection between heterochromatin and replication control, and show that heterochromatin factors affect timing of replication. These analyses implicate Shelterin in facultative heterochromatin assembly at late origins, which has important implications for the maintenance of genome stability and gene regulation. Whole cell extract DNA and DNA recovered from Taz1 or Rif1-immunoprecipitated chromatin of fission yeast were random-prime PCR amplified and labeled with Cy3 (whole cell extract DNA) or with Cy5 (IP DNA) and analyzed using custom 60mer Agilent array that tiles Schizosaccharomyces pombe genome in 300bp intervals alternately on both strands.
Project description:ChIP-chip analyses of lysine 9 dimethylated histone H3 in Shelterin mutants. Facultative heterochromatin regulates gene expression, but its assembly is poorly understood. Previously, we identified facultative heterochromatin islands in the fission yeast genome and found that RNA elimination machinery promotes island assembly at meiotic genes. Here, we report that Taz1, a component of the telomere protection complex Shelterin, is required to assemble heterochromatin islands at regions corresponding to late replication origins that are sites of double-strand break formation during meiosis. The loss of Taz1 and other Shelterin subunits, including Ccq1 that interacts with Clr4/Suv39h, abolishes heterochromatin at late origins and causes defective silencing of associated genes. Moreover, the late origin regulator Rif1 affects heterochromatin at Taz1-dependent islands and subtelomeric regions. We uncover a connection between heterochromatin and replication control, and show that heterochromatin factors affect timing of replication. These analyses implicate Shelterin in facultative heterochromatin assembly at late origins, which has important implications for the maintenance of genome stability and gene regulation. Whole cell extract DNA and DNA recovered from H3K9 dimethylated chromatin of fission yeast were random-prime PCR amplified and labeled with Cy3 (whole cell extract) or Cy5 (IP DNA) and analyzed using custom 60mer Agilent array that tiles Schizosaccharomyces pombe genome in 300bp intervals alternately on both strands.
Project description:THO/TREX is a conserved nuclear complex that functions in mRNP biogenesis and prevents transcription-associated recombination. Whether or not it has a ubiquitous role in the genome is an open question. ChIP-chip studies reveal that the Hpr1 component of THO and the Sub2 RNA-dependent ATPase have genome wide-distributions at active ORFs in yeast. In contrast to RNAPII, evenly distributed from promoter to termination regions, THO and Sub2 are absent at promoters and distributed in a sharp 5’→3’ gradient. Importantly, ChIP-chips reveal an over-recruitment of Rrm3 in active genes in THO mutants that is reduced by overexpression of RNase H1. Our work establishes a genome-wide function for THO-Sub2 in transcription elongation and mRNP biogenesis that function to prevent the accumulation of transcription-mediated replication obstacles, including R-loops.