Project description:Genomic locations of V5-tagged budding yeast Rif1 (including wilt-type and designer mutants) were analysed by ChIP-Seq. Mutants tested were rif1-7A and rif1-7E, in which Ser/Thr residues in the cluster of SQ/TQ sites were mutated to Ala or Glu, respectively. We also tested tested rif1-∆594, in which the C-terminal 594 amino acids were deleted.
Project description:Genomic locations of Myc-tagged budding yeast Rif1 (including wilt-type and designer mutants) were analysed by ChIP-Seq. Mutants tested were rif1-7A and rif1-7E, in which Ser/Thr residues in the cluster of SQ/TQ sites were mutated to Ala or Glu, respectively. The performance of RIF1-9V5 was also tested.
Project description:This experiment details ChIP sequencing to decipher the binding sites of the Rif1 protein in budding yeast. Rif1 binds most strongly to telomeres where its binding is mediated by Rap1. To reduce telomere binding and help reveal Rap1-independent binding sites, a truncation mutant of Rif1 lacking the Rap1 interaction domain was constructed and analysed. Binding was examined at various cell-cycle stages to elucidate the role of Rif1 in DNA replication and other chromosome transactions.
Project description:The Rif1 protein negatively regulates telomeric TG repeat length in the budding yeast S. cerevisiae, but how it prevents telomere over-extension is unknown. Rif1 was recently shown to control DNA replication by acting as a Protein Phosphatase 1 (PP1)-targeting subunit. Therefore we investigated whether Rif1 controls telomere length by targeting PP1 activity. We find that a Rif1 mutant that cannot interact with PP1 causes a long-telomere phenotype, similar to that of rif1∆ cells. Compromised PP1 function also causes telomere extension. Tethering PP1 at a specific telomere partially substitutes for Rif1 in limiting TG repeat length, confirming the importance of PP1 in telomere length control. Ablating Rif1-PP1 interaction leads to precocious activation of telomere-proximal replication origins and aberrantly early telomere replication. However, we find that Rif1 still limits telomere length even if nearby replication origins are deleted, indicating that effects of Rif1 on telomere length are not mediated through replication timing. Instead we find that, even at a telomere created after DNA synthesis during a mitotic block, Rif1-PP1 interaction is required to suppress telomere lengthening and prevent inappropriate recruitment of Tel1 kinase. Overall, our results show that Rif1 controls telomere length by recruiting PP1 to directly suppress telomerase-mediated TG repeat lengthening.
Project description:Carboxy-terminally tagged MOZ (Flag-V5-BIO tagged) was detected by ChIP-seq using anti-V5 antibody (Sigma, A7345) to precipitate chromatin associated with MOZ
Project description:Analysis of the expression of KH2 embryonic stem cells inducibly expressing V5 tagged Sox17 protein. Results provide information on the endodermal gene expression program activated after Sox17 expression in ES cells.
Project description:Analysis of the expression of KH2 embryonic stem cells inducibly expressing V5 tagged Sox17 protein. Results provide information on the endodermal gene expression program activated after Sox17 expression in ES cells. Total RNA extracted from 48 hours doxycycline induced compaired to non-induced Sox17-V5 expressing ES cells