Project description:Using a TIP-seq protocol (specifically isolating transposon insertion junctions) we determined that the Ty1 retrotransposon targets tRNA genes and, in particular, we determined that the transposon inserts into nucleosomal DNA in an asymmetric pattern.
Project description:Using a TIP-seq protocol (specifically isolating transposon insertion junctions) we determined that the Ty1 retrotransposon targets tRNA genes and, in particular, we determined that the transposon inserts into nucleosomal DNA in an asymmetric pattern. TIP-seq recovery of transposon insertion junctions in haploid and diploid yeast
Project description:In budding yeast, the selective integration of the Ty1 LTR retrotransposon upstream of RNA polymerase III (Pol III)-transcribed genes requires the interaction between the AC40, a common subunit of Pol III and Pol I, and Ty1 integrase (IN1). The AC40/IN1 interaction involves a short sequence, the targeting domain (TD), present in the C-terminal part of IN1. Chip-seq analysis using WT or mutated Ty1 integrase demonstrated that TD is responsible for the recruitment of IN1 at both Pol I and Pol III-transcribed genes. Moreover, the introduction of the C-terminal residues of Ty1 in the Ty5 retrotransposon, which preferentially integrates in heterochromatin at silent mating loci (HMR and HML) and near telomeres, leads to its retargeting at Pol III-transcribed genes.
Project description:A stochastic simulation of yeast translation using Total Asymmetric Simple Exclusion Principle (TASEP) principles, representing ribosomes, tRNAs, mRNAs, and a tRNA re-charging process involving aminoacyl tRNA synthetases
Project description:We report testis H3K4me3 and DMC1 enrichment at DSB hotspots in various mice to examine differences between infertile hybrid mice and mice that have been humanized at PRDM9, which have rescued fertility. We find that infertile mice have an excess of "asymmetric" DSB hotspots, where both H3K4me3 and DMC1 reads tend to originate from only one homologue. At these hotspots, we see an excess of DMC1 relative to H3K4me3, consistent with delayed DSB repair at these sites. See Davies et al. Nature 2016 for a complete summary.
Project description:Nucleosomes in active chromatin are dynamic, but whether they have distinct structural conformations is unknown. To identify nucleosomes with alternative structures genome-wide, we used H4S47C-anchored cleavage mapping, which revealed that nucleosomes at 5% of budding yeast nucleosome positions have asymmetric histone-DNA interactions. These asymmetric interactions are enriched at nucleosome positions that flank promoters. Micrococcal nuclease (MNase) sequence-based profiles of asymmetric nucleosome positions revealed a corresponding asymmetry in MNase protection near the dyad axis, suggesting that the loss of DNA contacts around H4S47 is accompanied by protection of the DNA from MNase. Chromatin immunoprecipitation mapping of selected nucleosome remodelers indicated that asymmetric nucleosomes are bound by the RSC chromatin remodeling complex, which is required for maintaining nucleosomes at asymmetric positions. These results imply that the asymmetric nucleosome-RSC complex is a metastable intermediate representing partial unwrapping and protection of nucleosomal DNA on one side of the dyad axis during chromatin remodeling. We have analyzed the chromatin landscape of the yeast genome using paired-end MNase-seq and the chromatin binding of yeast remodelers Swr1, Ino80 and RSC at base-pair resolution using native chromatin immunoprecipitation followed by sequencing (N-ChIP-seq).
Project description:To identify yeast proteins associated with Ty1 integrase (IN) that could regulate Ty1 replication, we co-purified IN partners using the tandem chromatin affinity purification procedure after in vivo cross-link (TChAP), which we developed previously (Nguyen et al. 2014). We first identified RNA Pol I and Pol III complexes and also a small subset of additional evolutionary conserved complexes, including PAF1 (Polymerase-Associated Factor 1),FACT (FAcilitates Chromatin Transcription), the proteasome and the CK2 kinase. We next confirmed that CK2 interacts with Ty1 integrase in vivo and repress Ty1 retromobility. We showed that Ty1 IN is a substrate of CK2 in vitro and identified 12 phosphorylated residues. In vivo approaches showed that only part of the protein was phosphorylated in the cells and did not demonstrate any direct evidence between Ty1 IN phosphorylation and retromobility inhibition.
Project description:Retroviral integration is catalyzed by a tetramer of integrase (IN) assembled on viral DNA ends in a stable complex, known as the intasome. How the intasome interfaces with chromosomal DNA, which exists in the form of nucleosomal arrays, is currently unknown. Here we show that the prototype foamy virus (PFV) intasome is proficient at stable capture of nucleosomes as targets for integration. Single-particle cryo-electron microscopy (EM) reveals a multivalent intasome-nucleosome interface involving both gyres of nucleosomal DNA and one H2A-H2B heterodimer. While the histone octamer remains intact, the DNA is lifted from the surface of the H2A-H2B heterodimer to allow integration at strongly preferred superhelix location (SHL) ±3.5 positions. Amino acid substitutions disrupting these contacts impinge on the ability of the intasome to engage nucleosomes in vitro and redistribute viral integration sites on the genomic scale. Our findings elucidate the molecular basis for nucleosome capture by the viral DNA recombination machinery and the underlying nucleosome plasticity that allows integration. Genomic positions of integration sites of WT and mutant PFV vectors in HT1080 cells were determined using ligation-mediated PCR and next generation sequencing. Integration sites of purified recombinant PFV intasome into deproteinized human genomic DNA were used as a reference dataset.