Project description:Chromatin immunoprecipitation of Set3 complex applied with tilling array chip ( ChIP on chip of either Set3-TAP or Snt1-TAP) analysis demonstrated that the genome-wide localization of Set3 complex in Saccharomyces cerevisiae
Project description:Viruses are obligate intracellular pathogens that depend on host factors to complete their infection cycle. Very little is known of which plant factors are required for successful Tomato spotted wilt orthotospovirus (TSWV) infection. The viral ribonucleoprotein (RNP) fraction from TSWV infected Nicotiana benthamiana plants was purified and its protein composition was analysed by proteomics by mass spectrometry to identify host proteins that co-purify with viral RNPs. Related, we expressed a TSWV replicon system in a non-host system, Bakers’ yeast (Saccharomyces cerevisiae), and purified as well the RNP fraction from yeast. Comparative proteomics was used to find common enriched proteins observed in both yeast and plant RNP fractions.
Project description:Crosslinking mass spectrometry (CLMS) analysis was conducted on the enriched Saccharomyces cerevisiae spindle pole body (SPB) preparation to explore the structure of the SPB, particularly focusing on its topology and the interactions between subunits.
Project description:Proteins from within the Saccharomyces cerevisiae Mediator transcription complex were knocked out and compared against wild type yeast using two-color oligo arrays.
Project description:As a part of a study on how kinetochors are assembled at the centromeres of the chromosomes, cross-linking/mass spectrometry has been applied to investigate interactions between Okp1/ Ame1 heterodimer, which is part of the COMA complex, and CENP-A from Saccharomyces cerevisiae.
Project description:The chromatin at origins of replication is thought to influence DNA replication initiation in eukaryotic genomes. However, it remains unclear how the chromatin composition controls the firing of early-efficient (EE) or late-inefficient (LI) origins. Here, we used site-specific recombination and single-locus chromatin isolation to purify EE and LI replication origins in Saccharomyces cerevisiae. Using mass spectrometry, we define the histone modification landscape and identify the protein composition of native chromatin regions surrounding the EE and LI replication start sites. In addition to the known origin interactors, we find novel origin-associated factors, such as the kinetochore-associated Ask1/DASH complex. Strikingly, we show that Ask1 regulates the replication timing control of specific origins in yeast. Thus, our unbiased approach identifies functionally-relevant proteomes at single-copy loci and would be widely applicable to provide an in-depth quantitative characterization of histone modification and protein interaction networks of chromatin at any genomic locus of interest.
Project description:Histone modifications coupled to transcription elongation play important roles in regulating the accuracy and efficiency of gene expression. The mono-ubiquitylation of a conserved lysine in H2B (K123 in Saccharomyces cerevisiae and K120 in humans) occurs co-transcriptionally and is required for initiating a histone modification cascade on active genes. H2BK123 ubiquitylation (H2BK123ub) requires the RNA polymerase II (RNAPII)-associated Paf1 transcription elongation complex (Paf1C). Through its Histone Modification Domain (HMD), the Rtf1 subunit of Paf1C directly interacts with the ubiquitin conjugase Rad6, leading to the stimulation of H2BK123ub in vivo and in vitro. To understand the molecular mechanisms that specifically target Rad6 to its histone substrate, we identified the site of interaction for the HMD on Rad6. Using in vitro crosslinking followed by mass spectrometry, we localized the primary contact surface for the HMD to the highly conserved N-terminal helix of Rad6. Using a combination of genetic and biochemical experiments, we identified separation-of-function mutations in RAD6 that greatly impair H2BK123 ubiquitylation but not other Rad6 functions. Finally, by employing RNA-sequencing as a sensitive approach for comparing mutant phenotypes, we show that mutating either side of the proposed Rad6-HMD interface yields strikingly similar transcriptome profiles that extensively overlap with those of a mutant that lacks the site of ubiquitylation in H2B. Our results fit a model in which a specific interface between a transcription elongation factor and a ubiquitin conjugase guides substrate selection toward a highly conserved chromatin target during active gene expression.