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1.  Vacuolar H+-ATPase Works in Parallel with the HOG Pathway To Adapt Saccharomyces cerevisiae Cells to Osmotic Stress 
Eukaryotic Cell  2012;11(3):282-291.
Hyperosmotic stress activates an array of cellular detoxification mechanisms, including the high-osmolarity glycerol (HOG) pathway. We report here that vacuolar H+-ATPase (V-ATPase) activity helps provide osmotic tolerance in Saccharomyces cerevisiae. V-ATPase subunit genes exhibit complex haploinsufficiency interactions with HOG pathway components. vma mutants lacking V-ATPase function are sensitive to high concentrations of salt and exhibit Hog1p activation even at low salt concentrations, as demonstrated by phosphorylation of Hog1p, a shift in Hog1-green fluorescent protein localization, transcriptional activation of a subset of HOG pathway effectors, and transcriptional inhibition of parallel mitogen-activated protein kinase pathway targets. vma2Δ hog1Δ and vma3Δ pbs2Δ double mutants have a synthetic growth phenotype, poor salt tolerance, and an aberrant, hyper-elongated morphology on solid media, accompanied by activation of a filamentous response element-LacZ construct, indicating cross talk into the filamentous growth pathway. Vacuoles isolated from wild-type cells briefly exposed to salt show higher levels of V-ATPase activity, and Na+/H+ exchange in isolated vacuolar vesicles suggests a biochemical basis for the genetic interactions observed. V-ATPase activity is upregulated during salt stress by increasing assembly of the catalytic V1 sector with the membrane-bound Vo sector. Together, these data suggest that the V-ATPase acts in parallel with the HOG pathway in order to mediate salt detoxification.
PMCID: PMC3294445  PMID: 22210831
2.  Chromatin architectures at fission yeast transcriptional promoters and replication origins 
Nucleic Acids Research  2012;40(15):7176-7189.
We have used micrococcal nuclease (MNase) digestion followed by deep sequencing in order to obtain a higher resolution map than previously available of nucleosome positions in the fission yeast, Schizosaccharomyces pombe. Our data confirm an unusually short average nucleosome repeat length, ∼152 bp, in fission yeast and that transcriptional start sites (TSSs) are associated with nucleosome-depleted regions (NDRs), ordered nucleosome arrays downstream and less regularly spaced upstream nucleosomes. In addition, we found enrichments for associated function in four of eight groups of genes clustered according to chromatin configurations near TSSs. At replication origins, our data revealed asymmetric localization of pre-replication complex (pre-RC) proteins within large NDRs—a feature that is conserved in fission and budding yeast and is therefore likely to be conserved in other eukaryotic organisms.
PMCID: PMC3424540  PMID: 22573177
3.  Standardized collection of MNase-seq experiments enables unbiased dataset comparisons 
BMC Molecular Biology  2012;13:15.
The organization of eukaryotic DNA into chromatin has a strong influence on the accessibility and regulation of genetic information. The locations and occupancies of a principle component of chromatin, nucleosomes, are typically assayed through use of enzymatic digestion with micrococcal nuclease (MNase). MNase is an endo-exo nuclease that preferentially digests naked DNA and the DNA in linkers between nucleosomes, thus enriching for nucleosome-associated DNA. To determine nucleosome organization genome-wide, DNA remaining from MNase digestion is sequenced using high-throughput sequencing technologies (MNase-seq). Unfortunately, the results of MNase-seq can vary dramatically due to technical differences and this confounds comparisons between MNase-seq experiments, such as examining condition-dependent chromatin organizations.
In this study we use MNase digestion simulations to demonstrate how MNase-seq signals can vary for different nucleosome configuration when experiments are performed with different extents of MNase digestion. Signal variation in these simulations reveals an important DNA sampling bias that results from a neighborhood effect of MNase digestion techniques. The presence of this neighborhood effect ultimately confounds comparisons between different MNase-seq experiments. To address this issue we present a standardized chromatin preparation which controls for technical variance between MNase-based chromatin preparations and enables the collection of similarly sampled (matched) chromatin populations. Standardized preparation of chromatin includes a normalization step for DNA input into MNase digestions and close matching of the extent of digestion between each chromatin preparation using gel densitometry analysis. The protocol also includes directions for successful pairing with multiplex sequencing reactions.
We validated our method by comparing the experiment-to-experiment variation between biological replicates of chromatin preparations from S. cerevisiae. Results from our matched preparation consistently produced MNase-seq datasets that were more closely correlated than other unstandardized approaches. Additionally, we validated the ability of our approach at enabling accurate downstream comparisons of chromatin structures, by comparing the specificity of detecting Tup1-dependent chromatin remodeling events in comparisons between matched and un-matched wild-type and tup1Δ MNase-seq datasets. Our matched MNase-seq datasets demonstrated a significant reduction in non-specific (technical) differences between experiments and were able to maximize the detection of biologically-relevant (Tup1-dependent) changes in chromatin structure.
PMCID: PMC3464627  PMID: 22559821
Next-generation sequencing; High-throughput sequencing; Chromatin; Nucleosomes; Histones; MNase-seq; Micrococcal nuclease (MNase)
4.  Tup1 stabilizes promoter nucleosome positioning and occupancy at transcriptionally plastic genes 
Nucleic Acids Research  2011;39(20):8803-8819.
Despite technical advances, the future of chromatin mapping studies requires an ability to draw accurate comparisons between different chromatin states to enhance our understanding of genome biology. In this study, we used matched chromatin preparations to enable specific and accurate comparisons of Saccharomyces cerevisiae chromatin structures in the presence and absence of the co-repressor protein Tup1. Analysis of wild-type and tup1 Δ chromatin data sets revealed unique organizational themes relating to the function of Tup1. Regulatory regions bound by Tup1 assumed a distinct chromatin architecture composed of a wide nucleosome-depleted region, low occupancy/poorly positioned promoter nucleosomes, a larger number and wider distribution of transcription factor-binding sites and downstream genes with enhanced transcription plasticity. Regions of Tup1-dependent chromatin structure were defined for the first time across the entire yeast genome and are shown to strongly overlap with activity of the chromatin remodeler Isw2. Additionally, Tup1-dependent chromatin structures are shown to relate to distinct biological processes and transcriptional states of regulated genes, including Tup1 stabilization of Minus 1 and Minus 2 promoter nucleosomes at actively repressed genes. Together these results help to enhance our mechanistic understanding of Tup1 regulation of chromatin structure and gene expression.
PMCID: PMC3203618  PMID: 21785133
5.  The Stress Response Factors Yap6, Cin5, Phd1, and Skn7 Direct Targeting of the Conserved Co-Repressor Tup1-Ssn6 in S. cerevisiae 
PLoS ONE  2011;6(4):e19060.
Maintaining the proper expression of the transcriptome during development or in response to a changing environment requires a delicate balance between transcriptional regulators with activating and repressing functions. The budding yeast transcriptional co-repressor Tup1-Ssn6 is a model for studying similar repressor complexes in multicellular eukaryotes. Tup1-Ssn6 does not bind DNA directly, but is directed to individual promoters by one or more DNA-binding proteins, referred to as Tup1 recruiters. This functional architecture allows the Tup1-Ssn6 to modulate the expression of genes required for the response to a variety of cellular stresses. To understand the targeting or the Tup1-Ssn6 complex, we determined the genomic distribution of Tup1 and Ssn6 by ChIP-chip. We found that most loci bound by Tup1-Ssn6 could not be explained by co-occupancy with a known recruiting cofactor and that deletion of individual known Tup1 recruiters did not significantly alter the Tup1 binding profile. These observations suggest that new Tup1 recruiting proteins remain to be discovered and that Tup1 recruitment typically depends on multiple recruiting cofactors. To identify new recruiting proteins, we computationally screened for factors with binding patterns similar to the observed Tup1-Ssn6 genomic distribution. Four top candidates, Cin5, Skn7, Phd1, and Yap6, all known to be associated with stress response gene regulation, were experimentally confirmed to physically interact with Tup1 and/or Ssn6. Incorporating these new recruitment cofactors with previously characterized cofactors now explains the majority of Tup1 targeting across the genome, and expands our understanding of the mechanism by which Tup1-Ssn6 is directed to its targets.
PMCID: PMC3084262  PMID: 21552514

Results 1-5 (5)