Publications by authors named "Fred Winston"

58 Publications

The biochemical and genetic discovery of the SAGA complex.

Biochim Biophys Acta Gene Regul Mech 2021 02 16;1864(2):194669. Epub 2020 Dec 16.

Department of Cell and Developmental Biology, Penn Epigenetics Institute, Department of Biology, Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States of America.

One of the major advances in our understanding of gene regulation in eukaryotes was the discovery of factors that regulate transcription by controlling chromatin structure. Prominent among these discoveries was the demonstration that Gcn5 is a histone acetyltransferase, establishing a direct connection between transcriptional activation and histone acetylation. This breakthrough was soon followed by the purification of a protein complex that contains Gcn5, the SAGA complex. In this article, we review the early genetic and biochemical experiments that led to the discovery of SAGA and the elucidation of its multiple activities.
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http://dx.doi.org/10.1016/j.bbagrm.2020.194669DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854503PMC
February 2021

The role of FACT in managing chromatin: disruption, assembly, or repair?

Nucleic Acids Res 2020 12;48(21):11929-11941

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

FACT (FAcilitates Chromatin Transcription) has long been considered to be a transcription elongation factor whose ability to destabilize nucleosomes promotes RNAPII progression on chromatin templates. However, this is just one function of this histone chaperone, as FACT also functions in DNA replication. While broadly conserved among eukaryotes and essential for viability in many organisms, dependence on FACT varies widely, with some differentiated cells proliferating normally in its absence. It is therefore unclear what the core functions of FACT are, whether they differ in different circumstances, and what makes FACT essential in some situations but not others. Here, we review recent advances and propose a unifying model for FACT activity. By analogy to DNA repair, we propose that the ability of FACT to both destabilize and assemble nucleosomes allows it to monitor and restore nucleosome integrity as part of a system of chromatin repair, in which disruptions in the packaging of DNA are sensed and returned to their normal state. The requirement for FACT then depends on the level of chromatin disruption occurring in the cell, and the cell's ability to tolerate packaging defects. The role of FACT in transcription would then be just one facet of a broader system for maintaining chromatin integrity.
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http://dx.doi.org/10.1093/nar/gkaa912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7708052PMC
December 2020

The conserved elongation factor Spn1 is required for normal transcription, histone modifications, and splicing in Saccharomyces cerevisiae.

Nucleic Acids Res 2020 10;48(18):10241-10258

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

Spn1/Iws1 is a conserved protein involved in transcription and chromatin dynamics, yet its general in vivo requirement for these functions is unknown. Using a Spn1 depletion system in Saccharomyces cerevisiae, we demonstrate that Spn1 broadly influences several aspects of gene expression on a genome-wide scale. We show that Spn1 is globally required for normal mRNA levels and for normal splicing of ribosomal protein transcripts. Furthermore, Spn1 maintains the localization of H3K36 and H3K4 methylation across the genome and is required for normal histone levels at highly expressed genes. Finally, we show that the association of Spn1 with the transcription machinery is strongly dependent on its binding partner, Spt6, while the association of Spt6 and Set2 with transcribed regions is partially dependent on Spn1. Taken together, our results show that Spn1 affects multiple aspects of gene expression and provide additional evidence that it functions as a histone chaperone in vivo.
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http://dx.doi.org/10.1093/nar/gkaa745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544207PMC
October 2020

Whole-Genome Sequencing of Yeast Cells.

Curr Protoc Mol Biol 2019 09;128(1):e103

Department of Genetics, Harvard Medical School, Boston, Massachusetts.

The budding yeast, Saccharomyces cerevisiae, has been widely used for genetic studies of fundamental cellular functions. The isolation and analysis of yeast mutants is a commonly used and powerful technique to identify the genes that are involved in a process of interest. Furthermore, natural genetic variation among wild yeast strains has been studied for analysis of polygenic traits by quantitative trait loci mapping. Whole-genome sequencing, often combined with bulk segregant analysis, is a powerful technique that helps determine the identity of mutations causing a phenotype. Here, we describe protocols for the construction of libraries for S. cerevisiae whole-genome sequencing. We also present a bioinformatic pipeline to determine the genetic variants in a yeast strain using whole-genome sequencing data. This pipeline can also be used for analyzing Schizosaccharomyces pombe mutants. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Generation of haploid spores for bulk segregant analysis Basic Protocol 2: Extraction of genomic DNA from yeast cells Basic Protocol 3: Shearing of genomic DNA for library preparation Basic Protocol 4: Construction and amplification of DNA libraries Support Protocol 1: Annealing oligonucleotides for forming Y-adapters Support Protocol 2: Size selection and cleanup using SPRI beads Basic Protocol 5: Identification of genomic variants from sequencing data.
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http://dx.doi.org/10.1002/cpmb.103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6741438PMC
September 2019

Auxin-Inducible Degron System for Depletion of Proteins in Saccharomyces cerevisiae.

Curr Protoc Mol Biol 2019 09;128(1):e104

Department of Genetics, Harvard Medical School, Boston, Massachusetts.

The auxin-inducible degron (AID) is a powerful tool that is used for depletion of proteins to study their function in vivo. This method can conditionally induce the degradation of any protein by the proteasome simply by the addition of the plant hormone auxin. This approach is particularly valuable to study the function of essential proteins. The protocols provided here describe the steps to construct the necessary strains and to optimize auxin-inducible depletion in Saccharomyces cerevisiae. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Construction of TIR1-expressing strains by transformation Basic Protocol 2: Tagging a yeast protein of interest with an auxin-inducible degron Support Protocol: Construction of depletion strains by genetic crosses Basic Protocol 3: Optimization for depletion of the auxin-inducible-degron-tagged protein.
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http://dx.doi.org/10.1002/cpmb.104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6741457PMC
September 2019

A conserved genetic interaction between Spt6 and Set2 regulates H3K36 methylation.

Nucleic Acids Res 2019 05;47(8):3888-3903

Department of Genetics, Harvard Medical School, Boston, MA, USA 02115.

The transcription elongation factor Spt6 and the H3K36 methyltransferase Set2 are both required for H3K36 methylation and transcriptional fidelity in Saccharomyces cerevisiae. However, the nature of the requirement for Spt6 has remained elusive. By selecting for suppressors of a transcriptional defect in an spt6 mutant, we have isolated several highly clustered, dominant SET2 mutations (SET2sup mutations) in a region encoding a proposed autoinhibitory domain. SET2sup mutations suppress the H3K36 methylation defect in the spt6 mutant, as well as in other mutants that impair H3K36 methylation. We also show that SET2sup mutations overcome the requirement for certain Set2 domains for H3K36 methylation. In vivo, SET2sup mutants have elevated levels of H3K36 methylation and the purified Set2sup mutant protein has greater enzymatic activityin vitro. ChIP-seq studies demonstrate that the H3K36 methylation defect in the spt6 mutant, as well as its suppression by a SET2sup mutation, occurs at a step following the recruitment of Set2 to chromatin. Other experiments show that a similar genetic relationship between Spt6 and Set2 exists in Schizosaccharomyces pombe. Taken together, our results suggest a conserved mechanism by which the Set2 autoinhibitory domain requires multiple Set2 interactions to ensure that H3K36 methylation occurs specifically on actively transcribed chromatin.
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http://dx.doi.org/10.1093/nar/gkz119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486648PMC
May 2019

Spt6 Is Required for the Fidelity of Promoter Selection.

Mol Cell 2018 11 11;72(4):687-699.e6. Epub 2018 Oct 11.

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Spt6 is a conserved factor that controls transcription and chromatin structure across the genome. Although Spt6 is viewed as an elongation factor, spt6 mutations in Saccharomyces cerevisiae allow elevated levels of transcripts from within coding regions, suggesting that Spt6 also controls initiation. To address the requirements for Spt6 in transcription and chromatin structure, we have combined four genome-wide approaches. Our results demonstrate that Spt6 represses transcription initiation at thousands of intragenic promoters. We characterize these intragenic promoters and find sequence features conserved with genic promoters. Finally, we show that Spt6 also regulates transcription initiation at most genic promoters and propose a model of initiation site competition to account for this. Together, our results demonstrate that Spt6 controls the fidelity of transcription initiation throughout the genome.
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http://dx.doi.org/10.1016/j.molcel.2018.09.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6239972PMC
November 2018

Spt5 Plays Vital Roles in the Control of Sense and Antisense Transcription Elongation.

Mol Cell 2017 Apr 30;66(1):77-88.e5. Epub 2017 Mar 30.

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Spt5 is an essential and conserved factor that functions in transcription and co-transcriptional processes. However, many aspects of the requirement for Spt5 in transcription are poorly understood. We have analyzed the consequences of Spt5 depletion in Schizosaccharomyces pombe using four genome-wide approaches. Our results demonstrate that Spt5 is crucial for a normal rate of RNA synthesis and distribution of RNAPII over transcription units. In the absence of Spt5, RNAPII localization changes dramatically, with reduced levels and a relative accumulation over the first ∼500 bp, suggesting that Spt5 is required for transcription past a barrier. Spt5 depletion also results in widespread antisense transcription initiating within this barrier region. Deletions of this region alter the distribution of RNAPII on the sense strand, suggesting that the barrier observed after Spt5 depletion is normally a site at which Spt5 stimulates elongation. Our results reveal a global requirement for Spt5 in transcription elongation.
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http://dx.doi.org/10.1016/j.molcel.2017.02.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5394798PMC
April 2017

Back to the Future: Mutant Hunts Are Still the Way To Go.

Genetics 2016 07;203(3):1007-10

Department of Molecular and Cell Biology, University of California, Berkeley, California 94720

Innumerable breakthroughs in many fundamental areas of biology have come from unbiased screens and selections for mutations, either across the genome or within a gene. However, long-standing hurdles to key elements of mutant hunts (mutagenesis, phenotypic characterization, and linkage of phenotype to genotype) have limited the organisms in which mutant hunts could be used. These hurdles are now being eliminated by an explosion of new technologies. We believe that a renewed emphasis on unbiased mutant hunts, in both existing model systems and in those where genetics is just now becoming feasible, will lead to new seminal discoveries and surprises.
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http://dx.doi.org/10.1534/genetics.115.180596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4937489PMC
July 2016

Analysis of Polygenic Mutants Suggests a Role for Mediator in Regulating Transcriptional Activation Distance in Saccharomyces cerevisiae.

Genetics 2015 Oct 17;201(2):599-612. Epub 2015 Aug 17.

Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115

Studies of natural populations of many organisms have shown that traits are often complex, caused by contributions of mutations in multiple genes. In contrast, genetic studies in the laboratory primarily focus on studying the phenotypes caused by mutations in a single gene. However, the single mutation approach may be limited with respect to the breadth and degree of new phenotypes that can be found. We have taken the approach of isolating complex, or polygenic mutants in the lab to study the regulation of transcriptional activation distance in yeast. While most aspects of eukaryotic transcription are conserved from yeast to human, transcriptional activation distance is not. In Saccharomyces cerevisiae, the upstream activating sequence (UAS) is generally found within 450 base pairs of the transcription start site (TSS) and when the UAS is moved too far away, activation no longer occurs. In contrast, metazoan enhancers can activate from as far as several hundred kilobases from the TSS. Previously, we identified single mutations that allow transcription activation to occur at a greater-than-normal distance from the GAL1 UAS. As the single mutant phenotypes were weak, we have now isolated polygenic mutants that possess strong long-distance phenotypes. By identification of the causative mutations we have accounted for most of the heritability of the phenotype in each strain and have provided evidence that the Mediator coactivator complex plays both positive and negative roles in the regulation of transcription activation distance.
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http://dx.doi.org/10.1534/genetics.115.181164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4596672PMC
October 2015

Spt6 regulates intragenic and antisense transcription, nucleosome positioning, and histone modifications genome-wide in fission yeast.

Mol Cell Biol 2013 Dec 7;33(24):4779-92. Epub 2013 Oct 7.

Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

Spt6 is a highly conserved histone chaperone that interacts directly with both RNA polymerase II and histones to regulate gene expression. To gain a comprehensive understanding of the roles of Spt6, we performed genome-wide analyses of transcription, chromatin structure, and histone modifications in a Schizosaccharomyces pombe spt6 mutant. Our results demonstrate dramatic changes to transcription and chromatin structure in the mutant, including elevated antisense transcripts at >70% of all genes and general loss of the +1 nucleosome. Furthermore, Spt6 is required for marks associated with active transcription, including trimethylation of histone H3 on lysine 4, previously observed in humans but not Saccharomyces cerevisiae, and lysine 36. Taken together, our results indicate that Spt6 is critical for the accuracy of transcription and the integrity of chromatin, likely via its direct interactions with RNA polymerase II and histones.
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http://dx.doi.org/10.1128/MCB.01068-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3889546PMC
December 2013

Conserved regulators of nucleolar size revealed by global phenotypic analyses.

Sci Signal 2013 Aug 20;6(289):ra70. Epub 2013 Aug 20.

1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

Regulation of cell growth is a fundamental process in development and disease that integrates a vast array of extra- and intracellular information. A central player in this process is RNA polymerase I (Pol I), which transcribes ribosomal RNA (rRNA) genes in the nucleolus. Rapidly growing cancer cells are characterized by increased Pol I-mediated transcription and, consequently, nucleolar hypertrophy. To map the genetic network underlying the regulation of nucleolar size and of Pol I-mediated transcription, we performed comparative, genome-wide loss-of-function analyses of nucleolar size in Saccharomyces cerevisiae and Drosophila melanogaster coupled with mass spectrometry-based analyses of the ribosomal DNA (rDNA) promoter. With this approach, we identified a set of conserved and nonconserved molecular complexes that control nucleolar size. Furthermore, we characterized a direct role of the histone information regulator (HIR) complex in repressing rRNA transcription in yeast. Our study provides a full-genome, cross-species analysis of a nuclear subcompartment and shows that this approach can identify conserved molecular modules.
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http://dx.doi.org/10.1126/scisignal.2004145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3964804PMC
August 2013

Cell-cycle perturbations suppress the slow-growth defect of spt10Δ mutants in Saccharomyces cerevisiae.

G3 (Bethesda) 2013 Mar 1;3(3):573-83. Epub 2013 Mar 1.

Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.

Spt10 is a putative acetyltransferase of Saccharomyces cerevisiae that directly activates the transcription of histone genes. Deletion of SPT10 causes a severe slow growth phenotype, showing that Spt10 is critical for normal cell division. To gain insight into the function of Spt10, we identified mutations that impair or improve the growth of spt10 null (spt10Δ) mutants. Mutations that cause lethality in combination with spt10Δ include particular components of the SAGA complex as well as asf1Δ and hir1Δ. Partial suppressors of the spt10Δ growth defect include mutations that perturb cell-cycle progression through the G1/S transition, S phase, and G2/M. Consistent with these results, slowing of cell-cycle progression by treatment with hydroxyurea or growth on medium containing glycerol as the carbon source also partially suppresses the spt10Δ slow-growth defect. In addition, mutations that impair the Lsm1-7-Pat1 complex, which regulates decapping of polyadenylated mRNAs, also partially suppress the spt10Δ growth defect. Interestingly, suppression of the spt10Δ growth defect is not accompanied by a restoration of normal histone mRNA levels. These findings suggest that Spt10 has multiple roles during cell division.
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http://dx.doi.org/10.1534/g3.112.005389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3583463PMC
March 2013

The Schizosaccharomyces pombe inv1+ regulatory region is unusually large and contains redundant cis-acting elements that function in a SAGA- and Swi/Snf-dependent fashion.

Eukaryot Cell 2012 Aug 15;11(8):1067-74. Epub 2012 Jun 15.

Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

The Schizosaccharomyces pombe inv1(+) gene encodes invertase, the enzyme required for hydrolysis of sucrose and raffinose. Transcription of inv1(+) is regulated by glucose levels, with transcription tightly repressed in high glucose and strongly induced in low glucose. To understand this regulation, we have analyzed the inv1(+) cis-regulatory region and the requirement for the trans-acting coactivators SAGA and Swi/Snf. Surprisingly, deletion of the entire 1-kilobase intergenic region between the inv1(+) TATA element and the upstream open reading frame SPCC191.10 does not significantly alter regulation of inv1(+) transcription. However, a longer deletion that extends through SPCC191.10 abolishes inv1(+) induction in low glucose. Additional analysis demonstrates that there are multiple, redundant regulatory regions spread over 1.5 kb 5' of inv1(+), including within SPCC191.10, that can confer glucose-mediated transcriptional regulation to inv1(+). Furthermore, SPCC191.10 can regulate inv1(+) transcription in an orientation-independent fashion and from a distance as great as 3 kb. With respect to trans-acting factors, both SAGA and Swi/Snf are recruited to SPCC191.10 and to other locations in the large inv1(+) regulatory region in a glucose-dependent fashion, and both are required for inv1(+) derepression. Taken together, these results demonstrate that inv1(+) regulation in S. pombe occurs via the use of multiple regulatory elements and that activation can occur over a great distance, even from elements within other open reading frames.
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http://dx.doi.org/10.1128/EC.00141-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3416058PMC
August 2012

A Screen for Germination Mutants in Saccharomyces cerevisiae.

G3 (Bethesda) 2011 Jul 1;1(2):143-9. Epub 2011 Jul 1.

Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115.

Spore germination in Saccharomyces cerevisiae is a process in which a quiescent cell begins to divide. During germination, the cell undergoes dramatic changes in cell wall and membrane composition, as well as in gene expression. To understand germination in greater detail, we screened the S. cerevisiae deletion set for germination mutants. Our results identified two genes, TRF4 and ERG6, that are required for normal germination on solid media. TRF4 is a member of the TRAMP complex that, together with the exosome, degrades RNA polymerase II transcripts. ERG6 encodes a key step in ergosterol biosynthesis. Taken together, these results demonstrate the complex nature of germination and two genes important in the process.
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http://dx.doi.org/10.1534/g3.111.000323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3276131PMC
July 2011

Chromatin and transcription in yeast.

Genetics 2012 Feb;190(2):351-87

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

Understanding the mechanisms by which chromatin structure controls eukaryotic transcription has been an intense area of investigation for the past 25 years. Many of the key discoveries that created the foundation for this field came from studies of Saccharomyces cerevisiae, including the discovery of the role of chromatin in transcriptional silencing, as well as the discovery of chromatin-remodeling factors and histone modification activities. Since that time, studies in yeast have continued to contribute in leading ways. This review article summarizes the large body of yeast studies in this field.
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http://dx.doi.org/10.1534/genetics.111.132266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3276623PMC
February 2012

Spt6 is required for heterochromatic silencing in the fission yeast Schizosaccharomyces pombe.

Mol Cell Biol 2011 Oct 15;31(20):4193-204. Epub 2011 Aug 15.

Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.

Spt6 is a conserved factor, critically required for several transcription- and chromatin-related processes. We now show that Spt6 and its binding partner, Iws1, are required for heterochromatic silencing in Schizosaccharomyces pombe. Our studies demonstrate that Spt6 is required for silencing of all heterochromatic loci and that an spt6 mutant has an unusual combination of heterochromatic phenotypes compared to previously studied silencing mutants. Unexpectedly, we find normal nucleosome positioning over heterochromatin and normal levels of histone H3K9 dimethylation at the endogenous pericentric repeats. However, we also find greatly reduced levels of H3K9 trimethylation, elevated levels of H3K14 acetylation, reduced recruitment of several silencing factors, and defects in heterochromatin spreading. Our evidence suggests that Spt6 plays a role at both the transcriptional and posttranscriptional levels; in an spt6 mutant, RNA polymerase II (RNAPII) occupancy at the pericentric regions is only modestly increased, while production of small interfering RNAs (siRNAs) is lost. Taken together, our results suggest that Spt6 is required for multiple steps in heterochromatic silencing by controlling chromatin, transcriptional, and posttranscriptional processes.
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http://dx.doi.org/10.1128/MCB.05568-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3187285PMC
October 2011

Tra1 has specific regulatory roles, rather than global functions, within the SAGA co-activator complex.

EMBO J 2011 Jun 3;30(14):2843-52. Epub 2011 Jun 3.

Department of Genetics, Harvard Medical School, Boston, MA, USA.

The SAGA complex is a conserved, multifunctional co-activator that has broad roles in eukaryotic transcription. Previous studies suggested that Tra1, the largest SAGA component, is required either for SAGA assembly or for SAGA recruitment by DNA-bound transcriptional activators. In contrast to Saccharomyces cerevisiae and mouse, a tra1Δ mutant is viable in Schizosaccharomyces pombe, allowing us to test these issues in vivo. We find that, in a tra1Δ mutant, SAGA assembles and is recruited to some, but not all, promoters. Consistent with these findings, Tra1 regulates the expression of only a subset of SAGA-dependent genes. We previously reported that the SAGA subunits Gcn5 and Spt8 have opposing regulatory roles during S. pombe sexual differentiation. We show here that, like Gcn5, Tra1 represses this pathway, although by a distinct mechanism. Thus, our study reveals that Tra1 has specific regulatory roles, rather than global functions, within SAGA.
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http://dx.doi.org/10.1038/emboj.2011.181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3160243PMC
June 2011

The Hog1 mitogen-activated protein kinase mediates a hypoxic response in Saccharomyces cerevisiae.

Genetics 2011 Jun 5;188(2):325-38. Epub 2011 Apr 5.

Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.

We have studied hypoxic induction of transcription by studying the seripauperin (PAU) genes of Saccharomyces cerevisiae. Previous studies showed that PAU induction requires the depletion of heme and is dependent upon the transcription factor Upc2. We have now identified additional factors required for PAU induction during hypoxia, including Hog1, a mitogen-activated protein kinase (MAPK) whose signaling pathway originates at the membrane. Our results have led to a model in which heme and ergosterol depletion alters membrane fluidity, thereby activating Hog1 for hypoxic induction. Hypoxic activation of Hog1 is distinct from its previously characterized response to osmotic stress, as the two conditions cause different transcriptional consequences. Furthermore, Hog1-dependent hypoxic activation is independent of the S. cerevisiae general stress response. In addition to Hog1, specific components of the SAGA coactivator complex, including Spt20 and Sgf73, are also required for PAU induction. Interestingly, the mammalian ortholog of Spt20, p38IP, has been previously shown to interact with the mammalian ortholog of Hog1, p38. Taken together, our results have uncovered a previously unknown hypoxic-response pathway that may be conserved throughout eukaryotes.
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http://dx.doi.org/10.1534/genetics.111.128322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3122313PMC
June 2011

Control of chromatin structure by spt6: different consequences in coding and regulatory regions.

Mol Cell Biol 2011 Feb 22;31(3):531-41. Epub 2010 Nov 22.

Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.

Spt6 is a highly conserved factor required for normal transcription and chromatin structure. To gain new insights into the roles of Spt6, we measured nucleosome occupancy along Saccharomyces cerevisiae chromosome III in an spt6 mutant. We found that the level of nucleosomes is greatly reduced across some, but not all, coding regions in an spt6 mutant, with nucleosome loss preferentially occurring over highly transcribed genes. This result provides strong support for recent studies that have suggested that transcription at low levels does not displace nucleosomes, while transcription at high levels does, and adds the idea that Spt6 is required for restoration of nucleosomes at the highly transcribed genes. Unexpectedly, our studies have also suggested that the spt6 effects on nucleosome levels across coding regions do not cause the spt6 effects on mRNA levels, suggesting that the role of Spt6 across coding regions is separate from its role in transcriptional regulation. In the case of the CHA1 gene, regulation by Spt6 likely occurs by controlling the position of the +1 nucleosome. These results, along with previous studies, suggest that Spt6 regulates transcription by controlling chromatin structure over regulatory regions, and its effects on nucleosome levels over coding regions likely serve an independent function.
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http://dx.doi.org/10.1128/MCB.01068-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3028613PMC
February 2011

The structure of an Iws1/Spt6 complex reveals an interaction domain conserved in TFIIS, Elongin A and Med26.

EMBO J 2010 Dec 5;29(23):3979-91. Epub 2010 Nov 5.

Département de Biologie et Génomique Structurales, IGBMC (Institut de Génétique et Biologie Moléculaire et Cellulaire), UDS, CNRS, INSERM, Illkirch Cedex, France.

Binding of elongation factor Spt6 to Iws1 provides an effective means for coupling eukaryotic mRNA synthesis, chromatin remodelling and mRNA export. We show that an N-terminal region of Spt6 (Spt6N) is responsible for interaction with Iws1. The crystallographic structures of Encephalitozoon cuniculi Iws1 and the Iws1/Spt6N complex reveal two conserved binding subdomains in Iws1. The first subdomain (one HEAT repeat; HEAT subdomain) is a putative phosphoprotein-binding site most likely involved in an Spt6-independent function of Iws1. The second subdomain (two ARM repeats; ARM subdomain) specifically recognizes a bipartite N-terminal region of Spt6. Mutations that alter this region of Spt6 cause severe phenotypes in vivo. Importantly, the ARM subdomain of Iws1 is conserved in several transcription factors, including TFIIS, Elongin A and Med26. We show that the homologous region in yeast TFIIS enables this factor to interact with SAGA and the Mediator subunits Spt8 and Med13, suggesting the molecular basis for TFIIS recruitment at promoters. Taken together, our results provide new structural information about the Iws1/Spt6 complex and reveal a novel interaction domain used for the formation of transcription networks.
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http://dx.doi.org/10.1038/emboj.2010.272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3020637PMC
December 2010

Spt10 and Spt21 are required for transcriptional silencing in Saccharomyces cerevisiae.

Eukaryot Cell 2011 Jan 5;10(1):118-29. Epub 2010 Nov 5.

Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.

In Saccharomyces cerevisiae, transcriptional silencing occurs at three classes of genomic regions: near the telomeres, at the silent mating type loci, and within the ribosomal DNA (rDNA) repeats. In all three cases, silencing depends upon several factors, including specific types of histone modifications. In this work we have investigated the roles in silencing for Spt10 and Spt21, two proteins previously shown to control transcription of particular histone genes. Building on a recent study showing that Spt10 is required for telomeric silencing, our results show that in both spt10 and spt21 mutants, silencing is reduced near telomeres and at HMLα, while it is increased at the rDNA. Both spt10 and spt21 mutations cause modest effects on Sir protein recruitment and histone modifications at telomeric regions, and they cause significant changes in chromatin structure, as judged by its accessibility to dam methylase. These silencing and chromatin changes are not seen upon deletion of HTA2-HTB2, the primary histone locus regulated by Spt10 and Spt21. These results suggest that Spt10 and Spt21 control silencing in S. cerevisiae by altering chromatin structure through roles beyond the control of histone gene expression.
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http://dx.doi.org/10.1128/EC.00246-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3019801PMC
January 2011

Noncanonical tandem SH2 enables interaction of elongation factor Spt6 with RNA polymerase II.

J Biol Chem 2010 Dec 6;285(49):38389-98. Epub 2010 Oct 6.

Département de Biologie et Génomique Structurales, Institut de Génétique et Biologie Moléculaire et Cellulaire, Université de Strasbourg, CNRS, INSERM, 1 rue Laurent Fries, B.P. 10142, 67404 Illkirch Cedex, France.

Src homology 2 (SH2) domains are mostly found in multicellular organisms where they recognize phosphotyrosine-containing signaling proteins. Spt6, a conserved transcription factor and putative histone chaperone, contains a C-terminal SH2 domain conserved from yeast to human. In mammals, this SH2 domain recognizes phosphoserines rather than phosphotyrosines and is essential for the recruitment of Spt6 by elongating RNA polymerase II (RNAPII), enabling Spt6 to participate in the coupling of transcription elongation, chromatin modulation, and mRNA export. We have determined the structure of the entire Spt6 C-terminal region from Antonospora locustae, revealing the presence of two highly conserved tandem SH2 domains rather than a single SH2 domain. Although the first SH2 domain has a canonical organization, the second SH2 domain is highly noncanonical and appears to be unique in the SH2 family. However, both SH2 domains have phosphate-binding determinants. Our biochemical and genetic data demonstrate that the complete tandem, but not the individual SH2 domains, are necessary and sufficient for the interaction of Spt6 with RNAPII and are important for Spt6 function in vivo. Furthermore, our data suggest that binding of RNAPII to the Spt6 tandem SH2 is more extensive than the mere recognition of a doubly phosphorylated C-terminal domain peptide by the tandem SH2. Taken together, our results show that Spt6 interaction with RNAPII via a novel arrangement of canonical and noncanonical SH2 domains is crucial for Spt6 function in vivo.
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http://dx.doi.org/10.1074/jbc.M110.146696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2992272PMC
December 2010

Alterations in DNA replication and histone levels promote histone gene amplification in Saccharomyces cerevisiae.

Genetics 2010 Apr 5;184(4):985-97. Epub 2010 Feb 5.

Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.

Gene amplification, a process that increases the copy number of a gene or a genomic region to two or more, is utilized by many organisms in response to environmental stress or decreased levels of a gene product. Our previous studies in Saccharomyces cerevisiae identified the amplification of a histone H2A-H2B gene pair, HTA2-HTB2, in response to the deletion of the other H2A-H2B gene pair, HTA1-HTB1. This amplification arises from a recombination event between two flanking Ty1 elements to form a new, stable circular chromosome and occurs at a frequency higher than has been observed for other Ty1-Ty1 recombination events. To understand the regulation of this amplification event, we screened the S. cerevisiae nonessential deletion set for mutations that alter the amplification frequency. Among the deletions that increase HTA2-HTB2 amplification frequency, we identified those that either decrease DNA replication fork progression (rrm3Delta, dpb3Delta, dpb4Delta, and clb5Delta) or that reduce histone H3-H4 levels (hht2-hhf2Delta). These two classes are related because reduced histone H3-H4 levels increase replication fork pauses, and impaired replication forks cause a reduction in histone levels. Consistent with our mutant screen, we found that the introduction of DNA replication stress by hydroxyurea induces the HTA2-HTB2 amplification event. Taken together, our results suggest that either reduced histone levels or slowed replication forks stimulate the HTA2-HTB2 amplification event, contributing to the restoration of normal chromatin structure.
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http://dx.doi.org/10.1534/genetics.109.113662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2865932PMC
April 2010

A transcription switch toggled by noncoding RNAs.

Authors:
Fred Winston

Proc Natl Acad Sci U S A 2009 Oct 21;106(43):18049-50. Epub 2009 Oct 21.

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

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http://dx.doi.org/10.1073/pnas.0910272106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2775307PMC
October 2009

The S. pombe SAGA complex controls the switch from proliferation to sexual differentiation through the opposing roles of its subunits Gcn5 and Spt8.

Genes Dev 2008 Nov;22(22):3184-95

Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.

The SAGA complex is a conserved multifunctional coactivator known to play broad roles in eukaryotic transcription. To gain new insights into its functions, we performed biochemical and genetic analyses of SAGA in the fission yeast, Schizosaccharomyces pombe. Purification of the S. pombe SAGA complex showed that its subunit composition is identical to that of Saccharomyces cerevisiae. Analysis of S. pombe SAGA mutants revealed that SAGA has two opposing roles regulating sexual differentiation. First, in nutrient-rich conditions, the SAGA histone acetyltransferase Gcn5 represses ste11(+), which encodes the master regulator of the mating pathway. In contrast, the SAGA subunit Spt8 is required for the induction of ste11(+) upon nutrient starvation. Chromatin immunoprecipitation experiments suggest that these regulatory effects are direct, as SAGA is physically associated with the ste11(+) promoter independent of nutrient levels. Genetic tests suggest that nutrient levels do cause a switch in SAGA function, as spt8Delta suppresses gcn5Delta with respect to ste11(+) derepression in rich medium, whereas the opposite relationship, gcn5Delta suppression of spt8Delta, occurs during starvation. Thus, SAGA plays distinct roles in the control of the switch from proliferation to differentiation in S. pombe through the dynamic and opposing activities of Gcn5 and Spt8.
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http://dx.doi.org/10.1101/gad.1719908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2593614PMC
November 2008

Chromatin- and transcription-related factors repress transcription from within coding regions throughout the Saccharomyces cerevisiae genome.

PLoS Biol 2008 Nov;6(11):e277

Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

Previous studies in Saccharomyces cerevisiae have demonstrated that cryptic promoters within coding regions activate transcription in particular mutants. We have performed a comprehensive analysis of cryptic transcription in order to identify factors that normally repress cryptic promoters, to determine the amount of cryptic transcription genome-wide, and to study the potential for expression of genetic information by cryptic transcription. Our results show that a large number of factors that control chromatin structure and transcription are required to repress cryptic transcription from at least 1,000 locations across the S. cerevisiae genome. Two results suggest that some cryptic transcripts are translated. First, as expected, many cryptic transcripts contain an ATG and an open reading frame of at least 100 codons. Second, several cryptic transcripts are translated into proteins. Furthermore, a subset of cryptic transcripts tested is transiently induced in wild-type cells following a nutritional shift, suggesting a possible physiological role in response to a change in growth conditions. Taken together, our results demonstrate that, during normal growth, the global integrity of gene expression is maintained by a wide range of factors and suggest that, under altered genetic or physiological conditions, the expression of alternative genetic information may occur.
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http://dx.doi.org/10.1371/journal.pbio.0060277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2581627PMC
November 2008

Fission yeast SWI/SNF and RSC complexes show compositional and functional differences from budding yeast.

Nat Struct Mol Biol 2008 Aug 11;15(8):873-80. Epub 2008 Jul 11.

Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.

SWI/SNF chromatin-remodeling complexes have crucial roles in transcription and other chromatin-related processes. The analysis of the two members of this class in Saccharomyces cerevisiae, SWI/SNF and RSC, has heavily contributed to our understanding of these complexes. To understand the in vivo functions of SWI/SNF and RSC in an evolutionarily distant organism, we have characterized these complexes in Schizosaccharomyces pombe. Although core components are conserved between the two yeasts, the compositions of S. pombe SWI/SNF and RSC differ from their S. cerevisiae counterparts and in some ways are more similar to metazoan complexes. Furthermore, several of the conserved proteins, including actin-like proteins, are markedly different between the two yeasts with respect to their requirement for viability. Finally, phenotypic and microarray analyses identified widespread requirements for SWI/SNF and RSC on transcription including strong evidence that SWI/SNF directly represses iron-transport genes.
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http://dx.doi.org/10.1038/nsmb.1452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2559950PMC
August 2008

EMS and UV mutagenesis in yeast.

Authors:
Fred Winston

Curr Protoc Mol Biol 2008 Apr;Chapter 13:Unit 13.3B

Harvard Medical School, Boston, Massachusetts, USA.

Many fundamental biological processes have been elucidated by the isolation and analysis of mutants that are defective in such processes. Therefore, the methods to generate mutants are of great importance in model organisms. This unit describes two protocols for mutagenesis of yeast-using ethyl methanesulfate (EMS) and ultraviolet (UV) light. Each of these methods has been used successfully for many years.
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http://dx.doi.org/10.1002/0471142727.mb1303bs82DOI Listing
April 2008