Evidence based advocacy.
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Academic Medical Centers↗
Biomedical subjects
F Winston
Publications and source records attributed to F Winston.
Histone H3 lysine 4 methylation is mediated by Set1 and required for cell growth and rDNA silencing in Saccharomyces cerevisiae.
Histone methylation is known to be associated with both transcriptionally active and repressive chromatin states. Recent studies have identified SET domain-containing proteins such as SUV39H1 and Clr4 as mediators of H3 lysine 9 (Lys9) methylation and heterochromatin formation. Interestingly, H3 Lys9 methylation is not observed from bulk histones isolated from asynchronous populations of Saccharomyces cerevisiae or Tetrahymena thermophila. In contrast, H3 lysine 4 (Lys4) methylation is a predominant modification in these smaller eukaryotes. To identify the responsible methyltransferase(s) and to gain insight into the function of H3 Lys4 methylation, we have developed a histone H3 Lys4 methyl-specific antiserum. With this antiserum, we show that deletion of SET1, but not of other putative SET domain-containing genes, in S. cerevisiae, results in the complete abolishment of H3 Lys4 methylation in vivo. Furthermore, loss of H3 Lys4 methylation in a set1 Delta strain can be rescued by SET1. Analysis of histone H3 mutations at Lys4 revealed a slow-growth defect similar to a set1 Delta strain. Chromatin immunoprecipitation assays show that H3 Lys4 methylation is present at the rDNA locus and that Set1-mediated H3 Lys4 methylation is required for repression of RNA polymerase II transcription within rDNA. Taken together, these data suggest that Set1-mediated H3 Lys4 methylation is required for normal cell growth and transcriptional silencing.
Animals↗
The S. cerevisiae SAGA complex functions in vivo as a coactivator for transcriptional activation by Gal4.
Previous studies demonstrated that the SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex facilitates the binding of TATA-binding protein (TBP) during transcriptional activation of the GAL1 gene of Saccharomyces cerevisiae. TBP binding was shown to require the SAGA components Spt3 and Spt20/Ada5, but not the SAGA component Gcn5. We have now examined whether SAGA is directly required as a coactivator in vivo by using chromatin immunoprecipitation analysis. Our results demonstrate that SAGA is physically recruited in vivo to the upstream activation sequence (UAS) regions of the galactose-inducible GAL genes. This recruitment is dependent on both induction by galactose and the Gal4 activation domain. Furthermore, we demonstrate that another well-characterized activator, Gal4-VP16, also recruits SAGA in vivo. Finally, we provide evidence that a specific interaction between Spt3 and TBP in vivo is important for Gal4 transcriptional activation at a step after SAGA recruitment. These results, taken together with previous studies, demonstrate a dependent pathway for the recruitment of TBP to GAL gene promoters consisting of the recruitment of SAGA by Gal4 and the subsequent recruitment of TBP by SAGA.
Acetyltransferases↗
NRG1 is required for glucose repression of the SUC2 and GAL genes of Saccharomyces cerevisiae.
BACKGROUND: Glucose repression of transcription in the yeast, Saccharomyces cerevisiae, has been shown to be controlled by several factors, including two repressors called Mig1 and Mig2. Past results suggest that other repressors may be involved in glucose repression. RESULTS: By a screen for factors that control transcription of the glucose-repressible SUC2 gene of S. cerevisiae, the NRG1 gene was identified. Analysis of an nrg1Delta mutant has demonstrated that mRNA levels are elevated at both the SUC2 and of the GAL genes of S. cerevisiae when cells are grown under normally glucose-repressing conditions. In addition, genetic interactions have been detected between nrg1Delta and other factors that control SUC2 transcription. CONCLUSIONS: The analysis of nrg1Delta demonstrates that Nrg1 plays a role in glucose repression of the SUC2 and GAL genes of S. cerevisiae. Thus, three repressors, Nrg1, Mig1, and Mig2, are involved as the downstream targets of the glucose signaling in S. cerevisiae.
Adenosine Triphosphatases↗
Control of eukaryotic transcription elongation.
The Spt4, Spt5, and Spt6 proteins are conserved eukaryotic transcription-elongation factors. Recent studies have provided the first evidence that they are generally required in multicellular eukaryotes, including during development and for viral gene expression.
Animals↗
Spt5 and spt6 are associated with active transcription and have characteristics of general elongation factors in D. melanogaster.
The Spt4, Spt5, and Spt6 proteins are conserved throughout eukaryotes and are believed to play critical and related roles in transcription. They have a positive role in transcription elongation in Saccharomyces cerevisiae and in the activation of transcription by the HIV Tat protein in human cells. In contrast, a complex of Spt4 and Spt5 is required in vitro for the inhibition of RNA polymerase II (Pol II) elongation by the drug DRB, suggesting also a negative role in vivo. To learn more about the function of the Spt4/Spt5 complex and Spt6 in vivo, we have identified Drosophila homologs of Spt5 and Spt6 and characterized their localization on Drosophila polytene chromosomes. We find that Spt5 and Spt6 localize extensively with the phosphorylated, actively elongating form of Pol II, to transcriptionally active sites during salivary gland development and upon heat shock. Furthermore, Spt5 and Spt6 do not colocalize widely with the unphosphorylated, nonelongating form of Pol II. These results strongly suggest that Spt5 and Spt6 play closely related roles associated with active transcription in vivo.
Animals↗
Redundant roles for the TFIID and SAGA complexes in global transcription.
The transcription factors TFIID and SAGA are multi-subunit complexes involved in transcription by RNA polymerase II. TFIID and SAGA contain common TATA-binding protein (TBP)-associated factor (TAF(II)) subunits and each complex contains a subunit with histone acetyltransferase activity. These observations have raised questions about whether the functions of the two complexes in vivo are unique or overlapping. Here we use genome-wide expression analysis to investigate how expression of the yeast genome depends on both shared and unique subunits of these two complexes. We find that expression of most genes requires one or more of the common TAF(II) subunits, indicating that the functions of TFIID and SAGA are widely required for gene expression. Among the subunits shared by TFIID and SAGA are three histone-like TAF(II)s, which have been proposed to form a sub-complex and mediate a common function in global transcription. Unexpectedly, we find that the histone-like TAF(II)s have distinct roles in expression of the yeast genome. Most importantly, we show that the histone acetylase components of TFIID and SAGA (TAF(II)145 and Gcn5) are functionally redundant, indicating that expression of a large fraction of yeast genes can be regulated through the action of either complex.
Acetyltransferases↗
Histone H2A is required for normal centromere function in Saccharomyces cerevisiae.
Histones are structural and functional components of the eukaryotic chromosome, and their function is essential for normal cell cycle progression. In this work, we describe the characterization of two Saccharomyces cerevisiae cold-sensitive histone H2A mutants. Both mutants contain single amino acid replacements of residues predicted to be on the surface of the nucleosome and in close contact with DNA. We show that these H2A mutations cause an increase-in-ploidy phenotype, an increased rate of chromosome loss, and a defect in traversing the G(2)-M phase of the cell cycle. Moreover, these H2A mutations show genetic interactions with mutations in genes encoding kinetochore components. Finally, chromatin analysis of these H2A mutants has revealed an altered centromeric chromatin structure. Taken together, these results strongly suggest that histone H2A is required for proper centromere-kinetochore function during chromosome segregation.
Base Sequence↗
Whole-genome expression analysis of snf/swi mutants of Saccharomyces cerevisiae.
The Saccharomyces cerevisiae Snf/Swi complex has been previously demonstrated to control transcription and chromatin structure of particular genes in vivo and to remodel nucleosomes in vitro. We have performed whole-genome expression analysis, using DNA microarrays, to study mutants deleted for a gene encoding one conserved (Snf2) or one unconserved (Swi1) Snf/Swi component. This analysis was performed on cells grown in both rich and minimal media. The microarray results, combined with Northern blot, computational, and genetic analyses, show that snf2Delta and swi1Delta mutations cause similar effects on mRNA levels, that Snf/Swi controls some genes differently in rich and minimal media, and that Snf/Swi control is exerted at the level of individual genes rather than over larger chromosomal domains. In addition, this work shows that Snf/Swi controls mRNA levels of MATalpha-specific genes, likely via controlling transcription of the regulators MATalpha1 and MCM1. Finally, we provide evidence that Snf/Swi acts both as an activator and as a repressor of transcription, and that neither mode of control is an indirect effect of the other.
Acid Phosphatase↗
The Swi/Snf family nucleosome-remodeling complexes and transcriptional control.
The Swi/Snf family of nucleosome-remodeling complexes has been shown to play important roles in gene expression throughout eukaryotes. Genetic and biochemical studies previously suggested that Swi/Snf activates transcription by remodeling nucleosomes, thereby permitting increased access of transcription factors for their binding sites. Recent studies have identified additional Swi/Snf biochemical activities and have suggested possible mechanisms by which Swi/Snf is targeted to specific promoters. Surprisingly, studies have also revealed that, besides being necessary for activation, Swi/Snf is required for transcriptional repression of some genes. These analyses have transformed our understanding of the function of the Swi/Snf family of complexes and suggest that they control transcription in diverse ways.
Animals↗
The Spt components of SAGA facilitate TBP binding to a promoter at a post-activator-binding step in vivo.
The SAGA complex of Saccharomyces cerevisiae is required for the transcription of many RNA polymerase II-dependent genes. Previous studies have demonstrated that SAGA possesses histone acetyltransferase activity, catalyzed by the SAGA component Gcn5. However, the transcription of many genes, although SAGA dependent, is Gcn5 independent, suggesting the existence of distinct SAGA activities. We have studied the in vivo role of two other SAGA components, Spt3 and Spt20, at the well-characterized GAL1 promoter. Our results demonstrate that both Spt3 and Spt20 are required for the binding of TATA-binding protein but not of the activator Gal4 and that this role is Gcn5 independent. These results suggest a coactivator role for Spt3 and Spt20 in the recruitment of TBP.
Acetylation↗
The nucleosome remodeling complex, Snf/Swi, is required for the maintenance of transcription in vivo and is partially redundant with the histone acetyltransferase, Gcn5.
Snf/Swi, a nucleosome remodeling complex, is important for overcoming nucleosome-mediated repression of transcription in Saccharomyces cerevisiae. We have addressed the mechanism by which Snf/Swi controls transcription in vivo of an Snf/Swi-dependent promoter, that of the SUC2 gene. By single-cell analysis, our results show that Snf/Swi is required for activated levels of SUC2 expression in every cell of a population. In addition, Snf/Swi is required for maintenance of SUC2 transcription, suggesting that continuous chromatin remodeling is necessary to maintain an active transcriptional state. Finally, Snf/Swi and Gcn5, a histone acetyltransferase, have partially redundant roles in the control of SUC2 transcription, suggesting a functional overlap between two different mechanisms believed to overcome repression by nucleosomes, nucleosome remodeling and histone acetylation.
Acetyltransferases↗
Transcriptional activation by Gcn4p involves independent interactions with the SWI/SNF complex and the SRB/mediator.
Mutations in three subunits of the SWI/SNF complex and in the Med2p subunit of the SRB/mediator of pol II holoenzyme impaired Gcn4p-activated transcription of HIS3 without reducing Gcn4p-independent transcription of this gene. Recombinant Gcn4p interacted with SWI/SNF and SRB/mediator subunits in cell extracts in a manner dependent on the same hydrophobic clusters in the Gcn4p activation domain; however, higher concentrations of Gcn4p were required for binding to SWI/SNF versus SRB/mediator subunits. In addition, SRB/mediator and SWI/SNF subunits did not coimmunopreciptate from the extracts. These findings, together with the fact that Gcn4p specifically interacted with purified SWI/SNF, strongly suggest that Gcn4p independently recruits SWI/SNF and holoenzyme to its target promoters in the course of activating transcription.
DNA Polymerase II↗
Two functionally distinct forms of the RSC nucleosome-remodeling complex, containing essential AT hook, BAH, and bromodomains.
RSC is an essential 15 protein nucleosome-remodeling complex from S. cerevisiae. We have identified two closely related RSC members, Rsc1 and Rsc2. Biochemical analysis revealed Rsc1 and Rsc2 in distinct complexes, defining two forms of RSC. Genetic analysis has shown that Rsc1 and Rsc2 possess shared and unique functions. Rsc1 and Rsc2 each contain two bromodomains, a bromo-adjacent homology (BAH) domain, and an AT hook. One of the bromodomains, the BAH domain, and the AT hook are each essential for Rsc1 and Rsc2 functions, although they are not required for assembly into RSC complexes. Therefore, these domains are required for RSC function. Additional genetic analysis provides further evidence that RSC function is related to transcriptional control.
Adenosine Triphosphatases↗
The bromodomain: a chromatin-targeting module?
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Acetyltransferases↗
Specific components of the SAGA complex are required for Gcn4- and Gcr1-mediated activation of the his4-912delta promoter in Saccharomyces cerevisiae.
Mutations selected as suppressors of Ty or solo delta insertion mutations in Saccharomyces cerevisiae have identified several genes, SPT3, SPT7, SPT8, and SPT20, that encode components of the SAGA complex. However, the mechanism by which SAGA activates transcription of specific RNA polymerase II-dependent genes is unknown. We have conducted a fine-structure mutagenesis of one widely used SAGA-dependent promoter, the delta element of his4-912delta, to identify sequence elements important for its promoter activity. Our analysis has characterized three delta regions necessary for full promoter activity and accurate start site selection: an upstream activating sequence, a TATA region, and an initiator region. In addition, we have shown that factors present at the adjacent UASHIS4 (Gcn4, Bas1, and Pho2) also activate the delta promoter in his4-912delta. Our results suggest a model in which the delta promoter in his4-912delta is primarily activated by two factors: Gcr1 acting at the UASdelta and Gcn4 acting at the UASHIS4. Finally, we tested whether activation by either of these factors is dependent on components of the SAGA complex. Our results demonstrate that Spt3 and Spt20 are required for full delta promoter activity, but that Gcn5, another member of SAGA, is not required. Spt3 appears to be partially required for activation of his4-912delta by both Gcr1 and Gcn4. Thus, our work suggests that SAGA exerts a large effect on delta promoter activity through a combination of smaller effects on multiple factors.
Base Sequence↗
Functional organization of the yeast SAGA complex: distinct components involved in structural integrity, nucleosome acetylation, and TATA-binding protein interaction.
SAGA, a recently described protein complex in Saccharomyces cerevisiae, is important for transcription in vivo and possesses histone acetylation function. Here we report both biochemical and genetic analyses of members of three classes of transcription regulatory factors contained within the SAGA complex. We demonstrate a correlation between the phenotypic severity of SAGA mutants and SAGA structural integrity. Specifically, null mutations in the Gcn5/Ada2/Ada3 or Spt3/Spt8 classes cause moderate phenotypes and subtle structural alterations, while mutations in a third subgroup, Spt7/Spt20, as well as Ada1, disrupt the complex and cause severe phenotypes. Interestingly, double mutants (gcn5Delta spt3Delta and gcn5Delta spt8Delta) causing loss of a member of each of the moderate classes have severe phenotypes, similar to spt7Delta, spt20Delta, or ada1Delta mutants. In addition, we have investigated biochemical functions suggested by the moderate phenotypic classes and find that first, normal nucleosomal acetylation by SAGA requires a specific domain of Gcn5, termed the bromodomain. Deletion of this domain also causes specific transcriptional defects at the HIS3 promoter in vivo. Second, SAGA interacts with TBP, the TATA-binding protein, and this interaction requires Spt8 in vitro. Overall, our data demonstrate that SAGA harbors multiple, distinct transcription-related functions, including direct TBP interaction and nucleosomal histone acetylation. Loss of either of these causes slight impairment in vivo, but loss of both is highly detrimental to growth and transcription.
Acetylation↗
Characterization of a human homologue of the Saccharomyces cerevisiae transcription factor spt3 (SUPT3H).
Spt3 is a Saccharomyces cerevisiae transcription factor that is required in vivo for the transcription of a number of RNA polymerase II-transcribed genes. We report the cloning of the gene encoding the human homologue of Spt3, SUPT3H, and its initial functional analysis. The human and yeast Spt3 homologues share an overall identity of 30% that defines three conserved regions, suggesting possible functional domains. To determine whether SUPT3H is a true functional Spt3 homologue, we tested for complementation of an spt3Delta mutation in yeast. While expression of the full-length SUPT3H is unable to complement an spt3Delta mutation, expression of a human-yeast chimeric gene that contains 42% human sequences can partially complement an spt3Delta mutation. These data suggest that the transcriptional control carried out by Spt3 has been conserved from yeast to human.
Amino Acid Sequence↗