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David J Stillman

Publications and source records attributed to David J Stillman.

At least 19 recordsLinked to original sources
PubMed2006

Opposing roles for Set2 and yFACT in regulating TBP binding at promoters.

Previous work links histone methylation by Set2 with transcriptional elongation. yFACT (Spt16-Pob3 and Nhp6) reorganizes nucleosomes and functions in both transcriptional initiation and elongation. We show that growth defects caused by spt16 or pob3 mutations can be suppressed by deleting SET2, suggesting that Set2 and yFACT have opposing roles. Set2 methylates K36 of histone H3, and K36 substitutions also suppress yFACT mutations. In contrast, set1 enhances yFACT mutations. Methylation at H3 K4 by Set1 is required for set2 to suppress yFACT defects. We did not detect an elongation defect at an 8 kb ORF in yFACT mutants. Instead, pob3 mutants displayed reduced binding of both pol II and TBP to the GAL1 promoter. Importantly, both GAL1 transcription and promoter binding of pol II and TBP are significantly restored in the pob3 set2 double mutant. Defects caused by an spt16 mutation are enhanced by either TBP or TFIIA mutants. These synthetic defects are suppressed by set2, demonstrating that yFACT and Set2 oppose one another during transcriptional initiation at a step involving DNA binding by TBP and TFIIA.

Amino Acid Substitution↗
PubMed2006

SWI/SNF binding to the HO promoter requires histone acetylation and stimulates TATA-binding protein recruitment.

We use chromatin immunoprecipitation assays to show that the Gcn5 histone acetyltransferase in SAGA is required for SWI/SNF association with the HO promoter and that binding of SWI/SNF and SAGA are interdependent. Previous results showed that SWI/SNF binding to HO was Gcn5 independent, but that work used a strain with a mutation in the Ash1 daughter-specific repressor of HO expression. Here, we show that Ash1 functions as a repressor that inhibits SWI/SNF binding and that Gcn5 is required to overcome Ash1 repression in mother cells to allow HO transcription. Thus, Gcn5 facilitates SWI/SNF binding by antagonizing Ash1. Similarly, a mutation in SIN3, like an ash1 mutation, allows both HO expression and SWI/SNF binding in the absence of Gcn5. Although Ash1 has recently been identified in a Sin3-Rpd3 complex, our genetic analysis shows that Ash1 and Sin3 have distinct functions in regulating HO. Analysis of mutant strains shows that SWI/SNF binding and HO expression are correlated and regulated by histone acetylation. The defect in HO expression caused by a mutant SWI/SNF with a Swi2(E834K) substitution can be partially suppressed by ash1 or spt3 mutation or by a gain-of-function V71E substitution in the TATA-binding protein (TBP). Spt3 inhibits TBP binding at HO, and genetic analysis suggests that Spt3 and TBP(V71E) act in the same pathway, distinct from that of Ash1. We have detected SWI/SNF binding at the HO TATA region, and our results suggest that SWI/SNF, either directly or indirectly, facilitates TBP binding at HO.

Acetylation↗
PubMed2005

Genetic interactions between Nhp6 and Gcn5 with Mot1 and the Ccr4-Not complex that regulate binding of TATA-binding protein in Saccharomyces cerevisiae.

Our previous work suggests that the Nhp6 HMGB protein stimulates RNA polymerase II transcription via the TATA-binding protein TBP and that Nhp6 functions in the same functional pathway as the Gcn5 histone acetyltransferase. In this report we examine the genetic relationship between Nhp6 and Gcn5 with the Mot1 and Ccr4-Not complexes, both of which have been implicated in regulating DNA binding by TBP. We find that combining either a nhp6ab or a gcn5 mutation with mot1, ccr4, not4, or not5 mutations results in lethality. Combining spt15 point mutations (in TBP) with either mot1 or ccr4 also results in either a growth defect or lethality. Several of these synthetic lethalities can be suppressed by overexpression of TFIIA, TBP, or Nhp6, suggesting that these genes facilitate formation of the TBP-TFIIA-DNA complex. The growth defect of a not5 mutant can be suppressed by a mot1 mutant. HO gene expression is reduced by nhp6ab, gcn5, or mot1 mutations, and the additive decreases in HO mRNA levels in nhp6ab mot1 and gcn5 mot1 strains suggest different modes of action. Chromatin immunoprecipitation experiments show decreased binding of TBP to promoters in mot1 mutants and a further decrease when combined with either nhp6ab or gcn5 mutations.

Adenosine Triphosphatases↗
PubMed2005

ACE2, CBK1, and BUD4 in budding and cell separation.

Mutations in the RAM network genes, including CBK1, MOB2, KIC1, HYM1, and TAO3, cause defects in bud site selection, asymmetric apical growth, and mating projections. Additionally, these mutants show altered colony morphology, cell separation defects, and reduced CTS1 expression, phenotypes also seen by mutating the Ace2 transcription factor. We show that an ACE2 multicopy plasmid suppresses the latter three defects of RAM network mutations, demonstrating that Ace2 is downstream of the RAM network and suggesting that these phenotypes are caused by reduced expression of Ace2 target genes. We show that wild-type W303 strains have a bud4 mutation and that combining bud4 with either ace2 or cbk1 in haploids results in altered colony morphology. We describe a timed sedimentation assay that allows quantitation of cytokinesis defects and subtle changes in budding pattern and cell shape. Experiments examining budding patterns and sedimentation rates both show that Ace2 and Cbk1 have independent functions in addition to their common pathway in transcription of genes such as CTS1. SWI5 encodes a transcription factor paralogous to ACE2. Additive effects are seen in cbk1 swi5 strains, and we show that activation of some target genes, such as EGT2, requires either Swi5 or Ace2 with Cbk1. The relative roles and interactions of Ace2, Cbk1, and Bud4 in bud site selection, polarized growth, and cell separation are discussed.

Cell Cycle Proteins↗
PubMed2005

The yeast FACT complex has a role in transcriptional initiation.

A crucial step in eukaryotic transcriptional initiation is recognition of the promoter TATA by the TATA-binding protein (TBP), which then allows TFIIA and TFIIB to be recruited. However, nucleosomes block the interaction between TBP and DNA. We show that the yeast FACT complex (yFACT) promotes TBP binding to a TATA box in chromatin both in vivo and in vitro. The SPT16 gene encodes a subunit of yFACT, and we show that certain spt16 mutations are synthetically lethal with TBP mutants. Some of these genetic defects can be suppressed by TFIIA overexpression, strongly suggesting a role for yFACT in TBP-TFIIA complex formation in vivo. Mutations in the TOA2 subunit of TFIIA that disrupt TBP-TFIIA complex formation in vitro are also synthetically lethal with spt16. In some cases this spt16 toa2 lethality is suppressed by overexpression of TBP or the Nhp6 architectural transcription factor that is also a component of yFACT. The Spt3 protein in the SAGA complex has been shown to regulate TBP binding at certain promoters, and we show that some spt16 phenotypes can be suppressed by spt3 mutations. Chromatin immunoprecipitations show TBP binding to promoters is reduced in single spt16 and spt3 mutants but increases in the spt16 spt3 double mutant, reflecting the mutual suppression seen in the genetic assays. Finally, in vitro studies show that yFACT promotes TBP binding to a TATA sequence within a reconstituted nucleosome in a TFIIA-dependent manner. Thus, yFACT functions in establishing transcription initiation complexes in addition to the previously described role in elongation.

Cell Cycle Proteins↗
PubMed2004

The Zap1 transcriptional activator also acts as a repressor by binding downstream of the TATA box in ZRT2.

The zinc-responsive transcriptional activator Zap1 regulates the expression of both high- and low-affinity zinc uptake permeases encoded by the ZRT1 and ZRT2 genes. Zap1 mediates this response by binding to zinc-responsive elements (ZREs) located within the promoter regions of each gene. ZRT2 has a remarkably different expression profile in response to zinc compared to ZRT1. While ZRT1 is maximally induced during zinc limitation, ZRT2 is repressed in low zinc but remains induced upon zinc supplementation. In this study, we determined the mechanism underlying this paradoxical Zap1-dependent regulation of ZRT2. We demonstrate that a nonconsensus ZRE (ZRT2 ZRE3), which overlaps with one of the ZRT2 transcriptional start sites, is essential for repression of ZRT2 in low zinc and that Zap1 binds to ZRT2 ZRE3 with a low affinity. The low-affinity ZRE is also essential for the ZRT2 expression profile. These results indicate that the unusual pattern of ZRT2 regulation among Zap1 target genes involves the antagonistic effect of Zap1 binding to a low-affinity ZRE repressor site and high-affinity ZREs required for activation.

Binding Sites↗
PubMed2004

TATA-binding protein mutants that are lethal in the absence of the Nhp6 high-mobility-group protein.

The Saccharomyces cerevisiae Nhp6 protein is related to the high-mobility-group B family of architectural DNA-binding proteins that bind DNA nonspecifically but bend DNA sharply. Nhp6 is involved in transcriptional activation by both RNA polymerase II (Pol II) and Pol III. Our previous genetic studies have implicated Nhp6 in facilitating TATA-binding protein (TBP) binding to some Pol II promoters in vivo, and we have used a novel genetic screen to isolate 32 new mutations in TBP that are viable in wild-type cells but lethal in the absence of Nhp6. The TBP mutations that are lethal in the absence of Nhp6 cluster in three regions: on the upper surface of TBP that may have a regulatory role, near residues that contact Spt3, or near residues known to contact either TFIIA or Brf1 (in TFIIIB). The latter set of mutations suggests that Nhp6 becomes essential when a TBP mutant compromises its ability to interact with either TFIIA or Brf1. Importantly, the synthetic lethality for some of the TBP mutations is suppressed by a multicopy plasmid with SNR6 or by an spt3 mutation. It has been previously shown that nhp6ab mutants are defective in expressing SNR6, a Pol III-transcribed gene encoding the U6 splicing RNA. Chromatin immunoprecipitation experiments show that TBP binding to SNR6 is reduced in an nhp6ab mutant. Nhp6 interacts with Spt16/Pob3, the yeast equivalent of the FACT elongation complex, consistent with nhp6ab cells being extremely sensitive to 6-azauracil (6-AU). However, this 6-AU sensitivity can be suppressed by multicopy SNR6 or BRF1. Additionally, strains with SNR6 promoter mutations are sensitive to 6-AU, suggesting that decreased SNR6 RNA levels contribute to 6-AU sensitivity. These results challenge the widely held belief that 6-AU sensitivity results from a defect in transcriptional elongation.

DNA-Binding Proteins↗
PubMed2004

Role for Nhp6, Gcn5, and the Swi/Snf complex in stimulating formation of the TATA-binding protein-TFIIA-DNA complex.

The TATA-binding protein (TBP), TFIIA, and TFIIB interact with promoter DNA to form a complex required for transcriptional initiation, and many transcriptional regulators function by either stimulating or inhibiting formation of this complex. We have recently identified TBP mutants that are viable in wild-type cells but lethal in the absence of the Nhp6 architectural transcription factor. Here we show that many of these TBP mutants were also lethal in strains with disruptions of either GCN5, encoding the histone acetyltransferase in the SAGA complex, or SWI2, encoding the catalytic subunit of the Swi/Snf chromatin remodeling complex. These synthetic lethalities could be suppressed by overexpression of TOA1 and TOA2, the genes encoding TFIIA. We also used TFIIA mutants that eliminated in vitro interactions with TBP. These viable TFIIA mutants were lethal in strains lacking Gcn5, Swi2, or Nhp6. These lethalities could be suppressed by overexpression of TBP or Nhp6, suggesting that these coactivators stimulate formation of the TBP-TFIIA-DNA complex. In vitro studies have previously shown that TBP binds very poorly to a TATA sequence within a nucleosome but that Swi/Snf stimulates binding of TBP and TFIIA. In vitro binding experiments presented here show that histone acetylation facilitates TBP binding to a nucleosomal binding site and that Nhp6 stimulates formation of a TBP-TFIIA-DNA complex. Consistent with the idea that Nhp6, Gcn5, and Swi/Snf have overlapping functions in vivo, nhp6a nhp6b gcn5 mutants had a severe growth defect, and mutations in both nhp6a nhp6b swi2 and gcn5 swi2 strains were lethal.

Acetylation↗
PubMed2003

ACE2 is required for daughter cell-specific G1 delay in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells reproduce by budding to yield a mother cell and a smaller daughter cell. Although both mother and daughter begin G1 simultaneously, the mother cell progresses through G1 more rapidly. Daughter cell G1 delay has long been thought to be due to a requirement for attaining a certain critical cell size before passing the commitment point in the cell cycle known as START. We present an alternative model in which the daughter cell-specific Ace2 transcription factor delays G1 in daughter cells. Deletion of ACE2 produces daughter cells that proceed through G1 at the same rate as mother cells, whereas a mutant Ace2 protein that is not restricted to daughter cells delays G1 equally in both mothers and daughters. The differential in G1 length between mothers and daughters requires the Cln3 G1 cyclin, and CLN3-GFP reporter expression is reduced in daughters in an ACE2-dependent manner. Specific daughter delay elements in the CLN3 promoter are required for normal daughter G1 delay, and these elements bind to an unidentified 127-kDa protein. This DNA-binding activity is enhanced by deletion of ACE2. These results support a model in which daughter cell G1 delay is determined not by cell size but by an intrinsic property of the daughter cell generated by asymmetric cell division.

Base Sequence↗
PubMed2003

New 'marker swap' plasmids for converting selectable markers on budding yeast gene disruptions and plasmids.

Marker swap plasmids can be used to change markers for genes disrupted with nutritional markers in the yeast Saccharomyces cerevisiae. We describe 18 new marker swap plasmids, and we also review other plasmids available for marker conversions. All of these plasmids have long regions of flanking sequence identity, and thus the efficiency of homologous recombination mediated by marker conversion is very high. Marker swaps allow one to easily perform crosses to construct double mutant strains even if each of the disrupted strains contains the same marker, as is the case with the KanMX marker used in the yeast knockout collection. Marker swaps can also be used to change the selectable marker on plasmids, eliminating the need for subcloning.

Genetic Markers↗
PubMed2003

Regulation of TATA-binding protein binding by the SAGA complex and the Nhp6 high-mobility group protein.

Transcriptional activation of the yeast HO gene involves the sequential action of DNA-binding and chromatin-modifying factors. Here we examine the role of the SAGA complex and the Nhp6 architectural transcription factor in HO regulation. Our data suggest that these factors regulate binding of the TATA-binding protein (TBP) to the promoter. A gcn5 mutation, eliminating the histone acetyltransferase present in SAGA, reduces the transcription of HO, but expression is restored in a gcn5 spt3 double mutant. We conclude that the major role of Gcn5 in HO activation is to overcome repression by Spt3. Spt3 is also part of SAGA, and thus two proteins in the same regulatory complex can have opposing roles in transcriptional regulation. Chromatin immunoprecipitation experiments show that TBP binding to HO is very weak in wild-type cells but markedly increased in an spt3 mutant, indicating that Spt3 reduces HO expression by inhibiting TBP binding. In contrast, it has been shown previously that Spt3 stimulates TBP binding to the GAL1 promoter as well as GAL1 expression, and thus, Spt3 regulates these promoters differently. We also find genetic interactions between TBP and either Gcn5 or the high-mobility-group protein Nhp6, including multicopy suppression and synthetic lethality. These results suggest that, while Spt3 acts to inhibit TBP interaction with the HO promoter, Gcn5 and Nhp6 act to promote TBP binding. The result of these interactions is to limit TBP binding and HO expression to a short period within the cell cycle. Furthermore, the synthetic lethality resulting from combining a gcn5 mutation with specific TBP point mutations can be suppressed by the overexpression of transcription factor IIA (TFIIA), suggesting that histone acetylation by Gcn5 can stimulate transcription by promoting the formation of a TBP/TFIIA complex.

DNA-Binding Proteins↗
PubMed2002

Mutations in the pho2 (bas2) transcription factor that differentially affect activation with its partner proteins bas1, pho4, and swi5.

The yeast PHO2 gene encodes a homeodomain protein that exemplifies combinatorial control in transcriptional activation. Pho2 alone binds DNA in vitro with low affinity, but in vivo it activates transcription with at least three disparate DNA-binding proteins: the zinc finger protein Swi5, the helix-loop-helix factor Pho4, and Bas1, an myb-like activator. Pho2 + Swi5 activates HO, Pho2 + Pho4 activates PHO5, and Pho2 + Bas1 activates genes in the purine and histidine biosynthesis pathways. We have conducted a genetic screen and identified 23 single amino acid substitutions in Pho2 that differentially affect its ability to activate its specific target genes. Analysis of the mutations suggests that the central portion of Pho2 serves as protein-protein interactive surface, with a requirement for distinct amino acids for each partner protein.

Amino Acid Sequence↗
PubMed2002

Functional mapping of Bas2. Identification of activation and Bas1-interaction domains.

The transcriptional activator protein Bas2 is required to express more than 20 genes in pathways for purine nucleotide and histidine biosynthesis, phosphate utilization, and the HO endonuclease by acting with co-regulator proteins Bas1, Pho4, and Swi5. The role that Bas2 plays in transcriptional activation may be to unmask latent activation domains in the co-regulator and to promote ternary complex formation between Bas2, the co-regulator, and DNA. We show that Bas2 also contributes to transcriptional activation by providing an activation domain. We localize this domain in Bas2 to the C-terminal 156 amino acids using deletion analysis and fusion to a heterologous DNA binding domain. Additionally, we show that Bas2 makes direct contacts with Bas1. This interaction is detected by co-immunoprecipitation and by two-hybrid analysis. We localize the interaction region to the central portion of Bas2, from amino acids 112 to 404.

Binding Sites↗
PubMed2002

Regulation of Fas-associated death domain interactions by the death effector domain identified by a modified reverse two-hybrid screen.

The adapter protein FADD consists of two protein interaction domains and is an essential component of the death inducing signaling complex (DISC) that is formed by activated death receptors of the tumor necrosis factor (TNF) receptor family. The FADD death domain binds to activated receptors such as Fas or other adapters such as TRADD, whereas the FADD death effector domain binds to procaspase 8. Each domain can interact with its target in the absence of the other domain, and this has led to the idea that the two domains function independently. FADD death domain interactions with Fas and TRADD are thought to occur on the same surface; however, the regulation of these interactions is poorly understood. We developed a modified reverse two-hybrid method that can identify mutations, which inhibit some protein-protein interactions without affecting other interactions. Using this method, we identified mutations in FADD that prevent binding to Fas but do not affect binding to TRADD. Surprisingly, these mutations were in the death effector domain rather than the death domain. To test whether the mutants function in mammalian cells, we expressed wild type or mutant FADD molecules in FADD-deficient cells. Wild type FADD rescued both Fas ligand- and TNF-dependent signaling, whereas the FADD death effector domain mutants rescued only TNF signaling. These data indicate that in contrast to current models, the death effector domain of FADD is involved in interaction with Fas.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Correlation between chromatin association and transcriptional regulation for the Act3p/Arp4 nuclear actin-related protein of Saccharomyces cerevisiae.

Actin-related proteins (Arps), which share a basal structure with actin but have distinct functions, have been found in a wide variety of organisms. While their functions are not yet clear, some Arps are localized in the nucleus and are suggested to contribute to the regulation of transcription. An essential gene of Saccharomyces cerevisiae, Act3p/Arp4, encodes the first identified nuclear Arp, which has been shown to bind to core histones in vitro. Here we have analyzed the in vivo function of Act3p/Arp4 on the his4-912delta promoter. Chromatin immunoprecipitation assays show that Act3p/Arp4 is bound to the entire his4-912delta promoter region. Conditional act3/arp4 mutations affect transcription from the his4-912delta promoter, where decreased Act3p/Arp4 binding and a change in nuclease sensitivity of chromatin were observed, showing the involvement of Act3p/Arp4 in the regulation of gene expression through the organization of chromatin structure. Taken together with the presence of Act3p/Arp4 in chromatin remodeling and histone acetyltransferase complexes, it is suggested that Act3p/Arp4 functions in transcriptional regulation to recruit chromatin remodeling and histone acetyltransferase complexes onto chromatin.

Actins↗