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J L Workman

Publications and source records attributed to J L Workman.

At least 19 recordsLinked to original sources
PubMed2004

Proteomic analysis of chromatin-modifying complexes in Saccharomyces cerevisiae identifies novel subunits.

Epigenetics is the alteration of phenotype without affecting the genotype. An underlying molecular mechanism of epigenetics is the changes of chromatin structure by covalent histone modifications and nucleosome reorganization. In the yeast, Saccharomyces cerevisiae, two of the most well-studied macromolecular complexes that perform these epigenetic changes are the ATP-dependent Swi/Snf chromatin-remodelling complex and the SAGA histone acetyltransferase complex. To understand fully the mechanism by which these large protein complexes perform their functions in the cell, it is crucial that all the subunits of these complexes are identified. In an attempt to identify new subunits associated with SAGA and Swi/Snf, we used tandem affinity purification, followed by a multidimensional protein identification technology to analyse the subunit composition. Our analysis identified two novel proteins, one associated with SAGA, YPL047W (Sgf11), and another associated with Swi/Snf, Rtt102.

Adenosine Triphosphate↗
PubMed2001

The yeast SAS (something about silencing) protein complex contains a MYST-type putative acetyltransferase and functions with chromatin assembly factor ASF1.

It is well established that acetylation of histone and nonhistone proteins is intimately linked to transcriptional activation. However, loss of acetyltransferase activity has also been shown to cause silencing defects, implicating acetylation in gene silencing. The something about silencing (Sas) 2 protein of Saccharomyces cerevisiae, a member of the MYST (MOZ, Ybf2/Sas3, Sas2, and TIP60) acetyltransferase family, promotes silencing at HML and telomeres. Here we identify a ~450-kD SAS complex containing Sas2p, Sas4p, and the tf2f-related Sas5 protein. Mutations in the conserved acetyl-CoA binding motif of Sas2p are shown to disrupt the ability of Sas2p to mediate the silencing at HML and telomeres, providing evidence for an important role for the acetyltransferase activity of the SAS complex in silencing. Furthermore, the SAS complex is found to interact with chromatin assembly factor Asf1p, and asf1 mutants show silencing defects similar to mutants in the SAS complex. Thus, ASF1-dependent chromatin assembly may mediate the role of the SAS complex in silencing.

Acetyltransferases↗
PubMed2001

Histone H3 specific acetyltransferases are essential for cell cycle progression.

Longstanding observations suggest that acetylation and/or amino-terminal tail structure of histones H3 and H4 are critical for eukaryotic cells. For Saccharomyces cerevisiae, loss of a single H4-specific histone acetyltransferase (HAT), Esa1p, results in cell cycle defects and death. In contrast, although several yeast HAT complexes preferentially acetylate histone H3, the catalytic subunits of these complexes are not essential for viability. To resolve the apparent paradox between the significance of H3 versus H4 acetylation, we tested the hypothesis that H3 modification is essential, but is accomplished through combined activities of two enzymes. We observed that Sas3p and Gcn5p HAT complexes have overlapping patterns of acetylation. Simultaneous disruption of SAS3, the homolog of the MOZ leukemia gene, and GCN5, the hGCN5/PCAF homolog, is synthetically lethal due to loss of acetyltransferase activity. This key combination of activities is specific for these two HATs because neither is synthetically lethal with mutations of other MYST family or H3-specific acetyltransferases. Further, the combined loss of GCN5 and SAS3 functions results in an extensive, global loss of H3 acetylation and arrest in the G(2)/M phase of the cell cycle. The strikingly similar effect of loss of combined essential H3 HAT activities and the loss of a single essential H4 HAT underscores the fundamental biological significance of each of these chromatin-modifying activities.

Acetylation↗
PubMed2001

Promoter targeting of chromatin-modifying complexes.

The action of multi-subunit complexes that are able to overcome the repressive effects of chromatin is an important step in the regulation of eukaryotic gene expression. Identification of complexes that modify the structure of chromatin to help factors access the underlying DNA has enhanced our understanding of how some genes are controlled. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) represent one group of complexes that regulate the level of acetylation on the N-terminal tails of core histone proteins. The SWI/SNF complex is the prototype of a second group of complexes, which use the energy of ATP-hydrolysis to alter histone-DNA contacts, leading to changes in chromatin conformation. Genetic studies in yeast have revealed that some of these multi-subunit complexes interact in vivo to control transcription of a subset of genes. It has become apparent that some gene promoters require modifications by both types of complexes. An important question regarding these two types of complexes is how they are recruited to the promoters of genes that are dependent on their activity for their expression. This review will tie together many studies on promoter recruitment of both HATs and SWI/SNF. Emphasis will be placed on recent data that demonstrates functional interplay between these two types of chromatin-remodeling activities. In addition, this review summarizes recent data demonstrating the ability of repressors and corepressors to recruit histone deacetylase complexes. Interestingly, many subunits of chromatin-modifying complexes in humans have been implicated in the development of cancer. Thus, studying how these complexes work can help us better understand human diseases.

Acetyltransferases↗
PubMed2001

Recruitment of HAT complexes by direct activator interactions with the ATM-related Tra1 subunit.

Promoter-specific recruitment of histone acetyltransferase activity is often critical for transcriptional activation. We present a detailed study of the interaction between the histone acetyltransferase complexes SAGA and NuA4, and transcription activators. We demonstrate by affinity chromatography and photo-cross-linking label transfer that acidic activators directly interact with Tra1p, a shared subunit of SAGA and NuA4. Mutations within the COOH-terminus of Tra1p disrupted its interaction with activators and resulted in gene-specific transcriptional defects that correlated with lowered promoter-specific histone acetylation. These data demonstrate that the essential Tra1 protein serves as a common target for activators in both SAGA and NuA4 acetyltransferases.

Acetylation↗
PubMed2001

Histone acetyltransferase complexes stabilize swi/snf binding to promoter nucleosomes.

To investigate the function of SWI/SNF in site-specific chromatin remodeling at promoters, we have used a purified system to analyze its distribution, function, and retention following recruitment by a sequence-specific transcription activator. Activator recruitment of SWI/SNF bound the complex to promoter proximal nucleosomes and led to localized nucleosome disruption. However, retention of SWI/SNF on the promoter required either the continued binding of the transcription activator or acetylated histones. Histone acetylation by either the SAGA or NuA4 HAT complexes increased the retention of SWI/SNF on the promoter. These data illustrate a functional link between HAT complexes and the SWI/SNF chromatin remodeling complex and provide a mechanistic basis for the ordered recruitment of these complexes.

Acetyltransferases↗
PubMed2000

Sds3 (suppressor of defective silencing 3) is an integral component of the yeast Sin3[middle dot]Rpd3 histone deacetylase complex and is required for histone deacetylase activity.

SDS3 (suppressor of defective silencing 3) was originally identified in a screen for mutations that cause increased silencing of a crippled HMR silencer in a rap1 mutant background. In addition, sds3 mutants have phenotypes very similar to those seen in sin3 and rpd3 mutants, suggesting that it functions in the same genetic pathway. In this manuscript we demonstrate that Sds3p is an integral subunit of a previously identified high molecular weight Rpd3p.Sin3p containing yeast histone deacetylase complex. By analyzing an sds3Delta strain we show that, in the absence of Sds3p, Sin3p can be chromatographically separated from Rpd3p, indicating that Sds3p promotes the integrity of the complex. Moreover, the remaining Rpd3p complex in the sds3Delta strain had little or no histone deacetylase activity. Thus, Sds3p plays important roles in the integrity and catalytic activity of the Rpd3p.Sin3p complex.

Fungal Proteins↗
PubMed2000

Distribution of acetylated histones resulting from Gal4-VP16 recruitment of SAGA and NuA4 complexes.

We analyzed the targeting of histone acetyltransferase (HAT) complexes by DNA-binding activators during transcriptional activation and the resulting distribution of acetylated histones. An in vitro competition assay was developed to acetylate and transcribe a nucleosomal array template in the presence of excess non-specific chromatin, which mimics in vivo conditions. Stimulation of transcription from the nucleosomal array template under competitive conditions by the SAGA and NuA4 HAT complexes depended on the presence of the Gal4-VP16 activator, which recognizes sites in the promoter and directly interacts with these HATs. Importantly, the stimulation of transcription by SAGA and NuA4 depended on the presence of Gal4-VP16 during histone acetylation, and Gal4-VP16-bound nucleosomal templates were acetylated preferentially by SAGA and NuA4 relative to the competitor chromatin. While targeting of the SAGA complex led to H3 acetylation of promoter-proximal nucleosomes, targeting of the NuA4 complex led to a broader domain of H4 acetylation of >3 kbp. Thus, either promoter-proximal H3 acetylation by SAGA or broadly distributed acetylation of H4 by NuA4 activated transcription from chromatin templates.

Acetylation↗
PubMed2000

The something about silencing protein, Sas3, is the catalytic subunit of NuA3, a yTAF(II)30-containing HAT complex that interacts with the Spt16 subunit of the yeast CP (Cdc68/Pob3)-FACT complex.

We have purified and characterized a Gcn5-independent nucleosomal histone H3 HAT complex, NuA3 (Nucleosomal Acetyltransferase of histone H3). Peptide sequencing of proteins from the purified NuA3 complex identified Sas3 as the catalytic HAT subunit of the complex. Sas3 is the yeast homolog of the human MOZ oncogene. Sas3 is required for both the HAT activity and the integrity of the NuA3 complex. In addition, NuA3 contains the TBP- associated factor, yTAF(II)30, which is also a component of the TFIID, TFIIF, and SWI/SNF complexes. Sas3 mediates interaction of the NuA3 complex with Spt16 both in vivo and in vitro. Spt16 functions as a component of the yeast CP (Cdc68/Pob3) and mammalian FACT (facilitates chromatin transcription) complexes, which are involved in transcription elongation and DNA replication. This interaction suggests that the NuA3 complex might function in concert with FACT-CP to stimulate transcription or replication elongation through nucleosomes by providing a coupled acetyltransferase activity.

Acetyltransferases↗
PubMed2000

Promoter targeting and chromatin remodeling by the SWI/SNF complex.

The SWI/SNF complex is a 2 MDa multi-subunit DNA-dependent ATPase that contributes to the regulation of gene transcription by altering chromatin structure. Recent studies have revealed that the SWI/SNF complex is targeted to promoters via direct interactions with transcription activators and have provided insights into mechanisms by which the complex alters nucleosome structure and contributes to the remodeling of chromatin.

Adenosine Triphosphatases↗
PubMed2000

The many HATs of transcription coactivators.

Histone acetylation is closely linked to gene transcription. The identification of histone acetyltransferases (HATs) and the large multiprotein complexes in which they reside has yielded important insights into how these enzymes regulate transcription. The demonstration that HAT complexes interact with sequence-specific activator proteins illustrates how these complexes target specific genes. In addition to histones, some HATs can acetylate non-histone proteins suggesting multiple roles for these enzymes.

Acetyltransferases↗
PubMed2000

Recruitment of the SWI-SNF chromatin remodeling complex as a mechanism of gene activation by the glucocorticoid receptor tau1 activation domain.

The SWI-SNF complex has been shown to alter nucleosome conformation in an ATP-dependent manner, leading to increased accessibility of nucleosomal DNA to transcription factors. In this study, we show that the SWI-SNF complex can potentiate the activity of the glucocorticoid receptor (GR) through the N-terminal transactivation domain, tau1, in both yeast and mammalian cells. GR-tau1 can directly interact with purified SWI-SNF complex, and mutations in tau1 that affect the transactivation activity in vivo also directly affect tau1 interaction with SWI-SNF. Furthermore, the SWI-SNF complex can stimulate tau1-driven transcription from chromatin templates in vitro. Taken together, these results support a model in which the GR can directly recruit the SWI-SNF complex to target promoters during glucocorticoid-dependent gene activation. We also provide evidence that the SWI-SNF and SAGA complexes represent independent pathways of tau1-mediated activation but play overlapping roles that are able to compensate for one another under some conditions.

Amino Acid Sequence↗
PubMed1999

NuA4, an essential transcription adaptor/histone H4 acetyltransferase complex containing Esa1p and the ATM-related cofactor Tra1p.

Post-translational acetylation of histone H4 N-terminal tail in chromatin has been associated with several nuclear processes including transcription. We report the purification and characterization of a native multisubunit complex (NuA4) from yeast that acetylates nucleosomal histone H4. NuA4 has an apparent molecular mass of 1.3 MDa. All four conserved lysines of histone H4 can be acetylated by NuA4. We have identified the catalytic subunit of the complex as the product of ESA1, an essential gene required for cell cycle progression in yeast. Antibodies against Esa1p specifically immunoprecipitate NuA4 activity whereas the complex purified from a temperature-sensitive esa1 mutant loses its acetyltransferase activity at the restrictive temperature. Additionally, we have identified another subunit of the complex as the product of TRA1, an ATM-related essential gene homologous to human TRRAP, an essential cofactor for c-Myc- and E2F-mediated oncogenic transformation. Finally, the ability of NuA4 to stimulate GAL4-VP16-driven transcription from chromatin templates in vitro is also lost in the temperature-sensitive esa1 mutant. The function of the essential Esa1 protein as the HAT subunit of NuA4 and the presence of Tra1p, a putative transcription activator-interacting subunit, supports an essential link between nuclear H4 acetylation, transcriptional regulation and cell cycle control.

Acetylation↗
PubMed1999

Nucleosome mobilization catalysed by the yeast SWI/SNF complex.

The generation of a local chromatin topology conducive to transcription is a key step in gene regulation. The yeast SWI/SNF complex is the founding member of a family of ATP-dependent remodelling activities capable of altering chromatin structure both in vitro and in vivo. Despite its importance, the pathway by which the SWI/SNF complex disrupts chromatin structure is unknown. Here we use a model system to demonstrate that the yeast SWI/SNF complex can reposition nucleosomes in an ATP-dependent reaction that favours attachment of the histone octamer to an acceptor site on the same molecule of DNA (in cis). We show that SWI/SNF-mediated displacement of the histone octamer is effectively blocked by a barrier introduced into the DNA, suggesting that this redistribution involves sliding or tracking of nucleosomes along DNA, and that it is achieved by a catalytic mechanism. We conclude that SWI/SNF catalyses the redistribution of nucleosomes along DNA in cis, which may represent a general mechanism by which ATP-dependent chromatin remodelling occurs.

Catalysis↗
PubMed1999

Expanded lysine acetylation specificity of Gcn5 in native complexes.

The coactivator/adaptor protein Gcn5 is a conserved histone acetyltransferase, which functions as the catalytic subunit in multiple yeast transcriptional regulatory complexes. The ability of Gcn5 to acetylate nucleosomal histones is significantly reduced relative to its activity on free histones, where it predominantly modifies histone H3 at lysine 14. However, the association of Gcn5 in multisubunit complexes potentiates its nucleosomal histone acetyltransferase activity. Here, we show that the association of Gcn5 with other proteins in two native yeast complexes, Ada and SAGA (Spt-Ada-Gcn5-acetyltransferase), directly confers upon Gcn5 the ability to acetylate an expanded set of lysines on H3. Furthermore Ada and SAGA have overlapping, yet distinct, patterns of acetylation, suggesting that the association of specific subunits determines site specificity.

Acetylation↗
PubMed1999

Transcriptional analysis of purified histone acetyltransferase complexes.

Acetylation of lysine residues within the amino-terminal tails of the core histone proteins is strongly correlated to the regulation of gene transcription in vivo. To directly study the effects of histone acetylation on transcription, we have developed a biochemical system examining the regulation of RNA polymerase II-directed transcription by native histone acetyltransferases (HATs). For the promoter sequences investigated, it has been demonstrated that HATs facilitate transcription from nucleosomal DNA templates in an acetyl-CoA-dependent fashion but do not affect transcription from histone-free templates. Here, protocols are presented describing the in vitro assembly of evenly spaced nucleosomal arrays on DNA fragments harboring gene regulatory sequences and the use of these templates with purified HAT complexes in transcription assays.

Acetyltransferases↗