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In JoVE (1)
Other Publications (9)
Articles by Andrea A. Duina in JoVE
Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast
Andrea A. Duina1, Claire E. Turkal1
1Biology Department, Hendrix College
Other articles by Andrea A. Duina on PubMed
Molecular and Cellular Biology. Jan, 2004 | Pubmed ID: 14701730
We have isolated new histone H3 mutants in Saccharomyces cerevisiae that confer phenotypes indicative of transcriptional defects. Here we describe the characterization of one such mutant, encoded by the hht2-11 allele, which contains the single amino acid change L61W in the globular domain of H3. Whole-genome expression analyses show that the hht2-11 mutation confers pleiotropic transcriptional defects and that many of the genes it affects are normally controlled by the Swi/Snf chromatin remodeling complex. Furthermore, we show that Swi/Snf occupancy at two promoters, PHO84 and SER3, is reduced in hht2-11 mutants. Detailed studies of the PHO84 promoter suggest that the hht2-11 mutation impairs Swi/Snf association with chromatin in a direct fashion. Taken together, our results strongly suggest that the integrity of the globular domain of histone H3 is an important determinant in the ability of Swi/Snf to associate with chromatin.
Evidence That the Localization of the Elongation Factor Spt16 Across Transcribed Genes is Dependent Upon Histone H3 Integrity in Saccharomyces Cerevisiae
Genetics. Sep, 2007 | Pubmed ID: 17603125
A previous study of histone H3 in Saccharomyces cerevisiae identified a mutant with a single amino acid change, leucine 61 to tryptophan, that confers several transcriptional defects. We now present several lines of evidence that this H3 mutant, H3-L61W, is impaired at the level of transcription elongation, likely by altered interactions with the conserved factor Spt16, a subunit of the transcription elongation complex yFACT. First, a selection for suppressors of the H3-L61W cold-sensitive phenotype has identified novel mutations in the gene encoding Spt16. These genetic interactions are allele specific, suggesting a direct interaction between H3 and Spt16. Second, similar to several other elongation and chromatin mutants, including spt16 mutants, an H3-L61W mutant allows transcription from a cryptic promoter within the FLO8 coding region. Finally, chromatin-immunoprecipitation experiments show that in an H3-L61W mutant there is a dramatically altered profile of Spt16 association over transcribed regions, with reduced levels over 5'-coding regions and elevated levels over the 3' regions. Taken together, these and other results provide strong evidence that the integrity of histone H3 is crucial for ensuring proper distribution of Spt16 across transcribed genes and suggest a model for the mechanism by which Spt16 normally dissociates from DNA following transcription.
Uncoupling of the Patterns of Chromatin Association of Different Transcription Elongation Factors by a Histone H3 Mutant in Saccharomyces Cerevisiae
Eukaryotic Cell. Feb, 2009 | Pubmed ID: 19047363
The transcription elongation complexes yFACT, Spt4/Spt5, and Spt6/Iws1 were previously shown to follow similar patterns of association across transcribed genes in Saccharomyces cerevisiae. Using a histone H3 mutant, we now provide evidence that the mechanism of association of yFACT across genes is separable from that adopted by Spt4/Spt5 and Spt6/Iws1.
Mutant Versions of the S. Cerevisiae Transcription Elongation Factor Spt16 Define Regions of Spt16 That Functionally Interact with Histone H3
PloS One. 2011 | Pubmed ID: 21673966
In eukaryotic cells, the highly conserved FACT (FAcilitates Chromatin Transcription) complex plays important roles in several chromatin-based processes including transcription initiation and elongation. During transcription elongation, the FACT complex interacts directly with nucleosomes to facilitate histone removal upon RNA polymerase II (Pol II) passage and assists in the reconstitution of nucleosomes following Pol II passage. Although the contribution of the FACT complex to the process of transcription elongation has been well established, the mechanisms that govern interactions between FACT and chromatin still remain to be fully elucidated. Using the budding yeast Saccharomyces cerevisiae as a model system, we provide evidence that the middle domain of the FACT subunit Spt16--the Spt16-M domain--is involved in functional interactions with histone H3. Our results show that the Spt16-M domain plays a role in the prevention of cryptic intragenic transcription during transcription elongation and also suggest that the Spt16-M domain has a function in regulating dissociation of Spt16 from chromatin at the end of the transcription process. We also provide evidence for a role for the extreme carboxy terminus of Spt16 in functional interactions with histone H3. Taken together, our studies point to previously undescribed roles for the Spt16 M-domain and extreme carboxy terminus in regulating interactions between Spt16 and chromatin during the process of transcription elongation.
Histone Chaperones Spt6 and FACT: Similarities and Differences in Modes of Action at Transcribed Genes
Genetics Research International. 2011 | Pubmed ID: 22567361
The process of gene transcription requires the participation of a large number of factors that collectively promote the accurate and efficient expression of an organism's genetic information. In eukaryotic cells, a subset of these factors can control the chromatin environments across the regulatory and transcribed units of genes to modulate the transcription process and to ensure that the underlying genetic information is utilized properly. This article focuses on two such factors-the highly conserved histone chaperones Spt6 and FACT-that play critical roles in managing chromatin during the gene transcription process. These factors have related but distinct functions during transcription and several recent studies have provided exciting new insights into their mechanisms of action at transcribed genes. A discussion of their respective roles in regulating gene transcription, including their shared and unique contributions to this process, is presented.
A Nucleosomal Region Important for Ensuring Proper Interactions Between the Transcription Elongation Factor Spt16 and Transcribed Genes in Saccharomyces Cerevisiae
G3 (Bethesda, Md.). Jun, 2013 | Pubmed ID: 23576521
The highly conserved FACT (FAcilitates Chromatin Transactions) histone chaperone assists in the transcription elongation process first by facilitating the removal of histones in front of transcribing RNA polymerase II (Pol II) and then by contributing to nucleosome reassembly in the wake of Pol II passage. Whereas it is well established that FACT localizes across actively transcribed genes, the mechanisms that regulate FACT recruitment to and disengagement from chromatin during transcription still remain to be elucidated. Using the Saccharomyces cerevisiae model system, we previously showed that a histone H3 mutant--H3-L61W--greatly perturbs interactions between the yeast FACT (yFACT) complex and chromatin during transcription, resulting in a pronounced shift in yFACT occupancy toward the 3' ends of transcribed genes. In the present study we report that two histone H4 mutants-H4-R36A and H4-K31E-alter the association pattern of the yFACT subunit Spt16 across transcribed genes in a fashion similar to that seen for H3-L61W. Interestingly, H4-R36, H4-K31, and H3-L61 are in close proximity to each other on the side of the nucleosome. We also provide evidence that the H4-R36A and H3-L61W mutants impair proper Spt16-chromatin interactions by perturbing a common process. Collectively, our results suggest that a nucleosomal region encompassing the H4-R36, H4-K31, and H3-L61 residues plays an important role in ensuring proper association of yFACT across transcribed genes.
New Roles for Old Characters: an Educational Primer for Use with "Vps Factors Are Required for Efficient Transcription Elongation in Budding Yeast"
Genetics. May, 2013 | Pubmed ID: 23633140
An article from Alan Hinnebusch's laboratory in the March 2013 issue of GENETICS establishes an exciting new link between proteins with well-established roles in the endomembrane system and the process of transcription elongation. This Primer article provides tools needed for students to fully appreciate, analyze, and critically evaluate the experiments and interpretations of Gaur et al. (2013). The primer includes detailed descriptions of techniques used in the study, such as the chromatin immunoprecipitation assay and assays for transcription elongation, and it provides a framework to facilitate an understanding of how a combination of genetic, biochemical, and cell microscopy experimental approaches were used by the authors to converge on a single major conclusion. Suggestions for using this Primer article in an undergraduate or graduate-level course in conjunction with the original article to promote student learning are also presented.
Genetics. May, 2014 | Pubmed ID: 24807111
The budding yeast Saccharomyces cerevisiae is a powerful model organism for studying fundamental aspects of eukaryotic cell biology. This Primer article presents a brief historical perspective on the emergence of this organism as a premier experimental system over the course of the past century. An overview of the central features of the S. cerevisiae genome, including the nature of its genetic elements and general organization, is also provided. Some of the most common experimental tools and resources available to yeast geneticists are presented in a way designed to engage and challenge undergraduate and graduate students eager to learn more about the experimental amenability of budding yeast. Finally, a discussion of several major discoveries derived from yeast studies highlights the far-reaching impact that the yeast system has had and will continue to have on our understanding of a variety of cellular processes relevant to all eukaryotes, including humans.
A Systematic Mutational Analysis of a Histone H3 Residue in Budding Yeast Provides Insights into Chromatin Dynamics
G3 (Bethesda, Md.). Feb, 2015 | Pubmed ID: 25711831
In previous work using the Saccharomyces cerevisiae model system, a mutant version of histone H3-H3-L61W-was found to confer a variety of abnormal growth phenotypes and defects in specific aspects of the transcription process, including a pronounced alteration in the distribution pattern of the transcription elongation factor Spt16 across transcribed genes and promotion of cryptic transcription initiation within the FLO8 gene. To gain insights into the contribution of the H3-L61 residue to chromatin function, we have generated yeast strains expressing versions of histone H3 harboring all possible natural amino acid substitutions at position 61 (H3-L61X mutants) and tested them in a series of assays. We found that whereas 16 of the 19 H3-L61X mutants support viability when expressed as the sole source of histone H3 in cells, all 19 confer abnormal phenotypes ranging from very mild to severe, a finding that might in part explain the high degree of conservation of the H3-L61 residue among eukaryotes. An examination of the strength of the defects conferred by each H3-L61X mutant and the nature of the corresponding substituted residue provides insights into structural features of the nucleosome required for proper Spt16-gene interactions and for prevention of cryptic transcription initiation events. Finally, we provide evidence that the defects imparted by H3-L61X mutants on Spt16-gene interactions and on repression of intragenic transcription initiation are mechanistically related to each other.