RESUMO
OBJECTIVE: In past work in budding yeast, we identified a nucleosomal region required for proper interactions between the histone chaperone complex yFACT and transcribed genes. Specific histone mutations within this region cause a shift in yFACT occupancy towards the 3' end of genes, a defect that we have attributed to impaired yFACT dissociation from DNA following transcription. In this work we wished to assess the contributions of DNA sequences at the 3' end of genes in promoting yFACT dissociation upon transcription termination. RESULTS: We generated fourteen different alleles of the constitutively expressed yeast gene PMA1, each lacking a distinct DNA fragment across its 3' end, and assessed their effects on occupancy of the yFACT component Spt16. Whereas most of these alleles conferred no defects on Spt16 occupancy, one did cause a modest increase in Spt16 binding at the gene's 3' end. Interestingly, the same allele also caused minor retention of RNA Polymerase II (Pol II) and altered nucleosome occupancy across the same region of the gene. These results suggest that specific DNA sequences at the 3' ends of genes can play roles in promoting efficient yFACT and Pol II dissociation from genes and can also contribute to proper chromatin architecture.
Assuntos
Nucleossomos , RNA Polimerase II , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Nucleossomos/metabolismo , Nucleossomos/genética , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , Alelos , Sequência de Bases , Regulação Fúngica da Expressão Gênica , Transcrição GênicaRESUMO
Recent studies have unveiled an association between an L61R substitution within the human histone H3.3 protein and the presentation of neurodevelopmental disorders in two patients. In both cases, the mutation responsible for this substitution is encoded by one allele of the H3F3A gene and, if this mutation is indeed responsible for the disease phenotypes, it must act in a dominant fashion since the genomes of these patients also harbour three other alleles encoding wild-type histone H3.3. In our previous work in yeast, we have shown that most amino acid substitutions at H3-L61 cause an accumulation of the Spt16 component of the yFACT histone chaperone complex at the 3' end of transcribed genes, a defect we have attributed to impaired yFACT dissociation from chromatin following transcription. In those studies, however, the H3-L61R mutant had not been tested since it does not sustain viability when expressed as the sole source of histone H3 in cells. In the present work, we show that H3-L61R impairs proper Spt16 dissociation from genes when co-expressed with wild-type histone H3 in haploid cells as well as in diploid cells that express the mutant protein from one of four histone H3-encoding alleles. These results, combined with other studies linking loss of function mutations in human Spt16 and neurodevelopmental disorders, provide a possible molecular mechanism underlying the neurodevelopmental disorders seen in patients expressing the histone H3.3 L61R mutant.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Humanos , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/química , Saccharomycetales/genética , Saccharomycetales/metabolismo , Metilação de DNA , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Mutação , Nucleossomos/metabolismoRESUMO
FACT (FAcilitates Chromatin Transactions) is a highly conserved histone chaperone complex in eukaryotic cells that can interact and manipulate nucleosomes in order to promote a variety of DNA-based processes and to maintain the integrity of chromatin throughout the genome. Whereas key features of the physical interactions that occur between FACT and nucleosomes in vitro have been elucidated in recent years, less is known regarding FACT functional dynamics in vivo. Using the Saccharomyces cerevisiae system, we now provide evidence that at least at some genes dissociation of the FACT subunit Spt16 from their 3' ends is partially dependent on RNA Polymerase II (Pol II) termination. Combined with other studies, our results are consistent with a two-phase mechanism for FACT dissociation from genes, one that occurs upstream from Pol II dissociation and is Pol II termination-independent and the other that occurs further downstream and is dependent on Pol II termination.
Assuntos
RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Elongação da Transcrição/metabolismo , Saccharomyces cerevisiae/metabolismo , Terminação da Transcrição GenéticaRESUMO
Previous work in Saccharomyces cerevisiae identified three residues located in close proximity to each other on the side of the nucleosome whose integrity is required for proper association of the Spt16 component of the FACT complex across transcribed genes. In an effort to gain further insights into the parameters that control Spt16 interactions with genes in vivo, we tested the effects of additional histone mutants on Spt16 occupancy across two constitutively transcribed genes. These studies revealed that mutations in several charged residues in the vicinity of the three residues originally identified as important for Spt16-gene interactions also significantly perturb normal association of Spt16 across genes. Based on these and our previous findings, we propose that the charge landscape across the region encompassed by these residues, which we refer to as the Influences Spt16-Gene Interactions or ISGI region, is an important contributor to proper Spt16-gene interactions in vivo.
Assuntos
Histonas/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Alanina/genética , Substituição de Aminoácidos , Imunoprecipitação da Cromatina , Regulação Fúngica da Expressão Gênica , Histonas/química , Histonas/genética , Mutação , Nucleossomos/química , Nucleossomos/genética , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Fatores de Elongação da Transcrição/químicaRESUMO
We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.
Assuntos
Histonas/genética , Mutagênese Sítio-Dirigida/métodos , Reação em Cadeia da Polimerase/métodos , Saccharomycetales/genética , Alelos , Recombinação Homóloga , Fases de Leitura Aberta , Ácido Orótico/análogos & derivados , Ácido Orótico/farmacologia , Plasmídeos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomycetales/efeitos dos fármacosRESUMO
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.
Assuntos
Cromatina/metabolismo , Histonas/genética , Substituição de Aminoácidos , Mutação , Nucleossomos/química , Fenótipo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Iniciação da Transcrição Genética , Fatores de Elongação da Transcrição/genéticaRESUMO
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.
Assuntos
Biologia Molecular/métodos , Saccharomyces cerevisiae/genética , Animais , Cromossomos Fúngicos/genética , Farmacorresistência Fúngica , Genoma Fúngico/genética , Humanos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacosRESUMO
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.
Assuntos
Genética/educação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Elongação da Transcrição Genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
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.
Assuntos
Genes Fúngicos/genética , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/metabolismo , Aminoácidos/metabolismo , Histonas/metabolismo , Modelos Moleculares , Proteínas Mutantes/metabolismo , Mutação/genética , Fenótipo , Ligação Proteica/genética , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/citologiaRESUMO
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.
Assuntos
Histonas/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo , Cromatina/metabolismo , Histonas/genética , Proteínas Mutantes/genética , Proteínas Mutantes/isolamento & purificação , Fenótipo , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/isolamento & purificaçãoRESUMO
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.
RESUMO
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.
Assuntos
Cromatina/metabolismo , Histonas/metabolismo , Mutação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Cromatina/genética , Histonas/genética , Ligação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Elongação da Transcrição/genéticaRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/genética , Regulação Fúngica da Expressão Gênica , Histonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transcrição Gênica , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Histonas/metabolismo , Mutação/genética , Nucleossomos , Plasmídeos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Elongação da TranscriçãoRESUMO
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.