Trapping of yFACT at 3' ends of genes is not a universal characteristic of yeast versions of Bryant-Li-Bhoj syndrome histone H3 mutants.

Bryant-Li-Bhoj syndrome (BLBS) is associated with germline mutations in the genes encoding human histone H3.3. While to date 70 H3.

Assessing contributions of DNA sequences at the 3' end of a yeast gene on yFACT, RNA polymerase II, and nucleosome occupancy.

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.

Dominant effects of the histone mutant H3-L61R on Spt16-gene interactions in budding yeast.

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 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.

Evidence that dissociation of Spt16 from transcribed genes is partially dependent on RNA Polymerase II termination.

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 have been elucidated in recent years, less is known regarding FACT functional dynamics . Using the 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.

Charged residues on the side of the nucleosome contribute to normal Spt16-gene interactions in budding yeast.

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.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast.

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.

A systematic mutational analysis of a histone H3 residue in budding yeast provides insights into chromatin dynamics.

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.

Budding yeast for budding geneticists: a primer on the Saccharomyces cerevisiae model system.

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.

New roles for old characters: an educational primer for use with "Vps factors are required for efficient transcription elongation in budding yeast".

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).

A nucleosomal region important for ensuring proper interactions between the transcription elongation factor Spt16 and transcribed genes in Saccharomyces cerevisiae.

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.

Mutant versions of the S. cerevisiae transcription elongation factor Spt16 define regions of Spt16 that functionally interact with histone H3.

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.

Histone Chaperones Spt6 and FACT: Similarities and Differences in Modes of Action at Transcribed Genes.

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.

Uncoupling of the patterns of chromatin association of different transcription elongation factors by a histone H3 mutant in Saccharomyces cerevisiae.

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.

Evidence that the localization of the elongation factor Spt16 across transcribed genes is dependent upon histone H3 integrity in Saccharomyces cerevisiae.

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.

Analysis of a mutant histone H3 that perturbs the association of Swi/Snf with chromatin.

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.