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. 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

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. 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

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. 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

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

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 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

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

<p>Previous work in <i>Saccharomyces cerevisiae</i> 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 <i>in vivo</i>, 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 <i>in vivo.</i></p