7 research outputs found
Evidence that Spt2/Sin1, an HMG-Like Factor, Plays Roles in Transcription Elongation, Chromatin Structure, and Genome Stability in Saccharomyces cerevisiae
Spt2/Sin1 is a DNA binding protein with HMG-like domains that has been suggested to play a role in chromatin-mediated transcription in Saccharomyces cerevisiae. Previous studies have suggested models in which Spt2 plays an inhibitory role in the initiation of transcription of certain genes. In this work, we have taken several approaches to study Spt2 in greater detail. Our results have identified previously unknown genetic interactions between spt2Δ and mutations in genes encoding transcription elongation factors, including members of the PAF and HIR/HPC complexes. In addition, genome-wide and gene-specific chromatin immunoprecipitation analyses suggest that Spt2 is primarily associated with coding regions in a transcription-dependent fashion. Furthermore, our results show that Spt2, like other elongation factors, is required for the repression of transcription from a cryptic promoter within a coding region and that Spt2 is also required for repression of recombination within transcribed regions. Finally, we provide evidence that Spt2 plays a role in regulating the levels of histone H3 over transcribed regions. Taken together, our results suggest a direct link for Spt2 with transcription elongation, chromatin dynamics, and genome stability
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
Structure–function studies of histone H3/H4 tetramer maintenance during transcription by chaperone Spt2
Eaf1 Is the Platform for NuA4 Molecular Assembly That Evolutionarily Links Chromatin Acetylation to ATP-Dependent Exchange of Histone H2A Variants▿
Eaf1 (for Esa1-associated factor 1) and Eaf2 have been identified as stable subunits of NuA4, a yeast histone H4/H2A acetyltransferase complex implicated in gene regulation and DNA repair. While both SWI3-ADA2-N-CoR-TF IIIB domain-containing proteins are required for normal cell cycle progression, their depletion does not affect the global Esa1-dependent acetylation of histones. In contrast to all other subunits, Eaf1 is found exclusively associated with the NuA4 complex in vivo. It serves as a platform that coordinates the assembly of functional groups of subunits into the native NuA4 complex. Eaf1 shows structural similarities with human p400/Domino, a subunit of the NuA4-related TIP60 complex. On the other hand, p400 also possesses an SWI2/SNF2 family ATPase domain that is absent from the yeast NuA4 complex. This domain is highly related to the yeast Swr1 protein, which is responsible for the incorporation of histone variant H2AZ in chromatin. Since all of the components of the TIP60 complex are homologous to SWR1 or NuA4 subunits, we proposed that the human complex corresponds to a physical merge of two yeast complexes. p400 function in TIP60 then would be accomplished in yeast by cooperation between SWR1 and NuA4. In agreement with such a model, NuA4 and SWR1 mutants show strong genetic interactions, NuA4 affects histone H2AZ incorporation/acetylation in vivo, and both preset the PHO5 promoter for activation. Interestingly, the expression of a chimeric Eaf1-Swr1 protein recreates a single human-like complex in yeast cells. Our results identified the key central subunit for the structure and functions of the NuA4 histone acetyltransferase complex and functionally linked this activity with the histone variant H2AZ from yeast to human cells
Recruitment of the NuA4 complex poises the PHO5 promoter for chromatin remodeling and activation
The remodeling of the promoter chromatin structure is a key event for the induction of the PHO5 gene. Two DNA-binding proteins Pho2 and Pho4 are critical for this step. We found that the NuA4 histone acetyltransferase complex is essential for PHO5 transcriptional induction without affecting Pho4 translocation upon phosphate starvation. Our data also indicate that NuA4 is critical for the chromatin remodeling event that occurs over the PHO5 promoter prior to activation. Using Chromatin IP analysis, we found that Esa1-dependent histone H4 acetylation at the PHO5 promoter correlates with specific recruitment of the NuA4 complex to this locus under repressing conditions. We demonstrate that the homeodomain transcriptional activator Pho2 is responsible for this recruitment in vivo and interacts directly with the NuA4 complex. Finally, we show that Pho4 is unable to bind the PHO5 promoter without prior action of NuA4. These results indicate that, before induction, NuA4 complex recruitment by Pho2 is an essential event that presets the PHO5 promoter for subsequent binding by Pho4, chromatin remodeling and transcription