Architectural Transcription Factors and the SAGA Complex Function in Parallel Pathways To Activate Transcription

Abstract

Recent work has shown that transcription of the yeast HO gene involves the sequential recruitment of a series of transcription factors. We have performed a functional analysis of HO regulation by determining the ability of mutations in SIN1, SIN3, RPD3, and SIN4 negative regulators to permit HO expression in the absence of certain activators. Mutations in the SIN1 (‫؍‬SPT2) gene do not affect HO regulation, in contrast to results of other studies using an HO:lacZ reporter, and our data show that the regulatory properties of an HO:lacZ reporter differ from that of the native HO gene. Mutations in SIN3 and RPD3, which encode components of a histone deacetylase complex, show the same pattern of genetic suppression, and this suppression pattern differs from that seen in a sin4 mutant. The Sin4 protein is present in two transcriptional regulatory complexes, the RNA polymerase II holoenzyme/mediator and the SAGA histone acetylase complex. Our genetic analysis allows us to conclude that Swi/Snf chromatin remodeling complex has multiple roles in HO activation, and the data suggest that the ability of the SBF transcription factor to bind to the HO promoter may be affected by the acetylation state of the HO promoter. We also demonstrate that the Nhp6 architectural transcription factor, encoded by the redundant NHP6A and NHP6B genes, is required for HO expression. Suppression analysis with sin3, rpd3, and sin4 mutations suggests that Nhp6 and Gcn5 have similar functions. A gcn5 nhp6a nhp6b triple mutant is extremely sick, suggesting that the SAGA complex and the Nhp6 architectural transcription factors function in parallel pathways to activate transcription. We find that disruption of SIN4 allows this strain to grow at a reasonable rate, indicating a critical role for Sin4 in detecting structural changes in chromatin mediated by Gcn5 and Nhp6. These studies underscore the critical role of chromatin structure in regulating HO gene expression.The Saccharomyces cerevisiae HO gene encodes an endonuclease that is responsible for initiating mating type switching in yeast. The transcriptional regulation of HO is complex and has been the subject of intensive study (for reviews, see references 22 and 37). Recent studies have shown that transcription of specific genes can be affected by chromatin structure at the promoter (for reviews, see references 25, 28, 54, and 59). Chromatin structure plays an important role in regulation of HO transcription, as HO expression is altered by mutations in a number of important transcriptional regulators, including components of the Swi/Snf chromatin remodeling complex, the SAGA histone acetyltransferase complex, and the Sin3/Rpd3 histone deacetylase complex. GCN5, ADA2, and ADA3, which encode members of the SAGA histone acetyltransferase complex (18), are required for HO:lacZ expression (43), and native HO expression is also reduced in a gcn5 mutant (41). The yeast RPD3 gene encodes a histone deacetylase that is associated with Sin3 (26, 27). SIN3 and RPD3 are negative regulators of transcription...

SWI/SNF Binding to the HO Promoter Requires Histone Acetylation and Stimulates TATA-Binding Protein Recruitment

“…Importantly, the defect in HO expression in a gcn5 mutant can be suppressed by mutations in ASH1 (Fig. 2C), RPD3, or SIN3 (86). It follows from this model of Ash1 recruiting the large Sin3-Rpd3 complex to the promoter that the suppression of gcn5 provided by the sin3 and ash1 mutants should not be additive.…”

“…Our previous genetic analysis demonstrated that some mutations can suppress temporally downstream events, such as the lack of the SBF (Swi4/Swi6) factor, but not upstream factors, such as mutations in SWI/SNF (86). These results suggest that the regulatory pathway is not simply linear.…”

“…A sin3 mutation also allows HO to be expressed despite a gcn5 mutation (86) and raises the question of whether SWI/SNF binds to the HO promoter in the gcn5 sin3…”

“…Sin3 exists in a complex with the Rpd3 histone deacetylase (46,47), and genetic experiments have shown that sin3 and rpd3 mutations have similar phenotypes (80). We previously examined the abilities of suppressor mutations to allow HO expression in the absence of various activators (85,86). A sin3 mutation suppresses the requirement for the Swi5 DNA-binding protein or the Gcn5 HAT, but sin3 does not allow HO expression in the absence of Swi2 (in the SWI/SNF remodeler) or Swi6 (in the SBF DNA-binding protein).…”

Multiple Nhp6 Molecules Are Required to Recruit Spt16-Pob3 to Form yFACT Complexes and to Reorganize Nucleosomes

“…29). Mutations in yFACT subunits cause synthetic effects when combined with mutations in either histones or histone-modifying enzymes (9,11,30), so Nhp6 and yFACT activities could be affected by either histone modifications or altered histone sequences. As shown in Table I, nucleosomes assembled using endogenous chicken histone octamers required somewhat higher levels of Nhp6 to form yFACT⅐ nucleosome complexes than nucleosomes assembled with unmodified recombinant yeast histone octamers.…”