Peter R. Eriksson

Spt16–Pob3 and the HMG protein Nhp6 combine to form the nucleosome-binding factor SPN

Yeast Spt16/Cdc68 and Pob3 form a heterodimer that acts in both DNA replication and transcription. This is supported by studies of new alleles of SPT16 described here. We show that Spt16–Pob3 enhances HO transcription through a mechanism that is affected by chromatin modification, since some of the defects caused by mutations can be suppressed by deleting the histone deacetylase Rpd3. While otherwise conserved among many eukaryotes, Pob3 lacks the HMG1 DNA‐binding motif found in similar proteins such as the SSRP1 subunit of human FACT. SPT16 and POB3 display strong genetic interactions with NHP6A/B, which encodes an HMG1 motif, suggesting that these gene products function coordinately in vivo. While Spt16–Pob3 and Nhp6 do not appear to form stable heterotrimers, Nhp6 binds to nucleosomes and these Nhp6–nucleosomes can recruit Spt16–Pob3 to form SPN–nucleosomes. These complexes have altered electrophoretic mobility and a distinct pattern of enhanced sensitivity to DNase I. These results suggest that Spt16–Pob3 and Nhp6 cooperate to function as a novel nucleosome reorganizing factor.

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

ABSTRACT Recent work has shown that transcription of the yeastHO gene involves the sequential recruitment of a series of transcription factors. We have performed a functional analysis ofHO regulation by determining the ability of mutations inSIN1, SIN3, RPD3, andSIN4 negative regulators to permit HOexpression in the absence of certain activators. Mutations in theSIN1 (=SPT2) gene do not affect HOregulation, in contrast to results of other studies using anHO: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 andRPD3, 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 sin4mutant. 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 inHO 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 withsin3, 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 ofSIN4 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.

Regulation of Histone Gene Expression in Budding Yeast

We discuss the regulation of the histone genes of the budding yeast Saccharomyces cerevisiae. These include genes encoding the major core histones (H3, H4, H2A, and H2B), histone H1 (HHO1), H2AZ (HTZ1), and centromeric H3 (CSE4). Histone production is regulated during the cell cycle because the cell must replicate both its DNA during S phase and its chromatin. Consequently, the histone genes are activated in late G1 to provide sufficient core histones to assemble the replicated genome into chromatin. The major core histone genes are subject to both positive and negative regulation. The primary control system is positive, mediated by the histone gene-specific transcription activator, Spt10, through the histone upstream activating sequences (UAS) elements, with help from the major G1/S-phase activators, SBF (Swi4 cell cycle box binding factor) and perhaps MBF (MluI cell cycle box binding factor). Spt10 binds specifically to the histone UAS elements and contains a putative histone acetyltransferase domain. The negative system involves negative regulatory elements in the histone promoters, the RSC chromatin-remodeling complex, various histone chaperones [the histone regulatory (HIR) complex, Asf1, and Rtt106], and putative sequence-specific factors. The SWI/SNF chromatin-remodeling complex links the positive and negative systems. We propose that the negative system is a damping system that modulates the amount of transcription activated by Spt10 and SBF. We hypothesize that the negative system mediates negative feedback on the histone genes by histone proteins through the level of saturation of histone chaperones with histone. Thus, the negative system could communicate the degree of nucleosome assembly during DNA replication and the need to shut down the activating system under replication-stress conditions. We also discuss post-transcriptional regulation and dosage compensation of the histone genes.

Regulation of TATA-binding protein binding by the SAGA complex and the Nhp6 high-mobility group protein

ABSTRACT Transcriptional activation of the yeast HO gene involves the sequential action of DNA-binding and chromatin-modifying factors. Here we examine the role of the SAGA complex and the Nhp6 architectural transcription factor in HO regulation. Our data suggest that these factors regulate binding of the TATA-binding protein (TBP) to the promoter. A gcn5 mutation, eliminating the histone acetyltransferase present in SAGA, reduces the transcription of HO, but expression is restored in a gcn5 spt3 double mutant. We conclude that the major role of Gcn5 in HO activation is to overcome repression by Spt3. Spt3 is also part of SAGA, and thus two proteins in the same regulatory complex can have opposing roles in transcriptional regulation. Chromatin immunoprecipitation experiments show that TBP binding to HO is very weak in wild-type cells but markedly increased in an spt3 mutant, indicating that Spt3 reduces HO expression by inhibiting TBP binding. In contrast, it has been shown previously that Spt3 stimulates TBP binding to the GAL1 promoter as well as GAL1 expression, and thus, Spt3 regulates these promoters differently. We also find genetic interactions between TBP and either Gcn5 or the high-mobility-group protein Nhp6, including multicopy suppression and synthetic lethality. These results suggest that, while Spt3 acts to inhibit TBP interaction with the HO promoter, Gcn5 and Nhp6 act to promote TBP binding. The result of these interactions is to limit TBP binding and HO expression to a short period within the cell cycle. Furthermore, the synthetic lethality resulting from combining a gcn5 mutation with specific TBP point mutations can be suppressed by the overexpression of transcription factor IIA (TFIIA), suggesting that histone acetylation by Gcn5 can stimulate transcription by promoting the formation of a TBP/TFIIA complex.

Global Regulation by the Yeast Spt10 Protein Is Mediated through Chromatin Structure and the Histone Upstream Activating Sequence Elements

ABSTRACT The yeast SPT10 gene encodes a putative histone acetyltransferase (HAT) implicated as a global transcription regulator acting through basal promoters. Here we address the mechanism of this global regulation. Although microarray analysis confirmed that Spt10p is a global regulator, Spt10p was not detected at any of the most strongly affected genes in vivo. In contrast, the presence of Spt10p at the core histone gene promoters in vivo was confirmed. Since Spt10p activates the core histone genes, a shortage of histones could occur in spt10Δ cells, resulting in defective chromatin structure and a consequent activation of basal promoters. Consistent with this hypothesis, the spt10Δ phenotype can be rescued by extra copies of the histone genes and chromatin is poorly assembled in spt10Δ cells, as shown by irregular nucleosome spacing and reduced negative supercoiling of the endogenous 2μm plasmid. Furthermore, Spt10p binds specifically and highly cooperatively to pairs of upstream activating sequence elements in the core histone promoters [consensus sequence, (G/A)TTCCN6TTCNC], consistent with a direct role in histone gene regulation. No other high-affinity sites are predicted in the yeast genome. Thus, Spt10p is a sequence-specific activator of the histone genes, possessing a DNA-binding domain fused to a likely HAT domain.

TATA-Binding Protein Mutants That Are Lethal in the Absence of the Nhp6 High-Mobility-Group Protein

ABSTRACT The Saccharomyces cerevisiae Nhp6 protein is related to the high-mobility-group B family of architectural DNA-binding proteins that bind DNA nonspecifically but bend DNA sharply. Nhp6 is involved in transcriptional activation by both RNA polymerase II (Pol II) and Pol III. Our previous genetic studies have implicated Nhp6 in facilitating TATA-binding protein (TBP) binding to some Pol II promoters in vivo, and we have used a novel genetic screen to isolate 32 new mutations in TBP that are viable in wild-type cells but lethal in the absence of Nhp6. The TBP mutations that are lethal in the absence of Nhp6 cluster in three regions: on the upper surface of TBP that may have a regulatory role, near residues that contact Spt3, or near residues known to contact either TFIIA or Brf1 (in TFIIIB). The latter set of mutations suggests that Nhp6 becomes essential when a TBP mutant compromises its ability to interact with either TFIIA or Brf1. Importantly, the synthetic lethality for some of the TBP mutations is suppressed by a multicopy plasmid with SNR6 or by an spt3 mutation. It has been previously shown that nhp6ab mutants are defective in expressing SNR6, a Pol III-transcribed gene encoding the U6 splicing RNA. Chromatin immunoprecipitation experiments show that TBP binding to SNR6 is reduced in an nhp6ab mutant. Nhp6 interacts with Spt16/Pob3, the yeast equivalent of the FACT elongation complex, consistent with nhp6ab cells being extremely sensitive to 6-azauracil (6-AU). However, this 6-AU sensitivity can be suppressed by multicopy SNR6 or BRF1. Additionally, strains with SNR6 promoter mutations are sensitive to 6-AU, suggesting that decreased SNR6 RNA levels contribute to 6-AU sensitivity. These results challenge the widely held belief that 6-AU sensitivity results from a defect in transcriptional elongation.

Role for Nhp6, Gcn5, and the Swi/Snf Complex in Stimulating Formation of the TATA-Binding Protein-TFIIA-DNA Complex

ABSTRACT The TATA-binding protein (TBP), TFIIA, and TFIIB interact with promoter DNA to form a complex required for transcriptional initiation, and many transcriptional regulators function by either stimulating or inhibiting formation of this complex. We have recently identified TBP mutants that are viable in wild-type cells but lethal in the absence of the Nhp6 architectural transcription factor. Here we show that many of these TBP mutants were also lethal in strains with disruptions of either GCN5, encoding the histone acetyltransferase in the SAGA complex, or SWI2, encoding the catalytic subunit of the Swi/Snf chromatin remodeling complex. These synthetic lethalities could be suppressed by overexpression of TOA1 and TOA2, the genes encoding TFIIA. We also used TFIIA mutants that eliminated in vitro interactions with TBP. These viable TFIIA mutants were lethal in strains lacking Gcn5, Swi2, or Nhp6. These lethalities could be suppressed by overexpression of TBP or Nhp6, suggesting that these coactivators stimulate formation of the TBP-TFIIA-DNA complex. In vitro studies have previously shown that TBP binds very poorly to a TATA sequence within a nucleosome but that Swi/Snf stimulates binding of TBP and TFIIA. In vitro binding experiments presented here show that histone acetylation facilitates TBP binding to a nucleosomal binding site and that Nhp6 stimulates formation of a TBP-TFIIA-DNA complex. Consistent with the idea that Nhp6, Gcn5, and Swi/Snf have overlapping functions in vivo, nhp6a nhp6b gcn5 mutants had a severe growth defect, and mutations in both nhp6a nhp6b swi2 and gcn5 swi2 strains were lethal.

The ISW1 and CHD1 ATP-dependent chromatin remodelers compete to set nucleosome spacing in vivo

Abstract Adenosine triphosphate-dependent chromatin remodeling machines play a central role in gene regulation by manipulating chromatin structure. Most genes have a nucleosome-depleted region at the promoter and an array of regularly spaced nucleosomes phased relative to the transcription start site. In vitro, the three known yeast nucleosome spacing enzymes (CHD1, ISW1 and ISW2) form arrays with different spacing. We used genome-wide nucleosome sequencing to determine whether these enzymes space nucleosomes differently in vivo. We find that CHD1 and ISW1 compete to set the spacing on most genes, such that CHD1 dominates genes with shorter spacing and ISW1 dominates genes with longer spacing. In contrast, ISW2 plays a minor role, limited to transcriptionally inactive genes. Heavily transcribed genes show weak phasing and extreme spacing, either very short or very long, and are depleted of linker histone (H1). Genes with longer spacing are enriched in H1, which directs chromatin folding. We propose that CHD1 directs short spacing, resulting in eviction of H1 and chromatin unfolding, whereas ISW1 directs longer spacing, allowing H1 to bind and condense the chromatin. Thus, competition between the two remodelers to set the spacing on each gene may result in a highly dynamic chromatin structure.

Spt10 and Swi4 Control the Timing of Histone H2A/H2B Gene Activation in Budding Yeast

ABSTRACT The expression of the histone genes is regulated during the cell cycle to provide histones for nucleosome assembly during DNA replication. In budding yeast, histones H2A and H2B are expressed from divergent promoters at the HTA1-HTB1 and HTA2-HTB2 loci. Here, we show that the major activator of HTA1-HTB1 is Spt10, a sequence-specific DNA binding protein with a putative histone acetyltransferase (HAT) domain. Spt10 binds to two pairs of upstream activation sequence (UAS) elements in the HTA1-HTB1 promoter: UAS1 and UAS2 drive HTA1 expression, and UAS3 and UAS4 drive HTB1 expression. UAS3 and UAS4 also contain binding sites for the cell cycle regulator SBF (an Swi4-Swi6 heterodimer), which overlap the Spt10 binding sites. The binding of Spt10 and binding of SBF to UAS3 and UAS4 are mutually exclusive in vitro. Both SBF and Spt10 are bound in cells arrested with α-factor, apparently awaiting a signal to activate transcription. Soon after the removal of α-factor, SBF initiates a small, early peak of HTA1 and HTB1 transcription, which is followed by a much larger peak due to Spt10. Both activators dissociate from the HTA1-HTB1 promoter after expression has been activated. Thus, SBF and Spt10 cooperate to control the timing of HTA1-HTB1 expression.

The DNA-binding domain of the yeast Spt10p activator includes a zinc finger that is homologous to foamy virus integrase.

The yeast SPT10 gene encodes a putative histone acetyltransferase that binds specifically to pairs of upstream activating sequence (UAS) elements found only in the histone gene promoters. Here, we demonstrate that the DNA-binding domain of Spt10p is located between residues 283 and 396 and includes a His2-Cys2 zinc finger. The binding of Spt10p to the histone UAS is zinc-dependent and is disabled by a zinc finger mutation (C388S). The isolated DNA-binding domain binds to single histone UAS elements with high affinity. In contrast, full-length Spt10p binds with high affinity only to pairs of UAS elements with very strong positive cooperativity and is unable to bind to a single UAS element. This implies the presence of a “blocking” domain in full-length Spt10p, which forces it to search for a pair of UAS elements. Chromatin immunoprecipitation experiments indicate that, unlike wild-type Spt10p, the C388S protein does not bind to the promoter of the gene encoding histone H2A (HTA1) in vivo. The C388S mutant has a phenotype similar to that of the spt10Δ mutant: poor growth and global aberrations in gene expression. Thus, the C388S mutation disables the DNA-binding function of Spt10p in vitro and in vivo. The zinc finger of Spt10p is homologous to that of foamy virus integrase, perhaps suggesting that this integrase is also a sequence-specific DNA-binding protein.