Tim Formosa

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.

The Saccharomyces cerevisiae DNA polymerase alpha catalytic subunit interacts with Cdc68/Spt16 and with Pob3, a protein similar to an HMG1-like protein.

We have used DNA polymerase alpha affinity chromatography to identify factors involved in eukaryotic DNA replication in the yeast Saccharomyces cerevisiae. Two proteins that bound to the catalytic subunit of DNA polymerase alpha (Pol1 protein) are encoded by the essential genes CDC68/SPT16 and POB3. The binding of both proteins was enhanced when extracts lacking a previously characterized polymerase binding protein, Ctf4, were used. This finding suggests that Cdc68 and Pob3 may compete with Ctf4 for binding to Pol1. Pol1 and Pob3 were coimmunoprecipitated from whole-cell extracts with antiserum directed against Cdc68, and Pol1 was immunoprecipitated from whole-cell extracts with antiserum directed against the amino terminus of Pob3, suggesting that these proteins may form a complex in vivo. CDC68 also interacted genetically with POL1 and CTF4 mutations; the maximum permissive temperature of double mutants was lower than for any single mutant. Overexpression of Cdc68 in a pol1 mutant strain dramatically decreased cell viability, consistent with the formation or modulation of an essential complex by these proteins in vivo. A mutation in CDC68/SPT16 had previously been shown to cause pleiotropic effects on the regulation of transcription (J. A. Prendergrast et al., Genetics 124:81-90, 1990; E. A. Malone et al., Mol. Cell. Biol. 11:5710-5717, 1991; A. Rowley et al., Mol. Cell. Biol. 11:5718-5726, 1991), with a spectrum of phenotypes similar to those caused by mutations in the genes encoding histone proteins H2A and H2B (Malone et al., Mol. Cell. Biol. 11:5710-5717, 1991). We show that at the nonpermissive temperature, cdc68-1 mutants arrest as unbudded cells with a 1C DNA content, consistent with a possible role for Cdc68 in the prereplicative stage of the cell cycle. The cdc68-1 mutation caused elevated rates of chromosome fragment loss, a phenotype characteristic of genes whose native products are required for normal DNA metabolism. However, this mutation did not affect the rate of loss or recombination for two intact chromosomes, nor did it affect the retention of a low-copy-number plasmid. The previously uncharacterized Pob3 sequence has significant amino acid sequence similarity with an HMG1-like protein from vertebrates. Based on these results and because Cdc68 has been implicated as a regulator of chromatin structure, we postulate that polymerase alpha may interact with these proteins to gain access to its template or to origins of replication in vivo.

yFACT Induces Global Accessibility of Nucleosomal DNA without H2A-H2B Displacement

FACT has been proposed to function by displacing H2A-H2B dimers from nucleosomes to form hexasomes. Results described here with yeast FACT (yFACT) suggest instead that nucleosomes are reorganized to a form with the original composition but a looser, more dynamic structure. First, yFACT enhances hydroxyl radical accessibility and endonuclease digestion in vitro at sites throughout the nucleosome, not just in regions contacted by H2A-H2B. Accessibility increases dramatically, but the DNA remains partially protected. Second, increased nuclease sensitivity can occur without displacement of dimers from the nucleosome. Third, yFACT is required for eviction of nucleosomes from the GAL1-10 promoter during transcriptional activation in vivo, but the preferential reduction in dimer occupancy expected for hexasome formation is not observed. We propose that yFACT promotes a reversible transition between two nucleosomal forms, and that this activity contributes to the establishment and maintenance of the chromatin barrier as well as to overcoming it.

The role of FACT in making and breaking nucleosomes

FACT is a roughly 180kDa heterodimeric protein complex important for managing the properties of chromatin in eukaryotic cells. Chromatin is a repressive barrier that plays an important role in protecting genomic DNA and regulating access to it. This barrier must be temporarily removed during transcription, replication, and repair, but it also must be rapidly restored to the original state afterwards. Further, the properties of chromatin are dynamic and must be adjusted as conditions dictate. FACT was identified as a factor that destabilizes nucleosomes in vitro, but it has now also been implicated as a central factor in the deposition of histones to form nucleosomes, as an exchange factor that swaps the histones within existing nucleosomes for variant forms, and as a tether that prevents histones from being displaced by the passage of RNA polymerases during transcription. FACT therefore plays central roles in building, maintaining, adjusting, and overcoming the chromatin barrier. This review summarizes recent results that have begun to reveal how FACT can promote what appear to be contradictory goals, using a simple set of binding activities to both enhance and diminish the stability of nucleosomes. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly.

Evidence that POB1, a Saccharomyces cerevisiae protein that binds to DNA polymerase alpha, acts in DNA metabolism in vivo.

Potential DNA replication accessory factors from the yeast Saccharomyces cerevisiae have previously been identified by their ability to bind to DNA polymerase alpha protein affinity matrices (J. Miles and T. Formosa, Proc. Natl. Acad. Sci. USA 89:1276-1280, 1992). We have now used genetic methods to characterize the gene encoding one of these DNA polymerase alpha-binding proteins (POB1) to determine whether it plays a role in DNA replication in vivo. We find that yeast cells lacking POB1 are viable but display a constellation of phenotypes indicating defective DNA metabolism. Populations of cells lacking POB1 accumulate abnormally high numbers of enlarged large-budded cells with a single nucleus at the neck of the bud. The average DNA content in a population of cells lacking POB1 is shifted toward the G2 value. These two phenotypes indicate that while the bulk of DNA replication is completed without POB1, mitosis is delayed. Deleting POB1 also causes elevated levels of both chromosome loss and genetic recombination, enhances the temperature sensitivity of cells with mutant DNA polymerase alpha genes, causes increased sensitivity to UV radiation in cells lacking a functional RAD9 checkpoint gene, and causes an increased probability of death in cells carrying a mutation in the MEC1 checkpoint gene. The sequence of the POB1 gene indicates that it is identical to the CTF4 (CHL15) gene identified previously in screens for mutations that diminish the fidelity of chromosome transmission. These phenotypes are consistent with defective DNA metabolism in cells lacking POB1 and strongly suggest that this DNA polymerase alpha-binding protein plays a role in accurately duplicating the genome in vivo.

Structure of a Blm10 complex reveals common mechanisms for proteasome binding and gate opening.

The proteasome is an abundant protease that is critically important for numerous cellular pathways. Proteasomes are activated in vitro by three known classes of proteins/complexes, including Blm10/PA200. Here, we report a 3.4 A resolution crystal structure of a proteasome-Blm10 complex, which reveals that Blm10 surrounds the proteasome entry pore in the 1.2 MDa complex to form a largely closed dome that is expected to restrict access of potential substrates. This architecture and the observation that Blm10 induces a disordered proteasome gate structure challenge the assumption that Blm10 functions as an activator of proteolysis in vivo. The Blm10 C terminus binds in the same manner as seen for 11S activators and inferred for 19S/PAN activators and indicates a unified model for gate opening. We also demonstrate that Blm10 acts to maintain mitochondrial function. Consistent with the structural data, the C-terminal residues of Blm10 are needed for this activity.

The yeast FACT complex has a role in transcriptional initiation

ABSTRACT A crucial step in eukaryotic transcriptional initiation is recognition of the promoter TATA by the TATA-binding protein (TBP), which then allows TFIIA and TFIIB to be recruited. However, nucleosomes block the interaction between TBP and DNA. We show that the yeast FACT complex (yFACT) promotes TBP binding to a TATA box in chromatin both in vivo and in vitro. The SPT16 gene encodes a subunit of yFACT, and we show that certain spt16 mutations are synthetically lethal with TBP mutants. Some of these genetic defects can be suppressed by TFIIA overexpression, strongly suggesting a role for yFACT in TBP-TFIIA complex formation in vivo. Mutations in the TOA2 subunit of TFIIA that disrupt TBP-TFIIA complex formation in vitro are also synthetically lethal with spt16. In some cases this spt16 toa2 lethality is suppressed by overexpression of TBP or the Nhp6 architectural transcription factor that is also a component of yFACT. The Spt3 protein in the SAGA complex has been shown to regulate TBP binding at certain promoters, and we show that some spt16 phenotypes can be suppressed by spt3 mutations. Chromatin immunoprecipitations show TBP binding to promoters is reduced in single spt16 and spt3 mutants but increases in the spt16 spt3 double mutant, reflecting the mutual suppression seen in the genetic assays. Finally, in vitro studies show that yFACT promotes TBP binding to a TATA sequence within a reconstituted nucleosome in a TFIIA-dependent manner. Thus, yFACT functions in establishing transcription initiation complexes in addition to the previously described role in elongation.

Structural Features of Nucleosomes Reorganized by Yeast FACT and Its HMG Box Component, Nhp6

ABSTRACT The Saccharomyces cerevisiae Spt16/Cdc68, Pob3, and Nhp6 proteins (SPN or yFACT) bind to and alter nucleosomes in vitro, providing a potential explanation for their importance in both transcription and replication in vivo. We show that nucleosomes bound by either Nhp6 alone or the yFACT complex remain largely intact and immobile but are significantly reorganized, as indicated by changes in the pattern of sensitivity to DNase I and enhanced digestion by some restriction endonucleases. In contrast, yFACT enhanced access to exonuclease III only at very high levels of enzyme, suggesting that the DNA near the entry and exit sites of nucleosomes is largely unperturbed and that the position of the histone octamers relative to the DNA is not altered during reorganization. DNase I sensitivity was enhanced at sites clustered near the center of the nucleosomal DNA, away from the entry and exit points, and the pattern of nuclease sensitivity was only mildly affected by the configuration of linker extensions, further indicating that linkers play only a minor role in the reorganization of nucleosomes by yFACT. The DNA in contact with H2A-H2B dimers is therefore the region whose nuclease sensitivity was the least affected by yFACT reorganization. The most dramatic changes in nucleosome structure occurred when Spt16-Pob3 and the HMG box protein Nhp6 were both present, but Nhp6 alone altered DNase I sensitivity at some specific sites, supporting an independent role for this class of proteins in the general management of chromatin properties. yFACT activity does not require ATP hydrolysis and does not alter the position of nucleosomes, indicating that it acts through a mechanism distinct from chromatin remodeling. The results presented here suggest instead that yFACT promotes polymerase progression by reorganizing nucleosome cores into a less inhibitory conformation in which the properties of DNA sequences near the center of the nucleosomes are altered.

Opposing roles for Set2 and yFACT in regulating TBP binding at promoters

Previous work links histone methylation by Set2 with transcriptional elongation. yFACT (Spt16–Pob3 and Nhp6) reorganizes nucleosomes and functions in both transcriptional initiation and elongation. We show that growth defects caused by spt16 or pob3 mutations can be suppressed by deleting SET2, suggesting that Set2 and yFACT have opposing roles. Set2 methylates K36 of histone H3, and K36 substitutions also suppress yFACT mutations. In contrast, set1 enhances yFACT mutations. Methylation at H3 K4 by Set1 is required for set2 to suppress yFACT defects. We did not detect an elongation defect at an 8 kb ORF in yFACT mutants. Instead, pob3 mutants displayed reduced binding of both pol II and TBP to the GAL1 promoter. Importantly, both GAL1 transcription and promoter binding of pol II and TBP are significantly restored in the pob3 set2 double mutant. Defects caused by an spt16 mutation are enhanced by either TBP or TFIIA mutants. These synthetic defects are suppressed by set2, demonstrating that yFACT and Set2 oppose one another during transcriptional initiation at a step involving DNA binding by TBP and TFIIA.

Structural and Functional Analysis of the Spt16p N-terminal Domain Reveals Overlapping Roles of yFACT Subunits

yFACT (heterodimers of Saccharomyces cerevisiae Spt16-Pob3 combined with Nhp6) binds to and alters the properties of nucleosomes. The essential function of yFACT is not disrupted by deletion of the N-terminal domain (NTD) of Spt16 or by mutation of the middle domain of Pob3, but either alteration makes yeast cells sensitive to DNA replication stress. We have determined the structure of the Spt16 NTD and find evidence for a conserved potential peptide-binding site. Pob3-M also contains a putative binding site, and we show that these two sites perform an overlapping essential function. We find that yFACT can bind the N-terminal tails of some histones and that this interaction is important for yFACT-nucleosome binding. However, neither the Spt16 NTD nor a key residue in the putative Pob3-M-binding site was required for interactions with histone N termini or for yFACT-mediated nucleosome reorganization in vitro. Instead, both potential binding sites interact functionally with the C-terminal docking domain of the histone H2A. yFACT therefore appears to make multiple contacts with different sites within nucleosomes, and these interactions are partially redundant with one another. The docking domain of H2A is identified as an important participant in maintaining stability during yFACT-mediated nucleosome reorganization, suggesting new models for the mechanism of this activity.