Shinya Takahata

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.

FACT and Asf1 Regulate Nucleosome Dynamics and Coactivator Binding at the HO Promoter

Transcriptional activators and coactivators overcome repression by chromatin, but regulation of chromatin disassembly and coactivator binding to promoters is poorly understood. Activation of the yeast HO gene follows the sequential binding of both sequence-specific DNA-binding proteins and coactivators during the cell cycle. Here, we show that the nucleosome disassembly occurs in waves both along the length of the promoter and during the cell cycle. Different chromatin modifiers are required for chromatin disassembly at different regions of the promoter, with Swi/Snf, the FACT chromatin reorganizer, and the Asf1 histone chaperone each required for nucleosome eviction at distinct promoter regions. FACT and Asf1 both bind to upstream elements of the HO promoter well before the gene is transcribed. The Swi/Snf, SAGA, and Mediator coactivators bind first to the far upstream promoter region and subsequently to a promoter proximal region, and FACT and Asf1 are both required for this coactivator re-recruitment.

Forkhead proteins control the outcome of transcription factor binding by antiactivation

Transcription factors with identical DNA‐binding specificity often activate different genes in vivo. Yeast Ace2 and Swi5 are such activators, with targets we classify as Swi5‐only, Ace2‐only, or both. We define two unique regulatory modes. Ace2 and Swi5 both bind in vitro to Swi5‐only genes such as HO, but only Swi5 binds and activates in vivo. In contrast, Ace2 and Swi5 both bind in vivo to Ace2‐only genes, such as CTS1, but promoter‐bound Swi5 fails to activate. We show that activation by Swi5 is prevented by the binding of the Forkhead factors Fkh1 and Fkh2, which recruit the Rpd3(Large) histone deacetylase complex to the CTS1 promoter. Global analysis shows that all Ace2‐only genes are bound by both Ace2 and Swi5, and also by Fkh1/2. Genes normally activated by either Ace2 or Swi5 can be converted to Ace2‐only genes by the insertion of Fkh‐binding sites. Thus Fkh proteins, which function initially to activate SWI5 and ACE2, subsequently function as Swi5‐specific antiactivators.

The E2F functional analogue SBF recruits the Rpd3(L) HDAC, via Whi5 and Stb1, and the FACT chromatin reorganizer, to yeast G1 cyclin promoters

Regulation of the CLN1 and CLN2 G1 cyclin genes controls cell cycle progression. The SBF activator binds to these promoters but is kept inactive by the Whi5 and Stb1 inhibitors. The Cdc28 cyclin‐dependent kinase phosphorylates Whi5, ending the inhibition. Our chromatin immunoprecipitation (ChIP) experiments show that SBF, Whi5 and Stb1 recruit both Cdc28 and the Rpd3(L) histone deacetylase to CLN promoters, extending the analogy with mammalian G1 cyclin promoters in which Rb recruits histone deacetylases. Finally, we show that the SBF subunit Swi6 recruits the FACT chromatin reorganizer to SBF‐ and MBF‐regulated genes. Mutations affecting FACT reduce the transient nucleosome eviction seen at these promoters during a normal cell cycle and also reduce expression. Temperature‐sensitive mutations affecting FACT and Cdc28 can be suppressed by disruption of STB1 and WHI5, suggesting that one critical function of FACT and Cdc28 is overcoming chromatin repression at G1 cyclin promoters. Thus, SBF recruits complexes to promoters that either enhance (FACT) or repress (Rpd3L) accessibility to chromatin, and also recruits the kinase that activates START.

A Role for Chd1 and Set2 in Negatively Regulating DNA Replication in Saccharomyces cerevisiae

Chromatin-modifying factors regulate both transcription and DNA replication. The yFACT chromatin-reorganizing complex is involved in both processes, and the sensitivity of some yFACT mutants to the replication inhibitor hydroxyurea (HU) is one indication of a replication role. This HU sensitivity can be suppressed by disruptions of the SET2 or CHD1 genes, encoding a histone H3(K36) methyltransferase and a chromatin remodeling factor, respectively. The additive effect of set2 and chd1 mutations in suppressing the HU sensitivity of yFACT mutants suggests that these two factors function in separate pathways. The HU suppression is not an indirect effect of altered regulation of ribonucleotide reductase induced by HU. set2 and chd1 mutations also suppress the HU sensitivity of mutations in other genes involved in DNA replication, including CDC2, CTF4, ORC2, and MEC1. Additionally, a chd1 mutation can suppress the lethality normally caused by disruption of either MEC1 or RAD53 DNA damage checkpoint genes, as well as the lethality seen when a mec1 sml1 mutant is exposed to low levels of HU. The pob3 defect in S-phase progression is suppressed by set2 or chd1 mutations, suggesting that Set2 and Chd1 have specific roles in negatively regulating DNA replication.

Different Genetic Functions for the Rpd3(L) and Rpd3(S) Complexes Suggest Competition between NuA4 and Rpd3(S)

ABSTRACT Rpd3(L) and Rpd3(S) are distinct multisubunit complexes containing the Rpd3 histone deacetylase. Disruption of the GCN5 histone acetyltransferase gene shows a strong synthetic phenotype when combined with either an sds3 mutation affecting only the Rpd3(L) complex or an rco1 mutation affecting only Rpd3(S). However, these synthetic growth defects are not seen in a gcn5 sds3 rco1 triple mutant, suggesting that the balance between Rpd3(L) and Rpd3(S) is critical in cells lacking Gcn5. Different genetic interactions are seen with mutations affecting the FACT chromatin reorganizing complex. An sds3 mutation affecting only Rpd3(L) has a synthetic defect with FACT mutants, while rco1 and eaf3 mutations affecting Rpd3(S) suppress FACT mutant phenotypes. Rpd3(L) therefore acts in concert with FACT, but Rpd3(S) opposes it. Combining FACT mutations with mutations in the Esa1 subunit of the NuA4 histone acetyltransferase results in synthetic growth defects, and these can be suppressed by an rco1 or set2 mutation. An rco1 mutation suppresses phenotypes caused by mutations in the ESA1 and ARP4 subunits of NuA4, while Rco1 overexpression exacerbates these defects. These results suggest a model in which NuA4 and Rpd3(S) compete. Chromatin immunoprecipitation experiments show that eliminating Rpd3(S) increases the amount of NuA4 binding to the ARG3 promoter during transcriptional activation and to the sites of DNA repair induced by a double-strand break. Our results suggest that the Rpd3(L) and Rpd3(S) complexes have distinct functions in vivo and that the relative amounts of the two forms alter the effectiveness of other chromatin-altering complexes, such as FACT and NuA4.

Coupling Phosphate Homeostasis to Cell Cycle-Specific Transcription: Mitotic Activation of Saccharomyces cerevisiae PHO5 by Mcm1 and Forkhead Proteins

ABSTRACT Cells devote considerable resources to nutrient homeostasis, involving nutrient surveillance, acquisition, and storage at physiologically relevant concentrations. Many Saccharomyces cerevisiae transcripts coding for proteins with nutrient uptake functions exhibit peak periodic accumulation during M phase, indicating that an important aspect of nutrient homeostasis involves transcriptional regulation. Inorganic phosphate is a central macronutrient that we have previously shown oscillates inversely with mitotic activation of PHO5. The mechanism of this periodic cell cycle expression remains unknown. To date, only two sequence-specific activators, Pho4 and Pho2, were known to induce PHO5 transcription. We provide here evidence that Mcm1, a MADS-box protein, is essential for PHO5 mitotic activation. In addition, we found that cells simultaneously lacking the forkhead proteins, Fkh1 and Fkh2, exhibited a 2.5-fold decrease in PHO5 expression. The Mcm1-Fkh2 complex, first shown to transactivate genes within the CLB2 cluster that drive G2/M progression, also associated directly at the PHO5 promoter in a cell cycle-dependent manner in chromatin immunoprecipitation assays. Sds3, a component specific to the Rpd3L histone deacetylase complex, was also recruited to PHO5 in G1. These findings provide (i) further mechanistic insight into PHO5 mitotic activation, (ii) demonstrate that Mcm1-Fkh2 can function combinatorially with other activators to yield late M/G1 induction, and (iii) couple the mitotic cell cycle progression machinery to cellular phosphate homeostasis.

Repressive Chromatin Affects Factor Binding at Yeast HO (Homothallic Switching) Promoter

Background: Expression of the yeast HO gene is tightly regulated, with a large complex promoter. Results: The Rpd3(L) histone deacetylase is recruited twice by distinct factors to different promoter regions. Conclusion: Chromatin represses the HO promoter, making multiple coactivators required for gene expression. Significance: Multiple factors contribute to the establishment of a tightly repressed, cell cycle-regulated promoter. The yeast HO gene is tightly regulated, with multiple activators and coactivators needed to overcome repressive chromatin structures that form over this promoter. Coactivator binding is strongly interdependent, as loss of one factor sharply reduces recruitment of other factors. The Rpd3(L) histone deacetylase is recruited to HO at two distinct times during the cell cycle, first by Ash1 to the URS1 region of the promoter and then by SBF/Whi5/Stb1 to URS2. SBF itself is localized to only a subset of its potential binding sites in URS2, and this localization takes longer and is less robust than at other SBF target genes, suggesting that binding to the HO promoter is limited by chromatin structures that dynamically change as the cell cycle progresses. Ash1 only binds at the URS1 region of the promoter, but an ash1 mutation results in markedly increased binding of SBF and Rpd3(L) at URS2, some 450 bp distant from the site of Ash1 binding, suggesting these two regions of the promoter interact. An ash1 mutation also results in increased coactivator recruitment, Swi/Snf and Mediator localization in the absence of the normally required Gcn5 histone acetyltransferase, and HO expression even in the presence of a taf1 mutation affecting TFIID activity that otherwise blocks HO transcription. Ash1 therefore appears to play a central role in generating the strongly repressive environment at the HO promoter, which limits the binding of several coactivators at URS2 and TATA region.

A Role for FACT in Repopulation of Nucleosomes at Inducible Genes

Xenobiotic drugs induce Pleiotropic Drug Resistance (PDR) genes via the orthologous Pdr1/Pdr3 transcription activators. We previously identified the Mediator transcription co-activator complex as a key target of Pdr1 orthologs and demonstrated that Pdr1 interacts directly with the Gal11/Med15 subunit of the Mediator complex. Based on an interaction between Pdr1 and the FACT complex, we show that strains with spt16 or pob3 mutations are sensitive to xenobiotic drugs and display diminished PDR gene induction. Although FACT acts during the activation of some genes by assisting in the nucleosomes eviction at promoters, PDR promoters already contain nucleosome-depleted regions (NDRs) before induction. To determine the function of FACT at PDR genes, we examined the kinetics of RNA accumulation and changes in nucleosome occupancy following exposure to a xenobiotic drug in wild type and FACT mutant yeast strains. In the presence of normal FACT, PDR genes are transcribed within 5 minutes of xenobiotic stimulation and transcription returns to basal levels by 30–40 min. Nucleosomes are constitutively depleted in the promoter regions, are lost from the open reading frames during transcription, and the ORFs are wholly repopulated with nucleosomes as transcription ceases. While FACT mutations cause minor delays in activation of PDR genes, much more pronounced and significant defects in nucleosome repopulation in the ORFs are observed in FACT mutants upon transcription termination. FACT therefore has a major role in nucleosome redeposition following cessation of transcription at the PDR genes, the opposite of its better-known function in nucleosome disassembly.

Inner nuclear membrane protein Lem2 augments heterochromatin formation in response to nutritional conditions

Inner nuclear membrane proteins interact with chromosomes in the nucleus and are important for chromosome activity. Lem2 and Man1 are conserved members of the LEM‐domain nuclear membrane protein family. Mutations of LEM‐domain proteins are associated with laminopathy, but their cellular functions remain unclear. Here, we report that Lem2 maintains genome stability in the fission yeast Schizosaccharomyces pombe. S. pombe cells disrupted for the lem2+ gene (lem2∆) showed slow growth and increased rate of the minichromosome loss. These phenotypes were prominent in the rich culture medium, but not in the minimum medium. Centromeric heterochromatin formation was augmented upon transfer to the rich medium in wild‐type cells. This augmentation of heterochromatin formation was impaired in lem2∆ cells. Notably, lem2∆ cells occasionally exhibited spontaneous duplication of genome sequences flanked by the long‐terminal repeats of retrotransposons. The resulting duplication of the lnp1+ gene, which encodes an endoplasmic reticulum membrane protein, suppressed lem2∆ phenotypes, whereas the lem2∆ lnp1∆ double mutant showed a severe growth defect. A combination of mutations in Lem2 and Bqt4, which encodes a nuclear membrane protein that anchors telomeres to the nuclear membrane, caused synthetic lethality. These genetic interactions imply that Lem2 cooperates with the nuclear membrane protein network to regulate genome stability.