The HMG-box module in FACT is critical for suppressing epigenetic variegation of heterochromatin in fission yeast.

Chromatin condensation state is the key for retrieving genetic information. High-mobility group protein (HMG) proteins exhibit DNA-binding and bending activities, playing an important role in the regulation of chromatin structure. We have shown that nucleosomes tightly packaged into heterochromatin undergo considerable dynamic histone H2A-H2B maintenance via the direct interaction between HP1/Swi6 and facilitate chromatin transcription (FACT), which is composed of the Spt16/Pob3 heterodimer and Nhp6.

A zinc-finger protein Moc3 functions as a transcription activator to promote RNAi-dependent constitutive heterochromatin establishment in fission yeast.

In fission yeast, Schizosaccharomyces pombe, constitutive heterochromatin defined by methylation of histone H3 lysine 9 (H3K9me) and its binding protein Swi6/HP1 localizes at the telomere, centromere, and mating-type loci. These loci contain DNA sequences called dg and dh, and the RNA interference (RNAi)-dependent system establishes and maintains heterochromatin at dg/dh. Bi-directional transcription at dg/dh induced by RNA polymerase II is critical in RNAi-dependent heterochromatin formation because the transcribed RNAs provide substrates for siRNA synthesis and a platform for assembling RNAi factors.

Opposing Roles of FACT for Euchromatin and Heterochromatin in Yeast.

DNA is stored in the nucleus of a cell in a folded state; however, only the necessary genetic information is extracted from the required group of genes. The key to extracting genetic information is chromatin ambivalence. Depending on the chromosomal region, chromatin is characterized into low-density "euchromatin" and high-density "heterochromatin", with various factors being involved in its regulation.

Histone variant H2A.Z plays multiple roles in the maintenance of heterochromatin integrity.

H2A.Z, an evolutionally well-conserved histone H2A variant, is involved in many biological processes. Although the function of H2A.

Two secured FACT recruitment mechanisms are essential for heterochromatin maintenance.

FACT (facilitate chromatin transcription) is involved in heterochromatic silencing, but its mechanisms and function remain unclear. We reveal that the Spt16 recruitment mechanism operates in two distinct ways in heterochromatin. First, Pob3 mediates Spt16 recruitment onto the heterochromatin through its Spt16 dimerization and tandem PH domains.

Trimethylguanosine synthase 1 (Tgs1) is involved in Swi6/HP1-independent siRNA production and establishment of heterochromatin in fission yeast.

In fission yeast, siRNA generated by RNA interference (RNAi) factors plays critical roles in establishment and maintenance of heterochromatin. To achieve efficient siRNA synthesis, RNAi factors assemble on heterochromatin via association with Swi6, a homologue of heterochromatin protein 1 (HP1), and heterochromatic noncoding RNA (hncRNA) retained on chromatin. In addition, spliceosomes formed on hncRNA introns recruit RNAi factors to hncRNA and heterochromatin.

Construction and characterization of a zinc-inducible gene expression vector in fission yeast.

Gene expression vectors are useful and important tools that are commonly used in a variety of experiments, including expression of foreign genes, functional analysis of genes of interest and complementation experiments. In this study, a hybrid promoter, combining the adh1 upstream activating sequence (UAS) of fission yeast and the GAL10 core promoter of budding yeast, was constructed to enable high level expression depending on the presence of zinc in culture medium for fission yeast. When the hybrid promoter was cloned on the multicopy plasmid, it was fully induced and repressed within 10 h in the presence and absence of zinc, respectively.

Regulation of ectopic heterochromatin-mediated epigenetic diversification by the JmjC family protein Epe1.

H3K9 methylation (H3K9me) is a conserved marker of heterochromatin, a transcriptionally silent chromatin structure. Knowledge of the mechanisms for regulating heterochromatin distribution is limited. The fission yeast JmjC domain-containing protein Epe1 localizes to heterochromatin mainly through its interaction with Swi6, a homologue of heterochromatin protein 1 (HP1), and directs JmjC-mediated H3K9me demethylation in vivo.

Transcriptional activation is weakened when Taf1p N-terminal domain 1 is substituted with its Drosophila counterpart in yeast TFIID.

Transcription factor II D (TFIID), a multiprotein complex consisting of TATA-binding protein (TBP) and 13-14 TBP-associated factors (Tafs), plays a central role in transcription and regulates nearly all class II genes. The N-terminal domain of Taf1p (TAND) can be divided into two subdomains, TAND1 and TAND2, which bind to the concave and convex surfaces of TBP, respectively. The interaction between TAND and TBP is thought to be regulated by TFIIA, activators and/or DNA during transcriptional activation, as the TAND1-bound form of TBP cannot bind to the TATA box.

H3K36 methylation state and associated silencing mechanisms.

Epigenetic marks determine cell fate via numerous reader proteins. H3K36 methylation is a common epigenetic mark that is thought to be associated with the activities of the RNA polymerase 2 C-terminal domain. We discuss a novel silencing mechanism regulated by Set2-dependent H3K36 methylation that involves exosome-dependent RNA processing.

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.

Histone H3K36 trimethylation is essential for multiple silencing mechanisms in fission yeast.

In budding yeast, Set2 catalyzes di- and trimethylation of H3K36 (H3K36me2 and H3K36me3) via an interaction between its Set2-Rpb1 interaction (SRI) domain and C-terminal repeats of RNA polymerase II (Pol2) phosphorylated at Ser2 and Ser5 (CTD-S2,5-P). H3K36me2 is sufficient for recruitment of the Rpd3S histone deacetylase complex to repress cryptic transcription from transcribed regions. In fission yeast, Set2 is also responsible for H3K36 methylation, which represses a subset of RNAs including heterochromatic and subtelomeric RNAs, at least in part via recruitment of Clr6 complex II, a homolog of Rpd3S.

A novel method for purification of the endogenously expressed fission yeast Set2 complex.

Chromatin-associated proteins are heterogeneously and dynamically composed. To gain a complete understanding of DNA packaging and basic nuclear functions, it is important to generate a comprehensive inventory of these proteins. However, biochemical purification of chromatin-associated proteins is difficult and is accompanied by concerns over complex stability, protein solubility and yield.

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.

Repressive chromatin affects factor binding at yeast HO (homothallic switching) 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.

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.

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.

Coupling phosphate homeostasis to cell cycle-specific transcription: mitotic activation of Saccharomyces cerevisiae PHO5 by Mcm1 and Forkhead proteins.

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.

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 genetic functions for the Rpd3(L) and Rpd3(S) complexes suggest competition between NuA4 and Rpd3(S).

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.

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.

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.

Functional silencing of TATA-binding protein (TBP) by a covalent linkage of the N-terminal domain of TBP-associated factor 1.

General transcription factor TFIID is comprised of TATA-binding protein (TBP) and TBP-associated factors (TAFs), together playing critical roles in regulation of transcription initiation. The TAF N-terminal domain (TAND) of yeast TAF1 containing two subdomains, TAND1 (residues 10-37) and TAND2 (residues 46-71), is sufficient to interact with TBP and suppress the TATA binding activity of TBP. However, the detailed structural analysis of the complex between yeast TBP and TAND12 (residues 6-71) was hindered by its poor solubility and stability in solution.

Autonomous function of the amino-terminal inhibitory domain of TAF1 in transcriptional regulation.

The general transcription factor TFIID is composed of TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). TFIID mediates the transcriptional activation of a subset of eukaryotic promoters. The N-terminal domain (TAND) of TAF1 protein (Taf1p) inhibits TBP by binding to its concave and convex surfaces.

Identification of a novel TATA element-binding protein binding region at the N terminus of the Saccharomyces cerevisiae TAF1 protein.

TFIID, a multiprotein complex composed of TATA element-binding protein (TBP) and 14 TBP-associated factors (TAFs), can directly recognize core promoter elements and mediate transcriptional activation. The TAF N-terminal domain (TAND) of TAF1 may play a significant role in these two principal TFIID functions by regulating the access of TBP to the TATA element. In yeast, TAND consists of two subdomains, TAND1 (10-37 amino acids (aa)) and TAND2 (46-71 aa), which interact with the concave and convex surfaces of TBP, respectively.