Summary FACT has been proposed to function by displacing an H2A–H2B dimer 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 does not require displacement of dimers from the nucleosome. Third, yFACT is required for eviction of nucleosomes from the GAL1-10 promoter and the adjacent ORF in vivo, but most sites do not exhibit the preferential reduction in dimer occupancy expected for hexasome formation. We propose that yFACT promotes a reversible transition between two nucleosomal forms and that this is useful both to overcome the repressive chromatin barrier and to establish and maintain this barrier.

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.

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 (lem2Δ) 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 lem2Δ cells. Notably, lem2Δ 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 lem2Δ phenotypes, whereas the lem2Δ lnp1Δ 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.

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.

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.

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. Here, we show that CTD-S2P-dependent interaction of fission yeast Set2 with Pol2 via the SRI domain is required for formation of H3K36me3, but not H3K36me2. H3K36me3 silenced heterochromatic and subtelomeric transcripts mainly through post-transcriptional and transcriptional mechanisms, respectively, whereas H3K36me2 was not enough for silencing. Clr6 complex II appeared not to be responsible for heterochromatic silencing by H3K36me3. Our results demonstrate that H3K36 methylation has multiple outputs in fission yeast; these findings provide insights into the distinct roles of H3K36 methylation in metazoans, which have different enzymes for synthesis of H3K36me1/2 and H3K36me3.

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.

Chromatin-modifying factors regulate both transcription and DNA replication. The yFACT chromatinreorganizing 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.