Regulation of transcriptional silencing in yeast by growth temperature.

Increasing evidence indicates that transcriptionally silent chromatin structure is dynamic and may change its conformation in response to external or internal stimuli. We show that growth temperature affects all three forms of transcriptional silencing in Saccharomyces cerevisiae. In general, increasing the temperature within the range of 23-37 degrees C strengthens HM and telomeric silencing but reduces rDNA silencing. High temperature (37 degrees C) can suppress the silencing defects of histone H4 mutants. We demonstrate that DNA at the silent HML locus becomes more and more negatively supercoiled as temperature increases in a Sir-dependent manner, which is indicative of enhanced silent chromatin. This enhancement of silent chromatin is not dependent on silencers and therefore does not require de novo assembly of silent chromatin. We also present evidence suggesting that MAP kinase-mediated Sir3p hyperphosphorylation, which plays a role in regulating silencing in response to certain stress conditions, is not involved in high temperature-induced strengthening of silencing. In addition, Pnc1p, a positive regulator of Sir2p activity, plays no role in thermal regulation of silencing. Therefore, growth temperature regulates transcriptional silencing by a novel mechanism.

Formation of boundaries of transcriptionally silent chromatin by nucleosome-excluding structures.

The eukaryotic genome is divided into chromosomal domains of distinct gene activities. Transcriptionally silent chromatin tends to encroach upon active chromatin. Barrier elements that can block the spread of silent chromatin have been documented, but the mechanisms of their function are not resolved. We show that the prokaryotic LexA protein can function as a barrier to the propagation of transcriptionally silent chromatin in yeast. The barrier function of LexA correlates with its ability to disrupt local chromatin structure. In accord with this, (CCGNN)(n) and poly(dA-dT), both of which do not favor nucleosome formation, can also act as efficient boundaries of silent chromatin. Moreover, we show that a Rap1p-binding barrier element also disrupts chromatin structure. These results demonstrate that nucleosome exclusion is one of the mechanisms for the establishment of boundaries of silent chromatin domains.

A targeted histone acetyltransferase can create a sizable region of hyperacetylated chromatin and counteract the propagation of transcriptionally silent chromatin.

Transcriptionally silent chromatin is associated with reduced histone acetylation and its propagation depends on histone hypoacetylation promoted by histone deacetylases. We show that tethered histone acetyltransferase (HAT) Esa1p or Gcn5p creates a segment of hyperacetylated chromatin that is at least 2.6 kb in size and counteracts transcriptional silencing that emanates from a silencer in yeast. Esa1p and Gcn5p counteract URA3 silencing even when they are targeted 1.7 kb downstream of the promoter and >2.0 kb from the silencer. The anti-silencing effect of a targeted HAT is strengthened by increasing the number of targeting sites, but impaired by events that enhance silencing. A tethered HAT can also counteract telomeric silencing. The anti-silencing effect of Gcn5p is abolished by a mutation that eliminated its HAT activity or by deleting the ADA2 gene encoding a structural component of Gcn5p-containing HAT complexes. These results demonstrate that a tethered HAT complex can create a sizable region of histone hyperacetylation and serve as a barrier to encroaching repressive chromatin.

RPD3 is required for the inactivation of yeast ribosomal DNA genes in stationary phase.

rRNA transcription in Saccharomyces cerevisiae is performed by RNA polymerase I and regulated by changes in growth conditions. During log phase, approximately 50% of the ribosomal DNA (rDNA) genes in each cell are transcribed and maintained in an open, psoralen-accessible conformation. During stationary phase, the percentage of open rDNA genes is greatly reduced. In this study we found that the Rpd3 histone deacetylase was required to inactivate (close) individual rDNA genes as cells entered stationary phase. Even though approximately 50% of the rDNA genes remained open during stationary phase in rpd3Delta mutants, overall rRNA synthesis was still reduced. Using electron microscopy of Miller chromatin spreads, we found that the number of RNA polymerases transcribing each open gene in the rpd3Delta mutant was significantly reduced when cells grew past log phase. Bulk levels of histone H3 and H4 acetylation were reduced during stationary phase in an RPD3-dependent manner. However, histone H3 and H4 acetylation was not significantly altered at the rDNA locus in an rpd3Delta mutant. Rpd3 therefore regulates the number of open rDNA repeats.

Telomeric and rDNA silencing in Saccharomyces cerevisiae are dependent on a nuclear NAD(+) salvage pathway.

The Sir2 protein is an NAD(+)-dependent protein deacetylase that is required for silencing at the silent mating-type loci, telomeres, and the ribosomal DNA (rDNA). Mutations in the NAD(+) salvage gene NPT1 weaken all three forms of silencing and also cause a reduction in the intracellular NAD(+) level. We now show that mutation of a highly conserved histidine residue in Npt1p results in a silencing defect, indicating that Npt1p enzymatic activity is required for silencing. Deletion of another NAD(+) salvage pathway gene called PNC1 caused a less severe silencing defect and did not significantly reduce the intracellular NAD(+) concentration. However, silencing in the absence of PNC1 was completely dependent on the import of nicotinic acid from the growth medium. Deletion of a gene in the de novo NAD(+) synthesis pathway BNA1 resulted in a significant rDNA silencing defect only on medium deficient in nicotinic acid, an NAD(+) precursor. By immunofluorescence microscopy, Myc-tagged Bna1p was localized throughout the whole cell in an asynchronously growing population. In contrast, Myc-tagged Npt1p was highly concentrated in the nucleus in approximately 40% of the cells, indicating that NAD(+) salvage occurs in the nucleus in a significant fraction of cells. We propose a model in which two components of the NAD(+) salvage pathway, Pnc1p and Npt1p, function together in recycling the nuclear nicotinamide generated by Sir2p deacetylase activity back into NAD(+).

RNA polymerase I propagates unidirectional spreading of rDNA silent chromatin.

The ribosomal DNA (rDNA) tandem array in Saccharomyces cerevisiae induces transcriptional silencing of RNA polymerase II-transcribed genes. This SIR2-dependent form of repression (rDNA silencing) also functions to limit rDNA recombination and is involved in life span control. In this report, we demonstrate that rDNA silencing spreads into the centromere-proximal unique sequence located downstream of RNA polymerase I (Pol I) transcription, but fails to enter the upstream telomere-proximal sequences. The spreading of silencing correlates with SIR2-dependent histone H3 and H4 deacetylation and can be extended by SIR2 overexpression. Surprisingly, rDNA silencing required transcription by RNA polymerase I and the direction of spreading was controlled by the direction of Pol I transcription.