Schematic map of ribosomal genes in S. cerevisiae. Horizontal black arrows, RNA transcripts. Filled black boxes, 35S and 5S coding units. Gray boxes, E-PRO and C-PRO promoters. White box, putative transcriptional enhancer. ARS, origin of DNA replication. Ellipses, positioned nucleosomes (Vogelauer et al., 1998). Thin horizontal white arrow, strand-specific oligonucleotide used in the RT-PCR reactions. Thick black lines, PCR amplicons produced in ChIP analyses.

Chromatin regulators control ERC formation. (A) Southern analysis of ERC species. DNA was isolated from the specified yeast strains or after 16 h treatment with 500 μM NAM, and probed with a radiolabeled rDNA sequence. ERCs are indicated by arrows. The differences in the migration of bands corresponding to ERCs are due to different durations of electrophoretic migration. (B) Quantification of ERC amount. Averages and SDs of at least three independent experiments are reported. Two-tailed t test was applied for statistical analysis. Asterisks indicate statistically relevant differences; α = 0.05. The p values are as follows: sir2, p = 0.0419; NAM, p = 0.0086; rpd3, p = 0.0454; hst1, p = 0.1388; hst2, p = 0.6499; hst3, p = 0.0083; hst4, p = 0.1529; nhp6ab, p = 0.0474.

ERC formation is stimulated by ncRNA transcription. (A) Schematic map of the rDNA unit. Thin horizontal black arrows, ncRNAs produced from E-PRO and C-PRO promoters. Horizontal white arrow, oligo NTS1-R used in the strand-specific RT-PCR. (B) Quantification of the ncRNA expression level. The histograms indicate the averages and SDs from three independent experiments. Two-tailed t-test was applied for statistical analysis. Asterisks indicate statistically relevant differences; α = 0.05. The p values are as follows: sir2, p = 0.0006; NAM, p = 0.002; rpd3, p = 0.0013; hst1, p = 0.0703; hst2, p = 0.1451; hst3, p = 0.0047; hst4, p = 0.125; nhp6ab, p = 0.0454.

The H4 acetylation profile is altered at the rDNA of histone deacetylases and mutants of high-mobility-group proteins. (A) Graphical representation of an rDNA unit. Horizontal thick black lines indicate the positions of PCR amplicons generated by the ChIP experiments (not to scale). (B) ChIP analysis of the histone H4 acetylation profile at rDNA. Chromatin from the specified strains was immunoprecipitated with antibodies against the C-terminal tail of histone H4 (H4 C-TERM) or against the histone H4 acetylated at lysines 5, 8, 12, and 16 (ACH4). The graphs show the acetylation enrichment, in the indicated rDNA regions, of the mutants relative to isogenic WT strain, reported as 1 (blue line). The lines show the average ratios and SD of technical replicates in a representative experiment out of two performed.

The alterations in H4 Lys-16 acetylation correlate with the observed changes in global H4 acetylation. (A) Schematic map of the rDNA repeat. The fragments amplified in the ChIP analyses are shown. (B) ChIP analysis of the histone H4 Lys-16 acetylation at rDNA. As in Figure 4B, except that the antibodies used were against the C-terminal tail of histone H4 (H4 C-TERM) or against H4 acetylated at Lys-16 (H4K16-ac).

Deacetylation of H4K16 is required for the suppression of ERC formation and ncRNA transcription. (A) Substitution of histone H4 Lys-16 with glutamine increases ERC production. Quantification of ERC amount produced by yeast strains bearing substitutions of H4K16 to glutamine, H4K16Q (left), or to arginine, H4K16R (right). ERC species were quantified and normalized as described in Figure 2 (n > 3; ±SD). Two-tailed t test was applied for statistical analysis. Asterisk indicates the statistically relevant difference, α = 0.05, between WT (PKY501) and H4K16Q (p = 0.0204) strains. (B) The H4K16Q mutant up-regulates ncRNA transcription at rDNA. Measure of the E-PRO–derived ncRNA expression level of H4K16Q (left), H4K16R (right), and the corresponding isogenic WT as in Figure 3 (n = 3; ±SD). Asterisk indicates the statistically relevant difference, α = 0.05, between WT (PKY501) and H4K16Q (p = 0.0264) strains.

The nhp6ab mutant has reduced SIR2 expression. (A) The nhp6ab double mutant leads to the loss of Sir2p from the rDNA locus. WT and nhp6ab strains were subjected to ChIP analysis with antibodies against Sir2p. The recovered DNA was amplified in two regions known to bind Sir2p—the enhancer (ENH) and the C-PRO promoter. (B) Sir2 mRNA expression is reduced in nhp6ab cells. The graph reports the Sir2 mRNA levels of nhp6ab and WT strains measured by RT-PCR (n = 3; ±SD). (C) The nhp6ab mutant cells have a decreased Sir2 protein level. Protein extracts from three independent cell cultures of WT (left) or nhp6ab (right) cells were analyzed by Western blot with anti-Sir2 and anti–α-tubulin antibodies.

SIR2 overexpression restores the WT level of ncRNA transcription and H4 acetylation in the nhp6ab mutant. (A) SIR2 overexpression in the nhp6ab mutant restores the levels of ncRNA transcription. WT and nhp6ab cells were transformed with the pAR44 plasmid (+) or with the empty vector (–). Quantification of ncRNA expression was performed as described in Figure 3 (n ≥ 4; ±SD). Two-tailed t test was applied for statistical analysis. Asterisks indicate the statistically relevant differences between nhp6ab cells with or without SIR2 overexpression (α = 0.01; p = 0.0015) or between WT cells with or without SIR2 overexpression (α = 0.01; p = 0.009). (B) SIR2 overexpression rescues the altered histone H4 acetylation of the nhp6ab mutant. Cells overexpressing (+) or not (–) SIR2, were grown in galactose and processed for ChIP analysis with antibodies against the C-terminal tail of histone H4 or against the acetylated histone H4 (ACH4). Quantification details and positions of PCR fragments are as in Figure 4. (C) The increased H4K16 acetylation of nhp6ab drops to the WT level after SIR2 overexpression. As in B, except that the antibodies were anti–acetyl histone H4-Lys-16 (H4K16-ac) and anti–H4 C-terminal tail (H4 C-TERM). (D) SIR2 overexpression does not reduce the ERC levels in the nhp6ab strain. DNA from cells containing the PAR44 plasmid (+) or the empty vector (–) was extracted and processed for ERC analysis as described in Figure 2 (n = 3; ±SD).