Schematic representation of the yeast rDNA locus. The position of the rDNA repeat cluster on chromosome XII with respect to the centromere (CEN) and telomeres (tel) is shown. Each rDNA repeat consists of the Pol I-transcribed 35S rRNA gene (precursor for the 18S, 5.8S, and 25S rRNAs), the RNA Pol III-transcribed 5S rRNA gene, and two intergenic spacer regions, IGS1 and -2. Arrows mark the transcription start sites and direction. The positions of three regulatory DNA elements—enhancer (ENH), core promoter (CP), and upstream element (UE)—and of the Pol I promoter-proximal Reb1 binding site are indicated.

UAF30 deletion alters the arrangement of structural chromatin components at the 35S rRNA genes. (A) Yeast strains (y617, y621, y954, y1145, y1171, y1172, y1690, and y1695) carrying a wild-type allele or a deletion (Δ) in UAF30 and expressing the indicated MNase fusion proteins (MN) were cultured and treated with formaldehyde as described in Materials and Methods. Crude nucleus preparations were subjected to chromatin endogenous cleavage (ChEC) in the absence or presence of calcium for the indicated times, as detailed in Materials and Methods. DNA was analyzed as described in the legend to Fig. 2A. Different symbols highlight changes in MNase accessibility, as described in the legend to Fig. 2A. (B) Profiles of DNA fragment patterns in individual samples obtained after 30 min (H2B-MN- and Hmo1-MN-expressing strains) or 60 min (other strains) of ChEC. Profile analysis was performed as described in the legend to Fig. 2B. Different symbols highlight changes in MNase accessibility as described in the legend to Fig. 2A.

UAF and CF bind to the Pol I promoter in the absence of Pol I or Rrn3 in vivo. (A) Yeast strains (y1143, y1144, y1148, y1149, y1707, and y1712) carrying a wild-type copy or a temperature-sensitive (ts) allele of RRN3 and expressing the indicated MNase fusion proteins (MN) were treated as described in the legend to Fig. 2D. Crude nuclei were subjected to ChEC analysis as described in the legend to Fig. 3A. (B, C) Yeast strains (y618, y1109, y1926, y1927, y1928, and y1929) carrying a wild-type allele (wt) or deletions (Δ) in RRN3, RRN7, RPA135, or UAF30 and expressing the indicated MNase fusion proteins (MN) were treated and subjected to ChEC analysis as described in the legend to Fig. 3A.

Deletion of UAF30 leads to loss of Sir2 from the rDNA locus. (A, B) Yeast strains (y1450, y1691, y1185, and y1346) carrying a wild-type allele or a deletion (Δ) in UAF30 or SIR2 and expressing the indicated MNase fusion proteins (MN) were treated and subjected to ChEC analysis as described in the legend to Fig. 3A. The positions of two TBP-MNase-mediated cuts at a bidirectional Pol II promoter within IGS1 are marked by filled dots. In panel A, bottom, the mem-brane was also hybridized with probe GIT1 detecting the HMR locus. (C) Profiles of DNA fragment patterns in individual samples obtained after 60 min of ChEC. Profile analysis was performed as described in the legend to Fig. 2B.

UAF organizes the rDNA chromatin structure. In a wild-type strain, UAF acts locally at the Pol I promoter by stimulating Pol I transcription and restricting access of other polymerases to a cryptic promoter located upstream of the Pol I initiation site. Protection of the cryptic promoter is lost upon deletion of UAF components (uafΔ), and Pol II and III gain access to this region. UAF is further required for efficient recruitment of Sir2 to rDNA and therefore for silencing of Pol II transcription. Thus, the specific transcriptional state of the 35S rRNA genes may determine the chromatin structure at the entire transcribed domain.

Deletion of components of the basal Pol I transcription machinery leads to drastic changes in chromatin structure at the rDNA locus. (A) Yeast strains (NOY505, NOY408-1a, NOY558, NOY604, NOY699, and y1120) carrying a wild-type allele (wt) or deletions (Δ) in RRN3, RRN7, RRN5, RPA135, or UAF30 were cultured and treated with formaldehyde, as described in Materials and Methods. Crude nucleus preparations were incubated in the absence (−) or presence of 0.05, 0.15, 0.3, or 1 U of micrococcal nuclease (MNase), as detailed in Materials and Methods. DNA was isolated and analyzed using a Southern blot by indirect end labeling. An autoradiogram is shown. Different symbols highlight changes in MNase accessibility and are referred to in the text. A cartoon of the genomic region analyzed, including the positions and names of the probes used for indirect end labeling, is depicted on the right. (B) Profiles of DNA fragment patterns in individual samples obtained after digestion with 0.15 U of MNase as depicted in panel A. The genotype of the yeast strains analyzed is indicated in the legend of each graph. The relative signal intensity in the respective lanes was determined as described in Materials and Methods and plotted against the distance of migration in the gel. Different symbols highlight changes in MNase accessibility, as shown in panel A. A cartoon of the genomic region is indicated below the graphs. (C) Yeast strains (NOY703 and y895) carrying deletions in RRN9 and RRN7 were analyzed as described in panel A, except that 0.25, 0.5, 1, and 2 units of MNase were used for digestion. (D) Yeast strains (CG379 and Ycc95) carrying a wild-type copy or a temperature-sensitive allele (ts) of RRN3 were cultured at the permissive (24°C) and the restrictive (37°C) temperatures, as described in Materials and Methods. Chromatin was analyzed as described in panel A.

UAF30 deletion leads to compositional and structural changes within Pol I promoter chromatin. (A to C) Yeast strains (y881, y1121, y1151, y1184, y1185, y1328, y1329, y1453, y1689, y1692, y881, y1196, y952, and y1177) carrying a wild-type allele or a deletion (Δ) in UAF30 and expressing the indicated MNase fusion proteins (MN) were treated and subjected to ChEC analysis as described in the legend to Fig. 3A. The positions of two TBP-MNase-mediated cuts at a bidirectional Pol II promoter within IGS1 are marked by filled dots. (D) Yeast strains (y618, y881, y1184, y1185, and y1453) expressing the indicated MNase fusion proteins (MN) were treated and subjected to ChEC analysis as described in the legend to Fig. 3A before psoralen cross-linking was performed. DNA was isolated, digested with XcmI and SacII, and analyzed in a Southern blot, with probe rDNp detecting a 2-kb fragment-encompassing promoter and 35S coding regions of the rRNA gene. Graphs at the bottom depict s- and f-band profiles for samples before or after 20 min of ChEC (for details, see Materials and Methods).

Pol II and III gain access to the Pol I promoter region in the UAF30 deletion mutant. (A) Yeast strains (y624, y1174, y1273, y1294, y1330, y1681, y1688, and y1694) carrying a wild-type allele or a deletion (Δ) in UAF30 and expressing the indicated MNase fusion proteins (MN) were treated and subjected to ChEC analysis as described in the legend to Fig. 3A. The positions of two Rpb3-MNase-mediated cuts at a bidirectional Pol II promoter within IGS1 are marked by filled dots. (B) Profiles of DNA fragment patterns in individual samples obtained after 30 min (Rpa190-MNase-expressing strains) or 60 min (other strains) of ChEC. Profile analysis was performed as described in the legend to Fig. 2B. (C) Yeast strains (y938, y945, y1560, y1566, y1708, and y1713) carrying a wild-type copy or a temperature-sensitive (ts) allele of RRN3 and expressing the indicated MNase fusion proteins (MN) were treated as described in the legend to Fig. 2D. Crude nuclei were subjected to ChEC analysis as described in the legend to Fig. 3A.

ChIP analysis confirms results obtained in ChEC experiments. (A to D) Yeast strains (y1184, y1185, y1328, y1329, y1450, y1453, y1689, and y1691) carrying a wild-type allele (wt) or a deletion (Δ) in UAF30 and expressing the proteins indicated in the upper left corner of each panel as fusion proteins with a C-terminal triple HA tag were subjected to ChIP experiments as described in Materials and Methods. The amounts of specific DNA fragments present in the input and retained on the beads were determined by quantitative PCR with primer pairs listed in Table 2. The bar graphs depict percentages of total input DNA retained after ChIP of the respective triple HA-tagged proteins. Error bars represent the standard deviations from three independent ChIP experiments, each of which was analyzed in triplicate quantitative PCRs. A cartoon representing the rDNA locus, depicting the locations of the different rDNA regions amplified in the quantitative PCRs, is shown at the top. prom, Pol I promoter.