Mutating UAF30 reduces the number of actively transcribed rDNA genes in the tandem array. (A) Miller spreads of WT (YRH4) and uaf30Δ (YRH165) strains at low magnification to visualize the entire nucleolar area. Active genes are found throughout the spread WT nucleolus, while few active genes (examples are indicated by arrows) are seen in the uaf30Δ mutant. (B) Psoralen cross-linking analysis of the proportion of rDNA genes that are actively transcribed (open) in exponentially growing WT, uaf30::Tn3 (PS1174), and uaf30Δ (YRH165) strains. A representative Southern blot is shown. Note the decrease in signal for the open genes (indicated by a dot) and an increase in the closed signal of the lower band. (C) Quantitation of the percentage of open rDNA genes from three independent psoralen cross-linking experiments. Standard deviations are indicated.

Analysis of Pol II transcription of rDNA genes. (A) Miller spread of Pol II transcription within nucleolar DNA of a PSW strain. Shown is an EM at two magnifications of nucleolar chromatin from an rrn9Δ rpa135Δ PSW strain (NOY897). The top panel shows a representative example of such regions, which are not seen in normal cells. The lower panel is the boxed region at higher magnification. Arrowheads mark two rRNA transcripts with terminal knobs. Bars, 1 μm. (B) Psoralen cross-linking analysis of WT (YSB521) and rrn5Δ PSW (YSB526) strains. A representative Southern blot is shown. Note the absence of a discernible band for the PSW strain in the gel region corresponding to an open chromatin structure (indicated by a dot). Fainter bands above the dots are the result of partial EcoRI digestion and are not indicative of the active (open species). The latter are represented by the dark lower band.

Uaf30p is required for proper UAF association with the rDNA promoter. (A) DNase I footprinting analysis of UAF complex purified from a WT (NOY798) or uaf30Δ (NOY1047) strain on the noncoding (top) DNA strand. The HindIII-BstBI fragment radioactively labeled on the top strand was incubated with increasing amounts of WT UAF (lane 2, 1 μl; lane 3, 3 μl; and lane 4, 5 μl) or UAF from uaf30Δ cells (lane 5, 3 μl; and lane 6, 5 μl). Lane 1 is the control reaction with labeled fragment without UAF. Lanes G, A, T, and C are dideoxy sequencing reactions from the pNOY3238 plasmid used for footprinting. Arrowheads at −48 and −107 indicate the extent of the WT UAF footprint on the rDNA promoter. DNase I hypersensitive sites at −77/−76, −100/−97, and −113/−110 are indicated. Sequencing reactions are from the same gel as the footprinting reactions but have different exposures. (B) ChIP analysis of TAP-tagged Rrn5 (UAF) and Rrn7 (CF) association with the rDNA promoter (NTS2) and the transcribed region (5′-ETS and 25S). Average ratios and standard deviations are calculated and plotted as in Fig. 3. Untagged strain, BY4741. Rrn5-TAP WT strain, YRH690. Rrn5-TAP uaf30Δ strain, YRH880. Rrn7-TAP WT strain, YRH691. Rrn7-TAP uaf30Δ strain, YRH884. (C) Western blot analysis of TAP-tagged Rrn5 and Rrn7 in the WT and uaf30Δ strain backgrounds used for the ChIP assays. Amounts of each tagged protein in the uaf30Δ mutant relative to the WT level are indicated.

High density of RNA polymerases on the rDNA genes of uaf30 mutants. (A) Representative rDNA genes from WT (YRH4), WT + rap (WT grown in the presence of 0.2 μg/ml rapamycin for 1 h), top1Δ (YRH160), uaf30::Tn3 (PS1-174), and uaf30Δ (YRH165) strains. The average number of polymerases (pols) per active gene is indicated, along with the standard deviation in parentheses. The polymerase density on genes from the uaf30 mutants was so high that it was difficult to determine an exact number of polymerases per gene. N, number of genes counted. Bar, 0.5 μm. (B) Pulsed-field gel electrophoresis of BamHI-digested yeast chromosome preparations. BamHI does not cut within the rDNA array and therefore releases it intact from chromosome XII. Following Southern blotting, the released array was detected using a ³²P-labeled rDNA-specific probe. 143c, control strain NOY1051 with an array size of 143 repeats. 42c, control strain NOY886 with an array size of 42 repeats. Other strains were the same as in panel A. (C) Reduced rDNA array size in an spt4Δ mutant derivative (YRH245) of YRH4 (WT).

Reduced overall association of Pol I with the rDNA tandem array in the absence of Uaf30. (A) Western blot analysis of TAP-tagged Rpa135 protein levels in three independent isolates of WT (YRH692) and uaf30Δ (YRH864) strains. The anti-protein A and antitubulin antibodies were used. The ratio of Rpa135-TAP to tubulin signal for each isolate is indicated at the bottom. (B) ChIP analysis of TAP-tagged Rpa135 association with the 35S rDNA region of the tandem array. PCR was used to amplify six segments spaced ∼750 bp apart (represented by horizontal bars 1 to 6). ACT1 was the negative control. The average ratios and standard deviations of the immunoprecipitated PCR signal to the input chromatin PCR signal are shown.

Testing of alternative mechanisms for inducing polymerase-dense rDNA genes. (A) Pol II transcribes individual rDNA genes in PSW cells at a very low rate and does not impede Pol I transcription in uaf30Δ strains. Representative active genes from NOY897 (rrn9Δ rpa135 PSW), PS1-174 (uaf30::Tn3), and YRH62 (uaf30::Tn3 rpd3Δ) are shown. Respectively, these three strains use Pol II only, Pol I and Pol II, and Pol I only to transcribe rDNA genes. Bar, 0.5 μm. (B) Sensitivity of WT (YRH282), uaf30::Tn3 (YRH420), and top1::Tn3 (JB1241) strains to 250 μg/ml 6-AU. Other control strains had 143 (NOY1051), 42 (NOY886), or 25 (NOY1071) rDNA copies. The SC-ura plates were incubated for 2 days and the 6-AU plates for 3 days. Strains were transformed with pRS416 to make them Ura⁺. (C) Tetrad dissections of a diploid strain (YRH641) that is heterozygous for the uaf30Δ::kanMX4 and top1Δ::kanMX4 mutations. Dissected spores from tetrads 1 to 5 were grown on YPD for the indicated number of days.