Table 1.
Strains used in this study.
Figure 1.
The SWI/SNF complex interacts genetically with Pol I.
(A) Deletion mutants of SNF6 (DAS647) were mated with rpa135(D784G) (NOY2172) or rpa49Δ (DAS50). Diploids were selected, sporulated, and resulting tetrads were dissected. Images of haploid colonies were made after 2 days incubation at 30°C. Segregant genotype is labeled below each image. (B) Genetic interactions between indicated strains are listed. Non-lethal interactions were confirmed to have predicted growth rates, taking into account the defects in growth present in parental haploid strains. “N.D.” indicates genetic combinations that were not analyzed.
Figure 2.
Yeast cells were cultured, crosslinked with formaldehyde, lysed, sonicated and immunoprecipitated with anti-Myc 9E10 or anti-HA 12CA5 antibody. (A) Positions of 8 pairs of primers used in real-time PCR analyses are indicated by horizontal bars under the schematic of an rDNA repeat. (B) Positive control [Rrn7-(MYC)13 (DAS303)] and negative control [WT (NOY396)] ChIP results. Y-axis represents the amount of rDNA immunoprecipitated relative to total rDNA in input samples. (C) Snf6p (DAS649) ChIP signal compared to negative control. (D) A135-(his)7-(HA)3 (DAS477) associates with the promoter and the entire coding region of rDNA. (E) Snf2p (DAS750) associates with the coding region of the rDNA. Data were quantified from at least 2 10-fold dilutions per sample from duplicate cultures. Error = ±1standard deviation.
Figure 3.
rRNA synthesis rate of snf6Δ is only 40% of WT despite similar Pol I occupancy.
(A) Yeast cells were cultured in SD-met media to an A600 ∼ 0.3. Then [methy-3H]methionine was incorporated into cells for 5 min followed by a 5 min chase with excess (500 µg/ml) cold methionine. RNA was isolated, run on an agarose gel, transferred to nitrocellulose membrane, and detected by autoradiography. Pol I transcription in snf6Δ was normalized to WT. Data were analyzed from 2 independent [methy-3H]methionine incorporation assays. Only one dataset is shown here. (B) Pol I occupancy of rDNA in snf6Δ resembles that of WT by ChIP except for a 2-fold increase of Pol I occupancy in snf6Δ at the 5′ end of the 18S gene (asterisk). An anti-A190 polyclonal antibody was used to immunoprecipitate Pol I complexes. The 8 primer pairs described for Figure 2 were used in real-time PCR to check the association of Pol I complex with rDNA. A ninth primer pair measured Pol I association with 5S rDNA. Data were quantified from at least 2 10-fold dilutions per sample from duplicate cultures. Error = ±1standard deviation.
Figure 4.
Miller chromatin spreads of WT and snf6Δ suggest similar Pol I occupancy of rRNA genes.
Direction of transcription is from left to right. rRNA transcripts per rDNA gene were quantified for these representative and many additional genes. For the genes shown, the number of rRNA transcripts was 51 for WT (DAS648), and was 44, 39, and 60 for the three snf6Δ (DAS647) genes (top to bottom).
Figure 5.
A small accumulation of Pol I complexes in the 5′ end of rDNA in snf6Δ.
(A) Distribution frequency for the number of polymerases per gene was revealed by EM analysis of Miller chromatin spreads in snf6Δ (DAS647) and WT (DAS648). (B) More than 100 rDNA genes from Miller chromatin spreads were analyzed in WT and snf6Δ. Pol I density and percentage of actively transcribed genes in snf6Δ and WT are similar. (C) Polymerase occupancy as a function of position within the transcribed region of rDNA in snf6Δ and WT cells. A small peak of Pol I occupancy in the 5′ end of rDNA in snf6Δ is indicated by an asterisk. All mappable genes in the dataset were analyzed, corresponding to >60 genes per strain and >2500 polymerases per strain. Schematic below the X-axis represents the Pol I transcribed region of the rDNA. (D) Same as C but an additional two WT control strains (NOY388 and BY4741) are plotted. BY4741 data are from El Hage et al. 2010 [34].