Transcription factor UAF, expansion and contraction of ribosomal DNA (rDNA) repeats, and RNA polymerase switch in transcription of yeast rDNA

Abstract

Strains of the yeast Saccharomyces cerevisiae defective in transcription factor UAF give rise to variants able to grow by transcribing endogenous ribosomal DNA (rDNA) by RNA polymerase II (Pol II). We have demonstrated that the switch to growth using the Pol II system consists of two steps: a mutational alteration in UAF and an expansion of chromosomal rDNA repeats. The first step, a single mutation in UAF, is sufficient to allow Pol II transcription of rDNA. In contrast to UAF mutations, mutations in Pol I or other Pol I transcription factors can not independently lead to Pol II transcription of rDNA, suggesting a specific role of UAF in preventing polymerase switch. The second step, expansion of chromosomal rDNA repeats to levels severalfold higher than the wild type, is required for efficient cell growth. Mutations in genes that affect recombination within the rDNA repeats, fob1 and sir2, decrease and increase, respectively, the frequency of switching to growth using Pol II, indicating that increased rDNA copy number is a cause rather than a consequence of the switch. Finally, we show that the switch to the Pol II system is accompanied by a striking alteration in the localization and morphology of the nucleolus. The altered state that uses Pol II for rDNA transcription is semistable and heritable through mitosis and meiosis. We discuss the significance of these observations in relation to the plasticity of rDNA tandem repeats and nucleolar structures as well as evolution of the Pol I machinery.

FIG. 1

Spot test of rrn9Δ N-PSW strain NOY703 and PSW variants derived from it. (A) Two independent colonies of NOY703 formed on a galactose plate were analyzed by spotting aliquots of 10-fold serial dilutions of suspension of colonies on YEP-galactose (Gal) and YEPD (Glu) (rows a and b). Several discrete colonies, which had just formed on YEPD by plating large numbers of NOY703 cells on glucose (similar to colonies shown in rows a and b), were combined and similarly analyzed (rows c and d). (B) Two independent colonies of NOY703 (rows a and b), NOY878 (rows c and d), and an N-PSW revertant derived from NOY878 (rows e and f) formed on galactose plates were analyzed by the spot test as for panel A. Plates were incubated at 30°C for 10 days.

FIG. 2

Comparison of chromosomal rDNA copy numbers of NOY556 (WT), NOY703 (Δ9,N-PSW), NOY878 (Δ9,PSW), NOY408-1a (ΔA135), and NOY408-1b (WT). DNA from these five strains were digested with HindIII and PstI, and the digests were subjected to agarose gel electrophoresis, followed by transfer to a nylon membrane and hybridization with a mixture of ³²P-labeled rDNA probe and SIR3 probe. An autoradiogram is shown with an inserted gap between lanes 3 and 4. Radioactivity in each DNA band was quantified with a PhosphorImager. The values for chromosomal rDNA (and plasmid rDNA) were first normalized to the values for reference SIR3 DNA. These values obtained for NOY703 (lane 2) and NOY878 (lane 3) were then divided by corresponding values for control strain NOY556 (lane 1), and the ratios calculated are shown below the pertinent bands. Similarly, the normalized values obtained for NOY408-1a (lane 4) were divided by corresponding values for control strain NOY408-1b (lane 5), and the ratios calculated are shown below the pertinent bands. It should be noted that growth of both NOY703 (lane 2) and NOY408-1a (lane 4) is achieved by transcription of GAL7-35S rDNA on helper plasmids (NOY103 and NOY102, respectively), and copy numbers of the helper plasmids were higher than those carried by the control strains due almost certainly to selection.

FIG. 3

Correlation between the sizes of chromosome XII as analyzed by CHEF electrophoresis and PSW/N-PSW phenotypes. (A) Chromosomal DNA was isolated from strains NOY505 (lane 1) and NOY703 (lane 2), PSW strain NOY878 (lane 3), an N-PSW strain derived spontaneously from NOY878 (lane 4), and strain NOY408-1a (lane 5). The size of chromosome XII was then analyzed by CHEF electrophoresis. Size markers (lane M) are Hansenula wingei chromosomes, and their sizes are indicated in megabase pairs. Left, chromosome patterns revealed by staining with ethidium bromide; middle and right, autoradiograms obtained after hybridization with a SIR3 probe and an rDNA probe, respectively. (B) Chromosomal DNA was isolated from the following strains and analyzed by CHEF electrophoresis as for panel A: NOY505 (lane 1), NOY703 (lane 2), NOY877 (lane 3) (NOY703 and NOY877 are parents [P] of diploids [D] shown in lanes 5 to 7), a diploid obtained after the cross of N-PSW strain NOY876 and N-PSW strain NOY769 (lane 4), three independent diploid clones obtained after the cross of N-PSW strain NOY703 and PSW strain NOY877 (lanes 5 to 7), a diploid strain obtained after the cross of two PSW strains NOY877 and NOY878 (lane 8), and two haploid segregants (D→H) from the cross of N-PSW strain NOY703 and PSW strain NOY877, one showing the N-PSW phenotype (lane 9) and the other showing the PSW phenotype (lane 10). Autoradiograms obtained after hybridization with a SIR3 probe and an rDNA probe, respectively, are shown. It should be noted that in panel B, a portion of the gel containing the initial sample plugs corresponding to lanes 1 to 5 was inadvertently lost before the gel was subjected to autoradiography. Therefore, radioactive signals in sample wells representing chromosome XII, which was present in incompletely digested cells or spheroplasts and failed to enter the gel, are not seen in these lanes.

FIG. 4

Effect of a fob1 mutation on the frequency of switch from the N-PSW to PSW states. (A) Chromosomal DNA was isolated from the following strains and analyzed by CHEF electrophoresis: NOY505 (lane 1), NOY703 (lane 2), four independent fob1 deletion isolates obtained from NOY703 by disruption of FOB1 (lanes 3 to 6), and a fob1 deletion isolate obtained from the control strain NOY505 (lane 7). The left and right panels show autoradiograms obtained after hybridization with a SIR3 probe and a rDNA probe, respectively. (B) Three independent colonies of NOY703 (Δ9, N-PSW) and those of a fob1Δ mutant derived from NOY703 were analyzed by spot test on YEP-galactose (Gal) and YEPD (Glu).

FIG. 5

Efficiency of switching from the N-PSW to PSW states. (A) Two independent colonies of strains carrying rrn9Δ (NOY703; N-PSW), rpa135Δ (NOY408-1a), and rrn9Δ rap135Δ (NOY896) were analyzed by spot test on YEP-galactose and YEPD. (B) Two independent colonies of strains carrying rrn6Δ (NOY566), rrn6Δ sir2Δ (NOY918), and rrn6Δ rrn5Δ (NOY919) were analyzed by spot test on YEP-galactose and YEPD. (C) N-PSW strains carrying rrn9Δ (NOY703), rrn9Δ sir2Δ (NOY901), rrn9Δ sir3Δ (NOY911), and rrn9Δ sir4Δ (NOY912) were grown on a YEP-galactose plate. Three single colonies from each strain were analyzed by spot test.

FIG. 6

EM analysis of the nucleolus in control strain NOY505 (A) and PSW strain NOY794 (B). The strains were grown in YEPD at 25°C to an A₆₀₀ of about 0.5, and samples were prepared for EM analysis. The nuclear envelope is marked with arrows to serve as a point of reference, and the nucleolus (electron-dense areas within the nucleus) is indicated as N. The vacuole is indicated as V. Bars, 1 μm.

FIG. 7

FISH analysis of rDNA in PSW strain NOY852 and control strain W303-1a. Yeast strains were analyzed for rDNA and DNA as described in Materials and Methods. Images of rDNA and DNA were pseudocolored green and red, respectively, giving overlapped regions yellow in overlay. Individual images are shown in black and white. Note that in the PSW strain, many of the DAPI-stained nuclei have a hole with decreased DAPI staining and that rDNA appears to surround these holes. In the control strain, such a hole surrounded by rDNA was rarely seen.

FIG. 8

Primer extension analysis of primary transcripts for detection of rDNA transcription by Pol II. RNA was prepared from the following strains growing on synthetic galactose medium supplemented with Casamino Acids, tryptophan, and adenosine (lanes G) and 1 h after shift from galactose to glucose synthetic medium with the same supplements (lanes D): W303-1a (lanes 1, 2, 9, and 10), NOY408-1a (lanes 3 and 4), NOY920 (lanes 5 and 6), NOY921 (lanes 7 and 8), NOY566 (lanes 11 and 12), NOY918 (lanes 13 and 14), and NOY919 (lanes 15 and 16). Primer extension reactions were done in parallel, but gel electrophoresis and autoradiography were done in two separate groups (shown in A and B) with the same WT samples included. Autoradiograms (exposure times, 12 h [A] and 18 h [B]) are shown. Lane 8′ is the same as lane 8 after a longer exposure, which was equivalent to ∼30 h. Positions indicated as +1, G, and P correspond to the start site for the Pol I rDNA promoter, that for the GAL7 promoter, and a major site (−29) among the 5′ ends identified for rDNA transcripts in PSW strains, respectively. Three independent experiments were carried out, and the amounts of Pol II-specific precursor rRNAs found in NOY921 in glucose (lane 8 or 8′) were 15.4% ± 2.0% of those in control PSW strain NOY920 (lane 6).