Cryptic transcripts from regions of repressed chromatin accumulate in strains with defects in the TRAMP complex or exosome
We speculated that transcripts may be generated from regions of repressed chromatin in the yeast genome but degraded by the TRAMP and exosome complexes. We therefore tested a telomeric region (TEL05L), the intergenic spacer (IGS) region of the rDNA repeat, a centromeric region (CEN3) and the silenced mating-type cassettes (MATa/α). In each case, we saw increased levels of a transcript in strains lacking Trf4, but not in single mutant strains lacking Trf5, Air1 or Air2 (, compare lane 2 with lanes 3–5).
Figure 2 Cryptic transcripts can be detected from regions of repressed chromatin in strains lacking TRAMP activity. (A) Northern analysis of TRAMP mutants grown in YPD at 25°C. RNA is resolved on a 1.2% agarose glyoxal gel (three upper panels) (more ...)
At many yeast telomeres, the terminal repeats are flanked by the ‘Y′ region', which is conserved in whole or in part at 16 yeast telomeres. At some telomeres, this region encodes a putative DNA helicase (Yamada et al, 1998
), designated Yel077c in the case of TEL05L
(). Strand-specific probes demonstrated that the transcript elevated in the trf4
Δ strain is an ncRNA expressed antisense to YEL077C
. The TEL05L
ncRNA is ~6.5 kb in length and was detected by a probe located within YEL074W
() and by a second probe located 2.8 kb further toward the chromosome end (data not shown), showing it to extend across the Y′ region. 5′ RACE generated a product that was enriched in trf4
Δ (arrow in ), which was cloned and sequenced. Three 5′ ends were identified, located 106–128 bp beyond the 3′ end of the open reading frame (ORF) of YEL077C
. The ncRNA therefore starts close to the chromosome end and runs antisense through the entire putative helicase ORF. Polymorphisms in sequenced products demonstrate that the ncRNA is transcribed from at least two telomeres (data not shown).
ncRNA was also elevated in a strain lacking Rrp6 (, lane 6), indicating that it is a target for exosome degradation. Depletion of Trf5 by growth of a trf4
strain on glucose medium (, lane 5) increased accumulation of the TEL05L
transcript relative to the trf4
Δ single mutant. Overexpression of Trf5, in GAL-trf5
strains grown in galactose medium, can suppress the phenotype of trf4
Δ strains on some TRAMP substrates (Houseley and Tollervey, 2006
), and this was the case for the TEL05L
transcript (, lane 4). We conclude that TRAMP4 and TRAMP5 both participate in degradation, with TRAMP4 probably playing the major role, and function with the exosome to degrade large ncRNAs generated from telomeric regions.
We have not further characterized the ~1.2 kb CEN3
transcript, and the structure of the MAT
loci prevents unambiguous assignment of the observed ~1.2 kb transcript to a silent cassette. However, connections between Trf4 and Top1 mutations and rDNA structure (see Introduction) suggested a functional link to IGS transcription, which was therefore further investigated. The ncRNA products of transcription from E-pro, IGS1-F and IGS1-R () are normally present at very low levels (Santangelo et al, 1988
) and we speculated that this might reflect rapid degradation involving the TRAMP and exosome complexes. To visualize these ncRNAs, northern blots of RNA from TRAMP and exosome mutants were probed with strand-specific probes to both rDNA intergenic spacer regions (). An sir2
Δ strain, which overexpresses IGS transcripts, was used as a positive control. A top1
Δ strain was also analyzed, as top1 trf4
double mutants were reported to show synthetic lethality and rDNA condensation phenotypes (Sadoff et al, 1995
; Castano et al, 1996
). Strains carrying sir2
Δ and top1
Δ showed elevated levels of three ncRNAs IGS1-R, IGS1-F and IGS2-R (, lanes 6 and 7).
Figure 3 IGS1-R is degraded by TRAMP and the exosome. (A) Locations of hybridization probes and ChIP primer sets used. (B) Northern analysis of TRAMP and exosome mutants and control strains grown in YPD at 25°C. Probes are (top to bottom) NTS1 HpaI, NTS2 (more ...)
The levels of IGS1-F and IGS2-R were unaltered in TRAMP or exosome mutants, whereas the level of IGS1-R was substantially increased in trf4Δ and to a lesser extent in air1Δ air2Δ and rrp6Δ strains. No stabilization of IGS1-R was seen in trf5Δ strains, and overexpression of Trf5 under GAL regulation did not suppress the trf4Δ phenotype (, lanes 3 and 4). This suggests that Trf5 does not efficiently target IGS1-R, even in the absence of Trf4. IGS1-R stabilization in the rrp6Δ exosome mutant strain was weaker than that in trf4Δ ( (lane 5) and D (lane 3)) and we therefore also examined the core exosome mutant mtr3-1 (). Even at permissive temperature (25°C), accumulation of IGS1-R was visible in the mtr3-1 strain. We conclude that TRAMP4 functions with both Rrp6 and the core exosome to degrade IGS1-R, whereas degradation of IGS1-F and IGS2-R presumably involves other activities.
Two major forms of IGS2-R of approximately 1.6 kb and 850 nt in length were detected (see also Li et al, 2006
). The longer IGS2-R species also hybridized to a downstream probe to IGS1, whereas the short species was not detected with this probe, indicating that it is 3′ truncated (data not shown). This would be consistent with termination of some IGS2-R transcripts around the location of the CAR, even in top1
Δ and sir2
Δ strains. A shorter probe directed against the 3′ region of IGS1-R (NTS1 short; see ) was also used. This region is extremely AT rich and the probe hybridized poorly; however, only the longer transcripts were detected by NTS1 short probe (, compare lanes 1–4 with 5–8). This indicates that the IGS1-R transcripts show 3′ heterogeneity.
To assess whether increased IGS1-R RNA reflects increased transcription or post-transcriptional stabilization, chromatin immunoprecipitation (ChIP) was performed to determine RNA Pol II occupancy in wild-type, trf4
Δ and top1
Δ cells () and sir2
Δ (data not shown). This revealed a clear peak of Pol II within the IGS1-R region in the wild-type strain. This peak was elevated in the top1
Δ strain, consistent with constitutive derepression (Bryk et al, 1997
). In contrast, the Pol II signal in the trf4
Δ strain was lower than that in the wild-type strain, despite elevated levels of the transcript.
We next wanted to determine how the TRAMP complex is recruited to the IGS1-R transcripts. The Nrd1–Nab3 heterodimer of RNA-binding proteins was reported to recruit the exosome to substrate RNAs (Arigo et al, 2006b
; Thiebaut et al, 2006
). The longest observed IGS1-R transcript contains 10 potential binding sites for Nab3 (UCUU) including two prominent clusters, and seven binding sites for Nrd1 (GUAA/G). The level of IGS1-R was therefore assessed in ts-lethal nab3-11
mutant strains (). IGS1-R was strongly stabilized in the nrd1-102
mutant, demonstrating that it is targeted by the Nrd1–Nab3 pathway. In contrast, the nab3-11
mutant conferred little or no stabilization (). Allele specificity has, however, previously been reported for mutations in these proteins (Conrad et al, 2000
) and the data do not demonstrate that Nrd1 is primarily responsible for targeting IGS1-R for degradation. On other transcripts, Nrd1–Nab3 are responsible for transcription termination (Steinmetz et al, 2001
; Arigo et al, 2006a
; Kim et al, 2006
; Thiebaut et al, 2006
). However, we saw no clear differences in the migration of the IGS1-R transcripts in the nrd1
mutants relative to the wild type (), suggesting that termination is not strongly impaired.
From the ChIP and northern data, we conclude that transcription of IGS2-R and IGS1-F is strongly repressed by a chromatin structure that requires Sir2 and Top1 for its maintenance. IGS1-R is more actively transcribed than IGS2-R or IGS1-F, but the resulting transcripts are rapidly degraded by a mechanism that requires the Nrd1/Nab3 complex, TRAMP4 and both the core exosome and Rrp6.
IGS1-R is polyadenylated and shows extensive 3′ heterogeneity
Primer extension was used to define the 5′ end of the IGS1-R transcript (). The 5′ end was mapped by running the primer extension products alongside a sequencing ladder on a 40 cm denaturing polyacrylamide gel (data not shown). The major 5′ end lies at +599 nt from the end of the 25S rRNA, with some secondary 5′ ends spanning about 20 bp. This position is 26 bp 3′ to that originally described (Santangelo et al, 1988
) and around +175 nt from the RFB.
Figure 4 IGS1-R is a 3′-heterogeneous transcript polyadenylated by Pap1. (A) Primer extension analysis of IGS1-R performed on poly(A)+ RNA from oligonucleotide NTS1 F3 (see C). (B) Ethidium-stained 6% acrylamide gel of 35-cycle 3′ (more ...)
Conventional analyses of polyadenylation using RNase H and oligo(dT) were complicated by the presence of two genome-encoded poly(A) tracts within IGS1-R (Supplementary Figures 1A and B
). However, the results were consistent with 3′ heterogeneity, as were the northern analyses shown in . Oligo-dT-directed 3′ RACE detected multiple transcripts (), showing that IGS1-R is polyadenylated even in trf4
Δ strains, and of 16 3′ RACE clones sequenced, 6 terminated in genomic encoded poly(A) tracts and the remaining 10 clones contained 9 different 3′ ends, demonstrating substantial 3′ heterogeneity ().
Depletion of Trf5 from the trf4Δ strain had little effect on transcript length (, lane 5), whereas transfer of a ts-lethal pap1-2 trf4Δ strain to 37°C resulted in shorter IGS1-R transcripts (, compare lanes 4 and 8). At permissive temperature, the abundance of IGS1-R transcripts was greater in the pap1-2 trf4Δ double mutant than in either single mutant (, compare lanes 2, 3 and 4). These results indicate that IGS1-R is polyadenylated by Pap1 and suggest that efficient polyadenylation promotes IGS1-R degradation.
We tested whether the polyadenylation activity of Trf4 is required for degradation of IGS1-R (). Plasmids expressing either Trf4 or the catalytically inactive Trf4-DADA (Vanacova et al, 2005
) fully suppressed the IGS1-R stabilization phenotype in the trf4
Δ strain. Recruitment of the exosome and IGS1-R degradation does not therefore require polyadenylation by Trf4 although the presence of the protein is necessary.
Trf4 functions in rDNA copy number control
TRAMP and exosome mutants were tested for alterations in rDNA copy number using pulsed field gel electrophoresis (PFGE) (). In the wild-type strain, chromosome XII (~60% of which is composed of rDNA repeats) showed a well-defined length corresponding to an rDNA array of ~200 repeats. In contrast, sir2
Δ and top1
Δ strains showed hyper-recombination phenotypes that caused chromosome XII to appear as smears (compare lane 1 with lanes 6 and 7 in and lanes 1–3 with lanes 7–9 and 13 and 14 ) (Christman et al, 1988
; Gottlieb and Esposito, 1989
; Bryk et al, 1997
). Strains lacking the TRAMP components, trf4
Δ or air1
Δ, showed sporadic deviations from the wild-type rDNA copy number, with the greatest effect in trf4
Δ strains. These deviations from wild-type repeat number had only limited penetrance, with three out of eight trf4
Δ clones analyzed showing a clear repeat number reduction and two strains showing repeat expansion. We observed no evidence of an rDNA hyper-recombination phenotype, which would be indicated by smearing of the rDNA band, in any trf4
Δ single mutant analyzed, in contrast to a previous report (Sadoff et al, 1995
Figure 5 Loss of Trf4 alters rDNA copy number. (A, B) Pulsed field analysis of rDNA copy number in TRAMP mutants and controls. Strains were grown to stationary phase in YPD, resolved on a 0.8% pulsed field gel and probed with 18S to highlight chromosome (more ...)
A METtrf4 top1
Δ strain was viable on restrictive high methionine medium, and multiple top1
Δ transformants isolated in four independent experiments were all growth-impaired but viable. This is in contrast to their reported synthetic lethality (Sadoff et al, 1995
; Castano et al, 1996
; Pan et al, 2006
), and may reflect differences in alleles or strain background. We also combined trf4
Δ with sir2
Δ, and growth of the double mutant strain was similar to that of the trf4
Δ single mutant. The double mutants of trf4
Δ with either sir2
Δ or top1
Δ showed large losses in rDNA repeat number (, lanes 10–12 and 15–17), and this phenotype was observed in all clones tested (at least four of each combination). In contrast, the combination of trf5
Δ with either sir2
Δ or top1
Δ had no apparent effect on rDNA repeat number (Supplementary Figure 2A
). We also tested the exosome mutant rrp6
Δ (Supplementary Figure 2B
). Loss of Rrp6 alone had no clear effect on repeat number (lane 2), but an rrp6
Δ double mutant showed reduced heterogeneity relative to top1
Δ single mutant strains (lanes 3–7).
To assess potential links between stability of the rDNA and transcription of IGS1-R, IGS1-F and IGS2-R, the trf4Δ sir2Δ and trf4Δ top1Δ double and single mutant strains were analyzed by northern hybridization () and Pol II ChIP ().
Levels of IGS1-R appeared to correlate with rDNA copy number instability, being higher in sir2
Δ and top1
Δ double-mutant strains than in the sir2
Δ and top1
Δ single mutants. In , the quantification of the northern data is normalized to rDNA repeat number. The levels of IGS1-F were highest in the sir2
Δ and top1
Δ single mutants, which showed the greatest repeat heterogeneity. In contrast, the levels of IGS2-R appeared principally dependent on the presence or absence of Sir2. The ratio of abundance of the ncRNAs was altered between top1
Δ and sir2
Δ strains expressing or lacking Trf4. It is possible that this has an effect on repeat number, although we have no direct evidence for this. Loss of Trf5 from either the sir2
Δ or top1
Δ strains had no clear effect on the levels of any of the IGS ncRNAs (Supplementary Figure 2C
ChIP for RNA Pol II at the IGS1-R locus () shows that Pol II occupancy is not significantly altered in top1Δ trf4Δ strains compared to top1Δ. Hence, the reduced heterogeneity of the rDNA repeats in the trf4Δ top1Δ double mutant relative to the top1Δ single mutant is not due to reduced transcription. This also confirms that the high accumulation of transcript is due to increased RNA stability in the absence of Trf4.
We conclude that deletion of TRF4, but not TRF5, from the sir2Δ or top1Δ strains leads to greatly increased levels of IGS1-R, due to increased RNA stability, and a drastic loss of rDNA repeats. Deletion of RRP6 from the top1Δ strain appeared to reduce the heterogeneity in rDNA repeat number without clearly decreasing average repeat length.
Trf4 catalytic activity is not required for repeat regulation
One explanation for the loss of rDNA repeats in trf4
Δ mutants would be that Trf4 is a DNA polymerase involved in rDNA replication, as previously proposed (Wang et al, 2000
). Were this the case, the polymerase activity of Trf4 would be required to allow the hyper-recombination observed in top1
To test this, we made use of the suppression of the rDNA repeat heterogeneity seen in top1Δ strains by loss of Trf4 (). We compared trf4Δ TOP1 (lanes 1, 4 and 7) and trf4Δ top1Δ (lanes 2–3, 5–6 and 8–9). These strains also carried plasmids lacking an insert (lanes 1–3), expressing intact Trf4 (lanes 4–6) or expressing the catalytically inactive Trf4-DADA (lanes 7–9). The top1Δ strains expressing wild-type Trf4 showed a hyper-recombination phenotype (lanes 5 and 6). Hyper-recombination was suppressed in the absence of Trf4 (lanes 2 and 3), but was clearly present when only Trf4-DADA was expressed (lanes 8 and 9). Western blotting confirmed that the mutant and wild-type Trf4 were expressed at similar levels (data not shown). In this experiment, strains were grown on minimal media to select for plasmid maintenance and recombination was less active than on complete YPD media used in the experiments shown in . Thus, the poly(A) polymerase activity of Trf4 is not required for IGS1-R degradation and is also not required for hyper-recombination.
Figure 6 The role of Trf4 in rDNA recombination. (A) Pulsed field analysis of trf4Δ top1Δ strains with Trf4 plasmids. One TOP1 and two top1Δ samples are shown in each case. Cells were grown in synthetic media and harvested at stationary (more ...)
rDNA recombination frequency is unaffected by loss of TRF4
These analyses do not resolve differences in recombination frequency and stability of the rDNA repeat tract. Recombination rates within the rDNA tract can be assessed by integrating a single-copy marker gene and scoring for its loss. The presence or absence of a functional MET25
gene can be scored by a colony color test on medium containing Pb2+
, on which met25
Δ colony sectors turn dark brown () (Smith and Boeke, 1997
). A MET25-GFP
construct was integrated into the IGS2 region of a single rDNA repeat in one strain of each genotype used in . Three independent MET25
insertion clones from each strain were then scored for colony sectoring phenotypes (). Comparison of sectoring levels in top1
Δ and top1
Δ shows that loss of TRF4
has little or no effect on recombination frequency. A transgene inserted at this location was previously shown to be repressed by Sir2 (Smith and Boeke, 1997
), and we confirmed this for our insert by western blotting (data not shown). Met25-GFP expression was lower in western blots from trf4
Δ strains than trf4
Δ strains (data not shown), showing that Sir2-dependent silencing was maintained. This indicates that hypoacetylation of H3 and H4 by Sir2 is not lost, since Sir2-dependent silencing requires its deacetylation activity (Li et al, 2006
In trf4Δ strains, transcription of the MET25-GFP reporter was frequently reduced, resulting in dark pigmentation (). This is consistent with the reduction of IGS1-F expression seen in sir2Δ trf4Δ strains compared to sir2Δ, but the effect was variable between fresh transformants and old cells. The sir2Δ trf4Δ strain proved hypersensitive to Pb2+ ions and could not be assessed in the sectoring assay.
Trf4 is recruited co-transcriptionally to IGS1
We hypothesized that the altered rDNA stability in trf4Δ strains is due to direct effects of Trf4 on the rDNA or chromatin rather than indirect consequences of defects in RNA processing. In this case, the role of IGS1-R transcription might be to recruit Trf4 to the rDNA in the vicinity of the RFB region. Were this model correct, we would detect an association of Trf4 with the rDNA IGS1-R region that is dependent on RNA Pol II transcription.
ChIP analysis of Trf4-Myc over the rDNA IGS regions () showed clear enrichment over IGS1-R. To confirm that this association was dependent on RNA Pol II transcription, we analyzed an rpb1-1
strain, which carries a fast-acting temperature-sensitive mutation in RNA Pol II (). The ChIP signal for Trf4 was substantially reduced across the IGS1-R region at the restrictive temperature, consistent with its recruitment by IGS1-R nascent transcripts, although the ChIP signal for Pol II showed greater reduction than the Trf4 signal (). To ensure that the protein context of the rDNA is not generally altered in this strain, lysates from this experiment were checked by ChIP for the presence of Sir2, which was only mildly reduced at 37°C (Supplementary Figure 3
Figure 7 Trf4 is recruited co-transcriptionally to the IGS1-R region of the rDNA. (A, B) ChIP analysis of Trf4 recruitment to the IGS in an rpb1-1 strain. rpb1-1 TRF4-13Myc cells were grown to mid-log at 25°C in YPD and half were shifted to 37°C (more ...)
These data indicate that Trf4 is recruited co-transcriptionally to the nascent IGS1-R transcripts, probably via Nrd1/Nab3. This is likely to be an important factor in allowing very rapid degradation of IGS1-R as soon as transcription termination generates a free 3′ end for exosome activity. This may also be the case for other cryptic ncRNA transcripts.
Alterations in rDNA copy number in trf4Δ strains do not reflect differences in cohesin recruitment
In order to address whether cohesin is displaced by IGS1-R accumulation in the trf4Δ strains, we performed ChIP analysis using a 13-Myc-tagged cohesin subunit Smc1. The Smc1-Myc fusion is the only form of Smc1 present in the cells, which showed no growth impairment. In addition, rDNA recombination rates were unaltered, as judged by the Pb2+ plate method described above (data not shown), indicating that the fusion protein is fully functional.
In the wild-type background, Smc1-Myc showed the expected distribution with a clear peak over the CAR (). Neither the distribution nor the intensity of the Smc1 ChIP signal was altered by the loss of Trf4. In contrast, the Smc1 ChIP signal was strongly reduced by loss of Top1, either in the presence or absence of Trf4. This makes it unlikely that the effects of Trf4 on rDNA copy number regulation are related to removal of cohesin from the IGS region.