1. Volume 29, Issue 3, Pages 313-323 (February 2008)
Transcription Termination and RNA Degradation Contribute to Silencing of RNA Polymerase II Transcription within Heterochromatin 
Lidia Vasiljeva, Minkyu Kim, Nihal Terzi, Luis M. Soares, Stephen Buratowski 
Molecular Cell 
Volume 29, Issue 3, Pages (February 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1
Endogenous RNAPII Transcription within rDNA NTS Regions Is Repressed by Nrd1 Complex and Exosome
(A) Mapping Nrd1- and Sir2-repressed transcripts. Schematic representation of rDNA unit showing NTS1, NTS2, 5S (arrowhead), and 35S precursor rRNA. The locations of the replication fork barrier (double triangle) and replication origin ARS (oval) are indicated. Positions of probes P1 (NTS1), P2 (NTS2), and 1–6 are shown as bars below the diagram, and Nrd1-regulated transcripts as gray arrows. The scale in kb is shown above. Total RNA (10 μg) from Pgal::NRD1 (DLY883) and sir2Δ (YF467) strains was resolved on 6% PAGE and analyzed by northern blot using probes 1–6. Strain DLY883 (Thiebaut et al., 2006) was grown on galactose or glucose for 2 hr to deplete Nrd1. The lower panel with probe 4 is a lighter exposure to show 5S RNA. Numbers on left indicate the positions of RNA markers in nucleotides.
(B) Accumulation of 5′ and 3′ ETS-containing RNA species in sir2Δ, Pgal::NRD1, and exosome mutant strains. Left panel, northern blot analysis of total RNA from WT (BY4741), lane 1; WT (DMY2800), lane 2; rrp6Δ (YSB2244), lane 3; Pgal::NRD1 (DLY883), lanes 4 and 5; rrp4-1 at 37°C (YRP1222), lane 6; and sir2Δ (YF467), lane 7. RNA was resolved on 1% agarose gels and 3′ ETS RNAs were visualized with probe 1. Positions of 25S, 18S, as well as the processing intermediates are indicated. Methylene-Blue-stained 25 and 18S rRNAs are shown below as a loading control. Right panel, northern blot analysis of total RNA from WT (BY4741), lane 1; Pgal::NRD1 (DLY883), lanes 2 and 3; rrp4-1 at 37°C (YRP1222), lane 4; and sir2Δ (YF467), lane 5.
(C) Northern blots of total RNA from WT (BY4741), lane 1; Pgal::NRD1 (DLY883), lanes 2–4; rrp4-1 (YRP1222) at 23°C and 37°C, lanes 5 and 6; Pgal::NAB3 (DLY889), lanes 7 and 8; sir2Δ (YF467), lane 9; and sen1-1 (FWY1), lane 10. Blots were hybridized with probe P1 (left panel) or P2 (right panel). Shift to glucose media was done for 2 or 6 hr to deplete Nrd1 and 14 hr for Nab3 depletion. 5 and 5.8S RNAs are shown as loading controls. Numbers on left indicate the positions of RNA markers in nucleotides.
(D) SIR2 mRNA level is elevated in Nrd1 and exosome mutants. Northern blot was performed with total RNA from WT (YF80 and YF81), lanes 1 and 2; Pgal::NRD1 (DLY883), lanes 3 and 4; rrp4-1 (YRP1222) at 23°C (lane 5) and 37°C (lane 6); tetOFF::DIS3, rrp6Δ (BSY1668, core exosome subunit DIS3 gene under the control of a Tet-repressible promoter combined with rrp6Δ, Dziembowski et al., 2007), lane 7; and rrp6Δ (BSY1669), lane 8. To deplete Dis3, cells were grown for 22 hr after addition of doxycycline. The membrane was probed for SIR2 mRNA. 25 and 18S rRNAs are shown as loading controls.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
A URA3 Reporter Integrated in rDNA Can Be Derepressed by Nrd1 and Rrp6 Mutants
(A) Schematic of rDNA unit (see Figure 1A for features) with position of the DMY2800 URA3 reporter shown. Gray arrows show positions and orientation of RNAPII transcripts.
(B) Derivatives of strain DMY2800 (YSB2243, YSB2246, YSB2245, YSB2244, and YSB2164; see Table S1) were assayed for growth with 10-fold serial dilutions on media containing (+URA) or lacking uracil (−URA) or containing 5-FOA. Lack of growth on FOA indicates URA3 expression.
(C) Northern blot analysis of URA3 expression in Nrd1-depleted, rrp6Δ, or sir2Δ cells. All strains carry the NTS2::URA3 reporter except for DMY2798 (lane 6), in which URA3 gene is inserted in a euchromatic region as a positive control for URA3 expression. The lower panel shows 5S rRNA as a loading control.
(D) NRD1, RRP6, and SIR2 mutations do not affect a euchromatic URA3 reporter (DMY2798). All strains carry the euchromatic URA3 reporter except for rows 3 and 4, which show NTS1::URA3 (DMY2804) and NTS2::URA3 (DMY2800) reporter strains as positive controls for repression.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Nrd1 and Exosome Mutants Derepress Telomeric Reporters
Strain UCC4602 and its derivatives (YSB2146, YSB2147, YSB2165, YSB2166, YSB2261, and YSB2265) contain URA3 and ADE2 reporters at telomeres of chromosomes VII and V, respectively.
(A) 10-fold serial dilutions of cells were plated on nonselective (+URA) or selective media (−URA and 5-FOA). URA3 repression is indicated by growth on 5-FOA.
(B) Northern blot analysis of URA3 expression in UCC4602 upon Nrd1 glucose depletion and sir2Δ cells. Nonrepressed control strain (DMY2798) has the URA3 gene at a euchromatic location. The lower panel shows 5S RNA as a loading control. Normalized URA3 levels were calculated relative to control DMY2798 and are listed below each lane.
(C) Cells were plated on medium with a low concentration of adenine. ADE2 expression is indicated by the red sectors.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Nrd1 Mediates Termination of RNAPII Transcription in NTS1
(A) Nrd1 crosslinks to the NTS1 CUT region. At the top is a schematic diagram of the rDNA repeat (as in Figure 1A) showing positions of the ChIP PCR products (black bars below with corresponding numbers, from Huang et al., 2006). ChIP was performed with Nrd1-TAP (YF817) and untagged (BY4741) strains. Lower panel shows PCR products, and middle panel shows data quantification as described in the Experimental Procedures.
(B) RNAPII crosslinks to the NTS1 CUT region and is stimulated by glucose. ChIP analysis with anti-Rpb3 antibody was carried out in Nrd1 WT (W303) cells grown on galactose and glucose media.
(C) Nrd1 depletion leads to defective RNAPII termination at the NTS1 CUT. ChIP with anti-Rpb3 antibody in Pgal::NRD1 (DLY883) cells grown either on galactose or on glucose for 2 hr is shown.
(D) RNAPII transcription occurs in transcriptionally active rDNA repeats. ChIP with anti-Rpb3 antibody in a strain containing reduced rDNA repeat number (25 copies all active, NOY1071) or a WT strain containing 190 copies of rDNA (NOY1064).
In all panels, error bars show standard deviation from three repetitions.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

6. Figure 5
Nrd1 Regulates Histone Modifications and Unequal Crossovers in rDNA
(A) The CUT promoter regions have lower nucleosome density. ChIP analysis with anti-H3 antibody was carried out for Nrd1 WT (W303) and Pgal::NRD1 (DLY883) cells grown on glucose media for 2 hr. Numbers at bottom refer to PCR products as diagrammed in Figure 4A.
(B) Loss of Nrd1 leads to higher levels of H3 K4 trimethylation. ChIP with anti-H3K4me3 antibody was carried out with the same chromatin used in (A). Signals were quantitated by a PhosphorImager and normalized to total H3.
(C) Loss of Nrd1 leads to higher levels of histone H4 acetylation. ChIP with anti-H4 tetra-acetyl antibody was carried out as in (B).
(D) Frequency of unequal rDNA crossovers is increased in Nrd1 mutants. Percent of the ADE2 gene loss (% marker loss) was calculated for a wild-type strain (WT, DMY3010), a sir2Δ strain (DMY3011), a Pgal::NRD1 strain containing plasmids with WT NRD1 (YSB2193), nrd1-102 (YSB2192), nrd1Δ2-165 (YSB2194), or a nrd1Δ::KAN strain with plasmids encoding WT NRD1 (YSB2205), nrd1-102 (YSB2207), nrd1Δ2-165 (YSB2206), or nrd1Δ (YSB2211). The relevant chromosomal genotype is shown above each bar while the plasmid encoded NRD1 allele is shown underneath. Error bars show standard deviation from three repetitions.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Multiple Levels for Silencing of Heterochromatic Genes in S. cerevisiae
1. Chromatin modifications (histone H4 and H3 deacetylation by Sir2) regulate initial recruitment of RNAPII to DNA. Acetyl groups are shown as flags.
2. Nrd1/Sen1 complex triggers termination of cryptic RNAPII transcription that does occur in heterochromatin.
3. The exosome contributes to repression by degrading RNA produced by RNAPII despite Sir2 and Nrd1 actions.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions