Association of the RENT complex with nontranscribed and coding regions of rDNA and a regional requirement for the replication fork block protein Fob1 in rDNA silencing

Abstract

Silencing within the yeast rDNA repeats inhibits hyperrecombination, represses transcription from foreign promoters, and extends replicative life span. rDNA silencing is mediated by a Sir2-containing complex called RENT (regulator of nucleolar silencing and telophase exit). We show that the Net1 (also called Cfi1) and Sir2 subunits of RENT localize primarily to two distinct regions within rDNA: in one of the nontranscribed spacers (NTS1) and around the Pol I promoter, extending into the 35S rRNA coding region. Binding to NTS1 overlaps the recombination hotspot and replication fork barrier elements, which have been shown previously to require the Fob1 protein for their activities. In cells lacking Fob1, silencing and the association of RENT subunits are abolished specifically at NTS1, while silencing and association at the Pol I promoter region are unaffected or increased. We find that Net1 and Sir2 are physically associated with Fob1 and subunits of RNA polymerase I. Together with the localization data, these results suggest the existence of two distinct modes for the recruitment of the RENT complex to rDNA and reveal a role for Fob1 in rDNA silencing and in the recruitment of the RENT complex. Furthermore, the Fob1-dependent associations of Net1 and Sir2 with the recombination hotspot region strongly suggest that Sir2 acts directly at this region to carry out its inhibitory effect on rDNA recombination and accelerated aging.

Figure 1.

Net1 and Sir2 associate primarily with the NTS1 and NTS2/18S regions of rDNA. (A) The physical structure of the tandemly repeating rDNA of S. cerevisiae is shown above, and a single 9.1-kb rDNA unit is shown expanded below. Each repeat yields a Pol I-transcribed 35S precursor rRNA (shown as a divided thick arrow) and a Pol III-transcribed 5S rRNA (arrowhead). The 35S coding regions are separated by a nontranscribed spacer (NTS), which is divided by the 5S gene into NTS1 and NTS2. Solid bars indicate the replication fork block region (RFB) and the Pol I transcription initiator region (TIR; Elion and Warner 1984, 1986; Brewer and Fangman 1988; Kobayashi et al. 1992). The locations of the replication fork barrier () and autonomously replicating sequences () are indicated. Vertical arrows indicate insertion sites of silencing reporters. (R1) NTS1::Ty1–mURA3; (R2) NTS2::Ty1–mURA3; (R3) NTS1::mURA3; (R4) NTS2::mURA3. PCR products analyzed in ChIP assays are indicated below the rDNA unit. (B) Representative graph showing the associations of Net1–TAP and Sir2–TAP within the rDNA repeat. Relative fold enrichment refers to the relative ratio of PCR products amplified from immunoprecipitated DNA to products from whole-cell extract DNA (see Materials and Methods). Positions along rDNA correspond to the physical map shown in A. (C) Examples of the ChIP data used to calculate enrichment at the 25S region of rDNA. The numbers below the panels refer to the PCR products shown in A. CUP1 primers were used as a control. WCE and IP refer to products amplified from whole-cell extracts and immunoprecipitated DNA, respectively. Untagged control (–), Net1–TAP (N), and Sir2–TAP (S) cells are indicated above lanes. (D) Same as in C but showing amplified DNA from the NTS1 region. (E) Same as in C but showing amplified DNA from the NTS2/18S region. (F) Multiplex PCR with rDNA and CUP1 primers showing rDNA enrichment. (G) Western blot showing the relative abundance of TAP-tagged proteins in whole-cell extracts (WCE) prepared from untagged (lane 1) or Net1–TAP, Sir2–TAP, or Fob1–TAP strains (lanes 2–4).

Figure 2.

H3 acetylation levels throughout rDNA are increased in sir2Δ cells. (A) Examples of the ChIP data used to determine the associations of diacetylated (K9/K14) histone H3 or Sir2 with rDNA (left panels) or telomeric (TEL) and mating-type loci (HMR-E) regions (right panels). (+) SIR2⁺ cells; (Δ) sir2Δ cells; numbers above the left panels refer to rDNA primers as indicated in Figure 1A. CUP1 and ACT1 primers were used as internal controls. (B, left) Quantification of ChIP experiments shows that the relative fold enrichment of diacetylated H3 increases throughout the rDNA in sir2Δ cells as compared with SIR2⁺ cells. rDNA primers are indicated below the graph and correspond to Figure 1A. (Right) H3 acetylation increases at telomeres and the silent mating-type loci in sir2Δ cells. (C, left) Association of Sir2 with rDNA in SIR2⁺ and sir2Δ cells showing that the highest levels of Sir2 are present at NTS1 and NTS2/18S. (Right) Association of Sir2 with telomeres and the silent mating-type loci is shown for comparison.

Figure 3.

Fob1 is required for rDNA silencing at NTS1 but not at NTS2/18S. Silencing was assessed by monitoring the growth of 10-fold serial dilutions of cells on –URA medium. Complete medium was used as a plating control. (A) Both FOB1 and SIR2 are required for Ty1–mURA3 silencing at NTS1. Silencing was assayed using strains containing a Ty1–mURA3 insertion either outside rDNA or at NTS1 (Smith and Boeke 1997). The approximate location of this reporter (R1) within rDNA is shown in Figure 1A. (B,C) In another reporter gene system, FOB1 and SIR2 are both required for silencing at NTS1 (B, R3 reporter), but only SIR2 is required for silencing at NTS2, near the 35S coding region (C, R4 reporter). See Figure 1A for the locations of R3 and R4 reporter genes. (D) The levels of Net1–TAP and Sir2–TAP proteins do not change in the absence of Fob1 as shown by Western blotting of whole-cell extracts. Actin is shown as a loading control.

Figure 4.

Fob1 is associated primarily with the NTS1 region. (A) Representative graph showing the association of Fob1–TAP across an rDNA repeat. Most of the protein is concentrated within NTS1. Two smaller peaks are observed at the 5S and near the start of the 35S rRNA genes. (B) Examples of ChIP data showing the association of Fob1–TAP with the 25S of rDNA. CUP1 is not significantly enriched in Fob1–TAP immunoprecipitations. (C) ChIP experiments showing the association of Fob1–TAP with the RFB region of NTS1. Labels are as in Figure 1. (F) Fob1–TAP cells. Primer reference numbers below the panels correspond to PCR products in A.

Figure 5.

Fob1 is required for the association of Net1 and Sir2 with NTS1. (A) Graph showing the association of Net1–TAP and Sir2–TAP with rDNA in fob1Δ cells. Net1 and Sir2 associate with the Pol I transcription initiation region and part of the 35S coding region. (B) Examples of ChIP experiments in fob1Δ cells showing reduced binding of both Net1–TAP and Sir2–TAP to the RFB region of NTS1. Panels show examples from both FOB1⁺ (upper set) and fob1Δ cells (lower set). CUP1 primers were used as a negative control. (C) Net1–TAP and Sir2–TAP associate with the NTS2/18S in both FOB1⁺ (upper set) and fob1Δ cells (lower set). Labels are as described in Figure 1, and locations of PCR products are shown below the graph in A.

Figure 6.

The RENT complex physically associates with Fob1. (A) Western blots showing that Net1–HA3 coprecipitates with Sir2 and Fob1–Myc13 (lane 5) from whole-cell extracts. Fob1–Myc13 also coprecipitates Net1–HA3 and Sir2 (lane 7). Actin serves as a loading control. (–) Untagged; (+) tagged. One percent of whole-cell extract (input) and 25% of bound material is shown for all panels. (B) Immunoprecipitation of Sir2 coprecipitates Net1–HA3 and Fob1–Myc13 (lane 4). (Δ) sir2Δ cells; (+) SIR2⁺ cells. (C) Western blots showing that Fob1 and Net1 can physically associate in vivo in the absence of Sir2 (lanes 3,4,7,8). (D, left) A Coomassie-stained gel of purified GST–Control (lane 1) and GST–Fob1 (lane 2) proteins. The control protein (UAP56) is a human protein involved in mRNA splicing. (Right) Western blot indicating that subunits of RENT from whole-cell yeast extracts associate specifically with GST–Fob1 (lane 5) but not with the GST–Control protein (lane 4). Neither GST fusion protein interacts with actin (Act1) or transcriptional repressors that do not participate in rDNA silencing (Sir3, Sir4, and Tup1).

Figure 7.

The RENT complex physically associates with RNA polymerase I. (A) Western blots showing that Net1–TAP coimmunoprecipitates with Sir2 (lanes 6,8) and the largest subunits of RNA polymerase I (Rpa135 and Rpa190, lanes 6,8) from whole-cell extracts. Act1 serves as a loading control. (–) Untagged; (+) tagged; (*) a cross-reacting band. One percent of whole-cell extract (input) and 25% of bound material are shown in A and B. (B) Western blots showing that Rpa135–TAP (lane 5) and Rpa190–TAP (lane 6) coimmunoprecipitate with Sir2.

Figure 8.

Two pathways for the recruitment of the RENT complex to rDNA. Fob1 recruits the RENT complex to the replication fork block region of NTS1. Silent chromatin generated by RENT then inhibits Fob1-dependent recombination. A Fob1-independent pathway, involving Pol I, recruits RENT to the NTS2/18S region. See text for details. Labels are as in Figure 1.