Deletion of the C-terminal 138 amino acids of Net1 significantly impairs growth

We established haploid yeast strains, expressing a series of Net1 truncation mutants C-terminally fused to GFP from the endogenous NET1 locus (Fig 2A, cartoon on the right, S1A Fig, western blot analysis). These yeast strains were subjected to growth analyses on solid medium and in liquid cultures (Fig 2A and 2B; S1 Dataset). A strain expressing full-length Net1 protein fused to GFP grew as well as a NET1 wild-type strain (Fig 2B and S1B Fig, compare NET1 with net^-GFP(1–1189)), indicating that the GFP tag likely did not interfere with important functions of the protein. C-terminal truncation of the Net1 sequence by only 138 amino acids led to a significant slow-growth phenotype (Fig 2A and 2B, compare net1^GFP(1–1051) with net^-GFP(1–1189)). Additional truncation affecting the integrity of the Sir2-binding domain of Net1 increased the doubling time (Fig 2B, net1^GFP(1–693), (1–455), and (1–341)). A strong growth defect, indistinguishable from that of a NET1 deletion strain, was observed when the C-terminal truncation affected the Cdc14-binding domain (Fig 2A and 2B, compare net1^GFP(1–233) with net1Δ). This suggested that the last 138 amino acids of Net1 (hereafter referred to as C-terminal region, CTR) harbor an important growth-supporting function.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. Yeast strains expressing Net1 with a C-terminal deletion are affected in growth but produce a nucleolar protein which still interacts with Cdc14 and Sir2.

(A, B) Diploid yeast strains (W13533, W13534, W13535, W13536, W13537, W13538, W13539, W11979) were sporulated yielding haploid progenies carrying a deletion of NET1 (net1Δ), or alleles for expression of (truncated) Net1^GFP fusion proteins encompassing the amino acids of the full-length Net1 as indicated on the left. All progenies expressed Nop56^mCherry fusion protein as nucleolar marker. A) Serial dilutions of cell suspensions of the haploid progenies of the diploid strains were spotted on XYD plates and incubated at 25°C for 2d before a photograph was taken. Cartoons of the respective Net1^GFP proteins are depicted on the right according to Fig 1B. B) Growth of individual haploid progenies of the diploid strains in YPD media at 30°C was determined using a TECAN plate reader system. Haploid wildtype strains y3290 and y3298 were cultured in parallel (NET1). The mean doubling time and standard deviation error was calculated from two to three independent biological and two independent technical replicates. The number (n) of independent measurements for the respective genotype is indicated on top of the bars. See S1 Dataset for raw data. C) Diploid yeast strains (W13776, W13777, W13778, W13779) were sporulated yielding haploid progenies expressing either Cdc14^myc or Sir2^myc and the indicated Net1^FLAG, Net1ΔCTR^FLAG, or the untagged wild-type Net1 protein. Whole-cell extracts (WCE) of the haploid strains were subjected to immuno-precipitation (IP) with anti-FLAG M2 antibody agarose (αFLAG-IP). Proteins were analyzed in a western blot with anti-Myc and anti-FLAG antibodies (αMyc,αFLAG). A fluorograph of the western blot membrane is shown. The size of molecular weight markers in kDa is indicated on the left. Positions of the tagged proteins are indicated on the right. D-E) Diploid yeast strains (W13533, W13534) were sporulated, yielding haploid progenies expressing Nop56^mCherry and either Net1^GFP, or Net1Δctr^GFP. Diploid parental strains (D, F) and haploid progenies (E, G) were subjected to live cell fluorescence microscopy. Data was collected for differential interference contrast (DIC, first lane of panels), mCherry, and GFP fluorescence (second, and third lane of panels, respectively). mCherry and GFP signals were merged in the fourth lane of panels. A scale bar (5μm) is shown in the first lane of panels.

https://doi.org/10.1371/journal.pgen.1008006.g002

A CTR deletion mutant still associates with Cdc14 and Sir2, and localizes to the nucleolus

A mutant Net1 protein lacking the CTR (hereafter referred to as Net1ΔCTR) still contains the regions important for Net1 binding to Cdc14 and Sir2 in the context of the RENT complex (Fig 1B) [29–31]. In good agreement, both FLAG-tagged full-length Net1 (Net1^FLAG) and Net1ΔCTR (Net1ΔCTR^FLAG) fusion proteins co-precipitated Myc-epitope-tagged Cdc14 and Sir2 (Cdc14^myc, Sir2^myc) with similar efficiencies from whole-cell extracts (Fig 2C, compare lanes 4 with 5, and 10 with 11; S1C Fig, full membrane). As expected, FLAG antibody-mediated precipitation of Myc-tagged Cdc14, or Sir2, was not observed when whole-cell extracts were prepared from strains that did not express FLAG fusion protein (Fig 2C, lanes 6 and 12). We then examined if the CTR was required for the reported nucleolar localization of Net1 [26–28]. Live cell imaging of haploid and diploid yeast strains co-expressing Net1^GFP fusion proteins and the nucleolar marker protein Nop56 fused to mCherry (Nop56^mCherry) confirmed co-localization of both proteins in the crescent shaped yeast nucleolus (Fig 2D and 2E). Net1ΔCTR^GFP showed clear nucleolar localization in a diploid yeast strain expressing a NET1 wild-type allele (Fig 2F). This strain was not compromised in growth (not shown), indicating that the net1Δctr^GFP allele had no dominant negative effect. A haploid strain expressing Net1ΔCTR^GFP was impaired in growth (Fig 2A and 2B), and showed abnormal cell morphology (Fig 2G, DIC), and de-localization of Nop56^mCherry over the whole nucleus (Fig 2G, mCherry). However, a subpopulation of Nop56^mCherry preferentially co-localized with Net1ΔCTR^GFP within a subnuclear compartment, likely the remainder of the nucleolus (Fig 2G, GFP and merge).

Expression of the CTR in trans rescues growth defects in net1Δctr and net1Δ strains

To analyze if the CTR is an independent functional domain, we tested whether expression of the CTR in trans could rescue the phenotypes observed in a net1Δctr strain. A cassette for constitutive overexpression of the CTR N-terminally fused to GFP (^GFPCTR) under the control of the TEF2 promoter was stably integrated in the LEU2 locus. Co-expression of ^GFPCTR in trans fully restored wild-type growth in net1Δctr and net1(1–455) strains, and largely suppressed the growth defect of a net1Δ strain (Fig 3A and 3B; S1 Dataset). This indicated that the CTR and the first 455 amino acids of Net1, containing the Cdc14 binding region (but lacking the Sir2 interacting domain) are sufficient to promote normal cell growth. Live cell imaging showed a preferential nucleolar localization of ^GFPCTR in all strains, although the overexpressed fusion protein also spread all over the cell (Fig 3C and 3D; S2B and S2C Fig, compare panels mCherry and GFP). In net1(1–455) and net1Δctr strains, overexpression of ^GFPCTR restored normal cell morphology as well as nucleolar localization of Nop56^mCherry (Figs 2G and 3D; S2A and S2B Fig compare panels DIC, and mCherry, respectively). However, cellular morphology was still significantly altered in a net1Δ strain overexpressing ^GFPCTR, in good correlation with the residual growth defect (S2C Fig, DIC).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 3. Expression of the CTR in trans rescues growth defects and Nop56 delocalization in net1Δctr strains.

(A, B) Diploid yeast strains (W12509, W12533, W13762) were sporulated. Haploid progenies carried a deletion of NET1 (net1Δ), or alleles for expression of the indicated truncated Net1 proteins. Where indicated (+) strains co-expressed chromosomally encoded ^GFPCTR. Growth analyses on plates and in liquid culture were performed with individual haploid progenies of the diploid strains as described in the legend to Fig 2A and 2B. Yeast strains carrying a NET1 wild-type allele (WT, K699 (A), and y3290 and y3298 (B)) were included as controls. C, D) The diploid yeast strain W15407 was sporulated, yielding haploid progenies carrying either a NET1 wild-type allele (C) or a net1Δctr allele (D), and expressing Nop56^mCherry and ^GFPCTR. These haploid strains were subjected to live cell fluorescence microscopy as described in the legend to Fig 2D.

https://doi.org/10.1371/journal.pgen.1008006.g003

As observed for expression of ^GFPCTR, expressing the CTR in fusion with a N-terminal FLAG-tag (^FLAGCTR) from a chromosomally integrated cassette suppressed the growth defect of net1Δctr strains (S2D Fig; S1 Dataset). Overexpression of the CTR did not affect the expression levels of Net1ΔCTR (S2E Fig).

Deletion of the CTR impairs Net1 association with the 35S rDNA promoter

Net1 and other RENT components associate with two rDNA regions, the 35S rDNA promoter region and the RFB at the 3’-end of the 35S rRNA gene [37]. To investigate if deletion of the CTR influenced this binding pattern, we performed Chromatin Endogenous Cleavage (ChEC) [43] and Chromatin Immuno-Precipitation (ChIP) analyses. Haploid yeast strains expressing either wild-type Net1 or Net1ΔCTR in fusion with micrococcal nuclease (MN) and a hemagglutinin (HA) epitope (Net1-MN^HA, ΔCTR-MN^HA) were created. For ChEC analyses, crude nuclei isolated from exponentially growing formaldehyde-crosslinked cells were treated with calcium to activate the MN. Cleavage events of the MN^HA-fusion proteins were mapped to the indicated genomic regions by Southern blot analysis using the indirect end-labeling technique [44]. Net1-MN^HA fusion protein mediated cleavages were observed both at the 35S rDNA promoter and at the RFB in good correlation with earlier results (Fig 4A, lanes 1–4 and 9–12) [41]. Net1ΔCTR-MN^HA-mediated cuts were strongly reduced at the 35S rDNA promoter region, whereas robust cleavages were still observed at the RFB (Fig 4A, lanes 5–8 and 13–16). In good agreement, Net1-MN^HA but not Net1ΔCTR-MN^HA co-precipitated 35S rDNA promoter fragments from cellular extracts in ChIP experiments (Fig 4B, compare ChIP of fragment 2, cartoon at the bottom for location of ChIP fragments in yeast rDNA). In contrast, co-precipitation of RFB fragments with Net1ΔCTR-MN^HA was unaltered or even slightly enhanced, when compared to ChIP with Net1-MN^HA (Fig 4B, compare ChIP of fragments 5, 6). As expected, no significant co-precipitation of rDNA fragments bearing the 5S rDNA, 18S rDNA, and 25S rDNA regions with the fusion proteins was observed (Fig 4B, ChIP of fragments 1, 3, 4). Thus, it can be concluded that the CTR is required for interaction of Net1 with the 35S rDNA promoter but does not affect Net1 association with the RFB, which is mediated by Fob1 [37]. In good correlation, Fob1 association with the RFB was unaltered or even slightly increased in net1Δctr strains when compared with NET1 strains (S3A and S3B Fig, compare lanes 9–12 with 13–15, compare ChIP of fragments 5, 6).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. 35S rDNA promoter association of RENT complex components is impaired in net1Δctr strains.

Haploid yeast strains expressing the indicated proteins C-terminally fused to MN^HA were subjected to ChEC, or ChIP experiments. A, C) Crude nuclei suspensions were subjected to ChEC in the presence of calcium for the indicated times (min ChEC), DNA was isolated, cut with XcmI, separated in a 1% agarose gel and subjected to Southern blot analysis by indirect end-labeling using the radiolabeled probes rDNp, IGS2. An autoradiograph of the membrane is shown. Cartoons at the sites depict a map of the detected genomic fragments including probe hybridization sites and important cis elements described in the legend to Fig 1A. Arrows at the top of the cartoons point to the uncut full-length XcmI fragments. An asterisk marks the site of a DNA double-strand break occurring at the 3’ end of the RFB in the absence of ChEC. B, D) fragmented chromatin was subjected to ChIP with the indicated MN^HA tagged fusion proteins using anti-HA (3F10) antibody. DNA co-precipitating with the tagged proteins and from input fractions was isolated and analyzed by quantitative PCR with different primer pairs amplifying distinct regions (1–6) within the rDNA locus indicated in the cartoon at the bottom. Bar graphs depict the efficiency of co-precipitation of the fragments as percentages of the input DNA (% ChIP). Mean values and standard deviation errors were derived from three independent ChIP experiments analyzed each in triplicate qPCR reactions (n = 9). A, B) ChEC and ChIP analyses with strains (y3041, y3042, y3043, y3044) expressing either Net1-MN^HA or Net1ΔCTR-MN^HA (ΔCTR-MN^HA). C, D) ChEC and ChIP analyses with strains (y3058, y3068, y3250), carrying a NET1 or a net1Δctr allele, and expressing Cdc14-MN^HA, in the absence or presence of a chromosomally integrated ^FLAGCTR expression cassette.

https://doi.org/10.1371/journal.pgen.1008006.g004

35S rDNA promoter association of RENT complex components is impaired in the net1Δctr strain

Cdc14 and Sir2 have rDNA association patterns very similar to that of Net1 [37,41,45]. Accordingly, Cdc14-MN^HA mediated ChEC in a NET1 wildtype strain led to cuts within the 35S rDNA promoter region and at the RFB, in good correlation with enrichment of these regions in ChIP experiments (Fig 4C, lanes 1–4, and 13–16; Fig 4D, ChIP of fragments 2, 5, 6). When expressed in a net1Δctr strain, Cdc14-MN^HA-mediated cleavages at the 35S rDNA promoter after ChEC were strongly reduced and the co-precipitation of this region in ChIP experiments was abolished (Fig 4C, lanes 5–8; Fig 4D, ChIP of fragment 2). In contrast, ChEC and ChIP indicated that the interaction of Cdc14 with the RFB was very similar to that observed in the NET1 strain (Fig 4C, lanes 17–20; Fig 4D, ChIP of fragments 5, 6). As for Cdc14-MN^HA, Sir2-MN^HA-mediated cleavages within the 35S rDNA promoter region in a net1Δctr strain were reduced when compared to cleavage events observed in a NET1 strain (S3C Fig, upper panel, compare lanes 1–4 with 5–8). As opposed to Cdc14-MN^HA, however, Sir2-MN^HA mediated cuts at the RFB in a net1Δctr strain were reduced, when compared with cleavages observed in a NET1 strain (S3C Fig, lower panel, compare lanes1-4 with 5–8).

Expression of the CTR in trans does not rescue 35S rDNA promoter interaction of RENT complex components in the net1Δctr strain

The impaired association of RENT complex components with the 35S rDNA promoter could be causative for the growth defect observed in the net1Δctr strain. Ectopic expression of ^FLAGCTR in net1Δctr strains restored wild-type growth (Fig 3A and 3B). Thus, it was tested if the interaction of Cdc14 with the 35S rDNA promoter could be re-established in this condition. ChEC and ChIP experiments were carried out in a net1Δctr strain expressing Cdc14-MN^HA from the endogenous locus and the ^FLAGCTR from a chromosomally integrated cassette. Expression of ^FLAGCTR resulted neither in significant Cdc14-MN^HA-mediated cleavage at the 35S rDNA promoter in ChEC experiments, nor did it lead to co-precipitation of 35S rDNA promoter fragments in ChIP (Fig 4C, lanes 9–12; Fig 4D, ChIP of fragment 2). Co-expression of ^FLAGCTR did not affect Cdc14-MN^HA association with the RFB (Fig 4C, lanes 21–24; Fig 4D, ChIP of fragments 5, 6). In agreement with the assumption that Cdc14 is recruited to the rDNA via its interaction with the N-terminus of Net1, very similar results were obtained in ChEC experiments with a ^FLAGCTR strain expressing Net1ΔCTR-MN^HA (S3D Fig, lanes 5–8).

Pol I association with 35S rRNA genes requires the CTR

The above experiments suggested, that the CTR plays a RENT complex-independent role supporting cellular growth, presumably by interacting with the 35S rDNA promoter. We therefore tested if the CTR harbors the Pol I transcription stimulating function of Net1. Haploid yeast strains carrying a NET1 or a net1Δctr allele and co-expressing MN^HA fusion proteins of the Pol I subunits Rpa43 or Rpa190 were subjected to ChEC and ChIP experiments. ChEC analyses revealed that Rpa43-MN^HA and Rpa190-MN^HA cleavage events at the 35S rDNA promoter region and at the 35S rDNA transcription termination site upstream of the RFB were reduced in net1Δctr strains, when compared to cleavages in NET1 strains (Fig 5A compare lanes 1–4 with lanes 5–8, and lanes 17–20 with lanes 21–24). This pointed to a lower occupancy of Pol I molecules within the 35S rRNA gene region. ChIP experiments strongly supported this finding, since co-precipitation of 35S rDNA fragments with the tagged Pol I subunits was substantially impaired in net1Δctr strains when compared with ChIP in NET1 strains (Fig 5B, ChIP of fragments 2–4). Importantly, ChEC and ChIP experiments indicated that expression of ^FLAGCTR in trans could fully restore Pol I association with the 35S rDNA in net1Δctr cells to wild-type levels, in good correlation with the re-establishment of normal cell growth in these strains (Fig 5A, compare lanes 9–12, and 25–28 with lanes 13–16, and 29–32, respectively; Fig 5B, ChIP of fragments 2–4; S3 Table, doubling times).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 5.

Robust Pol I association with 35S rRNA genes requires the CTR. (A, B) Haploid yeast strains (y3288, y3289, y3299, y3302, y3595, y3597, y3599, y3601) carrying a NET1 or a net1Δctr allele, and expressing Rpa43-MN^HA or Rpa190-MN^HA in the absence or presence of the Net1 ^FLAGCTR were subjected to ChEC (A) and ChIP (B) analyses as described in the legend to Fig 4.

https://doi.org/10.1371/journal.pgen.1008006.g005

The CTR interacts with many chromosomal loci

Since the CTR is required to recruit full-length Net1 to the 35S rDNA promoter (Fig 4A), we further investigated if the isolated CTR expressed in trans may interact with this chromosomal region. ChEC experiments were performed in NET1 or net1Δctr strains, expressing the CTR with an N-terminal FLAG tag and C-terminally fused to MN^HA (^FLAGCTR-MN^HA), under the control of the TEF2 promoter from a cassette integrated in the LEU2 locus. As observed for ^GFPCTR and ^FLAGCTR, expression of the ^FLAGCTR-MN^HA fusion protein suppressed the slow-growth phenotype of net1Δctr strains (S3 Table). Similar ^FLAGCTR-MN^HA-mediated cleavage events within the 35S rDNA promoter region were observed in both NET1 and net1Δctr strains (Fig 6A, upper panels, lanes 1–4, and 5–8). ^FLAGCTR-MN^HA-mediated cuts were also observed at the RFB and at further sites within IGS1 (Fig 6A, lower panels, lanes 1–4, and 5–8). Additionally, ^FLAGCTR-MN^HA-dependent cleavages were observed at accessible sites within intergenic sequences at other genomic loci, which were not cut by Net1-MN^HA (Fig 6B, lower panels, lanes 1–4, and 5–12). In accordance with the observed nuclear localization of ^GFPCTR (Fig 3C and 3D; S2B and S2C Fig), ChEC in the ^FLAGCTR-MN^HA-expressing strains eventually resulted in the complete degradation of genomic DNA, which did not occur in a Net1-MN^HA-expressing strain (Fig 6B, upper panels compare lanes 1–4 with lanes 5–12). Thus, upon overexpression of the CTR, the protein is available for DNA interactions within the entire nucleus, including the 35S rDNA promoter.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 6.

The CTR of Net1 interacts with many chromosomal loci and rescues slow growth in net1Δctr strains when fused to Rpa190. (A, B) Haploid yeast strains (y3087; y3157; y3160) carrying a NET1 or a net1Δctr allele and expressing CTR-MN^HA or Net1-MN^HA were subjected to ChEC analyses, as described in the legend to Fig 4. B) Agarose gels were stained with SYBR safe before transfer to the blotting membrane (upper panels), sizes of selected DNA fragments in the DNA marker (M) are given on the left. Lower panels, the membrane was hybridized with a radioactively labeled probe hybridizing with a XcmI fragment containing the NUP57/RPS23A loci as indicated in the cartoon on the left. C, D) Haploid yeast strains (y3673; y3674; y3746; y3747; y3748; y3749; y3756; y3757; y3758; y3759, y3290, y3298) carrying a NET1 or a net1Δctr allele and expressing CTR fusion proteins of Rpa190, or Rrn7 or the wild-type proteins, were subjected to growth analyses in liquid cultures (C), and on solid medium (D) as described in the legend to Fig 2A and 2B.

https://doi.org/10.1371/journal.pgen.1008006.g006

Expression of a Rpa190-CTR fusion protein suppresses the growth defect of a net1Δctr strain

The above data are compatible with a model in which the CTR assists Pol I recruitment at the 35S rDNA promoter. To further test this hypothesis experimentally, we fused the ^FLAGCTR to the C-terminus of Rpa190 and the CF component Rrn7 in net1Δctr strains. Whereas expression of Rrn7-^FLAGCTR could not significantly rescue the growth phenotype of net1Δctr strains, expression of Rpa190-^FLAGCTR fully suppressed slow growth (Fig 6C and 6D; S1 Dataset). The expression of CTR fusion proteins had no impact on growth in NET1 strains. This observation is in accordance with a Pol I-specific function of the CTR, but also indicates that simple tethering of the CTR to the 35S rDNA promoter may not be sufficient to obtain full stimulation of Pol I transcription.

Pol I associates with phosphorylated CTR

Previous work showed co-purification of Net1 with Pol I subunits [42,46]. Here, it was tested if the CTR of Net1 could associate with Pol I. Immuno-precipitation experiments using whole-cell extracts from a strain expressing Rpa43^myc and ^FLAGCTR with an anti-Myc antibody showed enrichment of the Myc-tagged bait protein and co-precipitation of a minor sub-population of ^FLAGCTR in western blot analysis (Fig 7A, lanes 1 and 3). As expected, ^FLAGCTR was not precipitated from whole-cell extracts of strains expressing the untagged Rpa43 protein (Fig 7A, lanes 2 and 4). ^FLAGCTR in whole-cell extracts migrated as a diffuse band around 30 kDa in SDS-PAGE, despite an estimated molecular weight of 18 kDa. Net1 is known to be phosphorylated and several phosphorylation sites have been identified within the CTR in proteomic studies (Fig 8A, asterisks mark described phosphorylation sites) [32,47–50]. To investigate if the aberrant migration behavior of the CTR could be explained by phosphorylation, ^FLAGCTR was enriched from whole-cell extracts using an anti-FLAG antibody matrix. The immobilized protein was incubated in the absence or presence of different concentrations of lambda protein phosphatase (λ PP). Western blot analysis revealed that the incubation with λ PP substantially increased the electrophoretic mobility of ^FLAGCTR (Fig 7B, lanes 3–5; S4B Fig, full membrane) suggesting that the CTR is phosphorylated in vivo.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 7.

The CTR is differentially phosphorylated in different growth states in vivo and CTR-dependent stimulation of Pol I transcription in vitro is decreased upon λ-protein phosphatase treatment. (A) The CTR associates with Pol I. Diploid yeast strains (W12903, W13491) were sporulated yielding haploid progenies expressing ^FLAGCTR and the wild-type Pol I subunit Rpa43, or Rpa43^myc. WCEs were prepared and subjected to IP experiments with anti-Myc antibody agarose followed by western blot analysis as described in the legend to Fig 2C. IgG on the right marks the positions of antibody chains in IPs. B) The CTR is hyperphosphorylated in vivo. ^FLAGCTR in WCE of the diploid yeast strain W13490 was bound to anti-FLAG M2 antibody agarose and treated with increasing amounts (grey triangle) of λ-protein phosphatase (λ-PP). Protein samples were analyzed by western blot as described in the legend to Fig 2C. C) The CTR is differentially phosphorylated in vivo. Haploid yeast strain y3739 expressing ^FLAGCTR was grown to exponential (exp), or to stationary phase (stat). WCE was prepared and subjected to western blot analysis with anti-FLAG antibody (αFLAG) as described in the legend to Fig 2C. Ponceau S stain of the membrane, and an image visualizing chemiluminescence signals upon immuno-detection (αFLAG) are shown. D) Recombinant CTR expressed in insect cells is hyperphosphorylated. Net1 CTR^TAP expressed in insect cells was bound to IgG-magnetic beads. Net1 CTR^TAP bound to the beads was either mock treated (-) or incubated with λ-PP (+). CTR^CBP fusion proteins were released by TEV protease cleavage and analyzed by SDS-PAGE and Coomassie staining. A photograph of the gel is shown. The size of molecular weight markers in kDa is indicated on the left. Positions of the tagged proteins as well as TEV protease are indicated on the right. E) CTR-dependent stimulation of Pol I transcription in vitro is decreased after λ-PP treatment. In vitro transcription reactions were carried out using Pol I purified from yeast, recombinant CF and Rrn3 purified from E. coli in the absence (-) or presence of increasing amounts (grey triangle) of CTR^CBP, either mock treated or incubated with λ-PP as described in (D). Radiolabeled RNA was isolated and separated by 6% acrylamide-urea gel electrophoresis. An autoradiograph is shown. The position of the specific transcript is indicated on the right.

https://doi.org/10.1371/journal.pgen.1008006.g007

Changes in post-translational covalent modifications of the CTR correlate with alterations in Pol I transcription of 35S rRNA genes in vivo

To investigate if the post-translational modification state of the CTR was subject to changes in vivo, whole-cell extracts were prepared from exponentially growing and stationary cells expressing ^FLAGCTR. Proteins were separated by SDS-PAGE and analyzed in a western blot. ^FLAGCTR in extracts from stationary cells had a higher mobility in SDS-PAGE than ^FLAGCTR in extracts from exponentially growing cells, suggesting alterations in the modification state (Fig 7C, αFLAG, compare “exp” with “stat”; S4C Fig, full membrane). Ponceau-red staining of the immunoblot membrane indicated that the overall mobility of cellular proteins was not altered in the different extracts (Fig 7C, Ponceau; S4C Fig). Ribosome biogenesis and Pol I transcription are downregulated when yeast cells grow to stationary phase [51,52]. Downregulation of Pol I transcription correlates with a decreased psoralen accessibility of the 35S rRNA gene region transcribed by Pol I [53–55]. As a control for downregulation of Pol I transcription in the above experiment, we subjected cells from the same cultures used for protein extraction to psoralen crosslinking analysis. In agreement with the expected alterations in Pol I transcription, psoralen accessibility of 35S rRNA genes was high in exponentially growing cells and negligible in stationary phase cells (S4D Fig, compare lanes 4 and 5).

λ protein phosphatase treatment decreases CTR-dependent stimulation of promoter-dependent Pol I transcription in vitro

Recombinant full-length Net1 has been shown to stimulate promoter-dependent Pol I transcription in vitro [42]. We tested if the CTR could be sufficient to promote Pol I stimulation in a reconstituted system. The CTR was expressed in insect cells with a C-terminal tandem affinity purification (TAP) tag (CTR^TAP) bound to IgG-sepharose and released as a calmodulin binding peptide (CBP) fusion protein (CTR^CBP) by cleavage with tobacco etch virus (TEV) protease (S4E Fig) [56]. As observed for the CTR expressed in exponentially growing yeast (Fig 7A–7C), purified recombinant CTR^CBP with a predicted size of 20 kDa migrated with an apparent molecular weight of 32 kDa in SDS-PAGE (Fig 7D; S4F Fig, lane1; S4E Fig, lane 3). λ PP treatment prior to TEV-mediated release from IgG-Sepharose significantly increased the mobility of the recombinant protein in SDS-PAGE when compared with the mobility of the mock-treated CTR^CBP, indicating that the protein was phosphorylated (Fig 7D compare lane 1 with lane 2; S4F Fig, full gel). λ PP-treated and mock-treated CTR^CBP were tested in promoter-dependent Pol I transcription in a minimal in vitro system consisting of Pol I purified from yeast and of recombinant Rrn3 and CF purified from E. coli [57]. The mock treated CTR^CBP could stimulate promoter-dependent Pol I transcription in a concentration-dependent manner up to 6-fold, whereas the λ PP treated CTR^CBP still led to a 2 to 3-fold stimulation (Fig 7E, compare lanes 1 and 2, with lanes 3–6 and lanes 7–10). Additionally, recombinant full-length Net1^TAP and Net1ΔCTR^TAPfusion proteins were purified from insect cells (S4G Fig) and tested in parallel with another preparation of CTR^CBP protein with and without λ PP treatment. The recombinant Net1^CBP was the strongest activator of Pol I transcription, whereas Net1ΔCTR^CBP still stimulated Pol I transcription to a similar extent as the purified CTR^CBP (S4H Fig). Thus, it is possible that Net1ΔCTR still partially supported Pol I transcription in vivo. In agreement with such hypothesis, residual Net1ΔCTR-MN^HA-mediated cleavage at the 35S rDNA promoter could still be observed upon ChEC (Fig 4A, lanes 5–8; S3D Fig, upper panel, lanes 5–8). In contrast, ChEC analyses performed with cells from a Net1(1–341)-MN^HA expressing strain did not lead to significant cuts within the 35S rDNA promoter (S3D Fig, upper panel, lanes 9–12). Net1(1–341)-MN^HA mediated cleavage at the RFB region, instead, was comparable–although not identical—to cleavage mediated by Net1-MN^HA or by Net1ΔCTR-MN^HA (S3D Fig, lower panels).

The acidic region of human UBF1 can in part functionally substitute for the CTR in the context of the Net1 protein

Whereas the full-length Net1 is not conserved in higher eukaryotes, local sequence alignment using the tool “matcher” of the EMBOSS platform revealed 25% identical and 43.5% similar residues within the acidic tail region (AR) of human UBF1 Pol I transcription factor (Fig 8A). An acidic serine/aspartate-rich sequence within the CTR showed the highest similarity with the UBF1 AR (Fig 8A, underlined amino acids). Assuming, that the CTR is hyperphosphorylated within this sequence, the similarity between the two regions might be even greater. To test if the UBF1 AR could functionally substitute for the Net1 CTR, yeast strains were created expressing different derivatives of Net1 in fusion with MN^HA (Fig 8B). Full-length Net1, Net1ΔCTR, or Net1 variants in which the CTR was replaced by the AR of human UBF1, an N-terminally extended AR (eAR), or a triplicated synthetic minimal VP16 activation domain (VP), respectively were analyzed. The VP16 activation domain triggers high-level Pol II-dependent transcription when recruited to gene promoters by a specific DNA binding domain in higher eukaryotes [58]. The expression of the respective MN^HA-fusion proteins in the above strains was verified by western blot analysis (Fig 8C; S5A Fig, full membrane). Six independently obtained clones of each of these strains were subjected to growth analyses together with clones of a strain expressing an untagged Net1 protein as a reference control (Fig 8D; S1 Dataset). Whereas all the strains expressing Net1 variants lacking the CTR grew slower than the Net1-MN^HA or Net1-expressing strains, Net1ΔCTR-AR-MN^HA and Net1ΔCTR-eAR-MN^HA-expressing strains grew slightly but statistically significantly faster than Net1ΔCTR-MN^HA-expressing strains. This indicated that the Net1-CTR and the UBF1-AR regions share a conserved function in promoting growth. In contrast, expression of Net1ΔCTR-VP-MN^HA did not significantly rescue the net1Δctr growth phenotype in those experiments (Fig 8D).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 8. The acidic region of human UBF1 shares conserved features with the CTR and can partially rescue the growth defect of net1Δctr strains.

(A) Local pairwise sequence alignment of human UBF1 (hUBF1) with Net1. Top, schematic representation of hUBF1 protein, depicting amino acid positions of full-length hUBF1, HMG boxes (grey rectangles), acidic tail region (AR, hexagon in light grey), and extended acidic tail region (eAR, black bar on the top). Bottom, sequence alignment of hUBF1 with Net1 using the “matcher” program EMBL-EBI Job Dispatcher framework (https://www.ebi.ac.uk/Tools/psa/emboss_matcher/). Identical (|), and similar (:) amino acid residues are indicated. Amino acid numbering of the full-length hUBF1 and yeast Net1 are indicated on the left, asterisks mark serine residues reported to be targets of phosphorylation (https://www.phosphosite.org/; https://www.yeastgenome.org), predicted CK2 phosphorylation sites are printed in red (http://www.cbs.dtu.dk/services/NetPhos/). A serine/aspartate rich amino acid stretch within the CTR is underlined. B-D) Diploid yeast strains were sporulated yielding haploid progenies expressing either wild-type Net1 or different Net1-MN^HA fusion proteins in which the CTR was either conserved (Net1-MN^HA) deleted (Net1ΔCTR-MN^HA), or replaced by the AR (Net1ΔCTR-AR-MN^HA), or eAR regions (Net1ΔCTR-eAR-MN^HA), or a synthetic, minimal transactivation domain of the viral VP16 protein (Net1ΔCTR-VP-MN^HA). B) Schematic representation of Net1-MN^HA fusion proteins expressed in the respective yeast strains. Net1 domains are depicted as described in the legend to Fig 1B, UBF1 domains are depicted as described in (A). The minimal transactivation domain of VP16 is depicted as a circle. C) WCE of overnight cultures of haploid progenies (y4065, y4067, y4069, y4071, y4073), expressing the indicated Net1-MN^HA fusion proteins were subjected to western blot analysis as described in the legend to Fig 2C, using anti-HA antibody (αHA, top panel), or a tubulin antibody (αtubulin, bottom panel). Images visualizing chemiluminescence on the membranes are shown. D) Growth analyses in liquid culture were performed with the indicated haploid derivatives of yeast strains (y4038, y4039, y4040, y4041, y4042, y4043, y4044, y4045, y4046) as described in the legend to Fig 2B. Differences in doubling times of Net1ΔCTR-AR-MN^HA and Net1ΔCTR-eAR-MN^HA strain relative to the Net1ΔCTR-MN^HA expressing strain were tested using the nonparametric Mann-Whitney U test; ***P<0.001, ****P<0.0001.

https://doi.org/10.1371/journal.pgen.1008006.g008

Oligonucleotides, plasmids and yeast strains

Unless noted otherwise, standard techniques were used for cloning of plasmids, and transformation of yeast cells [87–89]. Information about oligonucleotides, plasmids, and yeast strains used in this study can be found in S1–S3 Tables, including details on plasmid and strain construction. Plasmid sequences are available upon request. For the individual experiments in this study, yeast cells were grown at the indicated temperatures in either YPD (2% w/v) peptone, 1% (w/v) yeast extract, 2% glucose), YPAD (YPD including 100mg/l adenine), or XYD medium (YPD including 100mg/l adenine, 200mg/l tryptophan, and 10mM KH₂PO₄).

Growth analyses

Live cell fluorescence microscopy

Exponentially growing cells were harvested by centrifugation, transferred to glass slides and covered with an agarose slice (supplied with yeast nitrogen base, amino acids, and 2% glucose). Cells were observed at 20–22°C on an Axio Observer Z.1 confocal spinning disk (CSU-X1) microscope (Yokogawa; Carl Zeiss) using a Plan Apochromat 63×/1.40 oil differential interference contrast (DIC) M27 lens. Ten Z-stack images with an optical section spacing of 0.5 μm were acquired with a shutter speed of 200 ms using an AxioCam MRm camera (Carl Zeiss). Microscopy data was processed using ImageJ (National Institutes of Health, Bethesda, MD). Z-stacks of the region of interest (fluorescence channels) were projected using maximum-intensity projection. A single plane (DIC channel) was duplicated to visualize cell morphology.

Chromatin Endogenous Cleavage (ChEC), and Chromatin Immuno-Precipitation (ChIP)

For ChEC and ChIP experiments yeast strains expressing different genes as fusion proteins with a C-terminal MN followed by a triple HA-tag from their endogenous genomic location were grown in YP(A)D at 30°C to a final OD₆₀₀ of 0.5. ChEC analyses were performed as previously described [15]. All DNA samples were digested with XcmI prior to agarose gel electrophoresis and Southern blot analysis (see below). ChIP experiments were performed as described and analyzed by quantitative PCR [41]. Data was collected with a Rotor-Gene Q system (Qiagen). The cycle threshold values and amplification efficiency (E) for a defined primer pair for all input and ChIP samples of an experimental dataset were determined with the Rotor-Gene 6000 software (Qiagen) using the comparative quantification method. The cycle threshold (CT) value correlates with the Rotor-Gene software “take-off” value (tov). For each primer pair the efficiency (E) is calculated as an average from the fluorescence measurements for each individual sample. The percentage of co-precipitated DNA was determined by the formula %ChIP = ((1+E)^tov(ChIP)/(1+E)^tov(input))*100. Average and standard deviation errors for ChIP are derived from three independent ChIP experiments, each analyzed in triplicate qPCRs. The original qPCR data, including CT, “take off” and efficiency values, melting curve analysis, and mathematical operations are added as S2 Dataset.

Gel electrophoresis and blot analysis

SDS–PAGE and Western blot analysis was performed according to standard procedures [90,91]. Gels were either stained with Coomassie blue for detection of proteins or transferred to nitro cellulose (GE Healthcare) or PVDF membranes (Immobilon P, Roth) for subsequent immuno-detection. Transfer to membranes was verified by staining with Ponceau S. S4 Table contains a complete list of antibodies used for detection. Secondary antibodies coupled to IRDye 800 were detected with an LI-COR Odyssey Infrared Imaging System (LI-COR Bioscience). Secondary antibodies coupled to horse reddish peroxidase (HRPO) were visualized using BM Chemiluminescence Western Blotting Substrate (POD, Roche), and a LAS-3000 Chemiluminescence Imager.

Agarose gel electrophoresis and Southern blot analysis were performed as described [88], with the exception that transfer of nucleic acids onto nylon membranes (Positive Membrane, Qbiogen) by capillary transfer was performed with 1 M ammonium acetate [92]. S5 Table contains a list of templates used for the synthesis of radiolabeled hybridization probes generated using the RadPrime DNA labeling system (GE Healthcare).

Protein extraction, immuno-precipitation (IP) and λ-phosphatase treatment

For preparation of native protein extracts yeast cells were grown overnight in XYD media at 25°C to an OD₆₀₀ of 0.8. For preparation of yeast whole-cell extracts (WCE), cell equivalents corresponding to 10–20 OD₆₀₀ were harvested by centrifugation (805 rcf, 2 min) and washed once with ice-cold water. Lysis buffer (150 mM NaCl (for IPs with Net1^FLAG-fragments) or 350 mM KCl (IPs with Rpa43^myc), 50 mM Tris-HCl pH 7.5, 50 mM NaF, 5 mM EDTA, 0.1% IGEPAL CA-630, 60 mM β-glycerol phosphate) was added to yield a final volume of 150 μl cell suspension. After addition of 200μl of glass beads cells were lysed by shaking the mixture for 5 min at 4°C in a mixer mill (Retsch). After removal of cell debris by two consecutive centrifugation steps of 10 min and 15 min (16000 rcf, 4°C), equal volumes of supernatant and 2× SDS-sample buffer were mixed and incubated for 10 min at 100°C.

For immuno-precipitation of Myc-epitope tagged RNA polymerase I subunits, WCEs (adjusted to a total volume of 450μl) were incubated with anti-Myc antibodies (clone 9E10) for 2h at 4°C. Thereafter, 50 μl of protein A–agarose slurry (Santa Cruz Biotechnology Inc.) were added, and the mixtures were incubated on a rotator for 2 h at 4°C. For immuno-precipitation of FLAG-tagged Net1-fragments, WCEs (adjusted to a total volume of 450 μl) were incubated with 45μl of α-FLAG-beads (Sigma-Aldrich) on a rotator for 3 h at 4°C. Beads were collected by centrifugation and washed three times with 500 μl lysis buffer. After the last washing step, beads were resuspended in 1x SDS sample buffer and incubated for 10 min at 100°C.

After immuno-precipitation of FLAG-tagged CTR as described above, beads were collected by centrifugation and washed twice with 1ml of lysis buffer (without β-glycerol phosphate) and once with λ-phosphatase buffer (50 mM HEPES-KOH pH 7.5, 5 mM DTT, 2 mM MnCl₂). Beads were split in different samples and treated in the absence (control), or presence of 200, 400, and 2000 units of λ-phosphatase (New England Biolabs) in a total volume of 50μl λ-phosphatase buffer, and samples were incubated for 1 h at 37°C. Beads were collected by centrifugation and washed twice with 1 ml λ-phosphatase buffer. After the last washing step, beads were resuspended in 1x SDS-sample buffer and incubated for 10 min at 100°C.

All samples were analyzed by western blot as described above. The data presented in Figs 2C, 7A and 7B are representative for at least three independent experiments.

Yeast cell growth to stationary phase with subsequent protein and trimethyl psoralen crosslinking analysis

Single colonies of yeast strains were used to inoculate 3 ml of YPAD which were incubated for 2 days at 30°C under shaking. This culture was used to inoculate YPAD media to an OD₆₀₀ not higher than 0.1. Samples for denaturing protein extraction or trimethyl psoralen crosslinking analyses were withdrawn when the culture reached an OD600 of 0.5 (exponential phase) or 144 hours after this time point (stationary phase). For protein analysis cell equivalents of 0.5–3 OD₆₀₀, were subjected to protein extraction as described elsewhere [93]. For trimethyl-psoralen crosslinking analysis, cell equivalents of 25 OD₆₀₀ were treated as previously described [15]. All DNA samples were digested with EcoRI prior to agarose gel electrophoresis and Southern blot analysis (see above).

Purification of recombinant proteins and λ-phosphatase treatment

Purification of 6x histidine-tagged Rrn3, or 6x histidine-tagged core factor from E. coli, as well as TAP-tagged RNA Pol I from yeast have been described previously [57]. Recombinant expression of different Net1-TAP fusion proteins in baculovirus infected S. frugipedia SF21 cells was achieved according to a protocol described in [94–96]. For TAP-tag mediated purification, 50x10⁶ cells from a large scale infection were suspended in 30 ml TAP-lysis buffer (50 mM HEPES-KOH pH 7.5, 2.5 mM Mg-acetate, 0.001% (w/v) Tween, 0.2 M KCl, 5 mM 2-mercaptoethanol, 1 mM PMSF and 2 mM benzamidine) and whole-cell lysates were prepared as previously described [96]. To purify TAP tagged proteins from 30ml of extract were added to 75mg of magnetic beads (BcMag, Bioclone Inc.) coupled to rabbit IgGs (Sigma) prepared as described [97]. Further purification followed the protocol for TAP-tagged RNA Pol I from yeast [57].

Dephosphorylation of recombinant CTR-TAP fusion protein was performed similarly to the dephosphorylation of the FLAG-tagged CTR purified from yeast (see above). In brief, after binding TAP-tagged CTR to IgG-coated magnetic beads (see above), the affinity matrix was washed 3x with 1 ml TAP-lysis buffer, and 3x with 1 ml TAP-lysis buffer lacking PMSF and benzamidine. The magnetic beads were equilibrated with 1 ml λ-phosphatase buffer and split into two aliquots. Beads in each aliquot were suspended in a total volume of 50 μl λ-phosphatase buffer in the absence (mock control) or presence of 2000 u λ-phosphatase and incubated in a thermomixer (Eppendorf) for 1h at 30°C under shaking. The supernatant was discarded, and the beads were washed 3x with 1 ml TAP-lysis buffer lacking PMSF and benzamidine. Proteins bound to the beads were eluted by TEV cleavage as previously described [57]. This experiment has been repeated three times and a representative experiment is shown in Fig 7D.

Promoter-dependent RNA polymerase I in vitro transcription

In vitro transcription analysis was performed essentially as previously described [57], with minor changes. To allow formation of the initiation-competent Rrn3-Pol I complex, 5 nM RNA Pol I and 70 nM Rrn3 were incubated at 4°C for 60 min. The reaction was complemented with 10 nM CF, and 5 nM template DNA containing the 35S rDNA promoter. Additionally, potassium acetate was added to yield a constant salt concentration of 200 mM in the final transcription reaction. In some reactions affinity purified Net1 derivatives were included (5–20 nM for full-length Net1, and Net1ΔCTR, and 20–100 nM for CTR or dephosphorylated CTR). The reaction was eventually adjusted to 25μl containing 20 mM HEPES-KOH pH 7.8, 10 mM MgCl₂, 5 mM EGTA, 0.05 mM EDTA, 2.5 mM DTT, 0.2 mM ATP, 0.2 mM UTP, 0.2 mM GTP, 0.01 mM CTP including 1.25 μM α-³²P-CTP. Samples were incubated for 30 min at 24°C, before the reaction was stopped and processed as reported [57]. The data presented in Fig 7E and S4H Fig are representative for at least three independent experiments.