Increased protein synthesis during cell proliferation is accompanied by a compensatory increase in efficient ribosome production, but the mechanisms by which cells adapt to this requirement are not fully understood. In the present study, we demonstrate evidence that Pygo (Pygopus), a protein originally identified as a core component of the Wnt–β-catenin transcription complex is also involved in rRNA transcription during cancer cell growth. Pygo was detected in the nucleoli of several transformed cell lines and was associated with treacle and UBF (upstream binding factor), proteins that are essential for ribosome biogenesis in development and cancer. Pygo was also detected at the ribosomal gene promoter along with core components of the rDNA (ribosomal DNA) transcription complex. RNAi (RNA interference)-mediated depletion of hPygo2 (human Pygo 2) reduced histone H4 acetylation at the rDNA promoter, down-regulated rRNA production, and induced growth arrest in both p53-positive and -negative cells. In p53-positive cells, hPygo2 knockdown triggered the ribosomal stress pathway, culminating in p53-dependent growth arrest at G1-phase of the cell cycle. The results of the present study suggest a novel involvement of Pygo in the promotion of rRNA transcription in cancer cells.
- cancer cell growth mechanisms
- chromatin remodelling
- histone acetylation
- nucleolar stress
- ribosome biogenesis
Several ribosomes are required for each mRNA translated by a eukaryotic cell. Therefore a significant proportion of cellular resources are devoted to the production of ribosomes, especially during cell growth and division. An important initial step in ribosome biogenesis is the transcription of the 47S pre-rRNA (precursor rRNA) by Pol I (RNA polymerase I), a process finely tuned to respond to environmental cues such as nutrient availability and growth factors [1–3]. The recruitment of Pol I to sites of rRNA transcription is facilitated by structural and regulatory proteins such as UBF (upstream binding factor), SL-1 (selectivity factor 1) and treacle [also known as TCOF1 (Treacher Collins–Franceschetti syndrome 1)] . Once transcribed, the pre-rRNA undergoes a variety of post-transcriptional (maturation) modifications before being cleaved into smaller subunits. The cleaved rRNAs are assembled with a number of RPs (ribosomal proteins) into mature ribosomal subunits and exported from the nucleolus to the cytoplasm.
Although there are hundreds of rDNA (ribosomal DNA) gene repeats arrayed tandemly, approximately half are normally transcriptionally silent . Ribosome requirements are met either by adjusting the rate of transcription from active rDNA genes and/or by increasing the number of active repeats through epigenetic modulation. The mechanisms that form silent heterochromatic rDNA are well established, but much less is known about factors such as histone modification, which initiate or maintain actively transcribing euchromatic rDNA . For instance, how do cells that are actively engaged in proliferation, such as those required for development or malignant growth, epigenetically raise rRNA transcript production?
Pygo (Pygopus) proteins [7,8] function in chromatin remodelling for transcription at active Wnt target genes via interpretation of the histone code. Pygo binds to H3K4me3 (histone H3 trimethylated at Lys4) through its C-terminal PHD (plant homeodomain) [9,10], whereas its NHD (N-terminal homology domain) can recruit HAT (histone acetyltransferase) activity, which can augment target gene activation [11,12]. Those observations uniquely place Pygo in a position whereby it can relay histone marks (H3K4me3) by promoting further histone modification such as acetylation, that are associated with gene activation.
The chromatin remodelling function of Pygo may not be entirely restricted to Wnt signalling, as Pygo proteins have additional uncharacterized Wnt-independent roles in, for example, lens development  and malignant growth . In the present study, we asked what other Wnt-independent roles Pygo might have by investigating potential interactions with cellular proteins using well-established human cancer cell lines. The results of the present study suggest a novel requirement for hPygo2 (human Pygo 2) that would address how these proliferating cells adapt to their increased demand for ribosomes. In both in vivo and in vitro analyses, hPygo2 interacted with treacle, a nucleolarphosphoprotein required for ribosome biogenesis in neural crest-derived craniofacial structures . Additionally, hPygo2 was found to interact with UBF-1, a protein required for rDNApro (rDNA promoter) architecture and rRNA transcription  in the nucleoli of cancer cells and was associated with Pol I-associated transcription complexes at the rDNApros. Using RNAi (RNA interference), depletion of hPygo2 resulted in a reduction in histone H4 acetylation at rDNApros and reduced transcription of the pre-rRNA transcript in both p53-positive and -negative cells. In p53-positive cells, reduction in Pygo triggered the nucleolar stress response, resulting in activation of the RP–Mdm2 (murine double minute 2)–p53 pathway and subsequent cell-cycle arrest. The findings of the present study suggest a novel role for Pygo in ribosome biogenesis, and that Pygo promotes euchromatin formation at the vicinity of active rDNA genes, a requirement for proliferative growth.
MATERIALS AND METHODS
Cell lines, plasmids and antibodies
Cell lines were obtained from A.T.C.C. (Manassas, VA, U.S.A.), maintained in DMEM (Dulbecco's modified Eagle's medium), supplemented with 10% FBS (fetal bovine serum) (Invitrogen) and cultured at 37°C with 5% CO2. All GST (glutathione transferase)–hPygo2 constructs have been reported previously . A clone of tcof1 in pCMV-SPORT6 (GenBank® accession number BC016144), which lacks the last 74 amino acids, was purchased from Open Biosystems. A fragment containing an additional 33 C-terminal amino acids was amplified by PCR from normal human lung cDNA and inserted into pCMV-SPORT6 TCOF to generate a full-length clone encoding a protein of 1447 amino acids. All treacle/TCOF constructs were then PCR cloned from full-length pCMV-SPORT6 TCOF and inserted into the XhoI and XbaI sites of pCS2+ (primer sequences in Supplementary Table S1 at http://www.biochemj.org/bj/453/bj4530061add.htm). All constructs were verified by sequencing. Antisera raised against hPygo2 have been described elsewhere . Antibodies against treacle (H-90), UBF-1 (F-9 and H-300), p53 (DO-1), p21 (C-19) and RPA194 (H-300) were purchased from Santa Cruz Biotechnology; antibodies against phospho-Cdc2 (cell division cycle 2) (Tyr15), cyclin A (BF683), p21 (12D1) and cyclin B1 from Cell Signaling Technology; antibodies against hPygo2, H3K4me3, H3ac (acetylation at H3) and H4ac were from Millipore; antibodies against treacle, β-actin and BrdU (bromodeoxyuridine) from Sigma; and antibodies against fibrillarin and RPL11 were from Abcam. Other antibodies used include anti-β-tubulin (Pharmingen), anti-B23 (Zymed) and anti-Nop56 (Abnova).
GST pull-downs, IP (immunoprecipitation) and proteomics
GST pull-down and IP analyses were performed using reagents and buffer conditions essentially as described previously . For proteomic analysis, 1 mg of purified GST fusion protein and 200 mg of MCF-7 nuclear extract were incubated together for 3 h at 4°C, washed extensively, separated by SDS/PAGE (10% gel) and stained with Coomassie Brilliant Blue (Bio-Rad Laboratories). Proteins were excised from the gel and processed for tandem MS as described previously .
Cells were processed and stained for immunofluorescence as described previously . Detection of hPygo2 was performed using an anti-hPygo2 antibody (ABE109; Millipore). For the AMD (actinomycin D) experiments, HeLa cells were treated with 50 ng/ml AMD (Sigma) or ethanol carrier for 2 h at 37°C in 5% CO2 and then immediately processed. The BrUTP (bromouridine triphosphate; Sigma) incorporation assay was performed as described previously . Cells were subsequently incubated with BrdU and anti-hPygo2 antibodies (1:100 dilution in PBS with 0.05 units/ml RNase inhibitor) overnight at 4°C, stained with a mixture of Cy5 (indodicarbocyanine)-conjugated anti-(mouse IgG) and FITC-conjugated anti-(rabbit IgG) antibodies (Jackson Immunoresearch) and imaged on an Olympus FluoView FV1000 confocal microscope (Olympus).
Nuclei and nucleoli were isolated by sucrose density-gradient centrifugation as described previously , imaged using phase-contrast microscopy and immunoblotted for detection of cytoplasmic, nuclear and nucleolar markers. Extracts of whole cells, nuclei and nucleoli were prepared in Laemmli sample buffer.
ChIP (chromatin IP)
ChIP assays were performed as described previously . Briefly, sonication was performed on formaldehyde cross-linked cells to produce genomic DNA fragments of approximately 500 bp in size. Pre-cleared chromatin (400 μg) was subject to IP with 2 mg of antibodies against RPA194, UBF-1, treacle, hPygo2, H3K4me3, H3ac and H4ac. Antibodies against normal rabbit IgG and normal mouse IgG were used as negative IP controls. After washing, formaldehyde cross-links were reversed at 65°C. Samples were subsequently treated with RNase A and proteinase K. DNA was then purified using the QIAquick PCR purification kit (Qiagen).
For second-round ChIP, complexes were eluted after the first round of ChIP in 10 mM DTT (dithiothreitol) at 37°C for 30 min. Supernatant was then diluted 25 times with ChIP dilution buffer (1.1% Triton X-100, 0.01% SDS, 1.2 mM EDTA, 167 mM NaCl, 16.7 mM Tris, pH 8.1, and protease inhibitors) with 50 μg BSA (Sigma) and then subjected to a second round of ChIP. PCR was performed on the rDNApro using either the rDNApro primer sequences given in Supplementary Table S1 or primers recognizing the rDNA core promoter  and the ITS-1 (internal transcribed sequence 1) regions .
qPCR (quantitative real-time PCR)
qPCR was performed as described previously , using RT2 SYBR Green master mix (SABiosciences). Oligonucleotide primers targeting hPygo2  and 47S (Supplementary Table S1) were used and results were normalized to levels of β-actin mRNA. For ChIP–qPCR analysis, rDNApro occupancy was calculated relative to that of the input chromatin.
Cells were grown to approximately 25% confluency and siRNAs (small interfering RNAs) were transfected to a final concentration of 5 nM using Lipofectamine™ RNAiMAX (Invitrogen) according to the manufacturer's protocol. The two hPygo2-specific and NTC (non-targeting control) (a five base mismatch control of hPygo2-Z, mismatches are underlined in the sequence below) siRNAs were designed using the siDESIGN Center (http://www.thermoscientificbio.com/design-center/) and purchased from Dharmacon (Lafayette). The sense strand sequences were: 5′-GGAGACAGCUUUAGGGAAUUU-3′ (hPygo2-X), 5′-GGAGUGAGGUGAACGAUGAUU-3′ (hPygo2-Z) and 5′-GGACUGUGGUCAACCAUGUUU-3′ (NTC). The p53 siRNA was purchased as the TP53 siGENOME SMARTpool (Dharmacon).
HeLa cells were transfected with siRNAs 48 h before metabolic labelling using a modified protocol . Briefly, cells were washed with PBS and the medium was replaced with DMEM containing 5 μCi/ml 3H-labelled uridine and incubated for 30 min at 37°C. The labelling medium was then chased with DMEM containing 1 mg/ml unlabelled uridine. RNA was extracted 120 min later using TRI Reagent® (Ambion). Approximately 10 μg of RNA was separated on Mops formaldehyde gels, transferred on to nylon membranes, cross-linked, dried and sprayed with En3Hance (PerkinElmer) and exposed to autoradiography film.
Initially, HeLa cells were treated with siRNAs for 48 h. Cells were collected and fixed with 2% formaldehyde for 10 min at 37°C and then incubated in 90% methanol/PBS for 30 min at 4°C. Cells were then resuspended in PBS treated with 20 μg/ml RNase A for 20 min at 37°C, stained with 1 mg/ml propidium iodide and counted in a BD Biosciences FACSCalibur™ flow cytometer. P values were calculated using the paired two-tailed Student's t test.
hPygo2 interacts with treacle and UBF-1 and is detected in the nucleoli of cancer cells
In order to investigate novel functions of hPygo2, we analysed proteins from MCF-7 breast cancer cells that interacted with fusion proteins made between the hPygo2 domains and GST (Supplementary Figure S1A at http://www.biochemj.org/bj/453/bj4530061add.htm). MCF-7 cells were chosen because they express hPygo2 at a relatively high level and they required hPygo2 for growth . Several prominent protein bands associating with the NHD, the region that interacts with proteins involved in chromatin modulation and transcription [11,12,24,25], but not the PHD, the region that enables Pygo to fulfil its function in Wnt signalling, were isolated and analysed using tandem MS. Over 30% of the sequenced peptides derived from an isolate at approximately 205 kDa matched identically to the primary sequence of treacle, a protein required for the increase in rRNA production that supports proliferation of anterior neural crest cells [26,27] (Supplementary Figure S1B). The interaction of the NHD with peptides derived from treacle led us to hypothesize that previously reported growth requirements for hPygo2 in cancer cells might be related to a potential role in rRNA production.
In MCF-7 cells, endogenous treacle was detected in complexes that precipitated with hPygo2 antiserum, but not with pre-immune serum (Figure 1A, top panel). Conversely, hPygo2 was detected in complexes immunoprecipitated using antisera directed against treacle (Figure 1A, bottom panel). Using in vitro pull-down assays, full-length treacle (amino acids 1–1488) was recovered using GST–NHD (hPygo2 fragment 4, Supplementary Figure S2A at http://www.biochemj.org/bj/453/bj4530061add.htm), and the interaction of treacle with the NHD occurred within its C-terminal 493-amino-acid residues (treacle fragment 4, Figure S2B).
Similar to treacle , we tested whether hPygo2 interacted with UBF-1. In MCF-7 cells, endogenous UBF-1 was detected in complexes immunoprecipitated by an anti-hPygo2 antibody, but not with control IgG (Figure 1B, top panel). Conversely, hPygo2 was detected in complexes immunoprecipitated by an anti-UBF-1 antibody (Figure 1B, bottom panel). Consistent with its observed interaction with treacle and UBF-1, hPygo2 protein was enriched in the nucleolar compartment of fractionated HeLa cells along with two other well-described nucleolar proteins, UBF-1 and fibrillarin (Figure 1C).
Indirect immunofluorescence confirmed the nucleolar co-localization of hPygo2 with UBF-1 (Figure 2A) and treacle (Figure 2B) in MCF-7, HeLa and SKOV-3 cells. Furthermore, hPygo2 staining overlapped with newly incorporated BrUTP in nucleoli of HeLa and SKOV-3 cells, suggesting a possible association with de novo RNA synthesis (Figure 2C).
During mitosis, NORs (nucleolar organizing regions) are transcriptionally silent, but are still bound by core components of the rDNA transcription machinery such as UBF-1 [28–30]. Staining of mitotic HeLa cells revealed that hPygo2 co-localized with UBF-1 at specific NORs during prophase, but then did not appear with NORs during metaphase and telophase, suggesting a transient or dynamic nucleolar association of hPygo2 during cell division (Supplementary Figure S3 at http://www.biochemj.org/bj/453/bj4530061add.htm).
hPygo2 is uncoupled from core transcriptional rDNA components in AMD-treated cells
Production of the 47S pre-rRNA transcript is followed by its post-transcriptional processing via methylation and pseudouridylation for cleavage into the 28S, 18S and 5.8S rRNA subunits. Transcription and processing of the 47S pre-rRNA occurs in distinct nucleolar compartments. We used the Pol I inhibitor AMD to disrupt rDNA transcription and monitored its effects on the localization of hPygo2. At low concentrations, AMD inhibits actively transcribing rDNA transcriptional complexes and reversibly re-organizes nucleoli according to their functional compartments, the FCs (fibrillar centres), composed of the tandemly arrayed rDNA genes, the DFC (dense fibrillar component) and the GC (granular component), which contains rRNA undergoing post-transcriptional modification. This is observed by a restructuring of the FCs and DFCs into ‘nucleolar caps’, which appear as small blebs situated on the central body whose composition is assumed to be derived from the GC .
In control ethanol-treated HeLa cells, UBF-1 (Supplementary Figure S4A at http://www.biochemj.org/bj/453/bj4530061add.htm) and treacle (Supplementary Figure S4B) co-localized with hPygo2 in overlapping regions of the nucleoli. Fibrillarin is a component of snRNP (small nucleolar ribonucleoprotein) complexes that methylate 47S pre-RNA in a sequence-dependent manner . B23 is a nucleolar stress sensor required for downstream rRNA processing . In control ethanol-treated cells, hPygo2 was detected in regions of the nucleolus with fibrillarin (Supplementary Figure S4C) and B23 (Supplementary Figure S4D). These observations place hPygo2 in the nucleolus along with components of the core transcription machinery and the components involved in post-transcriptional processing.
In AMD-treated cells (Supplementary Figure S4, lower panels), hPygo2 was found mainly in the central body, whereas UBF-1 (Supplementary Figure S4A) and treacle (Supplementary Figure S4B) localized to the outer nucleolar caps, fibrillarin was localized to the inner nucleolar caps (Supplementary Figure S4C) and B23 was detected in the nucleoplasm (Supplementary Figure S4D). Although hPygo2 was not associated with nucleolar caps, it continued to remain associated with the nucleolus after AMD treatment, suggesting it is not likely to be a core component of the rDNA transcription machinery.
hPygo2 binds to rDNApro chromatin
The observations discussed above suggested that hPygo2 is not associated tightly enough with the core complexes to withstand the effects of AMD, even though they co-immunoprecipitated from cells. This is perhaps not surprising as hPygo2 has no DNA-binding ability, yet it might still indirectly interact with the promoter through the core complexes, a possibility we tested using ChIP assays. Sheared chromatin from HeLa cells was immunoprecipitated using anti-RPA194 (the large subunit of Pol I), anti-UBF-1, anti-treacle, anti-β-catenin and anti-hPygo2 antibodies and then subjected to PCR using oligonucleotide pairs complementary to the start of transcription on the rDNApro (Figure 3A). RPA194, UBF-1, treacle and hPygo2 immunoprecipitates all contained promoter sequences, but not control ITS-1 (Figure 3B). Furthermore, although second-round ChIP analysis confirmed the presence of all four proteins at the same region of the rDNApro (Figure 3C), hPygo2 only interacted with treacle and UBF-1, but not with Pol I or proteins involved in transcript processing (Figure 3D).
In separate experiments, the rDNA-specific primers did not amplify sequence from chromatin precipitated by anti-β-catenin antibodies (Supplementary Figure S5A at http://www.biochemj.org/bj/453/bj4530061add.htm). Furthermore, although active β-catenin was readily detectable at the Wnt response element of the Axin2 promoter following LiCl treatment of HeLa cells, it was not detectable at the rDNApro (Supplementary Figure S5B). Finally, we did not observe an interaction between β-catenin and UBF in vitro (Supplementary Figure S5C). Thus although Pygo is typically associated with the canonical Wnt transcription complex, it did not appear to rely on β-catenin for its localization to rDNA promoters.
hPygo2 recruits HAT activity to the rDNApro
Pygo was shown previously, through binding of its PHD to H3K4me3 marks, to recruit HAT activity to its conserved NHD at active Wnt target genes [9–12]. To address a possible role of hPygo2 in rDNA transcription, two hPygo2-targeting siRNAs were selected for loss-of-function studies. Transfection of both siRNAs resulted in an increase in p21 expression, whereas co-transfection of the hPygo2-coding region with the siRNA that targeted the 3′-UTR (untranslated region) (hPygo2-X) of hPygo2 restored the lower levels of p21 present in controls, verifying the specificity of the knockdown (Supplementary Figure S6A at http://www.biochemj.org/bj/453/bj4530061add.htm). Moreover, depletion of hPygo2 by siRNA clearly reduced the amount of nucleolar hPygo2 (Supplementary Figure S6B).
Using these siRNAs, we tested whether hPygo2 loss-of-function affected histone modifications at the rDNApro by ChIP–qPCR. Transfection of hPygo2 siRNA reduced the amount of hPygo2 bound to the rDNApro to approximately 41% compared with the NTC siRNA (Figure 4A). There was no effect on H3ac levels, but an increase was detected in H3K4me3 levels at the rDNApro, in spite of a slight decrease in the global levels of H3K4me3 (Supplementary Figure S7A at http://www.biochemj.org/bj/453/bj4530061add.htm), possibly reflecting a compensatory mechanism for hPygo2 recruitment. Notably, hPygo2 knockdown reduced H4ac levels occupying the promoter region to approximately 47% compared with the NTC siRNA, but did not significantly affect the binding of complex(es) containing Pol I or UBF (Figure 4A). Similar to H3K4me3, there was a detectable decrease in global levels of H4ac with hPygo2 siRNA treatment, but this was also accompanied by a decrease in H4 protein as well (Supplementary Figure S7B), consistent with an observed role for hPygo2 in histone gene expression . Thus although hPygo2 was not necessary for recruitment of transcriptional components, it appeared to be required to maintain acetylation of associated histones at the promoter during rDNA transcription.
To test whether the observed reduction in H4ac at the rDNApro had an effect on rRNA transcription, further loss-of-function experiments were performed. hPygo2 knockdown resulted in reductions in the 32S, 28S and 18S rRNAs as measured by 3H-labelled uridine incorporation (Figure 4B). Similarly, knockdown of hPygo2 reduced transcription from a transfected luciferase reporter gene under the control of the rDNApro  (pHrD-IRES-Luc; Figure 4C). To further confirm and quantitatively assess the transcription of the 47S pre-rRNA, qRT-PCR (quantitative reverse transcription–PCR) was performed on hPygo2 siRNA-treated cells. Treatment of both HeLa (Figure 5A) and SKOV-3 (Figure 6A) cells with hPygo2 siRNA showed a significant reduction hPygo2 mRNA levels as well as a significant reduction in the levels of the 47S rRNA. Together, these observations provided evidence that hPygo2 is involved in transcription of the 47S pre-rRNA by maintaining levels of H4ac.
Depletion of hPygo2 resulted in growth arrest by activation of the RP–Mdm2–p53 nucleolar stress response
Findings published previously indicated a strong growth requirement for hPygo2 in cancer [14,18], so we queried whether the potential role for hPygo2 in ribosome biogenesis was part of this requirement. Nucleolar stress-induced cell-cycle arrest is caused by disruptions in ribosome biogenesis resulting in the association of unincorporated RPs, such as RPL11, with the E3 ubiquitin ligase HDM2 (human double minute 2), thereby compromising its ability to inhibit p53 tumour suppressor and apoptotic function . We hypothesized that disruption of rDNA transcription by depletion of hPygo2 might trigger the nucleolar stress response.
It could be argued that the attenuation in rDNA transcription in response to treatment with hPygo2 siRNA was a secondary effect subsequent to disruption of cell-cycle progression. To test this possibility, we determined the effect of reducing both proteins on rRNA production using both hPygo2 and p53 siRNAs. Depletion of hPygo2 alone resulted in a decrease in 47S rRNA and an increase in p21 (Figure 5A). Co-transfection with both hPygo2 and p53 siRNAs resulted in a decrease in the p53 target gene p21 as expected. It did not, however, restore levels of 47S rRNA (Figure 5A), suggesting that the reduction in 47S rRNA by hPygo2 RNAi was independent of p53.
We next tested whether the observed increase in p53 by depletion of hPygo2 resulted from activation of the nucleolar stress response pathway . In cells treated with hPygo2 siRNA, RPL11 co-immunoprecipitated with HDM2, whereas in untreated (mock) and control (siNTC) siRNA-treated cells, we did not detect RPL11 protein in HDM2 immunoprecipitates (Figure 5B, upper panel). Interestingly, there was an increase in HDM2 levels in hPygo2-depleted cells (Figure 5B, ‘input’, lower panel), which was consistent with previous findings demonstrating HDM2 as a transcriptional target of p53 . Thus association of RPL11 with HDM2, following depletion of hPygo2 suggested that the nucleolar stress pathway was invoked.
The increase in p53 levels following hPygo2 knockdown resulted in cell-cycle arrest and a significant increase in the number of cells at G1-phase, accompanied by a decrease in the number of cells at G2/M-phase, compared with the NTC siRNA alone (Figure 5C). Likewise, growth arrest by hPygo2 depletion in HeLa cells was reversed compared with cells that were treated with control siRNAs, by co-depletion of p53 (Figure 5C). Alone, hPygo2 siRNA increased both p53 and the p53 target gene p21, but reduced the levels of the G2/M-phase markers phospho-Cdc2, cyclin A and cyclin B1 (Figure 5D). In contrast, co-depletion of hPygo2 and p53 reduced p21 expression and restored the cell-cycle progression markers phospho-Cdc2, cyclin A and cyclin B (Figure 5D). Together, these results suggested that hPygo2 depletion resulted in the accumulation of p53 and subsequent arrest at G1-phase through activation of the nucleolar stress response.
hPygo2 knockdown reduced rRNA transcription in p53-null cells
The above-mentioned experiments indicated that, although co-transfection of hPygo2 and p53 siRNAs had little effect on cell-cycle progression, there was a persistent reduction in 47S rRNA transcription (Figure 5A). Similarly, depletion of hPygo2 in the p53-null ovarian cancer cell line SKOV-3, which we demonstrated previously was sensitive to knockdown of hPygo2 , also caused a significant decrease in 47S rRNA (Figure 6A). Cell-cycle analysis of hPygo2-depleted SKOV-3 revealed an accumulation of cells in G2/M-phase (Figure 6B). The only cell-cycle marker with a detectable change after hPygo2 depletion was cyclin A, nevertheless its increased expression was consistent with the observed cell-cycle arrest at the G2/M-phase (Figure 6C). It is unclear whether G2/M-phase arrest in p53-null cells was the result of a nucleolar stress response similar to that of p53-positive cells. These observations, however, suggested that a primary effect of hPygo2 depletion was on rRNA production, leading to a subsequent negative effect on cell-cycle progression, providing evidence linking hPygo2 function to rDNA transcription.
The association of hPygo2 with ribosomal gene promoters and its participation in rRNA transcription, suggests a function for Pygo in ribosomal biogenesis during proliferative growth, in addition to its demonstrated role in canonical Wnt signalling. We suggest that hPygo2 plays a role in ribosomal gene transcription, specifically via its previously identified role, the acetylation of histone H4 , but at the rDNA gene promoter in lieu of, or more likely in addition to, Wnt target gene promoters/enhancers. This step would ostensibly maintain requisite levels of 47S pre-rRNA, necessary for cell division, and without which would lead to cell growth arrest.
Our data incorporate hPygo2 into models of active ribosomal gene transcription , perhaps as an adaptor protein for epigenetic modifiers required for histone acetylation. We propose that Pygo2 is present at the rDNApro through its interaction with both UBF-1 and treacle. The fact that Pygo2 does not make direct contact with Pol I further suggests a role in chromatin modification to augment transcription. It is possible that its recruitment to active sites or ribosomal gene transcription and may also depend on H3K4 methylation status at the vicinity of rDNApro.
The diverse requirements for hPygo2 in development suggest that its cellular function is context dependent, as demonstrated by pygo loss-of-function studies in mice. Transgenic animals deficient for mpygo2 (mouse pygo 2) die as neonates, but display a range of tissue and organ defects, some of which, such as in kidney and mammary gland morphogenesis, have been identified as canonical Wnt dependent [40–42]. The requirement for mpygo2 in lens formation, however, was demonstrated as Wnt-independent and there are other unattributed defects, such as overall growth agenesis and craniofacial defects, including cleft palate , the latter of which might be explained by a co-operative role for treacle and Pygo2 in ribosome biogenesis.
In support of our hypothesis that hPygo2 has an important function in ribosome biogenesis are our observations that depletion of hPygo2 triggered the nucleolar stress response, which could be reversed by knocking down p53, mirroring results found when treacle was knocked out by transgenesis in a mouse model for Treacher Collins syndrome [15,43]. The parallel between a requirement for treacle-dependent ribosome biogenesis in developing craniofacial cells and hPygo2-dependent ribosome biogenesis in cancer cells suggests that the interaction we observed between hPygo2 and treacle may be a conserved adaptation to rapid cell proliferation.
The localization of hPygo2 to the central nucleolar body and not to nucleolar caps after treatment with AMD, is not inconsistent with a role in active rDNA transcription. Although hPygo2 is not likely to be a core component of the rDNA transcription machinery, its interaction with the active transcription complex that we observed in untreated cells might be dynamic. Two observations that are relevant to this are: (i) Pygo is not localized to nucleolar caps after AMD treatment, but is still associated with the nucleolus; and (ii) Pygo does not co-localize with UBF at NORs during mitosis, when transcription is silenced [28–30], but does appear to be associated with the nucleolus during interphase.
The increased demand for ribosomes in cellular growth and proliferation requires that both RP and rRNA synthesis be amplified and precisely co-ordinated. Pygo may serve in this respect to augment or maintain Pol I-dependent transcription to keep in stride with RP production in cancer. Since hPygo2 is up-regulated in a variety of cancers [11,14,17,44,45], the findings from the present study suggest that targeting hPygo2 might be an effective therapeutic possibility, as this broadly applicable strategy would disrupt rDNA transcription, resulting in growth arrest.
Phillip Andrews performed most of the experiments and participated in writing the paper. Zhijian He performed the in vitro interaction assays and BrUTP incorporation assays. Youlian Tzenov performed the FACS and immunoblots in hPygo2-depleted SKOV-3 cells and the siRNA rescue experiment of hPygo2. Cathy Popadiuk provided initial intellectual input and co-supervised the project. Kenneth Kao was principal investigator for the project and wrote the paper.
This work was supported by grants from the Cancer Research Society, the Motorcycle Ride for Dad (Avalon Chapter) and the CIHR (Canadian Institutes of Health Research) [grant number MOP-14958].
We thank Laura Gillespie and Gary Paterno (Memorial University, NL, Canada) for reagents and for helpful discussions, Mark Kennedy (NCI-Frederick, MD, U.S.A.) for technical assistance and comments on the paper, Samson Jacob (Ohio State University, OH, U.S.A.) for providing us with the pHrD-IRES-Luc plasmid and Isabel Dominguez (Boston University, MA, U.S.A.) for the GST-β-catenin plasmid.
Abbreviations: AMD, actinomycin D; BrdU, bromodeoxyuridine; BrUTP, bromouridine triphosphate; Cdc2, cell division cycle 2; ChIP, chromatin immunoprecipitation; DFC, dense fibrillar component; DMEM, Dulbecco's modified Eagle's medium; FC, fibrillar centre; GC, granular component; GST, glutathione transferase; HAT, histone acetyltransferase; H3K4me3, histone H3 trimethylated at Lys4; HDM2, human double minute 2; hPygo2, human Pygopus 2; IP, immunoprecipitation; ITS-1, internal transcribed sequence 1; Mdm2, murine double minute 2; NHD, N-terminal homology domain; NOR, nucleolar organizing region; NTC, non-targeting control; PHD, plant homeodomain; Pol I, RNA polymerase I; pre-rRNA, precursor rRNA; Pygo, Pygopus; qPCR, quantitative real-time PCR; qRT-PCR, quantitative reverse transcription–PCR; rDNA, ribosomal DNA; rDNApro, ribosomal DNA promoter; RNAi, RNA interference; RP, ribosomal protein; siRNA, small interfering RNA; TCOF1, Treacher Collins–Franceschetti syndrome 1; UBF, upstream binding factor
- © The Authors Journal compilation © 2013 Biochemical Society