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Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I - PubMed

  • ️Fri Jan 01 2016

Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I

Randall A Dass et al. PLoS Genet. 2016.

Abstract

Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Wnt5a inhibits rRNA gene transcription and reduces the size of nucleoli.

(a) Semi-quantitative RT-PCR analysis of 47S pre-rRNA transcript levels in MCF7 cells treated with Wnt5a for the indicated times. Error bars indicate ± SD. *P < 0.05 (n = 3). (b) The inhibitory effect of Wnt5a treatment for 60 mins on 47S rRNA synthesis is prevented by pre-treatment (60 mins) with sFRP1 in MCF7 cells. *P < 0.01 (n = 3). (c) In situ nuclear run-on assay of 5-Fluorouridine (FUrd) incorporation into nascent transcripts. MCF7 cells were treated with Wnt5a for 20 min and FUrd was detected by immunofluorescence (green). Cells were co-stained for Fibrillarin (red) and DNA (blue) to highlight nucleolar incorporation of FUrd (yellow in merged image). Scale bar = 10 μm. Quantitation of nuclei with bright nucleolar incorporation of FUrd in vehicle- and Wnt5a-treated cells. Error bars indicate ± SD. *P < 0.0001 (n = 3). (d) Silver staining of nucleolar organizer regions (AgNOR) of MCF7 cells treated with vehicle, 200 ng/mL Wnt5a, or 1 μg/mL Actinomycin D (ActD) for 4 hours. Scale bar = 10 μm. Quantification of AgNOR staining showing that Wnt5a and ActD both reduce the area of nucleoli. Error bars indicate ± SD *P <0.05; (n = 3). (e) Confocal immunofluorescence of Ki-67 (red) in MCF7 cells treated with either vehicle or with 200 ng/mL Wnt5a for 4 hours. Scale bar = 10 μm (n = 3).

Fig 2
Fig 2. DVL1 accumulates in the nucleolus upon Wnt5a signaling.

(a) Immunofluorescence and confocal microscopy using antibodies to DVL1 (green) and Fibrillarin (red) merged with DNA (blue) in MCF7 cells and MCF7 cells stably expressing Wnt5a treated with vehicle or with ActD, 40 ng/mL for 4 hours. Scale bar, 10μm (n = 3). (b) Co-localization (yellow) of DVL1 (green) and Fibrillarin (red) indicates that DVL1 is present in nucleoli of human breast cell lines MCF7, BT549 and MDA-MB-231. Scale bar, 10μm (n = 3). (c) Immunofluorescence of DVL1 (green), Fibrillarin (red) with DNA stain (blue) in MCF7 cells shows increased accumulation of DVL1 in nucleoli within 15 to 60 min of treatment with 200 ng/mL Wnt5a treatment. Scale bar = 10 μm (n = 3).

Fig 3
Fig 3. DVL1 is recruited to the rRNA gene cassette upon Wnt5a signaling, while SIRT7 levels are reduced.

(a) Chromatin immunoprecipitation (ChIP) and qPCR analysis of DVL1 and UBF on rDNA chromatin isolated from MCF7 cells (Ctrl) and MCF7 cells stably expressing Wnt5a (Wnt5a). The rRNA gene promoter, 18S rDNA and 28S rDNA repeat, and IGSs were analyzed separately using antibodies against DVL1 and UBF (as indicated below the bar graphs). Values are presented as the percentage of input signal measured for each primer pair. Error bars indicate ± SD. (n = 3). (b) Equivalent ChIP and qPCR analysis of Pol I and UBF on chromatin isolated from the same cells. Error bars indicate ± SD (n = 3). (c) Equivalent ChIP and qPCR analysis of SIRT7 and H3K4me3 on chromatin isolated from the same cells. Error bars indicate ± SD. (n = 3). (d) Co-immunoprecipitation of DVL1 and SIRT7 with UBF from nuclear protein extracts of MCF7 cells (MCF7/Ctrl) or MCF7 cells stably expressing Wnt5a (MCF7/Wnt5a). Bound proteins were detected on immunoblots with antibodies against UBF, DVL1 and SIRT7. 12% of the input material is shown in Lanes 1 and 5, and 6% in Lanes 2 and 6. IP, immunoprecipitation; IB, immunoblotting. (e) Co-immunoprecipitation of DVL1 and UBF with Pol I from nuclear protein extracts of MCF7 cells (MCF7/Ctrl) or MCF7 cells stably expressing Wnt5a (MCF7/Wnt5a). Bound proteins were detected on immunoblots with antibodies against UBF and DVL1. Approximately 12% and 6% of the input material is shown in Lanes 1 and 5, and 2 and 6, respectively. IP, immuno-precipitation; IB, immuno-blotting (n = 3).

Fig 4
Fig 4. DVL1 is required for the reduction of rRNA synthesis mediated by Wnt5a.

(a) Immunofluorescence showing reduced nucleolar staining of DVL1 (green) in BT549 and MCF7 cells transduced with a DVL1 shRNA vector (shDVL1), compared to non-silencing control (shCtrl) Scale bar = 10 μm (n = 3). (b) Knock-down of DVL1 in MCF7 and BT549 cells results in elevated 47S pre-rRNA transcript levels. Relative 47S RNA levels were measured by RT-PCR in shCtrl and shDVL1 cells. Error bars indicate ± SD. *P < 0.01 (MCF7) (n = 3). (c) AgNOR staining of BT549 and MCF7 cells transduced with shDVL1 and shCtrl. Scale bar = 10 μm. (n = 3). Quantification of AgNOR staining showing that silencing of DVL1 increases the mean area of nucleoli in both BT549 and MCF7 cells. Image J software was used to compare the total area of AgNOR staining in fields containing equivalent numbers of cells. Error bars indicate ± SD. *P < 0.05 (n = 3). (d) Proliferation assay shows that MCF7 and BT549 cells proliferate more rapidly when subjected to DVL1 gene silencing by shRNA. Data points were obtained in triplicate (n = 3). (e) Upon knock-down of DVL1, BT549 and MCF7 cells are refractory to the inhibitory effect of Wnt5a on rRNA synthesis. Relative 47S RNA levels were measured in shCtrl cells and shDVL1 cells with and without treatment of Wnt5a for 15 min. Error bars indicate ± SD. *P < 0.05 (n = 3).

Fig 5
Fig 5. In mammary tumors, loss of Wnt5a expression led to increased nucleolar size, increased proliferation and changes in the distribution and expression level of the de-acetylase Sirt7.

(a) AgNOR staining of tumors of MMTV-PyMT/Wnt5a+/+ and MMTV-PyMT/Wnt5a-/-. Scale bar = 10μm. Quantification of nucleoli staining showing a larger area in MMTV-PyMT/Wnt5a+/+ and MMTV-PyMT/Wnt5a-/-. Error bars indicate ± SD. *P < 0.05 (n = 3). (b) Immunostaining of breast tumors from MMTV-PyMT/Wnt5a+/+ and MMTV-PyMT/Wnt5a-/- mice for Ki-67 and Sirt7. Scale bar = 240μm. (c) Survival curve showing that reduced expression of Wnt5a, DVL1 and SIRT7 correlates with lower survival of patients with breast cancer (TCGA data).

Fig 6
Fig 6. Model.

In breast cancer cells, Wnt5a functions as tumor suppressor by inducing Pol I transcriptional repression through nucleolar accumulation of the downstream effector DVL1. In the presence of Wnt5a, DVL1 rapidly accumulates in the nucleolus where it binds to the rDNA transcription unit. DVL1 recruitment may be facilitated via a direct interaction with UBF, with concomitant loss of SIRT7 from active rRNA genes. These compositional changes in Pol I lead to transcriptional inhibition. In this model, Wnt5a exerts its tumor suppressive effects by inducing nucleolar accumulation of DVL1 and attenuating rRNA synthesis.

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