Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I
Randall A. DassAishe A. SarshadBrittany B. CarsonJennifer M. FeenstraAmanpreet KaurAleš ObrdlíkMatthew ParksVarsha PrakashDamon LoveKristian PietrasRosa SerraScott C. BlanchardPiergiorgio PercipalleAnthony M.C. BrownC. Theresa Vincent
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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.Keywords:
ribosome biogenesis
RNA polymerase I
Transcription
RNA polymerase II
Myc is an oncoprotein that is involved in regulating both cell division and cell growth. Previous reports had indicated that Myc stimulated RNA polymerase III (Pol III), which synthesizes the 5 S ribosomal RNA (rRNA), as well as RNA polymerase II (Pol II), which synthesizes the transcripts encoding the ribosomal proteins (see Oskarsson and Trumpp). Three groups report that Myc also stimulates RNA polymerase I (Pol I), which synthesizes the transcript for the pre-rRNA transcript for the 28 S , 5.8 S , and 18 S rRNAs. Grewal et al . showed that in Drosophila , overexpression of dMyc increased rRNA transcript abundance, nucleolar size, and ribosome abundance, whereas hypomorphic alleles of dMyc resulted in decreases. These effects on ribosome biogenesis were specific in that overexpression of dp110 (the phosphoinositide 3-kinase catalytic subunit), which also causes increased cell size, did not increase ribosome biogenesis. Microarray analysis of larval transcripts indicated that dMyc stimulated the expression of a collection of genes involved in ribosome biogenesis, including the genes encoding Rpl135 (a Pol I subunit) and TIF-1A (a growth-regulated Pol I-associated factor). The importance of rRNA synthesis in mediating cell size increases in response to dMyc overexpression was reinforced by the inhibition of this effect of dMyc in Rpl135 −/− cells. Grandori et al. and Arabi et al. investigated the effect of Myc on mammalian cells. Both groups reported that, in contrast to flies, Myc directly bound the rDNA promoter, which encodes the rRNA transcript that produces the 28 S , 5.8 S , and 18 S rRNAs, through an E-box sequence. Grandori et al. also showed that c-Myc interacted with the TIF-1B (also known as SL1), which is a complex that is required for Pol I transcription. When c-Myc was abundant, more TIF-1B was bound to the rDNA promoter, and when c-Myc was deficient, less TIF-1B was bound. Both groups also reported localization of c-Myc to nucleoli under certain circumstances: inhibition of the proteasome (Arabi et al. and Grandori et al. ) and following serum addition after a period of deprivation (Grandori et al. ). These three reports provide evidence that Myc is a regulator of Pol I, directly in mammals and indirectly (through regulation of gene expression) in flies. Thus, Myc appears situated to be a master regulator of ribosome biogenesis through its effects on each of the three RNA polymerases. T. Oskarsson, A. Trumpp, The Myc trilogy: lord of RNA polymerases. Nat. Cell Biol. 7 , 215-217 (2005). [PubMed] S. S. Grewal, L. Li, A. Orian, R. N. Eisenman, B. A. Edgar, Myc-dependent regulation of ribosomal RNA synthesis during Drosophila development. Nat. Cell Biol. 7 , 295-302 (2005). [PubMed] C. Grandori, N. Gomez-Roman, Z. A. Felton-Edkins, C. Ngouenet, D. A. Galloway, R. N. Eisenman, R. J. White, c-Myc binds to human ribosomal DNA and stimulates transcription of rRNA genes by RNA polymerase I. Nat. Cell Biol. 7 , 311-318 (2005). [PubMed] A. Arabi, S. Wu, K. Ridderstråle, H. Bierhoff, C. Shiue, K. Fatyol, S. Fahlén, P. Hydbring, O. Söderberg, I. Grummt, L.-G. Larsson, A. P. H. Wright, c-Myc associates with ribosomal DNA and activates RNA polymerase I transcription. Nat. Cell Biol. 7 , 303-310 (2005). [PubMed]
ribosome biogenesis
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RNA polymerase III
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Fibrillarin
ribosome biogenesis
Nucleolin
RNA polymerase I
Small nucleolar RNA
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It is long been known that cancer and non-cancer cells can be distinguished on the basis of their nucleolar morphologies. As early as the 19th century, it was reported that cancer cells have larger and more irregularly shaped nucleoli. Since then, pathologists have used nucleolar morphology to predict the clinical outcome [1]. Nucleolar morphology is altered due to the up-regulation of ribosomal gene transcription. Within nucleoli, ribosomal genes (rDNA) are transcribed by RNA polymerase I (pol I). The pre-ribosomal RNA (pre-rRNA) transcripts are subsequently modified and processed into the mature 18S, 5.8S and 28S rRNAs. 5S rRNA is transcribed by RNA polymerase III in the nucleoplasm. Together with the ribosomal proteins, the 5S rRNA is imported into the nucleolus where 40S and 60S ribosomal subunits are assembled prior to export to the cytoplasm [1, 2].
Oncogenes such as c-Myc can both directly and indirectly upregulate rDNA transcription, while tumour suppressors like p53 and Rb suppress ribosome biogenesis. Mutations in these genes not only result in deregulated cell cycle control, but also upregulated ribosome biogenesis. In addition to ribosome biogenesis, the nucleolus is a key cellular stress sensor and plays a central role in p53 activation [1, 2].
The increased translational capacity of cancer cells enables them to maintain higher proliferation rates. As stated by Ruggero, “compared with normal cells, cancer cells may be addicted to increases in ribosome biogenesis and number” [1]. This provides new therapeutic opportunities. As it turns out many chemotherapeutic drugs used in cancer treatment already inhibit ribosome biogenesis. In one recent survey it was shown that 20 out of 36 drugs in clinical use inhibit ribosome biogenesis [3]. Most of these drugs were originally designed to target highly proliferating cells by damaging DNA, interfering with DNA synthesis or with mitosis. These targeting modalities of these drugs also lead to toxicity in normal highly proliferating tissues. An example is ActinomycinD (AMD), a DNA intercalator which has a preference for GC-rich DNA sequences. As rDNA has above average GC-richness and because of its open chromatin conformation, low concentrations of AMD preferentially inhibit RNA polymerase I transcription and upon prolonged exposure causes genome wide DNA damage. Alkylating drugs like cisplatin and oxaliplatin or topoisomerases poisons like camptothecin inhibit pol I transcription. The degree to which inhibition of ribosome biogenesis contributes to the efficacy of these drugs is difficult to establish [3]. This raises an important question. Can targeting ribosome biogenesis without DNA damage offer any therapeutic potential? Two recently described drugs CX-5461 and BMH-21 are now providing evidence that inhibition of ribosome biogenesis by targeting transcription of rDNA by pol I has promising therapeutic potential. CX-5461 was designed to specifically inhibit pol I transcription by disrupting pre-initiation complex formation at the rDNA promoter. CX-5461 has been shown to activate p53 via nucleolar stress. It induces autophagy as well as senescence in a multiple types of cancer cells in a p53-dependent manner. Especially in leukaemia and lymphoma cells, treatment with CX-5461 induces p53-dependent apoptosis, while normal cells tolerate it [4, 5].
Whether the drug also induces DNA damage was not fully addressed, but it was demonstrated that it could induce cell death in cells lacking ATM - a key mediator of DNA double strand break responses. However, more recently Laiho and colleagues have shown that at high concentrations, CX-5461 does induce a γH2AX response, raising concerns about DNA damage [6].
BMH-21 was identified in a screen performed by Laiho and colleagues aimed at identifying novel p53 activators. Like AMD, BMH-21 is a DNA intercalator with preference for GC rich sequences [7]. Continuing the parallel with AMD, BMH-21 is a potent and specific inhibitor rDNA transcription and induces nucleolar reorganisation often referred to as nucleolar capping. Interestingly, transcription inhibition was followed by the degradation of the main pol I subunit, RPA194, by the proteasome [6]. In contrast with AMD, initial indications were that BMH-21 did not appear to induce DNA damage as evidenced by the lack of a γH2AX response [7]. Inhibition of transcription by BMH-21 causes nucleolar stress, resulting in decreased proliferation and cell death. P53 is activated in BMH-21 treated cells but is not required for its anti-proliferative effects. Intriguingly, it appears that cancer cells with high demands for ribosome biogenesis are selectively targeted [6].
The current publication in Oncotarget now rules out any role for DNA damage signalling and repair pathways in the BMH-21 response. Moreover, BMH-21 derivatives that can induce DNA damage display lower efficiency in inducing nucleolar stress and inhibiting proliferation [8]. The central importance of this study is that it finally uncouples DNA damage and nucleolar stress and reveals an Achilles heel in cancer cells, their addiction to ribosome biogenesis.
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Abstract During RNA polymerase II (RNA Pol II) transcription, the chromatin structure undergoes dynamic changes, including opening and closing of the nucleosome to enhance transcription elongation and fidelity. These changes are mediated by transcription elongation factors, including Spt6, the FACT complex, and the Set2-Rpd3S HDAC pathway. These factors not only contribute to RNA Pol II elongation, reset the repressive chromatin structures after RNA Pol II has passed, thereby inhibiting aberrant transcription initiation from the internal cryptic promoters within gene bodies. Notably, the internal cryptic promoters of infrequently transcribed genes are sensitive to such chromatin-based regulation but those of hyperactive genes are not. To determine why, the weak core promoters of genes that generate cryptic transcripts in cells lacking transcription elongation factors (e.g. STE11) were replaced with those from more active genes. Interestingly, as core promoter activity increased, activation of internal cryptic promoter dropped. This associated with loss of active histone modifications at the internal cryptic promoter. Moreover, environmental changes and transcription elongation factor mutations that downregulated the core promoters of highly active genes concomitantly increased their cryptic transcription. We therefore propose that the chromatin-based regulation of internal cryptic promoters is mediated by core promoter strength as well as transcription elongation factors.
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The nucleolus is the cellular site of ribosome biosynthesis. At this site, active ribosomal DNA (rDNA) genes are rapidly transcribed by RNA polymerase I (pol I) molecules. Recent advances in our understanding of the pol I transcription system have indicated that regulation of ribosomal RNA (rRNA) synthesis is a critical factor in cell growth. Importantly, the same signaling networks that control cell growth and proliferation and are deregulated in cancer appear to control pol I transcription. Therefore, the study of the biochemical basis for growth regulation of pol I transcription can provide basic information about the nuclear signaling network. Hopefully, this information may facilitate the search for drugs that can inhibit the growth of tumor cells by blocking pol I activation. In addition to its function in ribosome biogenesis, recent studies have revealed the prominent role of the nucleolus in cell senescence. These findings have stimulated a new wave of research on the functional relationship between the nucleolus and aging. The aim of this review is to provide an overview of some current topics in the area of nucleolus biology, and it has been written for a general readership.
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Recent genome-wide studies in metazoans have shown that RNA polymerase II (Pol II) accumulates to high densities on many promoters at a rate-limited step in transcription. However, the status of this Pol II remains an area of debate. Here, we compare quantitative outputs of a global run-on sequencing assay and chromatin immunoprecipitation sequencing assays and demonstrate that the majority of the Pol II on Drosophila promoters is transcriptionally engaged; very little exists in a preinitiation or arrested complex. These promoter-proximal polymerases are inhibited from further elongation by detergent-sensitive factors, and knockdown of negative elongation factor, NELF, reduces their levels. These results not only solidify the notion that pausing occurs at most promoters, but demonstrate that it is the major rate-limiting step in early transcription at these promoters. Finally, the divergent elongation complexes seen at mammalian promoters are far less prevalent in Drosophila, and this specificity in orientation correlates with directional core promoter elements, which are abundant in Drosophila.
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Summary Beginning with transcription of ribosomal RNA (rRNA) by RNA Polymerase (Pol) I in the nucleolus, ribosome biogenesis is intimately tied to cell growth and proliferation. Perturbation of ribosome biogenesis has been previously shown to affect nucleolar structure, yet the underlying mechanism is unknown. We generated loss-of-function mouse mutants of Pol I subunits, Polr1a, Polr1b, Polr1c and Polr1d , thereby genetically inhibiting rRNA transcription and ribosome biogenesis. Pol I mutant embryos are preimplantation lethal and have fewer nucleoli. Using hiPSCs triple labeled for the three nucleolar compartments, we observe two phenotypes upon Pol I inhibition: a single condensed nucleolus, and fragmented nucleoli. We find that when rRNA transcription is inhibited, the viscosity of the granular compartment of the nucleolus is increased disrupting its liquid-liquid phase separation properties, which results in a condensed nucleolus. Taken together, our data suggests that Pol I function and rRNA transcription are required for maintaining nucleolar structure and integrity.
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Protein-coding genes in trypanosomes occur in polycistronic transcription units (PTUs). How RNA polymerase II (Pol II) initiates transcription of PTUs has not been resolved; the current model favors chromatin modifications inducing transcription rather than sequence-specific promoters. Here, we uncover core promoters by functional characterization of Pol II peaks identified by chromatin immunoprecipitation sequencing (ChIP-seq). Two distinct promoters are located between divergent PTUs, each driving unidirectional transcription. Detailed analysis identifies a 75-bp promoter that is necessary and sufficient to drive full reporter expression and contains functional motifs. Analysis of further promoters suggests transcription initiation is regulated and promoters are either focused or dispersed. In contrast to the previous model of unregulated and promoter-independent transcription initiation, we find that sequence-specific promoters determine the initiation of Pol II transcription of protein-coding genes PTUs. These findings in Trypanosoma brucei suggest that in addition of chromatin modifications, promoter motifs-based regulation of gene expression is deeply conserved among eukaryotes.
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