Publications by authors named "Frédéric Lessard"

21 Publications

  • Page 1 of 1

A hydride transfer complex reprograms NAD metabolism and bypasses senescence.

Mol Cell 2021 Sep;81(18):3848-3865.e19

CRCHUM, 900 Saint-Denis St, Montréal, QC H2X 0A9, Canada; Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, QC H3C 3J7, Canada. Electronic address:

Metabolic rewiring and redox balance play pivotal roles in cancer. Cellular senescence is a barrier for tumorigenesis circumvented in cancer cells by poorly understood mechanisms. We report a multi-enzymatic complex that reprograms NAD metabolism by transferring reducing equivalents from NADH to NADP. This hydride transfer complex (HTC) is assembled by malate dehydrogenase 1, malic enzyme 1, and cytosolic pyruvate carboxylase. HTC is found in phase-separated bodies in the cytosol of cancer or hypoxic cells and can be assembled in vitro with recombinant proteins. HTC is repressed in senescent cells but induced by p53 inactivation. HTC enzymes are highly expressed in mouse and human prostate cancer models, and their inactivation triggers senescence. Exogenous expression of HTC is sufficient to bypass senescence, rescue cells from complex I inhibitors, and cooperate with oncogenic RAS to transform primary cells. Altogether, we provide evidence for a new multi-enzymatic complex that reprograms metabolism and overcomes cellular senescence.
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http://dx.doi.org/10.1016/j.molcel.2021.08.028DOI Listing
September 2021

New Insights into CDK Regulators: Novel Opportunities for Cancer Therapy.

Trends Cell Biol 2021 05 3;31(5):331-344. Epub 2021 Mar 3.

Department of Biochemistry and Molecular Medicine, University of Montreal, Montreal, QC, H3C 3J7, Canada. Electronic address:

Cyclins and their catalytic partners, the cyclin-dependent kinases (CDKs), control the transition between different phases of the cell cycle. CDK/cyclin activity is regulated by CDK inhibitors (CKIs), currently comprising the CDK-interacting protein/kinase inhibitory protein (CIP/KIP) family and the inhibitor of kinase (INK) family. Recent studies have identified a third group of CKIs, called ribosomal protein-inhibiting CDKs (RPICs). RPICs were discovered in the context of cellular senescence, a stable cell cycle arrest with tumor-suppressing abilities. RPICs accumulate in the nonribosomal fraction of senescent cells due to a decrease in rRNA biogenesis. Accordingly, RPICs are often downregulated in human cancers together with other ribosomal proteins, the tumor-suppressor functions of which are still under study. In this review, we discuss unique therapies that have been developed to target CDK activity in the context of cancer treatment or senescence-associated pathologies, providing novel tools for precision medicine.
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http://dx.doi.org/10.1016/j.tcb.2021.01.010DOI Listing
May 2021

Proximity-dependent Mapping of the Androgen Receptor Identifies Kruppel-like Factor 4 as a Functional Partner.

Mol Cell Proteomics 2021 Feb 26;20:100064. Epub 2021 Feb 26.

Centre de recherche du Centre Hospitalier Universitaire (CHU) de Québec-Université Laval, Axe Oncologie, Québec, Quebec, Canada; Centre de recherche sur le cancer de l'Université Laval, Québec, Quebec, Canada; PROTEO-Quebec Network for Research on Protein Function, Engineering, and Applications, Québec, Quebec, Canada; Department of Molecular Biology, Medical Biochemistry and Pathology, Faculté de Médecine, Université Laval, Québec, Quebec, Canada. Electronic address:

Prostate cancer (PCa) is the most frequently diagnosed cancer in men and the third cause of cancer mortality. PCa initiation and growth are driven by the androgen receptor (AR). The AR is activated by androgens such as testosterone and controls prostatic cell proliferation and survival. Here, we report an AR signaling network generated using BioID proximity labeling proteomics in androgen-dependent LAPC4 cells. We identified 31 AR-associated proteins in nonstimulated cells. Strikingly, the AR signaling network increased to 182 and 200 proteins, upon 24 h or 72 h of androgenic stimulation, respectively, for a total of 267 nonredundant AR-associated candidates. Among the latter group, we identified 213 proteins that were not previously reported in databases. Many of these new AR-associated proteins are involved in DNA metabolism, RNA processing, and RNA polymerase II transcription. Moreover, we identified 44 transcription factors, including the Kru¨ppel-like factor 4 (KLF4), which were found interacting in androgen-stimulated cells. Interestingly, KLF4 repressed the well-characterized AR-dependent transcription of the KLK3 (PSA) gene; AR and KLF4 also colocalized genome-wide. Taken together, our data report an expanded high-confidence proximity network for AR, which will be instrumental to further dissect the molecular mechanisms underlying androgen signaling in PCa cells.
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http://dx.doi.org/10.1016/j.mcpro.2021.100064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8050775PMC
February 2021

The chemotherapeutic agent CX-5461 irreversibly blocks RNA polymerase I initiation and promoter release to cause nucleolar disruption, DNA damage and cell inviability.

NAR Cancer 2020 Dec 6;2(4):zcaa032. Epub 2020 Nov 6.

Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre (CRCHU de Québec-Université Laval), Québec, QC, G1R 3S3, Canada.

In the search for drugs to effectively treat cancer, the last 10 years have seen a resurgence of interest in targeting ribosome biogenesis. CX-5461 is a potential inhibitor of ribosomal RNA synthesis that is now showing promise in phase I trials as a chemotherapeutic agent for a range of malignancies. Here, we show that CX-5461 irreversibly inhibits ribosomal RNA transcription by arresting RNA polymerase I (RPI/Pol1/PolR1) in a transcription initiation complex. CX-5461 does not achieve this by preventing formation of the pre-initiation complex nor does it affect the promoter recruitment of the SL1 TBP complex or the HMGB-box upstream binding factor (UBF/UBTF). CX-5461 also does not prevent the subsequent recruitment of the initiation-competent RPI-Rrn3 complex. Rather, CX-5461 blocks promoter release of RPI-Rrn3, which remains irreversibly locked in the pre-initiation complex even after extensive drug removal. Unexpectedly, this results in an unproductive mode of RPI recruitment that correlates with the onset of nucleolar stress, inhibition of DNA replication, genome-wide DNA damage and cellular senescence. Our data demonstrate that the cytotoxicity of CX-5461 is at least in part the result of an irreversible inhibition of RPI transcription initiation and hence are of direct relevance to the design of improved strategies of chemotherapy.
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http://dx.doi.org/10.1093/narcan/zcaa032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7646227PMC
December 2020

SOCS1: phosphorylation, dimerization and tumor suppression.

Oncoscience 2019 Nov 23;6(11-12):386-389. Epub 2019 Dec 23.

Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Québec, Canada.

Suppressor of cytokine signaling (SOCS) family members are upregulated following JAK-STAT pathway activation by cytokines. SOCS proteins are recognized inhibitors of cytokine signaling playing roles in cell growth and differentiation. Moreover, SOCS1 and SOCS3 have been shown to be involved in tumor suppression through their ability to interact with p53 leading to the activation of its transcriptional program and showing the implication of SOCS family members in the regulation of apoptosis, ferroptosis and senescence. More recently, we demonstrated that the SRC family of non-receptor tyrosine kinases (SFK) can phosphorylate SOCS1 leading to its homodimerization and inhibiting its interaction with p53. Then, we reactivated the SOCS1-p53 tumor suppressor axis with the SFK inhibitor dasatinib in combination with the p53 activating compound PRIMA. This work suggests new avenues for cancer treatment and leaves open several new questions that deserve to be addressed.
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http://dx.doi.org/10.18632/oncoscience.495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6959930PMC
November 2019

NFE2L3 Controls Colon Cancer Cell Growth through Regulation of DUX4, a CDK1 Inhibitor.

Cell Rep 2019 11;29(6):1469-1481.e9

Lady Davis Institute for Medical Research, McGill University, Montreal, QC H3T 1E2, Canada; Department of Medicine, McGill University, Montreal, QC H3T 1E2, Canada; Department of Physiology, McGill University, Montreal, QC H3T 1E2, Canada. Electronic address:

Constitutive nuclear factor κB (NF-κB) activation is a hallmark of colon tumor growth. Cyclin-dependent kinases (CDKs) are critical cell-cycle regulators, and inhibition of CDK activity has been used successfully as anticancer therapy. Here, we show that the NFE2L3 transcription factor functions as a key regulator in a pathway that links NF-κB signaling to the control of CDK1 activity, thereby driving colon cancer cell proliferation. We found that NFE2L3 expression is regulated by the RELA subunit of NF-κB and that NFE2L3 levels are elevated in patients with colon adenocarcinoma when compared with normal adjacent tissue. Silencing of NFE2L3 significantly decreases colon cancer cell proliferation in vitro and tumor growth in vivo. NFE2L3 knockdown results in increased levels of double homeobox factor 4 (DUX4), which functions as a direct inhibitor of CDK1. The discovered oncogenic pathway governing cell-cycle progression may open up unique avenues for precision cancer therapy.
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http://dx.doi.org/10.1016/j.celrep.2019.09.087DOI Listing
November 2019

The Short N-Terminal Repeats of Transcription Termination Factor 1 Contain Semi-Redundant Nucleolar Localization Signals and P19-ARF Tumor Suppressor Binding Sites.

Yale J Biol Med 2019 09 20;92(3):385-396. Epub 2019 Sep 20.

Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre (Axe Cancer, CR-CHU de Québec), Quebec, QC, Canada.

The p14/p19 (ARF) tumor suppressor provides an important link in the activation of p53 (TP53) by inhibiting its targeted degradation via the E3 ligases MDM2/HDM2. However, ARF also limits tumor growth by directly inhibiting ribosomal RNA synthesis and processing. Initial studies of the ARF tumor suppressor were compounded by overlap between the INK4A and ARF genes encoded by the CDKN2A locus, but mouse models of pure ARF-loss and its inactivation in human cancers identified it as a distinct tumor suppressor even in the absence of p53. We previously demonstrated that both human and mouse ARF interact with Transcription Termination Factor 1 (TTF1, TTF-I), an essential factor implicated in transcription termination and silencing of the ribosomal RNA genes. Accumulation of ARF upon oncogenic stress was shown to inhibit ribosomal RNA synthesis by depleting nucleolar TTF1. Here we have mapped the functional nucleolar localization sequences (NoLS) of mouse TTF1 and the sequences responsible for interaction with ARF. We find that both sequences lie within the 25 amino acid N-terminal repeats of TTF1. Nucleolar localization depends on semi-redundant lysine-arginine motifs in each repeat and to a minor extent on binding to target DNA sequences by the Myb homology domain of TTF1. While nucleolar localization of TTF1 predominantly correlates with its interaction with ARF, NoLS activity and ARF binding are mediated by distinct sequences within each N-terminal repeat. The data suggest that the N-terminal repeats of mouse TTF1, and by analogy those of human TTF1, cooperate to mediate both nucleolar localization and ARF binding.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6747939PMC
September 2019

Phosphorylation of SOCS1 Inhibits the SOCS1-p53 Tumor Suppressor Axis.

Cancer Res 2019 07 17;79(13):3306-3319. Epub 2019 May 17.

Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Québec, Canada.

Expression of the suppressor of cytokine signaling-1 (SOCS1) is inactivated in hematopoietic and solid cancers by promoter methylation, miRNA-mediated silencing, and mutations. Paradoxically, SOCS1 is also overexpressed in many human cancers. We report here that the ability of SOCS1 to interact with p53 and regulate cellular senescence depends on a structural motif that includes tyrosine (Y)80 in the SH2 domain of SOCS1. Mutations in this motif are found at low frequency in some human cancers, and substitution of Y80 by a phosphomimetic residue inhibits p53-SOCS1 interaction and its functional consequences, including stimulation of p53 transcriptional activity, growth arrest, and cellular senescence. Mass spectrometry confirmed SOCS1 Y80 phosphorylation in cells, and a new mAb was generated to detect its presence in tissues by IHC. A tyrosine kinase library screen identified the SRC family as Y80-SOCS1 kinases. SRC family kinase inhibitors potentiated the SOCS1-p53 pathway and reinforced SOCS1-induced senescence. Samples from human lymphomas that often overexpress SOCS1 also displayed SRC family kinase activation, constitutive phosphorylation of SOCS1 on Y80, and SOCS1 cytoplasmic localization. Collectively, these results reveal a mechanism that inactivates the SOCS1-p53 senescence pathway and suggest that inhibition of SRC family kinases as personalized treatment in patients with lymphomas may be successful. SIGNIFICANCE: These findings show that SOCS1 phosphorylation by the SRC family inhibits its tumor-suppressive activity, indicating that patients with increased SOCS1 phosphorylation may benefit from SRC family kinase inhibitors.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-1503DOI Listing
July 2019

Ribosomal protein RPL22/eL22 regulates the cell cycle by acting as an inhibitor of the CDK4-cyclin D complex.

Cell Cycle 2019 Mar - Apr;18(6-7):759-770. Epub 2019 Mar 28.

a Department of Biochemistry and Molecular Medicine , Université de Montréal , Montréal , Québec , Canada.

Senescence is a tumor suppressor program characterized by a stable growth arrest while maintaining cell viability. Senescence-associated ribogenesis defects (SARD) have been shown to regulate senescence through the ability of the ribosomal protein S14 (RPS14 or uS11) to bind and inhibit the cyclin-dependent kinase 4 (CDK4). Here we report another ribosomal protein that binds and inhibits CDK4 in senescent cells: L22 (RPL22 or eL22). Enforcing the expression of RPL22/eL22 is sufficient to induce an RB and p53-dependent cellular senescent phenotype in human fibroblasts. Mechanistically, RPL22/eL22 can interact with and inhibit CDK4-Cyclin D1 to decrease RB phosphorylation both in vitro and in cells. Briefly, we show that ribosome-free RPL22/eL22 causes a cell cycle arrest which could be relevant during situations of nucleolar stress such as cellular senescence or the response to cancer chemotherapy.
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http://dx.doi.org/10.1080/15384101.2019.1593708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6464582PMC
April 2020

Ribosomal Proteins Control Tumor Suppressor Pathways in Response to Nucleolar Stress.

Bioessays 2019 03 1;41(3):e1800183. Epub 2019 Feb 1.

Department of Biochemistry and Molecular Medicine, Université de Montréal, C.P. 6128, Succ. Centre-Ville, Montréal, Québec H3C 3J7, Canada.

Ribosome biogenesis includes the making and processing of ribosomal RNAs, the biosynthesis of ribosomal proteins from their mRNAs in the cytosol and their transport to the nucleolus to assemble pre-ribosomal particles. Several stresses including cellular senescence reduce nucleolar rRNA synthesis and maturation increasing the availability of ribosome-free ribosomal proteins. Several ribosomal proteins can activate the p53 tumor suppressor pathway but cells without p53 can still arrest their proliferation in response to an imbalance between ribosomal proteins and mature rRNA production. Recent results on senescence-associated ribogenesis defects (SARD) show that the ribosomal protein S14 (RPS14 or uS11) can act as a CDK4/6 inhibitor linking ribosome biogenesis defects to the main engine of cell cycle progression. This work offers new insights into the regulation of the cell cycle and suggests novel avenues to design anticancer drugs.
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http://dx.doi.org/10.1002/bies.201800183DOI Listing
March 2019

Senescence-associated ribosome biogenesis defects contributes to cell cycle arrest through the Rb pathway.

Nat Cell Biol 2018 07 25;20(7):789-799. Epub 2018 Jun 25.

Department of Biochemistry and Molecular Medicine, Université de Montréal, Montreal, Quebec, Canada.

Cellular senescence is a tumour suppressor programme characterized by a stable cell cycle arrest. Here we report that cellular senescence triggered by a variety of stimuli leads to diminished ribosome biogenesis and the accumulation of both rRNA precursors and ribosomal proteins. These defects were associated with reduced expression of several ribosome biogenesis factors, the knockdown of which was also sufficient to induce senescence. Genetic analysis revealed that Rb but not p53 was required for the senescence response to altered ribosome biogenesis. Mechanistically, the ribosomal protein S14 (RPS14 or uS11) accumulates in the soluble non-ribosomal fraction of senescent cells, where it binds and inhibits CDK4 (cyclin-dependent kinase 4). Overexpression of RPS14 is sufficient to inhibit Rb phosphorylation, inducing cell cycle arrest and senescence. Here we describe a mechanism for maintaining the senescent cell cycle arrest that may be relevant for cancer therapy, as well as biomarkers to identify senescent cells.
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http://dx.doi.org/10.1038/s41556-018-0127-yDOI Listing
July 2018

A CDK4/6-Dependent Epigenetic Mechanism Protects Cancer Cells from PML-induced Senescence.

Cancer Res 2016 06 29;76(11):3252-64. Epub 2016 Mar 29.

Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Québec, Canada.

Promyelocytic leukemia (PML) plays a tumor suppressive role by inducing cellular senescence in response to oncogenic stress. However, tumor cell lines fail to engage in complete senescence upon PML activation. In this study, we investigated the mechanisms underlying resistance to PML-induced senescence. Here, we report that activation of the cyclin-dependent kinases CDK4 and CDK6 are essential and sufficient to impair senescence induced by PML expression. Disrupting CDK function by RNA interference or pharmacological inhibition restored senescence in tumor cells and diminished their tumorigenic potential in mouse xenograft models. Complete senescence correlated with an increase in autophagy, repression of E2F target genes, and an gene expression signature of blocked DNA methylation. Accordingly, treatment of tumor cells with inhibitors of DNA methylation reversed resistance to PML-induced senescence. Further, CDK inhibition with palbociclib promoted autophagy-dependent degradation of the DNA methyltransferase DNMT1. Lastly, we found that CDK4 interacted with and phosphorylated DNMT1 in vitro, suggesting that CDK activity is required for its stabilization. Taken together, our findings highlight a potentially valuable feature of CDK4/6 inhibitors as epigenetic modulators to facilitate activation of senescence programs in tumor cells. Cancer Res; 76(11); 3252-64. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-2347DOI Listing
June 2016

Permanent farnesylation of lamin A mutants linked to progeria impairs its phosphorylation at serine 22 during interphase.

Aging (Albany NY) 2016 Feb;8(2):366-81

Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Québec, H3C 3J7, Canada.

Mutants of lamin A cause diseases including the Hutchinson-Gilford progeria syndrome (HGPS) characterized by premature aging. Lamin A undergoes a series of processing reactions, including farnesylation and proteolytic cleavage of the farnesylated C-terminal domain. The role of cleavage is unknown but mutations that affect this reaction lead to progeria. Here we show that interphase serine 22 phosphorylation of endogenous mutant lamin A (progerin) is defective in cells from HGPS patients. This defect can be mimicked by expressing progerin in human cells and prevented by inhibition of farnesylation. Furthermore, serine 22 phosphorylation of non-farnesylated progerin was enhanced by a mutation that disrupts lamin A head to tail interactions. The phosphorylation of lamin A or non-farnesylated progerin was associated to the formation of spherical intranuclear lamin A droplets that accumulate protein kinases of the CDK family capable of phosphorylating lamin A at serine 22. CDK inhibitors compromised the turnover of progerin, accelerated senescence of HGPS cells and reversed the effects of FTI on progerin levels. We discuss a model of progeria where faulty serine 22 phosphorylation compromises phase separation of lamin A polymers, leading to accumulation of functionally impaired lamin A structures.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4789588PMC
http://dx.doi.org/10.18632/aging.100903DOI Listing
February 2016

Mutant lamin A links prophase to a p53 independent senescence program.

Cell Cycle 2015 Aug 1;14(15):2408-21. Epub 2015 Jun 1.

a Département de Biochimie ; Université de Montréal ; C.P. 6128; Succ. Center-Ville; Montréal , QC Canada.

Expression of oncogenes or short telomeres can trigger an anticancer response known as cellular senescence activating the p53 and RB tumor suppressor pathways. This mechanism is switched off in most tumor cells by mutations in p53 and RB signaling pathways. Surprisingly, p53 disabled tumor cells could be forced into senescence by expression of a mutant allele of the nuclear envelope protein lamin A. The pro-senescence lamin A mutant contains a deletion in the sequence required for processing by the protease ZMPSTE24 leading to accumulation of farnesylated lamin A in the nuclear envelope. In addition, the serine at position 22, a target for CDK1-dependent phosphorylation, was mutated to alanine, preventing CDK1-catalyzed nuclear envelope disassembly. The accumulation of this mutant lamin A compromised prophase to prometaphase transition leading to invaginations of the nuclear lamina, nuclear fragmentation and impaired chromosome condensation. Cells exited this impaired mitosis without cytokinesis and re-replicated their DNA ultimately arresting in interphase as polyploid cells with features of cellular senescence including increased expression of inflammatory gene products and a significant reduction of tumorigenicity in vivo.
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http://dx.doi.org/10.1080/15384101.2015.1053671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4614274PMC
August 2015

Conditional inactivation of Upstream Binding Factor reveals its epigenetic functions and the existence of a somatic nucleolar precursor body.

PLoS Genet 2014 Aug 14;10(8):e1004505. Epub 2014 Aug 14.

Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre, Edifice St Patrick, Québec, Québec, Canada; Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Laval University, Québec, Québec, Canada.

Upstream Binding Factor (UBF) is a unique multi-HMGB-box protein first identified as a co-factor in RNA polymerase I (RPI/PolI) transcription. However, its poor DNA sequence selectivity and its ability to generate nucleosome-like nucleoprotein complexes suggest a more generalized role in chromatin structure. We previously showed that extensive depletion of UBF reduced the number of actively transcribed ribosomal RNA (rRNA) genes, but had little effect on rRNA synthesis rates or cell proliferation, leaving open the question of its requirement for RPI transcription. Using gene deletion in mouse, we now show that UBF is essential for embryo development beyond morula. Conditional deletion in cell cultures reveals that UBF is also essential for transcription of the rRNA genes and that it defines the active chromatin conformation of both gene and enhancer sequences. Loss of UBF prevents formation of the SL1/TIF1B pre-initiation complex and recruitment of the RPI-Rrn3/TIF1A complex. It is also accompanied by recruitment of H3K9me3, canonical histone H1 and HP1α, but not by de novo DNA methylation. Further, genes retain penta-acetyl H4 and H2A.Z, suggesting that even in the absence of UBF the rRNA genes can maintain a potentially active state. In contrast to canonical histone H1, binding of H1.4 is dependent on UBF, strongly suggesting that it plays a positive role in gene activity. Unexpectedly, arrest of rRNA synthesis does not suppress transcription of the 5S, tRNA or snRNA genes, nor expression of the several hundred mRNA genes implicated in ribosome biogenesis. Thus, rRNA gene activity does not coordinate global gene expression for ribosome biogenesis. Loss of UBF also unexpectedly induced the formation in cells of a large sub-nuclear structure resembling the nucleolar precursor body (NPB) of oocytes and early embryos. These somatic NPBs contain rRNA synthesis and processing factors but do not associate with the rRNA gene loci (NORs).
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http://dx.doi.org/10.1371/journal.pgen.1004505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4133168PMC
August 2014

Cellular senescence and protein degradation: breaking down cancer.

Cell Cycle 2014 27;13(12):1840-58. Epub 2014 May 27.

Department of Biochemistry and Molecular Medicine; Université de Montréal; Montréal, Québec, Canada.

Autophagy and the ubiquitin-proteasome pathway (UPP) are the major protein degradation systems in eukaryotic cells. Whereas the former mediate a bulk nonspecific degradation, the UPP allows a rapid degradation of specific proteins. Both systems have been shown to play a role in tumorigenesis, and the interest in developing therapeutic agents inhibiting protein degradation is steadily growing. However, emerging data point to a critical role for autophagy in cellular senescence, an established tumor suppressor mechanism. Recently, a selective protein degradation process mediated by the UPP was also shown to contribute to the senescence phenotype. This process is tightly regulated by E3 ubiquitin ligases, deubiquitinases, and several post-translational modifications of target proteins. Illustrating the complexity of UPP, more than 600 human genes have been shown to encode E3 ubiquitin ligases, a number which exceeds that of the protein kinases. Nevertheless, our knowledge of proteasome-dependent protein degradation as a regulated process in cellular contexts such as cancer and senescence remains very limited. Here we discuss the implications of protein degradation in senescence and attempt to relate this function to the protein degradation pattern observed in cancer cells.
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http://dx.doi.org/10.4161/cc.29335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4111748PMC
January 2015

Tumor suppressor activity of the ERK/MAPK pathway by promoting selective protein degradation.

Genes Dev 2013 Apr 18;27(8):900-15. Epub 2013 Apr 18.

Département de Biochimie, Université de Montréal, Montréal, Québec H3C 3J7, Canada.

Constitutive activation of growth factor signaling pathways paradoxically triggers a cell cycle arrest known as cellular senescence. In primary cells expressing oncogenic ras, this mechanism effectively prevents cell transformation. Surprisingly, attenuation of ERK/MAP kinase signaling by genetic inactivation of Erk2, RNAi-mediated knockdown of ERK1 or ERK2, or MEK inhibitors prevented the activation of the senescence mechanism, allowing oncogenic ras to transform primary cells. Mechanistically, ERK-mediated senescence involved the proteasome-dependent degradation of proteins required for cell cycle progression, mitochondrial functions, cell migration, RNA metabolism, and cell signaling. This senescence-associated protein degradation (SAPD) was observed not only in cells expressing ectopic ras, but also in cells that senesced due to short telomeres. Individual RNAi-mediated inactivation of SAPD targets was sufficient to restore senescence in cells transformed by oncogenic ras or trigger senescence in normal cells. Conversely, the anti-senescence viral oncoproteins E1A, E6, and E7 prevented SAPD. In human prostate neoplasms, high levels of phosphorylated ERK were found in benign lesions, correlating with other senescence markers and low levels of STAT3, one of the SAPD targets. We thus identified a mechanism that links aberrant activation of growth signaling pathways and short telomeres to protein degradation and cellular senescence.
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http://dx.doi.org/10.1101/gad.203984.112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3650227PMC
April 2013

The cellular abundance of the essential transcription termination factor TTF-I regulates ribosome biogenesis and is determined by MDM2 ubiquitinylation.

Nucleic Acids Res 2012 Jul 1;40(12):5357-67. Epub 2012 Mar 1.

Cancer Research Centre and Department of Molecular Biology, Medical Biochemistry and Pathology of Laval University, CHUQ Research Centre, Pavillon St Patrick, 9 rue McMahon, Québec, G1R 3S3 Québec, Canada.

The ARF tumour suppressor stabilizes p53 by negatively regulating the E3 ubiquitin ligase MDM2 to promote cell cycle arrest and cell death. However, ARF is also able to arrest cell proliferation by inhibiting ribosome biogenesis. In greater part this is achieved by targeting the transcription termination factor I (TTF-I) for nucleolar export, leading to an inhibition of both ribosomal RNA synthesis and processing. We now show that in the absence of ARF, TTF-I is ubiquitinylated by MDM2. MDM2 interacts directly with TTF-I and regulates its cellular abundance by targeting it for degradation by the proteasome. Enhanced TTF-I levels inhibit ribosome biogenesis by suppressing ribosomal RNA synthesis and processing, strongly suggesting that exact TTF-I levels are critical for efficient ribosome biogenesis. We further show that concomitant with its ability to displace TTF-I from the nucleolus, ARF inhibits MDM2 ubiquitinylation of TTF-I by competitively binding to a site overlapping the MDM2 interaction site. Thus, both the sub-nuclear localization and the abundance of TTF-I are key regulators of ribosome biogenesis.
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http://dx.doi.org/10.1093/nar/gks198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3384320PMC
July 2012

A model for the topology of active ribosomal RNA genes.

EMBO Rep 2011 Mar 18;12(3):231-7. Epub 2011 Feb 18.

Science Faculty, Department of Molecular Biology, Nijmegen Centre for Molecular Life Sciences, Radboud University (274), PO Box 9101, Nijmegen 6500 HB, The Netherlands.

The Christmas tree view of active ribosomal RNA (rRNA) genes suggests a gene topology in which a large number of nascent rRNA transcripts are prevented from intertwining. The way in which this is achieved has remained unclear. By using a combination of chromatin immunoprecipitation and chromosome conformation capture techniques, we show that the promoter, upstream region and terminator R3 of active rRNA genes are held together spatially throughout the cell cycle, forming a stable core around which the transcribed region is organized. We suggest a new core-helix model for the topology of rRNA genes, that provides a structural basis for the productive synthesis or rRNA.
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http://dx.doi.org/10.1038/embor.2011.8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059908PMC
March 2011

The ARF tumor suppressor controls ribosome biogenesis by regulating the RNA polymerase I transcription factor TTF-I.

Mol Cell 2010 May;38(4):539-50

Cancer Research Centre, Department of Molecular Biology, Medical Biochemistry, and Pathology of Laval University, CHUQ Research Centre, Québec, Québec, Canada.

The p14/p19(ARF) (ARF) product of the CDKN2A gene displays tumor suppressor activity both in the presence and absence of p53/TP53. In p53-negative cells, ARF arrests cell proliferation, at least in part, by suppressing ribosomal RNA synthesis. We show that ARF does this by controlling the subnuclear localization of the RNA polymerase I transcription termination factor, TTF-I. TTF-I shuttles between nucleoplasm and nucleolus with the aid of the chaperone NPM/B23 and a nucleolar localization sequence within its N-terminal regulatory domain. ARF inhibits nucleolar import of TTF-I by binding to this nucleolar localization sequence, causing the accumulation of TTF-I in the nucleoplasm. Depletion of TTF-I recapitulates the effects of ARF on ribosomal RNA synthesis and is rescued by the introduction of a TTF-I transgene. Thus, our data delineate the pathway by which ARF regulates ribosomal RNA synthesis and provide a compelling explanation for the role of NPM.
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http://dx.doi.org/10.1016/j.molcel.2010.03.015DOI Listing
May 2010

Loss of human ribosomal gene CpG methylation enhances cryptic RNA polymerase II transcription and disrupts ribosomal RNA processing.

Mol Cell 2009 Aug;35(4):414-25

Cancer Research Centre, CHUQ-HDQ Research Centre, Pavillon St. Patrick, 9 Rue McMahon, Québec, QC G1R 3S3, Canada.

Epigenetic methyl-CpG silencing of the ribosomal RNA (rRNA) genes is thought to downregulate rRNA synthesis in mammals. In contrast, we now show that CpG methylation in fact positively influences rRNA synthesis and processing. Human HCT116 cells, inactivated for DNMT1 and DNMT3b or treated with aza-dC, lack CpG methylation and reactivate a large fraction of normally silent rRNA genes. Unexpectedly, these cells display reduced rRNA synthesis and processing and accumulate unprocessed 45S rRNA. Reactivation of the rRNA genes is associated with their cryptic transcription by RNA polymerase II. Ectopic expression of cryptic rRNA gene transcripts recapitulates the defects associated with loss of CpG methylation. The data demonstrate that rRNA gene silencing prevents cryptic RNA polymerase II transcription of these genes. Lack of silencing leads to the partial disruption of rRNA synthesis and rRNA processing, providing an explanation for the cytotoxic effects of loss of CpG methylation.
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http://dx.doi.org/10.1016/j.molcel.2009.07.008DOI Listing
August 2009
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