Paule Séité

Human tumor suppressor p14 ARF negatively regulates rRNA transcription and inhibits UBF1 transcription factor phosphorylation

The nucleolar Arf protein has been shown to regulate cell cycle through both p53-dependent and -independent pathways. In addition to the well-characterized Arf-mdm2-p53 pathway, several partners of Arf have recently been described that could participate in alternative regulation process. Among those is the nucleolar protein B23/NPM, involved in the sequential maturation of rRNA. p19ARF can interact with B23/NPM in high molecular complexes and partially inhibit the cleavage of the 32S rRNA, whereas the human p14ARF protein has been shown to participate in the degradation of NPM/B23 by the proteasome. These data led to define Arf as a negative regulator of ribosomal RNA maturation. Our recent finding that the human p14ARF protein was able to specifically interact with the rRNA promoter in a p53-independent context, led us to analyse in vitro and in vivo the consequences of this interaction. Luciferase assay and pulse-chase experiments demonstrated that the rRNA transcription was strongly reduced upon p14ARF overexpression. Investigations on potential interactions between p14ARF and the transcription machinery proteins demonstrated that the upstream binding factor (UBF), required for the initiation of the transcriptional complex, was a new partner of the p14ARF protein. We next examined the phosphorylation status of UBF as UBF phosphorylation is required to recruit on the promoter factors involved in the transcriptional complex. Upon p14ARF overexpression, UBF was found hypophosphorylated, thus unable to efficiently recruit the transcription complex. Taken together, these data define a new p53-independent pathway that could regulate cell cycle through the negative control of rRNA transcription.

Human Arf tumor suppressor specifically interacts with chromatin containing the promoter of rRNA genes

The tumor suppressor Arf (Alternative Reading Frame) protein (p14ARF in human and p19ARF in mouse) is mainly located in the nucleolus consistent with its subcellular localization, the protein has been shown to specifically interact with 5.8S rRNA and with B23/Nucleophosmin and to regulate ribosome biogenesis. Here, we show that the p14ARF protein interacts with chromatin and is recovered by chromatin immunoprecipitation (ChIP) in a fraction that contains a DNA sequence of the rRNA gene promoter. In addition, topoisomerase I (Topo I) that has been shown to interact with p14ARF coprecipitates with p14ARF containing chromatin. These data, in view of the function for Topo I in rRNA transcription, are consistent with a role for the p14ARF-Topo I complex in rRNA transcription and/or maturation.

ErbB380 kDa, a nuclear variant of the ErbB3 receptor, binds to the Cyclin D1 promoter to activate cell proliferation but is negatively controlled by p14ARF

EGFR family members are tyrosine kinase transmembrane receptors that, in response to specific extracellular ligands, activate cytoplasmic pathways involved in cell proliferation, migration and differentiation. More recently, a pivotal role for EGF receptors has emerged, through the description of their nuclear localization.We report here the characterization of a nuclear variant of the kinase-defective ErbB3 receptor, ErbB3(80 kDa), spanning the intracytoplasmic domain of the receptor. We assessed the putative transcriptional functions of ErbB3(80 KDa) in cancer cells, through the regulation of the proliferative Cyclin D1 gene, an already known target of the ErbB3 cytoplasmic signaling. We demonstrate here that the binding of ErbB3(80 KDa) on the promoter activates Cyclin D1 transcription and subsequent protein expression, leading to an increased cell proliferation. This mechanism can be balanced in response to the ectopic expression of the tumor suppressor p14ARF that physically interacts with ErbB3(100 kDa) and sequesters it into nucleoli. Our data also show that ErbB3(80 kDa) increases the transcription of proliferative genes even though the cytoplasmic pathways are not activated. This nuclear ErbB3 pathway and the target genes concerned need to be further studied. Indeed, such mechanism could explain the tumor relapse observed in response to treatments aimed at blocking the receptor activation in response to ligand binding.

Delineation of the domains required for physical and functional interaction of p14ARF with human topoisomerase I.

We recently reported an interaction between the p14ARF protein and human topoisomerase I (Topo I) resulting in the stimulation of the relaxation activity of Topo I. Our data showed that the complex between the two proteins was located within the nucleolus. In the present work, we have investigated the regions of p14ARF involved in this interaction by using targeted point mutagenesis and deletion mutants. A region encompassing exon 2-encoded sequence was required for physical binding of p14ARF to Topo I as well as for stimulatory activity of the enzyme. Exon 1β-encoded segment was not implicated in the interaction. Moreover, among p14ARF point mutants selected for their high conservation among different mammalian species, mutant p14ARF (RR87, 88AA) did not stimulate Topo I in spite of its association with the enzyme, suggesting its direct implication in the functional activity of ARF. In contrast, one mutant, p14ARF (R71A), was more efficient than wild-type protein to activate Topo I, suggesting that this residue is a key element to modulate Topo I activity. Finally, only ARF–Topo I complexes containing p14ARF exon 2 segment were found to be localized in the nucleolus, suggesting that this subnuclear location is linked to the biological function of the ARF–Topo I complex.

Atypical nuclear localization of VIP receptors in glioma cell lines and patients.

An increasing number of G protein-coupled receptors, like receptors for vasoactive intestinal peptide (VIP), are found in cell nucleus. As VIP receptors are involved in the regulation of glioma cell proliferation and migration, we investigated the expression and the nuclear localization of the VIP receptors VPAC1 and VPAC2 in this cancer. First, by applying Western blot and immunofluorescence detection in three human glioblastoma (GBM) cell lines, we observed a strong nuclear staining for the VPAC1 receptor and a weak nuclear VPAC2 receptor staining. Second, immunohistochemical staining of VPAC1 and VPAC2 on tissue microarrays (TMA) showed that the two receptors were expressed in normal brain and glioma tissues. Expression in the non-nuclear compartment of the two receptors significantly increased with the grade of the tumors. Analysis of nuclear staining revealed a significant increase of VPAC1 staining with glioma grade, with up to 50% of GBM displaying strong VPAC1 nuclear staining, whereas nuclear VPAC2 staining remained marginal. The increase in VPAC receptor expression with glioma grades and the enhanced nuclear localization of the VPAC1 receptors in GBM might be of importance for glioma progression.

Human papillomavirus 16 oncoprotein E7 stimulates UBF1-mediated rDNA gene transcription, inhibiting a p53-independent activity of p14ARF.

High-risk human papillomavirus oncoproteins E6 and E7 play a major role in HPV-related cancers. One of the main functions of E7 is the degradation of pRb, while E6 promotes the degradation of p53, inactivating the p14ARF-p53 pathway. pRb and p14ARF can repress ribosomal DNA (rDNA) transcription in part by targeting the Upstream Binding Factor 1 (UBF1), a key factor in the activation of RNA polymerase I machinery. We showed, through ectopic expression and siRNA silencing of p14ARF and/or E7, that E7 stimulates UBF1-mediated rDNA gene transcription, partly because of increased levels of phosphorylated UBF1, preventing the inhibitory function of p14ARF. Unexpectedly, activation of rDNA gene transcription was higher in cells co-expressing p14ARF and E7, compared to cells expressing E7 alone. We did not find a difference in P-UBF1 levels that could explain this data. However, p14ARF expression induced E7 to accumulate into the nucleolus, where rDNA transcription takes place, providing an opportunity for E7 to interact with nucleolar proteins involved in this process. GST-pull down and co-immunoprecipitation assays showed interactions between p14ARF, UBF1 and E7, although p14ARF and E7 are not able to directly interact. Co-expression of a pRb-binding-deficient mutant (E7C24G) and p14ARF resulted in EC24G nucleolar accumulation, but not in a significant higher activation of rDNA transcription, suggesting that the inactivation of pRb is involved in this phenomenon. Thus, p14ARF fails to prevent E7-mediated UBF1 phosphorylation, but could facilitate nucleolar pRb inactivation by targeting E7 to the nucleolus. While others have reported that p19ARF, the mouse homologue of p14ARF, inhibits some functions of E7, we showed that E7 inhibits a p53-independent function of p14ARF. These results point to a mutually functional interaction between p14ARF and E7 that might partly explain why the sustained p14ARF expression observed in most cervical pre-malignant lesions and malignancies may be ineffective.

In vitro and in vivo analysis of the interaction between 5.8S rRNA and ARF protein reveal a new difference between murine p19ARF and human p14ARF.

Both human and murine ARF proteins have been recently reported to impair rRNA maturation and ribosomes biogenesis through a p53-independent pathway. A specific interaction has been established between 5.8S rRNA and the murine p19ARF specie. We report here, by use of both in vitro and ChIP-RNA assays, the absence of any interaction between the human p14ARF and the homologous 5.8S rRNA. Our data are not consistent with the involvement of a 5.8S–p14ARF complex in ribosome biogenesis in man. Rather they suggest that the human protein does not require such an interaction to achieve a similar function. This result is a new argument in favour of functional differences between human and murine ARF proteins.

Integrated and Functional Genomics Analysis Validates the Relevance of the Nuclear Variant ErbB380kDa in Prostate Cancer Progression.

The EGF-family of tyrosine-kinase receptors activates cytoplasmic pathways involved in cell proliferation, migration and differentiation in response to specific extracellular ligands. Beside these canonical pathways, the nuclear localization of the ErbB receptors in primary tumours and cancer cell lines led to investigate their role as transcriptional regulators of cancer genes. The nuclear localization of ErbB3 has been reported in various cancer tissues and cell lines but the nuclear functions and the putative correlation with tumour progression and resistance to therapy remain unclear. We first assessed ErbB3 expression in normal and tumour prostate tissues. The nuclear staining was mainly due to an isoform matching the C-terminus domain of the full length ErbB3185kDa receptor. Nuclear staining was also restricted to cancer cells and was increased in advanced castration-resistant prostate cancer when compared to localized tumours, suggesting it could be involved in the progression of prostate cancer up to the terminal castration-resistant stage. ChIP-on-chip experiments were performed on immortalized and tumour cell lines selected upon characterization of endogenous nuclear expression of an ErbB380kDa isoform. Among the 1840 target promoters identified, 26 were selected before ErbB380kDa-dependent gene expression was evaluated by real-time quantitative RT-PCR, providing evidence that ErbB380kDa exerted transcriptional control on those genes. Some targets are already known to be involved in prostate cancer progression even though no link was previously established with ErbB3 membrane and/or nuclear signalling. Many others, not yet associated with prostate cancer, could provide new therapeutic possibilities for patients expressing ErbB380kDa. Detecting ErbB380kDa could thus constitute a useful marker of prognosis and response to therapy.