Publications de l’équipe

Résumé

Résumé

Mdm4, a protein related to the ubiquitin-ligase Mdm2, is an essential inhibitor of tumor suppressor protein p53. In both human and mouse cells, the Mdm4 gene encodes two major transcripts: one encodes the full-length oncoprotein (designated below as Mdm4-FL), whereas the other, resulting from a variant splicing that skips exon 6, encodes the shorter isoform Mdm4-S. Importantly, increased Mdm4-S mRNA levels were observed in several human cancers, and correlated with poor survival. However, the role of Mdm4-S in cancer progression remains controversial, because the Mdm4-S protein appeared to be a potent p53 inhibitor when overexpressed, but the splice variant also leads to a decrease in Mdm4-FL expression. To unambiguously determine the physiological impact of the Mdm4-S splice variant, we generated a mouse model with a targeted deletion of the Mdm4 exon 6, thereby creating an obligatory exon skipping. The mutant allele (Mdm4(ΔE6)) prevented the expression of Mdm4-FL, but also led to increased Mdm4-S mRNA levels. Mice homozygous for this allele died during embryonic development, but were rescued by a concomitant p53 deficiency. Furthermore in a hypomorphic p53(ΔP/ΔP) context, the Mdm4(ΔE6) allele led to p53 activation and delayed the growth of oncogene-induced tumors. We next determined the effect of Mdm4(+/ΔE6) heterozygosity in a hypermorphic p53(+/Δ31) genetic background, recently shown to be extremely sensitive to Mdm4 activity. Mdm4(+/ΔE6) p53(+/Δ31) pups were born, but suffered from aplastic anemia and died before weaning, again indicating an increased p53 activity. Our results demonstrate that the main effect of a skipping of Mdm4 exon 6 is not the synthesis of the Mdm4-S protein, but rather a decrease in Mdm4-FL expression. These and other data suggest that increased Mdm4-S mRNA levels might correlate with more aggressive cancers without encoding significant amounts of a potential oncoprotein. Hypotheses that may account for this apparent paradox are discussed.

Résumé

Human pluripotent stem cells (hPSCs) display extensive epigenetic instability, particularly on the X chromosome. In this study, we show that, in hPSCs, the inactive X chromosome has a specific heterochromatin landscape that predisposes it to erosion of X chromosome inactivation (XCI), a process that occurs spontaneously in hPSCs. Heterochromatin remodeling and gene reactivation occur in a non-random fashion and are confined to specific H3K27me3-enriched domains, leaving H3K9me3-marked regions unaffected. Using single-cell monitoring of XCI erosion, we show that this instability only occurs in pluripotent cells. We also provide evidence that loss of XIST expression is not the primary cause of XCI instability and that gene reactivation from the inactive X (Xi) precedes loss of XIST coating. Notably, expression and coating by the long non-coding RNA XACT are early events in XCI erosion and, therefore, may play a role in mediating this process.

Résumé

Hoyeraal-Hreidarsson syndrome (HHS) is a severe form of Dyskeratosis congenita characterized by developmental defects, bone marrow failure and immunodeficiency and has been associated with telomere dysfunction. Recently, mutations in Regulator of Telomere ELongation helicase 1 (RTEL1), a helicase first identified in Mus musculus as being responsible for the maintenance of long telomeres, have been identified in several HHS patients. Here we show that RTEL1 is required for the export and the correct cytoplasmic trafficking of the small nuclear (sn) RNA pre-U2, a component of the major spliceosome complex. RTEL1-HHS cells show abnormal subcellular partitioning of pre-U2, defects in the recycling of ribonucleotide proteins (RNP) in the cytoplasm and splicing defects. While most of these phenotypes can be suppressed by re-expressing the wild-type protein in RTEL1-HHS cells, expression of RTEL1 mutated variants in immortalized cells provokes cytoplasmic mislocalizations of pre-U2 and other RNP components, as well as splicing defects, thus phenocopying RTEL1-HHS cellular defects. Strikingly, expression of a cytoplasmic form of RTEL1 is sufficient to correct RNP mislocalizations both in RTEL1-HHS cells and in cells expressing nuclear mutated forms of RTEL1. This work unravels completely unanticipated roles for RTEL1 in RNP trafficking and strongly suggests that defects in RNP biogenesis pathways contribute to the pathology of HHS.

Résumé

Eukaryotic cells respond to DNA double-strand breaks (DSBs) by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA), which arises upon nucleolytic degradation (resection) of the DSB. Emerging evidences indicate that RNA-processing factors play critical, yet poorly understood, roles in genomic stability. Here, we provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. The lack of Xrn1 inhibits ssDNA generation at the DSB by preventing the loading of the MRX complex. By contrast, DSB resection is not affected in the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by homologous recombination (HR), suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Noteworthy, deep transcriptome analyses do not identify common misregulated gene expression that could explain the observed phenotypes. Our results provide a novel link between RNA processing and genome stability.