Non-coding murine centromeric transcripts associate with and potentiate Aurora B kinase

Non-coding RNAs are emerging as key players in many fundamental biological processes, including specification of higher-order chromatin structure. We examined the implication of RNA transcribed from mouse centromeric minor satellite repeats in the formation and function of centromere-associated complexes. Here we show that the levels of minor satellite RNA vary during cell-cycle progression, peaking in G2/M phase, concomitant with accumulation of proteins of the chromosomal passenger complex near the centromere. Consistent with this, we describe that murine minor satellite RNA are components of CENP-A-associated centromeric fractions and associate with proteins of the chromosomal passenger complex Aurora B and Survivin at the onset of mitosis. Interactions of endogenous Aurora B with CENP-A and Survivin are sensitive to RNaseA. Likewise, the kinase activity of Aurora B requires an RNA component. More importantly, Aurora B kinase activity can be potentiated by minor satellite RNA. In addition, decreased Aurora B activity after RNA depletion can be specifically rescued by restitution of these transcripts. Together, our data provide new functional evidence for minor satellite transcripts as key partners and regulators of the mitotic kinase Aurora B

Steroid receptor RNA activator protein binds to and counteracts SRA RNA-mediated activation of MyoD and muscle differentiation

The steroid receptor RNA activator (SRA) has the unusual property to function as both a non-coding RNA (ncRNA) and a protein SRAP. SRA ncRNA is known to increase the activity of a range of nuclear receptors as well as the master regulator of muscle differentiation MyoD. The contribution of SRA to either a ncRNA or a protein is influenced by alternative splicing of the first intron, the retention of which disrupts the SRAP open reading frame. We reported here that the ratio between non-coding and coding SRA isoforms increased during myogenic differentiation of human satellite cells but not myotonic dystrophy patient satellite cells, in which differentiation capacity is affected. Using constructs that exclusively produce SRA ncRNA or SRAP, we demonstrated that whereas SRA ncRNA was indeed an enhancer of myogenic differentiation and myogenic conversion of non-muscle cells through the co-activation of MyoD activity, SRAP prevented this SRA RNA-dependant co-activation. Interestingly, the SRAP inhibitory effect is mediated through the interaction of SRAP with its RNA counterpart via its RRM-like domain interacting with the functional sub-structure of SRA RNA, STR7. This study thus provides a new model for SRA-mediated regulation of MyoD transcriptional activity in the promotion of normal muscle differentiation, which takes into account the nature of SRA molecules present

Parp1 Localizes within the Dnmt1 Promoter and Protects Its Unmethylated State by Its Enzymatic Activity

Aberrant hypermethylation of CpG islands in housekeeping gene promoters and widespread genome hypomethylation are typical events occurring in cancer cells. The molecular mechanisms behind these cancer-related changes in DNA methylation patterns are not well understood. Two questions are particularly important: (i) how are CpG islands protected from methylation in normal cells, and how is this protection compromised in cancer cells, and (ii) how does the genome-wide demethylation in cancer cells occur. The latter question is especially intriguing since so far no DNA demethylase enzyme has been found.Our data show that the absence of ADP-ribose polymers (PARs), caused by ectopic over-expression of poly(ADP-ribose) glycohydrolase (PARG) in L929 mouse fibroblast cells leads to aberrant methylation of the CpG island in the promoter of the Dnmt1 gene, which in turn shuts down its transcription. The transcriptional silencing of Dnmt1 may be responsible for the widespread passive hypomethylation of genomic DNA which we detect on the example of pericentromeric repeat sequences. Chromatin immunoprecipitation results show that in normal cells the Dnmt1 promoter is occupied by poly(ADP-ribosyl)ated Parp1, suggesting that PARylated Parp1 plays a role in protecting the promoter from methylation.In conclusion, the genome methylation pattern following PARG over-expression mirrors the pattern characteristic of cancer cells, supporting our idea that the right balance between Parp/Parg activities maintains the DNA methylation patterns in normal cells. The finding that in normal cells Parp1 and ADP-ribose polymers localize on the Dnmt1 promoter raises the possibility that PARylated Parp1 marks those sequences in the genome that must remain unmethylated and protects them from methylation, thus playing a role in the epigenetic regulation of gene expression

The Human Proteins MBD5 and MBD6 Associate with Heterochromatin but They Do Not Bind Methylated DNA

BACKGROUND: MBD5 and MBD6 are two uncharacterized mammalian proteins that contain a putative Methyl-Binding Domain (MBD). In the proteins MBD1, MBD2, MBD4, and MeCP2, this domain allows the specific recognition of DNA containing methylated cytosine; as a consequence, the proteins serve as interpreters of DNA methylation, an essential epigenetic mark. It is unknown whether MBD5 or MBD6 also bind methylated DNA; this question has interest for basic research, but also practical consequences for human health, as MBD5 deletions are the likely cause of certain cases of mental retardation. PRINCIPAL FINDINGS: Here we report the first functional characterization of MBD5 and MBD6. We have observed that the proteins colocalize with heterochromatin in cultured cells, and that this localization requires the integrity of their MBD. However, heterochromatic localization is maintained in cells with severely decreased levels of DNA methylation. In vitro, neither MBD5 nor MBD6 binds any of the methylated sequences DNA that were tested. CONCLUSIONS: Our data suggest that MBD5 and MBD6 are unlikely to be methyl-binding proteins, yet they may contribute to the formation or function of heterochromatin. One isoform of MBD5 is highly expressed in oocytes, which suggests a possible role in epigenetic reprogramming after fertilization

Epigenetic regulation of centromeric chromatin: old dogs, new tricks?

The assembly of just a single kinetochore at the centromere of each sister chromatid is essential for accurate chromosome segregation during cell division. Surprisingly, despite their vital function, centromeres show considerable plasticity with respect to their chromosomal locations and activity. The establishment and maintenance of centromeric chromatin, and therefore the location of kinetochores, is epigenetically regulated. The histone H3 variant CENP-A is the key determinant of centromere identity and kinetochore assembly. Recent studies have identified many factors that affect CENP-A localization, but their precise roles in this process are unknown. We build on these advances and on new information about the timing of CENP-A assembly during the cell cycle to propose new models for how centromeric chromatin is established and propagated

Ebselen oxide and derivatives are new allosteric HER2 inhibitors for HER2 ‐positive cancers

International audienceHuman epidermal growth factor receptor 2 (ErbB2/HER2) is a tyrosine kinase receptor that is overexpressed in 25% of primary human breast cancers, as well as in multiple other cancers. HER2-targeted therapies improved progression-free and overall survival in patients with HER2+ breast cancers. However, associated resistance mechanisms and toxicity highlight the need for new therapeutic approaches for these cancers. We recently established that, in normal cells, HER2 is stabilized in a catalytically repressed state by direct interaction with members of the ezrin/radixin/moesin (ERM) family. In HER2-overexpressing tumors, the low expression of moesin contributes to the aberrant activation of HER2. Through a screen designed to find moesin-mimicking compounds, we identified ebselen oxide. We show that ebselen oxide, and some derivatives, conferred an efficient allosteric inhibition of overexpressed HER2, as well as mutated and truncated oncogenic forms of HER2, which are resistant to current therapies. Ebselen oxide selectively inhibited anchorage-dependent and -independent proliferation of HER2+ cancer cells and showed a significant benefit in combination with current anti-HER2 therapeutic agents. Finally, ebselen oxide significantly blocked HER2+ breast tumor progression in vivo. Collectively, these data provide evidence that ebselen oxide is a newly identified allosteric inhibitor of HER2 to be considered for therapeutic intervention on HER2+ cancers

Le Méningocoque, cet illustre méconnu

International audienceNeisseria meningitidis (meningococcus) is a Gram-negative bacterium responsible for two devastating forms of invasive diseases: purpura fulminans and meningitis. Since the first description of the epidemic nature of the illness at the dawn of the nineteenth century, the scientific knowledge of meningococcal infection has increased greatly. Major advances have been made in the management of the disease with the advent of antimicrobial therapy and the implementation of meningococcal vaccines. More recently, an extensive knowledge has been accumulated on meningococcal interaction with its human host, revealing key processes involved in disease progression and new promising therapeutic approaches.Neisseria meningitidis (méningocoque) est une bactérie à Gram négatif responsable de deux formes gravissimes de maladies invasives : le purpura fulminans et la méningite. Depuis la première description du caractère épidémique de la maladie à l'aube du 19e siècle, les connaissances scientifiques sur les infections méningococciques ont considérablement augmenté. Des progrès majeurs ont été réalisés dans la gestion de la maladie avec l'avènement des agents antimicrobiens et le développement de vaccins contre le méningocoque. De nombreuses connaissances ont récemment été accumulées sur son interaction avec l'être humain, son unique hôte, révélant les processus clés impliqués dans la progression de la maladie et de nouvelles approches thérapeutiques prometteuses