Mutant mice lacking alternatively spliced p53 isoforms unveil Ackr4 as a male-specific prognostic factor in Myc-driven B-cell lymphomas.

The Trp53 gene encodes several isoforms of elusive biological significance. Here, we show that mice lacking the Trp53 alternatively spliced (AS) exon, thereby expressing the canonical p53 protein but not isoforms with the AS C-terminus, have unexpectedly lost a male-specific protection against Myc-induced B-cell lymphomas. Lymphomagenesis was delayed in Trp53 +/+Eμ-Myc males compared to Trp53 ΔAS/ΔAS Eμ-Myc males, but also compared to Trp53 +/+Eμ-Myc and Trp53 ΔAS/ΔAS Eμ-Myc females. Pre-tumoral splenic cells from Trp53 +/+Eμ-Myc males exhibited a higher expression of Ackr4, encoding an atypical chemokine receptor with tumor suppressive effects. We identified Ackr4 as a p53 target gene whose p53-mediated transactivation is inhibited by estrogens, and as a male-specific factor of good prognosis relevant for murine Eμ-Myc-induced and human Burkitt lymphomas. Furthermore, the knockout of ACKR4 increased the chemokine-guided migration of Burkitt lymphoma cells. These data demonstrate the functional relevance of alternatively spliced p53 isoforms and reveal sex disparities in Myc-driven lymphomagenesis. </p

Mécanismes de réparations d’une cassure double-brin et résection au sein d’un microsatellite humain

Microsatellites are tandem repeats of a motif between one and nine base pairs. These repeats are found ubiquitously in all organisms and are particularly abundant in eukaryotic organisms. All these repeats are capable of forming secondary structures in vitro and possibly in vivo. Some microsatellites are prone to expansion, leading to many neurodegenerative diseases in humans such as myotonic dystrophy type 1 (DM1), the most frequently transmitted neurodegenerative disease. The onset and severity of symptoms are positively correlated with the number of repeats located in the 3'UTR of the DMPK gene. In previous work in the laboratory, a TALE nuclease (TALEN) was developed to introduce a double-strand break into a microsatellite (GTC)n from a DM1 patient. Understanding the mechanisms leading to repeat contraction in yeast is necessary to understand the mechanisms in humans. Thus, experiments were conducted in cells with altered CBD repair systems showing that RAD51, POL32 and DNL4 were not required for CBD repair within microsatellites. Only RAD50 and RAD52 appear to be required, indicating that the cell repairs CBDs in repeated regions by single-strand annealing. The objective of this thesis was to study the role of several genes (MRE11, EXO1, SGS1, DNA2, SAE2, RIF1 and RIF2), involved in the resection and repair of a single CBD within a CTG repeat region, in yeast.Les microsatellites sont des répétitions en tandem d’un motif compris entre une et neuf paires de bases. Ces répétitions retrouvées dans tous les organismes de façon ubiquitaire, sont particulièrement abondantes dans les organismes eucaryotes. Toutes ces répétitions sont capables de former des structures secondaires in vitro et possiblement in vivo. Certains microsatellites sont enclins à une expansion, conduisant à de nombreuses maladies neurodégénératives chez l’homme telle que la dystrophie myotonique de type 1 (DM1), maladie neurodégénérative la plus fréquemment transmise. L’apparition et la sévérité des symptômes sont positivement corrélée avec le nombre de répétitions, localisées dans le 3’UTR du gène DMPK. Dans des travaux précédents du laboratoire, une TALE nucléase (TALEN) a été élaborée dans le but d’introduire une cassure double-brin au sein d’un microsatellite (CTG)n provenant d’un patient DM1. La compréhension des mécanismes conduisant à la contraction des répétitions chez la levure est nécessaire si l’on souhaite en comprendre les mécanismes chez l’homme. Ainsi, des expériences ont été menées dans des cellules dont les systèmes de réparation des CDB ont été altérés, montrant que RAD51, POL32 et DNL4 n’étaient pas nécessaires à la réparation des CDB au sein des microsatellites. Seul RAD50 et RAD52 semblent nécessaires, indiquant que la cellule répare les CDB dans les régions répétées par single-strand annealing. L’objectif de cette thèse a été d’étudier le rôle de plusieurs gènes (MRE11, EXO1, SGS1, DNA2, SAE2, RIF1 et RIF2), impliqués dans la résection et la réparation d’une unique CDB au sein d’une région répétée CTG, chez la levure

Mécanismes de réparations d’une cassure double-brin et résection au sein d’un microsatellite humain

Microsatellites are tandem repeats of a motif between one and nine base pairs. These repeats are found ubiquitously in all organisms and are particularly abundant in eukaryotic organisms. All these repeats are capable of forming secondary structures in vitro and possibly in vivo. Some microsatellites are prone to expansion, leading to many neurodegenerative diseases in humans such as myotonic dystrophy type 1 (DM1), the most frequently transmitted neurodegenerative disease. The onset and severity of symptoms are positively correlated with the number of repeats located in the 3'UTR of the DMPK gene. In previous work in the laboratory, a TALE nuclease (TALEN) was developed to introduce a double-strand break into a microsatellite (GTC)n from a DM1 patient. Understanding the mechanisms leading to repeat contraction in yeast is necessary to understand the mechanisms in humans. Thus, experiments were conducted in cells with altered CBD repair systems showing that RAD51, POL32 and DNL4 were not required for CBD repair within microsatellites. Only RAD50 and RAD52 appear to be required, indicating that the cell repairs CBDs in repeated regions by single-strand annealing. The objective of this thesis was to study the role of several genes (MRE11, EXO1, SGS1, DNA2, SAE2, RIF1 and RIF2), involved in the resection and repair of a single CBD within a CTG repeat region, in yeast.Les microsatellites sont des répétitions en tandem d’un motif compris entre une et neuf paires de bases. Ces répétitions retrouvées dans tous les organismes de façon ubiquitaire, sont particulièrement abondantes dans les organismes eucaryotes. Toutes ces répétitions sont capables de former des structures secondaires in vitro et possiblement in vivo. Certains microsatellites sont enclins à une expansion, conduisant à de nombreuses maladies neurodégénératives chez l’homme telle que la dystrophie myotonique de type 1 (DM1), maladie neurodégénérative la plus fréquemment transmise. L’apparition et la sévérité des symptômes sont positivement corrélée avec le nombre de répétitions, localisées dans le 3’UTR du gène DMPK. Dans des travaux précédents du laboratoire, une TALE nucléase (TALEN) a été élaborée dans le but d’introduire une cassure double-brin au sein d’un microsatellite (CTG)n provenant d’un patient DM1. La compréhension des mécanismes conduisant à la contraction des répétitions chez la levure est nécessaire si l’on souhaite en comprendre les mécanismes chez l’homme. Ainsi, des expériences ont été menées dans des cellules dont les systèmes de réparation des CDB ont été altérés, montrant que RAD51, POL32 et DNL4 n’étaient pas nécessaires à la réparation des CDB au sein des microsatellites. Seul RAD50 et RAD52 semblent nécessaires, indiquant que la cellule répare les CDB dans les régions répétées par single-strand annealing. L’objectif de cette thèse a été d’étudier le rôle de plusieurs gènes (MRE11, EXO1, SGS1, DNA2, SAE2, RIF1 et RIF2), impliqués dans la résection et la réparation d’une unique CDB au sein d’une région répétée CTG, chez la levure

A fast, sensitive and cost-effective method for nucleic acid detection using non-radioactive probes

International audienceNucleic acid detection and quantification using a labeled DNA probe is a very common molecular biology procedure. Here, we describe a new method, based on commonly used laboratory solutions, for nucleic acid hybridization and detection with digoxigenin-labeled DNA probes. The protocol described is faster, more sensitive and much cheaper than a standard protocol using commercial solutions. Comparison with a classical radioactive detection method shows that the latter exhibits less background and shows a greater linear response. Hence, the proposed protocol may be routinely performed for qualitative detection of nucleic acid, but when precise signal quantitation needs to be obtained, radioactive probe hybridization associated to phosphorimaging technology is more reliable

Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal deletions

International audienceMicrosatellites are short tandem repeats, ubiquitous in all eukaryotes and represent ~2% of the human genome. Among them, trinucleotide repeats are responsible for more than two dozen neurological and developmental disorders. Targeting microsatellites with dedicated DNA endonucleases could become a viable option for patients affected with dramatic neurodegenerative disorders. Here, we used the Streptococcus pyogenes Cas9 to induce a double-strand break within the expanded CTG repeat involved in myotonic dystrophy type 1, integrated in a yeast chromosome. Repair of this double-strand break generated unexpected large chromosomal deletions around the repeat tract. These deletions depended on RAD50, RAD52, DNL4 and SAE2, and both non-homologous end-joining and single-strand annealing pathways were involved. Resection and repair of the double-strand break (DSB) were totally abolished in a rad50Δ strain, whereas they were impaired in a sae2Δ mutant, only on the DSB end containing most of the repeat tract. This observation demonstrates that Sae2 plays significant different roles in resecting a DSB end containing a repeated and structured sequence as compared to a non-repeated DSB end. In addition, we also discovered that gene conversion was less efficient when the DSB could be repaired using a homologous template, suggesting that the trinucleotide repeat may interfere with gene conversion too. Altogether, these data show that SpCas9 may not be the best choice when inducing a double-strand break at or near a microsatellite, especially in mammalian genomes that contain many more dispersed repeated elements than the yeast genome

Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal rearrangements

Microsatellites are short tandem repeats, ubiquitous in all eukaryotes and represent ∼2% of the human genome. Among them, trinucleotide repeats are responsible for more than two dozen neurological and developmental disorders. Targeting microsatellites with dedicated DNA endonucleases could become a viable option for patients affected with dramatic neurodegenerative disorders. Here, we used the Streptococcus pyogenes Cas9 to induce a double-strand break within the expanded CTG repeat involved in myotonic dystrophy type 1, integrated in a yeast chromosome. Repair of this double-strand break generated unexpected large chromosomal rearrangements around the repeat tract. These rearrangements depended on RAD52, DNL4 and SAE2, and both non-homologous end-joining and single-strand annealing pathways were involved. Resection and repair of the double-strand break (DSB) were totally abolished in a rad50Δ strain, whereas they were impaired in a sae2Δ mutant, only on the DSB end containing most of the repeat tract. This proved that Sae2 plays significant different roles in resecting a DSB end containing a repeated and structured sequence as compared to a non-repeated DSB end.In addition, we also discovered that gene conversion was less efficient when the DSB could be repaired using a homologous template, suggesting that the trinucleotide repeat may interfer with gene conversion too. Altogether, these data show that SpCas9 is probably not a good choice when inducing a double-strand break at or near a microsatellite, especially in mammalian genomes that contain many more dispersed repeated elements than the yeast genome