MG132-induced progerin clearance is mediated by autophagy activation and splicing regulation.

Hutchinson-Gilford progeria syndrome (HGPS) is a lethal premature and accelerated aging disease caused by a de novo point mutation in LMNA encoding A-type lamins. Progerin, a truncated and toxic prelamin A issued from aberrant splicing, accumulates in HGPS cells' nuclei and is a hallmark of the disease. Small amounts of progerin are also produced during normal aging. We show that progerin is sequestered into abnormally shaped promyelocytic nuclear bodies, identified as novel biomarkers in late passage HGPS cell lines. We found that the proteasome inhibitor MG132 induces progerin degradation through macroautophagy and strongly reduces progerin production through downregulation of SRSF-1 and SRSF-5 accumulation, controlling prelamin A mRNA aberrant splicing. MG132 treatment improves cellular HGPS phenotypes. MG132 injection in skeletal muscle of LmnaG609G/G609G mice locally reduces SRSF-1 expression and progerin levels. Altogether, we demonstrate progerin reduction based on MG132 dual action and shed light on a promising class of molecules toward a potential therapy for children with HGPS.

Splicing-directed therapy in a new mouse model of human accelerated aging.

Hutchinson-Gilford progeria syndrome (HGPS) is caused by a point mutation in the LMNA gene that activates a cryptic donor splice site and yields a truncated form of prelamin A called progerin. Small amounts of progerin are also produced during normal aging. Studies with mouse models of HGPS have allowed the recent development of the first therapeutic approaches for this disease. However, none of these earlier works have addressed the aberrant and pathogenic LMNA splicing observed in HGPS patients because of the lack of an appropriate mouse model. Here, we report a genetically modified mouse strain that carries the HGPS mutation. These mice accumulate progerin, present histological and transcriptional alterations characteristic of progeroid models, and phenocopy the main clinical manifestations of human HGPS, including shortened life span and bone and cardiovascular aberrations. Using this animal model, we have developed an antisense morpholino-based therapy that prevents the pathogenic Lmna splicing, markedly reducing the accumulation of progerin and its associated nuclear defects. Treatment of mutant mice with these morpholinos led to a marked amelioration of their progeroid phenotype and substantially extended their life span, supporting the effectiveness of antisense oligonucleotide-based therapies for treating human diseases of accelerated aging.

A naturally occurring human minidysferlin protein repairs sarcolemmal lesions in a mouse model of dysferlinopathy.

Dysferlinopathies are autosomal recessive, progressive muscle dystrophies caused by mutations in DYSF, leading to a loss or a severe reduction of dysferlin, a key protein in sarcolemmal repair. Currently, no etiological treatment is available for patients affected with dysferlinopathy. As for other muscular dystrophies, gene therapy approaches based on recombinant adeno-associated virus (rAAV) vectors are promising options. However, because dysferlin messenger RNA is far above the natural packaging size of rAAV, full-length dysferlin gene transfer would be problematic. In a patient presenting with a late-onset moderate dysferlinopathy, we identified a large homozygous deletion, leading to the production of a natural "minidysferlin" protein. Using rAAV-mediated gene transfer into muscle, we demonstrated targeting of the minidysferlin to the muscle membrane and efficient repair of sarcolemmal lesions in a mouse model of dysferlinopathy. Thus, as previously demonstrated in the case of dystrophin, a deletion mutant of the dysferlin gene is also functional, suggesting that dysferlin's structure is modular. This minidysferlin protein could be used as part of a therapeutic strategy for patients affected with dysferlinopathies.

In human pachytene spermatocytes, SUMO protein is restricted to the constitutive heterochromatin.

SUMO-1, a ubiquitin-like protein, is covalently bound to many proteins, leading to chromatin inactivation and transcriptional repression. The high concentration of SUMO-1 on the XY body in rodents suggests that this protein has an important role in facultative heterochromatin organization. In human, the precise role of SUMO in chromatin/heterochromatin organization remains to be defined. Here we describe the SUMO-1 distribution, during human male meiosis, in relation to the formation of the different types of heterochromatin. We show that, during late pachynema, SUMO-1 appears on the constitutive heterochromatin, but is excluded from the XY body facultative heterochromatin. At the SUMO-1 labelled areas, the presence of HP1alpha protein, as well as of trimethylated H3-K9 and H4-K20 histone modifications, supports a role for SUMO-1 in constitutive heterochromatin organization. We also establish that, on the constitutive heterochromatin, H4-K20me3 staining progressively decreases as SUMO-1 staining increases, suggesting that core histone(s), and histone H4 in particular, are direct targets for sumoylation. Our results also suggest that, in the context of global histone H4 hyperacetylation that precedes the histone-to-protamine transition at postmeiotic stages of spermatogenesis, histone H4 sumoylation may represent an important epigenetic marker replacing methylation on the constitutive heterochromatin.

Recurrent rearrangements in the proximal 15q11-q14 region: a new breakpoint cluster specific to unbalanced translocations.

Unbalanced translocations, that involve the proximal chromosome 15 long arm and the telomeric region of a partner chromosome, result in a karyotype of 45 chromosomes with monosomy of the proximal 15q imprinted region. Here, we present our analysis of eight such unbalanced translocations that, depending on the parental origin of the rearranged chromosome, were associated with either Prader-Willi or Angelman syndrome. First, using FISH with specific BAC clones, we characterized the chromosome 15 breakpoint of each translocation and demonstrate that four of them are clustered in a small 460 kb interval located in the proximal 15q14 band. Second, analyzing the sequence of this region, we demonstrate the proximity of a low-copy repeat 15 (LCR15)-duplicon element that is known to facilitate recombination events at meiosis and to promote rearrangements. The presence, in this region, of both a cluster of translocation breakpoints and a LCR15-duplicon element defines a new breakpoint cluster (BP6), which, to our knowledge, is the most distal breakpoint cluster described in proximal 15q. Third, we demonstrate that the breakpoints for other rearrangements including large inv dup (15) chromosomes do not map to BP6, suggesting that it is specific to translocations. Finally, the translocation breakpoints located within BP6 result in very large proximal 15q deletions providing new informative genotype-phenotype correlations.

PML nuclear bodies are highly organised DNA-protein structures with a function in heterochromatin remodelling at the G2 phase.

We have recently demonstrated that heterochromatin HP1 proteins are aberrantly distributed in lymphocytes of patients with immunodeficiency, centromeric instability and facial dysmorphy (ICF) syndrome. The three HP1 proteins accumulate in one giant body over the 1qh and 16qh juxtacentromeric heterochromatins, which are hypomethylated in ICF. The presence of PML (promyelocytic leukaemia) protein within this body suggests it to be a giant PML nuclear body (PML-NB). The structural integrity of PML-NBs is of major importance for normal cell functioning. Nevertheless, the structural organisation and the functions of these nuclear bodies remain unclear. Here, we take advantage of the large size of the giant body to demonstrate that it contains a core of satellite DNA with proteins being organised in ordered concentric layers forming a sphere around it. We extend these results to normal PML-NBs and propose a model for the general organisation of these structures at the G2 phase. Moreover, based on the presence of satellite DNA and the proteins HP1, BRCA1, ATRX and DAXX within the PML-NBs, we propose that these structures have a specific function: the re-establishment of the condensed heterochromatic state on late-replicated satellite DNA. Our findings that chromatin-remodelling proteins fail to accumulate around satellite DNA in PML-deficient NB4 cells support a central role for PML protein in this cellular function.

Screening for subtelomeric rearrangements using automated fluorescent genotyping of microsatellite markers: a Lebanese study.

A screening for submicroscopic rearrangements using specific polymorphic microsatellite markers from the subtelomeric regions of all chromosome arms was performed in 34 independent Lebanese families, including 45 patients with idiopathic mental retardation plus additional features. Five cryptic rearrangements were found in five different families, but subsequent FISH studies confirmed only three of those, showing a proportion of nearly 9% of subtelomeric rearrangements in our population. Two patients presented a de novo deletion from paternal origin, one involving telomere 3p, and another telomere 7p. An unbalanced paternally inherited translocation was detected in two patients from the same family resulting in both trisomy for telomere 5q and monosomy for telomere 6p.

Subcellular distribution of HP1 proteins is altered in ICF syndrome.

The Immunodeficiency, Centromeric instability, and Facial (ICF) syndrome is a rare autosomal recessive disorder that results from mutations in the DNMT3B gene, encoding a DNA-methyltransferase that acts on GC-rich satellite DNAs. This syndrome is characterized by immunodeficiency, facial dysmorphy, mental retardation of variable severity and chromosomal abnormalities that essentially involve juxtacentromeric heterochromatin of chromosomes 1 and 16. These abnormalities demonstrate that hypomethylation of satellite DNA can induce alterations in the structure of heterochromatin. In order to investigate the effect of DNA hypomethylation on heterochromatin organization, we analyzed the in vivo distribution of HP1 proteins, essential components of heterochromatin, in three ICF patients. We observed that, in a large proportion of ICF G2 nuclei, all HP1 isoforms show an aberrant signal concentrated into a prominent bright focus that co-localizes with the undercondensed 1qh or 16qh heterochromatin. We found that SP100, SUMO-1 and other proteins from the promyelocytic leukemia nuclear bodies (NBs) form a large body that co-localizes with the HP1 signal. This is the first description of altered nuclear distribution of HP1 proteins in the constitutional ICF syndrome. Our results show that satellite DNA hypomethylation does not prevent HP1 proteins from associating with heterochromatin. They suggest that, at G2 phase, HP1 proteins are involved in the heterochromatin condensation and may therefore remain concentrated at these sites until the condensation is complete. They also indicate that proteins from the NB could play a role in this process. Finally, satellite DNA length polymorphism could affect the efficiency of heterochromatin condensation and thus contribute to the variability of the ICF phenotype.

Mutation in the 5' alternatively spliced region of the XNP/ATR-X gene causes Chudley-Lowry syndrome.

The Chudley-Lowry syndrome (ChLS, MIM 309490) is an X-linked recessive condition characterized by moderate to severe mental retardation, short stature, mild obesity, hypogonadism, and distinctive facial features characterized by depressed nasal bridge, anteverted nares, inverted-V-shaped upper lip, and macrostomia. The original Chudley-Lowry family consists of three affected males in two generations. Linkage analysis had localized the gene to a large interval, Xp21-Xq26 and an obligate carrier was demonstrated to have highly skewed X inactivation. The combination of the clinical phenotype, consistent with that of the patients with ATR-X syndrome, the skewed X-inactivation pattern in a carrier female, as well as the mapping interval including band Xq13.3, prompted us to consider the XNP/ATR-X gene being involved in this syndrome. Using RT-PCR analysis, we screened the entire XNP/ATR-X gene and found a mutation in exon 2 (c.109C > T) giving rise to a stop codon at position 37 (p.R37X). Western blot and immunocytochemical analyses using a specific monoclonal antibody directed against XNP/ATR-X showed the protein to be present in lymphoblastoid cells from one affected male, despite the premature stop codon. To explain these discordant results, we further analyzed the 5' region of the XNP/ATR-X gene and found three alternative transcripts, which differ in the presence or absence of exon 2, and the length of exon 1. Our data suggest that ChLS is allelic to the ATR-X syndrome with its less severe phenotype being due to the presence of some XNP/ATR-X protein.

TSPY, the candidate gonadoblastoma gene on the human Y chromosome, has a widely expressed homologue on the X - implications for Y chromosome evolution.

TSPY, a candidate gene for a factor that promotes gonadoblastoma formation (GBY), is a testis-specific multicopy gene family in the male-specific region of the human Y (MSY) chromosome. Although it was originally proposed that male-specific genes on the Y originated from a transposed copy of an autosomal gene (Lahn & Page 1999b), at least two male-specific genes (RBMY and SRY) descended from a formerly recombining X-Y identical gene pair. Here we show that a TSPY homologue with similar gene structure lies in conserved positions, close to SMCX, on the X chromosome in human (TSPX ) and mouse (Tspx). TSPX is widely expressed and subject to X inactivation. TSPX and TSPY therefore evolved from an identical gene pair on the original mammalian sex chromosomes. This supports the hypothesis that even male-specific genes on the Y chromosome may have their origin in ubiquitously expressed genes on the X. It also strengthens the case for TSPY as a candidate for GBY, since independent functional studies link TSPX to cell cycle regulation.

Mechanism of intrachromosomal triplications 15q11-q13: a new clinical report.

We describe here a patient with intrachromosomal triplication 15q11-q13, a rare chromosomal event associated with severe mental retardation and intractable epilepsy. Cytogenetic studies including FISH on interphasic nuclei showed that the middle segment of the triplication was inverted in orientation. Molecular analyses demonstrated that the rearrangement was of maternal origin. Based on these cytogenetic and molecular data and those of the nine cases reported in the literature, we discuss the mechanistic origins of these triplications. We present several arguments for the mechanism involving two U-type exchanges occurring simultaneously at the pachytene stage of meiosis.

A human interstitial telomere associates in vivo with specific TRF2 and TIN2 proteins.

Mammalian telomeres are composed of long arrays of TTAGGG repeats that form a nucleoprotein complex which protects the chromosome ends. Human telomere function is known to require two TTAGGG repeat factors, TRF1 and TRF2, and several interacting proteins, but the mechanism by which the DNA/protein complex prevents end to end fusion in vivo has not been elucidated. In order to better understand the role of specific telomere-associated proteins in the organisation of chromosome ends, we have studied a patient with a rare chromosome rearrangement that has given rise to an interstitial telomere. Using specific antibodies and immuno-FISH on unfixed metaphase chromosomes, we show that the proteins TRF2 and TIN2 (TIN2 interacts with TRF1) co-localise with the interstitial TTAGGG repeats. Our results demonstrate, for the first time in humans, that TRF2 and TIN2 proteins associate with interstitial duplex TTAGGG repeats, in vivo. They confirm that double stranded-telomeric repeats, even when complexed with specific proteins, are not sufficient to create a functional telomere. Finally, they suggest a possible role for proteins in stabilising interstitial TTAGGG repeats.

Wdr12, a mouse gene encoding a novel WD-Repeat Protein with a notchless-like amino-terminal domain.

The WD-repeat protein family consists of a large group of structurally related yet functionally diverse proteins found predominantly in eukaryotic cells. These factors contain several (4-16) copies of a recognizable amino-acid sequence motif (the WD unit) thought to be organized into a "propeller-like" structure involved in protein-protein regulatory interactions. Here, we report the cloning of a mouse cDNA, referred to as Wdr12, which encodes a novel WD-repeat protein of 423 amino acids. The WDR12 protein was predicted to contain seven WD units and a nuclear localization signal located within a protruding peptide between the third and fourth WD domains. The amino-terminal region shows similarity to that of the Notchless WD repeat protein. Sequence comparisons revealed WDR12 orthologs in various eukaryotic species. Wdr12 seems to correspond to a single-copy gene in the mouse genome, located within the C1-C2 bands of chromosome 1. These data, together with the results of Wdr12 gene expression studies and evidence of in vitro binding of WDR12 to the cytoplasmic domain of Notch1, led us to postulate a function for the WDR12 protein in the modulation of Notch signaling activity.