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.

Integration of H-2Z1, a somatosensory cortex-expressed transgene, interferes with the expression of the Satb1 and Tbc1d5 flanking genes and affects the differentiation of a subset of cortical interneurons.

H-2Z1 is an enhancer trap transgenic mouse line in which the lacZ reporter delineates the somatosensory area of the cerebral cortex where it is expressed in a subset of layer IV neurons. In the search of somatosensory specific genes or regulatory sequences, we mapped the H-2Z1 transgene insertion site to chromosome 17, 100 and 460 kb away from Tbc1d5 and Satb1 flanking genes. We show here that insertion of the H-2Z1 transgene results in three distinct outcomes. First, a genetic background-sensitive expression of lacZ in several brain and body structures. While four genes in a 1 Mb region around the insertion are expressed in the barrel cortex, H-2Z1 expression resembles more that of its two direct neighbors. Moreover, H-2Z1 closely reports most of the body and brain expression sites of the Satb1 chromatin remodeling gene including tooth buds, thymic epithelium, pontine nuclei, fastigial cerebellar nuclei, and cerebral cortex. Second, the H-2Z1 transgene causes insertional mutagenesis of Tbc1d5 and Satb1, leading to a strong decrease in their expressions. Finally, insertion of H-2Z1 affects the differentiation of a subset of cortical GABAergic interneurons, a possible consequence of downregulation of Satb1 expression. Thus, the H-2Z1 "somatosensory" transgene is inserted in the regulatory landscape of two genes highly expressed in the developing somatosensory cortex and reports for a subdomain of their expression profiles. Together, our data suggest that regulation of H-2Z1 expression results from local and remote genetic interactions.

Neuromuscular defects and breathing disorders in a new mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is caused by insufficient levels of the survival motor neuron (SMN) protein leading to muscle paralysis and respiratory failure. In mouse, introducing the human SMN2 gene partially rescues Smn(-)(/)(-) embryonic lethality. However current models were either too severe or nearly unaffected precluding convenient drug testing for SMA. We report here new SMN2;Smn(-/-) lines carrying one to four copies of the human SMN2 gene. Mice carrying three SMN2 copies exhibited an intermediate phenotype with delayed appearance of motor defects and developmental breathing disorders reminiscent of those found in severe SMA patients. Although normal at birth, at 7 days of age respiratory rate was decreased and apnea frequency was increased in SMA mice in parallel with the appearance of neuromuscular junction defects in the diaphragm. With median survival of 15 days and postnatal onset of neurodegeneration, these mice could be an important tool for evaluating new therapeutics.

Hes1 is expressed in the second heart field and is required for outflow tract development.

BACKGROUND: Rapid growth of the embryonic heart occurs by addition of progenitor cells of the second heart field to the poles of the elongating heart tube. Failure or perturbation of this process leads to congenital heart defects. In order to provide further insight into second heart field development we characterized the insertion site of a transgene expressed in the second heart field and outflow tract as the result of an integration site position effect. RESULTS: Here we show that the integration site of the A17-Myf5-nlacZ-T55 transgene lies upstream of Hes1, encoding a basic helix-loop-helix containing transcriptional repressor required for the maintenance of diverse progenitor cell populations during embryonic development. Transgene expression in a subset of Hes1 expression sites, including the CNS, pharyngeal epithelia, pericardium, limb bud and lung endoderm suggests that Hes1 is the endogenous target of regulatory elements trapped by the transgene. Hes1 is expressed in pharyngeal endoderm and mesoderm including the second heart field. Analysis of Hes1 mutant hearts at embryonic day 15.5 reveals outflow tract alignment defects including ventricular septal defects and overriding aorta. At earlier developmental stages, Hes1 mutant embryos display defects in second heart field proliferation, a reduction in cardiac neural crest cells and failure to completely extend the outflow tract. CONCLUSIONS: Hes1 is expressed in cardiac progenitor cells in the early embryo and is required for development of the arterial pole of the heart.

Centrosome overduplication and mitotic instability in PKD2 transgenic lines.

Polycystin-2 (PC-2), a protein encoded by PKD2 and involved in autosomal dominant polycystic kidney disease (ADPKD), is a non-selective cationic channel recently implicated in the function of primary cilia. We recently constructed a new animal model in the form of a transgenic mouse with a BAC-containing human PKD2 inserted in its genome. Two transgenic mouse lines overexpressing human PKD2 showed mitotic instability. Fibroblasts from these transgenic mouse lines have abnormal chromosomal numbers. These lines also have supernumerary centrosomes. PC-2 overexpression is associated with mitotic instability and centrosome overduplication. PC-2 therefore seems to play a role in centrosome duplication, and this hypothesis is being evaluated in other models.

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.

The del22q11.2 candidate gene Tbx1 controls regional outflow tract identity and coronary artery patterning.

TBX1, encoding a T-box containing transcription factor, is the major candidate gene for del22q11.2 or DiGeorge syndrome, characterized by craniofacial and cardiovascular defects including tetralogy of Fallot and common arterial trunk. Mice lacking Tbx1 have severe defects in the development of pharyngeal derivatives including cardiac progenitor cells of the second heart field that contribute to the arterial pole of the heart. The outflow tract of Tbx1 mutant embryos is short and narrow resulting in common arterial trunk. Here we show by a series of genetic crosses using transgene markers of second heart field derived myocardium and coronary endothelial cells that a subdomain of myocardium normally observed at the base of the pulmonary trunk is reduced and malpositioned in Tbx1 mutant hearts. This defect is associated with anomalous coronary artery patterning. Both right and left coronary ostia form predominantly at the right/ventral sinus in mutant hearts, proximal coronary arteries coursing across the normally coronary free ventral region of the heart. We have identified Semaphorin3c as a Tbx1-dependent gene expressed in subpulmonary myocardium. Our results implicate second heart field development in coronary artery patterning and provide new insights into the association between conotruncal defects and coronary artery anomalies.

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.

SRPX2 mutations in disorders of language cortex and cognition.

The rolandic and sylvian fissures divide the human cerebral hemispheres and the adjacent areas participate in speech processing. The relationship of rolandic (sylvian) seizure disorders with speech and cognitive impairments is well known, albeit poorly understood. We have identified the Xq22 gene SRPX2 as being responsible for rolandic seizures (RSs) associated with oral and speech dyspraxia and mental retardation (MR). SRPX2 is a secreted sushi-repeat containing protein expressed in neurons of the human adult brain, including the rolandic area. The disease-causing mutation (N327S) resulted in gain-of-glycosylation of the secreted mutant protein. A second mutation (Y72S) was identified within the first sushi domain of SRPX2 in a male with RSs and bilateral perisylvian polymicrogyria and his female relatives with mild MR or unaffected carrier status. In cultured cells, both mutations were associated with altered patterns of intracellular processing, suggesting protein misfolding. In the murine brain, Srpx2 protein expression appeared in neurons at birth. The involvement of SRPX2 in these disorders suggests an important role for SRPX2 in the perisylvian region critical for language and cognitive development.

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.

Genomic organization and the tissue distribution of alternatively spliced isoforms of the mouse Spatial gene.

BACKGROUND: The stromal component of the thymic microenvironment is critical for T lymphocyte generation. Thymocyte differentiation involves a cascade of coordinated stromal genes controlling thymocyte survival, lineage commitment and selection. The "Stromal Protein Associated with Thymii And Lymph-node" (Spatial) gene encodes a putative transcription factor which may be involved in T-cell development. In the testis, the Spatial gene is also expressed by round spermatids during spermatogenesis. RESULTS: The Spatial gene maps to the B3-B4 region of murine chromosome 10 corresponding to the human syntenic region 10q22.1. The mouse Spatial genomic DNA is organised into 10 exons and is alternatively spliced to generate two short isoforms (Spatial-alpha and -gamma) and two other long isoforms (Spatial-delta and -epsilon) comprising 5 additional exons on the 3' site. Here, we report the cloning of a new short isoform, Spatial-beta, which differs from other isoforms by an additional alternative exon of 69 bases. This new exon encodes an interesting proline-rich signature that could confer to the 34 kDa Spatial-beta protein a particular function. By quantitative TaqMan RT-PCR, we have shown that the short isoforms are highly expressed in the thymus while the long isoforms are highly expressed in the testis. We further examined the inter-species conservation of Spatial between several mammals and identified that the protein which is rich in proline and positive amino acids, is highly conserved. CONCLUSIONS: The Spatial gene generates at least five alternative spliced variants: three short isoforms (Spatial-alpha, -beta and -gamma) highly expressed in the thymus and two long isoforms (Spatial-delta and -epsilon) highly expressed in the testis. These alternative spliced variants could have a tissue specific function.

Molecular cloning and characterization of mouse LITAF cDNA: role in the regulation of tumor necrosis factor-alpha (TNF-alpha) gene expression.

The inflammatory response to bacteria and bacterial products, such as lipopolysaccharides (LPSs), is mediated by a variety of secreted factors, but cytotoxic effects of LPS have been ascribed to the tumor necrosis factor alpha (TNF-alpha) activity. TNF-alpha is probably the most pleiotropic cytokine and, given the deleterious effects to the host of this factor, it has been postulated that its expression must be tightly regulated. Our laboratory has recently isolated, cloned and characterized a novel human transcription factor named LITAF or LPS-induced TNF-alpha factor. The present study reports the isolation, cloning and characterization of the mouse LITAF cDNA. Chromosomal localization revealed that mouse LITAF mapped to mouse chromosome 16, in a region highly homologous with the area on which human LITAF was previously located. Northern blot analysis shows that mouse LITAF is already expressed at embryonic day 7 of development, and is highly expressed in adult liver, heart and kidney. Moreover, upon LPS stimulation, we show that: (i) LITAF expression is increased in a mouse monocyte/macrophage cell line; and (ii) TNF-alpha expression is reduced in ES cell-derived macrophages lacking one copy of LITAF gene. Taken together, these results highlight the important role of LITAF in the regulation of TNF-alpha gene expression and suggest a potential role of LITAF in mouse organogenesis.

A human-mouse chimera of the alpha3alpha4alpha5(IV) collagen protomer rescues the renal phenotype in Col4a3-/- Alport mice.

Collagen IV is a major structural component of basement membranes. In the glomerular basement membrane (GBM) of the kidney, the alpha3, alpha4, and alpha5(IV) collagen chains form a distinct network that is essential for the long-term stability of the glomerular filtration barrier, and is absent in most patients affected with Alport syndrome, a progressive inherited nephropathy associated with mutation in COL4A3, COL4A4, or COL4A5 genes. To investigate, in vivo, the regulation of the expression, assembly, and function of the alpha3alpha4alpha5(IV) protomer, we have generated a yeast artificial chromosome transgenic line of mice carrying the human COL4A3-COL4A4 locus. Transgenic mice expressed the human alpha3 and alpha4(IV) chains in a tissue-specific manner. In the kidney, when expressed onto a Col4a3(-/-) background, the human alpha3(IV) chain restored the expression of and co-assembled with the mouse alpha4 and alpha5(IV) chains specifically at sites where the human alpha3(IV) was expressed, demonstrating that the expression of all three chains is required for network assembly. The co-assembly of the human and mouse chains into a hybrid network in the GBM restores a functional GBM and rescues the Alport phenotype, providing further evidence that defective assembly of the alpha3-alpha4-alpha5(IV) protomer, caused by mutations in any of the three chains, is the pathogenic mechanism responsible for the disease. This line of mice, humanized for the alpha3(IV) collagen chain, will also provide a valuable model for studying the pathogenesis of Goodpasture syndrome, an autoimmune disease caused by antibodies against this chain.

Cloning and characterization of the mouse homologue of the human dendritic cell maturation marker CD208/DC-LAMP.

DC-LAMP, a member of the lysosomal-associated membrane protein (LAMP) family, is specifically expressed by human dendritic cells (DC) upon activation and therefore serves as marker of human DC maturation. DC-LAMP is detected first in activated human DC within MHC class II molecules-containing compartments just before the translocation of MHC class II-peptide complexes to the cell surface, suggesting a possible involvement in this process. The present study describes the cloning and characterization of mouse DC-LAMP, whose predicted protein sequence is over 50% identical to the human counterpart. The mouse DC-LAMP gene spans over 25 kb and shares syntenic chromosomal localization (16B2-B4 and 3q26) and conserved organization with the human DC-LAMP gene. Analysis of mouse DC-LAMP mRNA and protein revealed the expression in lung peripheral cells, but also its unexpected absence from mouse lymphoid organs and from mouse DC activated either in vitro or in vivo. In conclusion, mouse DC-LAMP is not a marker of mature mouse DC and this observation raises new questions regarding the role of human DC-LAMP in human DC.

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.

Synteny comparison between apes and human using fine-mapping of the genome.

Comparing the genomes of the great apes and human should provide novel information concerning the origins of humankind. Relative to the great apes, the human karyotype has one fewer chromosome pair, as human chromosome 2 derived from the telomeric fusion of two ancestral primate chromosomes. To identify the genomic rearrangements that accompanied human speciation, we initiated a comparative study between human, chimpanzee, and gorilla. Using the HAPPY mapping method, an acellular adaptation of the radiation hybrid method, we mapped a few hundred markers on the human, chimpanzee, and gorilla genomes. This allowed us to identify several chromosome rearrangements, in particular a pericentric inversion and a translocation. We precisely localized the synteny breakpoint that led to the formation of human chromosome 2. This breakpoint was confirmed by FISH mapping.