[Show abstract][Hide abstract] ABSTRACT: The Arp2/3 complex is essential for actin filament nucleation in a variety of cellular processes. The activation of the Arp2/3 complex is mediated by nucleation-promoting factors, such as the Wiskott-Aldrich syndrome family proteins, which share a WCA (WH2 domain, central region, acidic region) catalytic module at the C-terminal region, required for Arp2/3 activation, but diverge at the N-terminal region, required for binding to specific activators. Here, we report the characterization of WASH, a new member of the WAS family that has nucleation-promoting factor activity and recently has been demonstrated to play a role in endosomal sorting. We found that overexpression of the WASH-WCA domain induced disruption of the actin cytoskeleton, whereas overexpression of full-length WASH in mammalian cells did not affect stress fiber organization. Furthermore, our analysis has revealed that nerve growth factor treatment of PC12 cells overexpressing full-length WASH leads to disruption of the actin cytoskeleton. We have also found that WASH interacts through its N-terminal region with BLOS2, a centrosomal protein belonging to the BLOC-1 complex that functions as a scaffolding factor in the biogenesis of lysosome-related organelles. In addition to BLOS2, WASH also interacts with centrosomal gamma-tubulin and with pallidin, an additional component of the BLOC-1 complex. Collectively, our data propose that WASH is a bimodular protein in which the C terminus is involved in Arp2/3-mediated actin nucleation, whereas the N-terminal portion is required for its regulation and localization in the cells. Moreover, our data suggest that WASH is also a component of the BLOC-1 complex that is associated with the centrosomes.
Preview · Article · Mar 2010 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: The Incontinentia Pigmenti (IP) locus contains the IKBKG/NEMO/IKKgamma gene and its truncated pseudogene copy, IKBKGP/deltaNEMO. The major genetic defect in IP is a heterozygous exon4_10 IKBKG deletion (IKBKGdel) caused by a recombination between two consecutive MER67B repeats. We analyzed 91 IP females carrying the IKBKGdel, 59 of whom carrying de novo mutations (65%). In eight parents, we found two recurrent nonpathological variants of IP locus, which were also present as rare polymorphism in control population: the IKBKGPdel, corresponding to the exon4_10 deletion in the pseudogene, and the MER67Bdup, that replicates the exon4_10 region downstream of the normal IKBKG gene. Using quantitative DNA analysis and microsatellite mapping, we established that both variants might promote the generation of the pathological IKBKGdel. Indeed, in family IP-516, the exon4_10 deletion was repositioned in the same allele from the pseudogene to the gene, whereas in family IP-688, the MER67Bdup generated the pathological IKBKGdel by recombination between two direct nonadjacent MER67Bs. Moreover, we found an instance of somatic recombination in a MER67Bdup variant, creating the IKBKGdel in an IP male. Our data suggest that the IP locus undergoes recombination producing recurrent variants that might be "at risk" of generating de novo IKBKGdel by NAHR during either meiotic or mitotic division.
[Show abstract][Hide abstract] ABSTRACT: Otosclerosis is the single most common cause of hearing impairment among adult Caucasians. Little is known about its aetiology and its molecular aspects. Until now, genetic linkage in Otosclerosis has been demonstrated in an Indian family and a Belgian family, showing the presence of two Otosclerosis loci, OTSC1 and OTSC2, respectively. Linkage analysis has never been applied to Italian otosclerotic families. We have collected four multigenerational Italian otosclerotic families that show dominant transmission for the pathology. Here, we report a detailed audiological analysis of these families and a genetic linkage study on the OTSC1 and OTSC2 loci. Statistical analysis revealed the absence of linkage between the disease in our families and the OTSC1 and OTSC2 loci. These data strongly suggest the presence of one or more additional loci for Otosclerosis, which still need to be defined. Sumario La otocsclerosis es la causa mas comun de problemas auditivos entre los adullos caucásicos. Poco se conoce acerca de su etiología y de sus aspectos moleculares. Hasta ahora, los enlaces genéticos en la Otoesclerosis han sido demostrados en familias de la India y Bélgica mostrando la prescncia de dos loci de Otoesclerosis, OTSC1 y ORSC2, respectivamente. Nunca se ha realizado el análisis de enlaces en familias con otoeselerosis en Italia. Nosotros coleclamos los datos multigeneracionales de cuatro familias italianas con Otoesclerosis, que mostraron transmisión dominante de esa patologia. En este trabajo reportamos el análisis audiológico detallado de estas familias y el estudio de enlaces genéticos con los loci OTSC1 y ORSC2. El análisis estadistico reveló la auscncia de relación entre la enfermedad en las cuatro familias y los loci OTSC1 y OTSC2. Estos datos sugieren fuertemente la presencia de uno o más loci adicionales para la Otoesclerosis, que aún es necesario definir.
No preview · Article · Jul 2009 · International Journal of Audiology
[Show abstract][Hide abstract] ABSTRACT: The subtelomeric regions of human chromosomes exhibit an extraordinary plasticity. To date, due to the high GC content and to the presence of telomeric repeats, the subtelomeric sequences are underrepresented in the genomic libraries and consequently their sequences are incomplete in the finished human genome sequence, and still much remains to be learned about subtelomere organization, evolution and function. Indeed, only in recent years, several studies have disclosed, within human subtelomeres, novel gene family members.
During a project aimed to analyze genes located in the telomeric region of the long arm of the human X chromosome, we have identified a novel transcript family, DDX11L, members of which map to 1pter, 2q13/14.1, 2qter, 3qter, 6pter, 9pter/9qter, 11pter, 12pter, 15qter, 16pter, 17pter, 19pter, 20pter/20qter, Xpter/Xqter and Yqter. Furthermore, we partially sequenced the underrepresented subtelomeres of human chromosomes showing a common evolutionary origin.
Our data indicate that an ancestral gene, originated as a rearranged portion of the primate DDX11 gene, and propagated along many subtelomeric locations, is emerging within subtelomeres of human chromosomes, defining a novel gene family. These findings support the possibility that the high plasticity of these regions, sites of DNA exchange among different chromosomes, could trigger the emergence of new genes.
[Show abstract][Hide abstract] ABSTRACT: Carnitine is a molecule with well-documented pleiotropic functions whose biosynthesis involves four catalytic steps. Here, we report a detailed analysis of the expression and transcriptional control of TMLH gene, which codifies for the first enzyme of carnitine biosynthesis. TMLH maps at the extreme end of Xq28, a chromosomal region of high genomic instability. By 5' and 3' RACE, we identified and mapped two alternative 5' TMLH first exons and seven alternative 3'-splice variants, which are spread over a genomic region of about 250 kb. While the two alternative 5' exons have different expression profiles, all the 3' alternative forms are ubiquitously expressed. Reporter assays revealed that the 3'-UTRs of each TMLH isoform might influence its own expression at post-transcriptional level. In addition, we identified a highly conserved promoter region of TMLH. Functional analysis of this region showed the presence of a CpG island, whose methylation-status could control the level of TMLH transcription. Finally, by mRNA in situ hybridization, we found that TMLH expression is present at E12.5 dpc in the mouse liver, lung and brain, and is then maintained in the postnatal brain with a specific neuronal pattern. Collectively, our data highlight a tight transcriptional and post-transcriptional control of TMLH expression.
[Show abstract][Hide abstract] ABSTRACT: The association between premature ovarian failure (POF) and the FMR1 repeat number (41> CGGn< 200) has been widely investigated. Current findings suggest that the risk estimation for POF can be calculated in the offspring of women with pre-mutated FMR1 alleles.
We describe the coexistence in a large Italian kindred of Fragile X syndrome and familial POF in females with ovarian dysfunctions who carried normal or expanded FMR1 alleles. Genetic analysis of the FMR1 gene in over three generations of females revealed that six carried pre-mutated alleles (61-200), of which two were also affected by POF. However a young woman, who presented a severe ovarian failure with early onset, carried normal FMR1 alleles (<40). The coexistence within the same family of two dysfunctional ovarian conditions, one FMR1-related and one not FMR1-related, suggests that the complexity of familial POF conditions is larger than expected.
Our case study represents a helpful observation and will provide familial cases with heterogeneous etiology that could be further studied when candidate genes in addition to the FMR1 premutation will be available.
Full-text · Article · Apr 2007 · BMC Medical Genetics
[Show abstract][Hide abstract] ABSTRACT: Cognitive impairments are heterogeneous conditions, and it is estimated that 10% may be caused by a defect of mental function genes on the X chromosome. One of those genes is Aristaless related homeobox (ARX) encoding a polyA-rich homeobox transcription factor essential for cerebral patterning and its mutations cause different neurologic disorders. We reported on the clinical and genetic analysis of an Italian family with X-linked mental retardation (XLMR) and intra-familial heterogeneity, and provided insight into its molecular defect.
We carried out on linkage-candidate gene studies in a new MRX family (MRX87). All coding regions and exon-intron boundaries of ARX gene were analysed by direct sequencing.
MRX87 patients had moderate to profound cognition impairment and a combination of minor congenital anomalies. The disease locus, MRX87, was mapped between DXS7104 and DXS1214, placing it in Xp22-p21 interval, a hot spot region for mental handicap. An in frame duplication of 24 bp (ARXdup24) in the second polyAlanine tract (polyA_II) in ARX was identified.
Our study underlines the role of ARXdup24 as a critical mutational site causing mental retardation linked to Xp22. Phenotypic heterogeneity of MRX87 patients represents a new observation relevant to the functional consequences of polyAlanine expansions enriching the puzzling complexity of ARXdup24-linked diseases.
Full-text · Article · Feb 2007 · BMC Medical Genetics
[Show abstract][Hide abstract] ABSTRACT: Deletions of Xq chromosome are reported for a number of familial conditions exhibiting premature ovarian failure (POF) and early menopause (EM).
We describe the inheritance of an interstitial deletion of the long arm of the X chromosome associated with either POF or EM in the same family. Cytogenetic studies and heterozygosity mapping by quantitative fluorescent PCR revealed a 46,X,del(X)(q26.2-q28) karyotype in a POF female, in her EM mother, and also in her aborted fetus with severe cardiopathy. Applying a microsatellite approach, we have narrowed the extension of an identical interstitial deletion located between DXS1187 and DXS1073. These data, in line with other mapped deletions, single out the proximal Xq28 as the region most frequently involved in ovarian failure. We also propose that other factors may influence the phenotypic effect of this alteration. Indeed, skewed X inactivation has been ascertained in EM and POF to be associated with different X haplotypes.
Our analysis indicates that Xq26.2-q28 deletion is responsible for gonad dysgenesis in a family with EM/POF. The dissimilar deletion penetrance may be due to epigenetic modifications of other X genes that can contribute to human reproduction, highlighting that ovarian failure should be considered as a multifactorial disease.
Full-text · Article · Mar 2006 · Human Reproduction
[Show abstract][Hide abstract] ABSTRACT: epsilon-N-Trimethyllysine hydroxylase (TMLH) (EC 220.127.116.11) is a non-heme-ferrous iron hydroxylase, Fe(++) and 2-oxoglutarate (2OG) dependent, catalyzing the first of four enzymatic reactions of the highly conserved carnitine biosynthetic pathway. Otherwise from all the other enzymes of carnitine biosynthesis, TMLH was found to be associated to the mitochondrial fraction. We here report molecular cloning of two alternative spliced forms of TMLH, which appear ubiquitously expressed in human adult and fetal tissues. The deduced proteins are designated TMLH-a and TMLH-b, and contain 421 and 399 amino acids, respectively. They share the first N-terminal 332 amino acids, including a mitochondrial targeting signal, but diverge at the C-terminal end. TMLH-a and TMLH-b exogenous expression in COS-1 cells shows that the first 15 amino acids are necessary and sufficient for mitochondrial import. Furthermore, comparative evolutionary analysis of the C-terminal portion of TMLH-a identifies a conserved domain characterized by a key triad of residues, His242-Glu244-His389 predicted to bind 2OG end. This sequence is conserved in the TMLH enzyme from all species but is partially substituted by a unique sequence in the TMLH-b variant. Indeed, TMLH-b is not functional by itself as well as a TMLH-H389L mutant produced by site directed mutagenesis. As great interest, we found that TMLH-b and TMLH-H389L, individually co-expressed with TMLH-a in COS-1 cells, negatively affect TMLH activity. Therefore, our studies on the TMLH alternative form provide relevant novel information, first that the C-terminal region of TMLH contains the main determinants for its enzymatic activity including a key H389 residue, and second that TMLH-b could act as a crucial physiological negative regulator of TMLH.
No preview · Article · Sep 2005 · Journal of Cellular Physiology
[Show abstract][Hide abstract] ABSTRACT: The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.
[Show abstract][Hide abstract] ABSTRACT: Incontinentia Pigmenti (IP) is an X-linked genodermatosis that is lethal for males and present in females with abnormal skin pigmentation and high variable clinical signs, including retinal detachment, anodontia, alopecia, nail dystrophy and nervous system defects. The NF-kappaB essential modulator (NEMO) gene, responsible for IP, encodes the regulatory subunit of the IkappaB kinase (IKK) complex required for nuclear factor kappaB (NF-kappaB) activation. We analyzed the NEMO gene in 122 IP patients and identified mutations in 83 (36 familiar and 47 sporadic cases). The recurrent NEMO exon 4-10 deletion that is the major cause of the disease was present in 73 females (59.8%). In addition 10 point alterations (8.2% of females) were identified: three frameshift, three nonsense, three missense and one in-frame deletion of a single amino acid. We measured the effects of these NEMO point-mutations on NF-kappaB signaling in nemo(-/-) deficient murine pre-B cells. A mutation in the N-terminal domain, required for IKK assembly, reduced but did not abolish NF-kappaB activation following lipopolysaccharide stimulation. Mutations that disrupt the C-terminal domain, required for the recruitment of upstream factors, showed lower or no NF-kappaB activation. A phenotype score based on clinical features of our IP patients was applied for summarizing disease severity. The score did not correlate with mutation type or domain affected indicating that other factors influence the severity of IP. Such a factor is likely to be X-inactivation. Indeed, 64% of our patients have extremely skewed X-inactivation pattern (>/=80 : 20). Overall IP pathogenesis thus depends on a combination of X-inactivation and protein domain that recruit upstream factors and activate NF-kappaB.
No preview · Article · Aug 2004 · Human Molecular Genetics
[Show abstract][Hide abstract] ABSTRACT: In eukaryotes, interactions among the alpha-helical coiled-coil domains (CCDs) of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a pivotal role in mediating the fusion among vesicles and target membranes. Surface residues of such CCDs are major candidates to regulate the specificity of membrane fusion, as they may alter local charge at the interaction layers and surface of the fusion complex, possibly modulating its formation and/or the binding of non-SNARE regulatory factors. Based on alternate patterns in surface residues, we have identified two motifs which group vesicular SNAREs in two novel subfamilies: RG-SNAREs and RD-SNAREs. The RG-SNARE CCD is common to all members of the widely conserved family of long VAMPs or longins and to yeast and non-neuronal VAMPs, possibly mediating "basic" fusion mechanisms; instead, only synaptobrevins from Bilateria share an RD-SNARE CCD, which is likely to mediate interactions to specific, yet unknown, regulatory factors and/or be the landmark of rapid fusion reactions like that mediating the release of neurotransmitters.
Full-text · Article · Jun 2004 · Biology of the Cell
[Show abstract][Hide abstract] ABSTRACT: We recently published the genomic characterization of the STAT5A and STAT5B paralogous genes that are located head to head in the 17q21 chromosome and share large regions of sequence identity. We here demonstrate by transient in vitro transfection that STAT5A and STAT5B promoters are able to direct comparable levels of transcription. The expression of basal promoters is enhanced after Sp1 up-regulation in HeLa and SL2 cells while DNA methylation associated to the recruitment of MeCP2 methyl CpG binding protein down-regulates STAT5A and B promoters by interfering with Sp1-induced transcription. In addition, cross-species sequence comparison identified a bi-directional negative cis-acting regulatory element located in the STAT5 intergenic region.
[Show abstract][Hide abstract] ABSTRACT: The synaptobrevin-like 1 (SYBL1) gene is ubiquitously expressed and codes for an unusual member of the v-SNAREs molecules implicated in cellular exocytosis.
This X-linked gene has the peculiarity of also being present on the Y chromosome in a transcriptional inactive status. Moreover,
although ubiquitous, the function of SYBL1 is prominent in specific tissues, such as brain. As a first insight into the molecular mechanisms controlling SYBL1 expression, in this report we describe the extent and role of SYBL1 upstream regions and characterize the binding of trans-acting factors. In vivo foot-printing experiments identify three protected regions. Band shift and transient reporter gene assays indicate a strong
role of two of these evolutionary conserved regions in regulating SYBL1 transcription. Because one site is the classical CAAT box, we characterized the binding to the other site of the mammalian
homologues of the selenocysteine tRNA gene transcription activating factor (Staf) family, zinc-finger transcription factors,
and their role in regulating SYBL1 expression. The results reported here clarify that a Staf-zinc finger family factor, together with the CAAT factor, is the
major nuclear protein bound to the SYBL1 promoter region and is responsible for its regulation in HeLa cells, thus identifying the basic control of SYBL1 transcription. In vivo binding of Staf proteins to the SYBL1 promoter is confirmed by chromatin immunoprecipitation assays. Our results identify a fourth mRNA promoter stimulated by
a member of the Staf-zinc finger family, the function of which on mRNA polymerase II promoters is still very poorly understood.
Full-text · Article · Mar 2004 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Otosclerosis is the single most common cause of hearing impairment among adult caucasians. Little is known about its aetiology and its molecular aspects. Until now, genetic linkage in otosclerosis has been demonstrated in an Indian family and a Belgian family, showing the presence of two otosclerosis loci, OTSC1 and OTSC2, respectively. Linkage analysis has never been applied to Italian otosclerotic families. We have collected four multigenerational Italian otosclerotic families that show dominant transmission for the pathology. Here, we report a detailed audiological analysis of these families and a genetic linkage study on the OTSC1 and OTSC2 loci. Statistical analysis revealed the absence of linkage between the disease in our families and the OTSC1 and OTSC2 loci. These data strongly suggest the presence of one or more additional loci for otosclerosis, which still need to be defined.
No preview · Article · Jan 2004 · International Journal of Audiology
[Show abstract][Hide abstract] ABSTRACT: Understanding and harnessing cellular potency are fundamental in biology and are also critical to the future therapeutic use of stem cells. Transcriptome analysis of these pluripotent cells is a first step towards such goals. Starting with sources that include oocytes, blastocysts, and embryonic and adult stem cells, we obtained 249,200 high-quality EST sequences and clustered them with public sequences to produce an index of approximately 30,000 total mouse genes that includes 977 previously unidentified genes. Analysis of gene expression levels by EST frequency identifies genes that characterize preimplantation embryos, embryonic stem cells, and adult stem cells, thus providing potential markers as well as clues to the functional features of these cells. Principal component analysis identified a set of 88 genes whose average expression levels decrease from oocytes to blastocysts, stem cells, postimplantation embryos, and finally to newborn tissues. This can be a first step towards a possible definition of a molecular scale of cellular potency. The sequences and cDNA clones recovered in this work provide a comprehensive resource for genes functioning in early mouse embryos and stem cells. The nonrestricted community access to the resource can accelerate a wide range of research, particularly in reproductive and regenerative medicine.
[Show abstract][Hide abstract] ABSTRACT: Microsatellite instability (MSI) has been reported in endometrial carcinoma (EC) and in colorectal carcinoma (CRC), primarily as a result of defective DNA mismatch repair (MMR). The MMR gene hMLH1 commonly is inactivated in both EC and CRC. In the current study, epigenetic mechanisms involved in hMLH1 inactivation have been investigated to further elucidate the role of these mechanisms in the pathogenesis of EC and CRC.
Polymerase chain reaction (PCR)-based microsatellite analysis performed on paraffin-embedded tissues was used to select 42 sporadic carcinomas (21 ECs and 21 CRCs) with MSI. Immunohistochemistry (IHC), using the anti-hMLH1 antibody, and mutation analysis, using denaturing high-performance liquid chromatography and automated sequencing, were performed on unstable carcinoma samples. Methylation analysis, using modified protocols for bisulfite treatment and methylation-specific PCR (MSP), was performed on DNA from archival tissue samples.
No MSI-positive tumor samples with normal hMLH1 immunostaining (n = 7) exhibited hMLH1 promoter methylation, whereas 8 of 35 unstable cases with loss of hMLH1 expression (23%) exhibited MSP amplification. Among analyzed cases, germ-line mutations of hMLH1 were found in 4 of 20 unmethylated samples (20%) and in 0 of 8 methylated samples. Bisulfite sequencing of amplification products from methylated samples demonstrated that almost all CpG dinucleotides within the hMLH1 promoter elements underwent methylation.
Although an MMR gene other than hMLH1 may be responsible for genetic instability in MSI-positive/IHC-positive tumors, the presence of MSP amplification and allelic deletions within the hMLH1 locus in subsets of MSI-positive/IHC-negative cases strongly suggests that hMLH1 promoter methylation may contribute to the inactivation of both hMLH1 alleles. Bisulfite analysis suggests that the mechanisms of hMLH1 silencing may depend on CpG density rather than site-specific methylation. Cancer 2003;98:1540-6.