RESUMO
Neural development is accomplished by differentiation events leading to metabolic reprogramming. Glycosphingolipid metabolism is reprogrammed during neural development with a switch from globo- to ganglio-series glycosphingolipid production. Failure to execute this glycosphingolipid switch leads to neurodevelopmental disorders in humans, indicating that glycosphingolipids are key players in this process. Nevertheless, both the molecular mechanisms that control the glycosphingolipid switch and its function in neurodevelopment are poorly understood. Here, we describe a self-contained circuit that controls glycosphingolipid reprogramming and neural differentiation. We find that globo-series glycosphingolipids repress the epigenetic regulator of neuronal gene expression AUTS2. AUTS2 in turn binds and activates the promoter of the first and rate-limiting ganglioside-producing enzyme GM3 synthase, thus fostering the synthesis of gangliosides. By this mechanism, the globo-AUTS2 axis controls glycosphingolipid reprogramming and neural gene expression during neural differentiation, which involves this circuit in neurodevelopment and its defects in neuropathology.
Assuntos
Diferenciação Celular/fisiologia , Reprogramação Celular/fisiologia , Glicoesfingolipídeos/metabolismo , Neurogênese/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Reprogramação Celular/efeitos dos fármacos , Proteínas do Citoesqueleto , Epigenômica , Gangliosídeos/metabolismo , Expressão Gênica , Inativação Gênica , Glicoesfingolipídeos/farmacologia , Células HeLa , Histonas/metabolismo , Humanos , Transtornos do Neurodesenvolvimento , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Neurônios/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas/genética , Proteínas/metabolismo , Sialiltransferases/genética , Sialiltransferases/metabolismo , Fatores de TranscriçãoRESUMO
Facioscapulohumeral muscular dystrophy (FSHD) is characterized by incomplete penetrance and intra-familial clinical variability. The disease has been associated with the genetic and epigenetic features of the D4Z4 repetitive elements at 4q35. Recently, D4Z4 hypomethylation has been proposed as a reliable marker in the FSHD diagnosis. We exploited the Italian Registry for FSHD, in which FSHD families are classified using the Clinical Comprehensive Evaluation Form (CCEF). A total of 122 index cases showing a classical FSHD phenotype (CCEF, category A) and 110 relatives were selected to test with the receiver operating characteristic (ROC) curve, the diagnostic and predictive value of D4Z4 methylation. Moreover, we performed DNA methylation analysis in selected large families with reduced penetrance characterized by the co-presence of subjects carriers of one D4Z4 reduced allele with no signs of disease or presenting the classic FSHD clinical phenotype. We observed a wide variability in the D4Z4 methylation levels among index cases revealing no association with clinical manifestation or disease severity. By extending the analysis to family members, we revealed the low predictive value of D4Z4 methylation in detecting the affected condition. In view of the variability in D4Z4 methylation profiles observed in our large cohort, we conclude that D4Z4 methylation does not mirror the clinical expression of FSHD. We recommend that measurement of this epigenetic mark must be interpreted with caution in clinical practice.
Assuntos
Epigênese Genética , Epigenômica , Estudos de Associação Genética , Genótipo , Distrofia Muscular Facioescapuloumeral/diagnóstico , Distrofia Muscular Facioescapuloumeral/genética , Fenótipo , Alelos , Variação Biológica da População , Metilação de DNA , Epigenômica/métodos , Família , Predisposição Genética para Doença , Humanos , Linhagem , Curva ROCRESUMO
Hypomorphic mutations in DNA-methyltransferase DNMT3B cause majority of the rare disorder Immunodeficiency, Centromere instability and Facial anomalies syndrome cases (ICF1). By unspecified mechanisms, mutant-DNMT3B interferes with lymphoid-specific pathways resulting in immune response defects. Interestingly, recent findings report that DNMT3B shapes intragenic CpG-methylation of highly-transcribed genes. However, how the DNMT3B-dependent epigenetic network modulates transcription and whether ICF1-specific mutations impair this process remains unknown. We performed a transcriptomic and epigenomic study in patient-derived B-cell lines to investigate the genome-scale effects of DNMT3B dysfunction. We highlighted that altered intragenic CpG-methylation impairs multiple aspects of transcriptional regulation, like alternative TSS usage, antisense transcription and exon splicing. These defects preferentially associate with changes of intragenic H3K4me3 and at lesser extent of H3K27me3 and H3K36me3. In addition, we highlighted a novel DNMT3B activity in modulating the self-regulatory circuit of sense-antisense pairs and the exon skipping during alternative splicing, through interacting with RNA molecules. Strikingly, altered transcription affects disease relevant genes, as for instance the memory-B cell marker CD27 and PTPRC genes, providing us with biological insights into the ICF1-syndrome pathogenesis. Our genome-scale approach sheds light on the mechanisms still poorly understood of the intragenic function of DNMT3B and DNA methylation in gene expression regulation.
Assuntos
Processamento Alternativo , Anorexia/genética , Caquexia/genética , DNA (Citosina-5-)-Metiltransferases/genética , Anormalidades do Olho/genética , Histonas/genética , Síndromes de Imunodeficiência/genética , Mutação , RNA Mensageiro/genética , Dermatopatias/genética , Anorexia/imunologia , Anorexia/patologia , Linfócitos B/imunologia , Linfócitos B/patologia , Caquexia/imunologia , Caquexia/patologia , Linhagem Celular Transformada , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/imunologia , Metilação de DNA , Epigênese Genética , Anormalidades do Olho/imunologia , Anormalidades do Olho/patologia , Fácies , Feminino , Histonas/imunologia , Humanos , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/patologia , Memória Imunológica , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/imunologia , Masculino , Regiões Promotoras Genéticas , RNA Mensageiro/imunologia , Dermatopatias/imunologia , Dermatopatias/patologia , Transcrição Gênica , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , DNA Metiltransferase 3BRESUMO
Methyl-CpG binding protein 2 (MeCP2) is a multi-function factor involved in locus-specific transcriptional modulation and the regulation of genome architecture, e.g., pericentric heterochromatin (PCH) organization. MECP2 mutations are responsible for Rett syndrome (RTT), a devastating postnatal neurodevelopmental disorder, the pathogenetic mechanisms of which are still unknown. MeCP2, together with Alpha-thalassemia/mental retardation syndrome X-linked protein (ATRX), accumulates at chromocenters, which are repressive PCH domains. As with MECP2, mutations in ATRX cause ATR-X syndrome which is associated with severe intellectual disability. We exploited two murine embryonic stem cell lines, in which the expression of MeCP2 or ATRX is abolished. Through immunostaining, chromatin immunoprecipitation and western blot, we show that MeCP2 and ATRX are reciprocally dependent both for their expression and targeting to chromocenters. Moreover, ATRX plays a role in the accumulation of members of the heterochromatin protein 1 (HP1) family at PCH and, as MeCP2, modulates their expression. Furthermore, ATRX and HP1 targeting to chromocenters depends on an RNA component. 3D-DNA fluorescence in situ hybridization (FISH) highlighted, for the first time, a contribution of ATRX in MeCP2-mediated chromocenter clustering during neural differentiation. Overall, we provide a detailed dissection of the functional interplay between MeCP2 and ATRX in higher-order PCH organization in neurons. Our findings suggest molecular defects common to RTT and ATR-X syndrome, including an alteration in PCH.
Assuntos
Diferenciação Celular/fisiologia , Heterocromatina/metabolismo , Proteína 2 de Ligação a Metil-CpG/metabolismo , Neurônios/metabolismo , Proteína Nuclear Ligada ao X/metabolismo , Animais , Diferenciação Celular/genética , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Modelos Animais de Doenças , Células-Tronco Embrionárias , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Heterocromatina/química , Heterocromatina/genética , Hibridização in Situ Fluorescente , Deficiência Intelectual/genética , Deficiência Intelectual Ligada ao Cromossomo X/genética , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Mutação , Síndrome de Rett/genética , Proteína Nuclear Ligada ao X/química , Proteína Nuclear Ligada ao X/genética , Talassemia alfa/genéticaRESUMO
X chromosome inactivation (XCI) is the phenomenon by which mammals compensate for dosage of X-linked genes in females (XX) versus males (XY). XCI patterns can be random or show extreme skewing, and can modify the mode of inheritance of X-driven phenotypes, which contributes to the variability of human pathologies. Recent findings have shown reversibility of the XCI process, which has opened new avenues in the approaches used for the treatment of X-linked diseases.
Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/genética , Inativação do Cromossomo X/genética , Animais , Humanos , Síndrome de Rett/genéticaRESUMO
BACKGROUND: Mecp2 null mice model Rett syndrome (RTT) a human neurological disorder affecting females after apparent normal pre- and peri-natal developmental periods. Neuroanatomical studies in cerebral cortex of RTT mouse models revealed delayed maturation of neuronal morphology and autonomous as well as non-cell autonomous reduction in dendritic complexity of postnatal cortical neurons. However, both morphometric parameters and high-resolution expression profile of cortical neurons at embryonic developmental stage have not yet been studied. Here we address these topics by using embryonic neuronal primary cultures from Mecp2 loss of function mouse model. RESULTS: We show that embryonic primary cortical neurons of Mecp2 null mice display reduced neurite complexity possibly reflecting transcriptional changes. We used RNA-sequencing coupled with a bioinformatics comparative approach to identify and remove the contribution of variable and hard to quantify non-neuronal brain cells present in our in vitro cell cultures. CONCLUSIONS: Our results support the need to investigate both Mecp2 morphological as well as molecular effect in neurons since prenatal developmental stage, long time before onset of Rett symptoms.
Assuntos
Encéfalo/patologia , Proteína 2 de Ligação a Metil-CpG/genética , Síndrome de Rett/embriologia , Síndrome de Rett/genética , Animais , Astrócitos/metabolismo , Encéfalo/embriologia , Encéfalo/metabolismo , Córtex Cerebral/metabolismo , Biologia Computacional , Dendritos/metabolismo , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Camundongos , Neuroglia/metabolismo , Neurônios/citologia , Síndrome de Rett/patologia , Análise de Sequência de RNARESUMO
Rett syndrome (RTT) is a rare neurodevelopmental disorder affecting almost exclusively females, caused in the overwhelming majority of the cases by loss-of-function mutations in the gene encoding methyl-CpG binding protein 2 (MECP2). High circulating levels of oxidative stress (OS) markers in patients suggest the involvement of OS in the RTT pathogenesis. To investigate the occurrence of oxidative brain damage in Mecp2 mutant mouse models, several OS markers were evaluated in whole brains of Mecp2-null (pre-symptomatic, symptomatic, and rescued) and Mecp2-308 mutated (pre-symptomatic and symptomatic) mice, and compared to those of wild type littermates. Selected OS markers included non-protein-bound iron, isoprostanes (F2-isoprostanes, F4-neuroprostanes, F2-dihomo-isoprostanes) and 4-hydroxy-2-nonenal protein adducts. Our findings indicate that oxidative brain damage 1) occurs in both Mecp2-null (both -/y and stop/y) and Mecp2-308 (both 308/y males and 308/+ females) mouse models of RTT; 2) precedes the onset of symptoms in both Mecp2-null and Mecp2-308 models; and 3) is rescued by Mecp2 brain specific gene reactivation. Our data provide direct evidence of the link between Mecp2 deficiency, oxidative stress and RTT pathology, as demonstrated by the rescue of the brain oxidative homeostasis following brain-specifically Mecp2-reactivated mice. The present study indicates that oxidative brain damage is a previously unrecognized hallmark feature of murine RTT, and suggests that Mecp2 is involved in the protection of the brain from oxidative stress.
Assuntos
Lesões Encefálicas/etiologia , Proteína 2 de Ligação a Metil-CpG/genética , Mutação/genética , Estresse Oxidativo/fisiologia , Síndrome de Rett/complicações , Síndrome de Rett/genética , Aldeídos/metabolismo , Análise de Variância , Animais , Ácido Araquidônico/metabolismo , Lesões Encefálicas/sangue , Lesões Encefálicas/patologia , Modelos Animais de Doenças , Ácidos Docosa-Hexaenoicos/metabolismo , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Isoprostanos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nestina/genética , Neuroprostanos/metabolismo , Síndrome de Rett/sangueRESUMO
Vesicular (v)- and target (t)-SNARE proteins assemble in SNARE complex to mediate membrane fusion. Tetanus neurotoxin-insensitive vesicular-associated membrane protein (TI-VAMP/VAMP7), a vesicular SNARE expressed in several cell types including neurons, was previously shown to play a major role in exocytosis involved in neurite growth in cultured neurons. Here we generated a complete constitutive knock-out by deleting the exon 3 of Vamp7. Loss of TI-VAMP expression did not lead to any striking developmental or neurological defect. Knock-out mice displayed decreased brain weight and increased third ventricle volume. Axon growth appeared normal in cultured knock-out neurons. Behavioral characterization unraveled that TI-VAMP knock-out was associated with increased anxiety. Our results thus suggest compensatory mechanisms allowing the TI-VAMP knock-out mice to fulfill major developmental processes. The phenotypic traits unraveled here further indicate an unexpected role of TI-VAMP-mediated vesicular traffic in anxiety and suggest a role for TI-VAMP in higher brain functions.
Assuntos
Ansiedade/genética , Metaloendopeptidases , Proteínas R-SNARE/deficiência , Proteínas R-SNARE/genética , Toxina Tetânica , Animais , Ansiedade/etiologia , Ansiedade/psicologia , Células COS , Células Cultivadas , Chlorocebus aethiops , Masculino , Metaloendopeptidases/administração & dosagem , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Coelhos , Toxina Tetânica/administração & dosagemRESUMO
Rett syndrome (RTT, online MIM 312750) is a devastating neurodevelopmental disorder characterized by motor and cognitive disabilities. It is mainly caused by pathogenetic variants in the X-linked MECP2 gene, encoding an epigenetic factor crucial for brain functioning. Despite intensive studies, the RTT pathogenetic mechanism remains to be fully elucidated. Impaired vascular function has been previously reported in RTT mouse models; however, whether an altered brain vascular homeostasis and the subsequent blood-brain barrier (BBB) breakdown occur in RTT and contribute to the disease-related cognitive impairment is still unknown. Interestingly, in symptomatic Mecp2-null (Mecp2-/y, Mecp2tm1.1Bird) mice, we found enhanced BBB permeability associated with an aberrant expression of the tight junction proteins Ocln and Cldn-5 in different brain areas, in terms of both transcript and protein levels. Additionally, Mecp2-null mice showed an altered expression of different genes encoding factors with a role in the BBB structure and function, such as Cldn3, Cldn12, Mpdz, Jam2, and Aqp4. With this study, we provide the first evidence of impaired BBB integrity in RTT and highlight a potential new molecular hallmark of the disease that might open new perspectives for the setting-up of novel therapeutic strategies.
Assuntos
Síndrome de Rett , Camundongos , Animais , Síndrome de Rett/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Camundongos Knockout , Camundongos Endogâmicos C57BL , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismoRESUMO
BACKGROUND: Bovine papillomaviruses (BPVs) types 1 and 2 are the only known papillomaviruses able to jump the species. In fact, BPVs 1/2 induce neoplasia in their natural bovine host but infection is also associated to neoplastic skin lesions in equids termed sarcoids. The equine sarcoid is considered to be the most common equine cutaneous tumour worldwide for which no effective therapy is available. Very little is known about the molecular mechanisms underlying tumourigenesis, although genes contributing to sarcoid development have been identified. Several studies associate the development of cancer to the loss of function of a number of oncosuppressor genes. In this study the putative role of O6-methylguanine-DNA methyltrasferase (MGMT) was investigated for sarcoids. The expression of the oncosuppressor protein was assessed in normal and sarcoid cells and tissues. In addition, the DNA methylation profile was analysed to assess the role of epigenetic mechanism in regulation of MGMT expression. RESULTS: A group of 15 equine sarcoids and two primary sarcoid cell lines (fibroblasts) were analyzed for the expression of MGMT protein by immunohistochemistry, immunofluorescence and Western blotting techniques. The sarcoid cell line EqSO4b and the tumour samples showed a reduction or absence of MGMT expression. To investigate the causes of deregulated MGMT expression, ten samples were analyzed for the DNA methylation profile of the CpG island associated to the MGMT promoter. The analysis of 73 CpGs encompassing the region of interest showed in 1 out of 10 (10%) sarcoids a pronouncedly altered methylation profile when compared to the control epidermal sample. Similarily the EqSO4b cell line showed an altered MGMT methylation pattern in comparison to normal fibroblasts. CONCLUSION: As previously demonstrated for the oncosuppressor gene FHIT, analysis of MGMT expression in sarcoid tissues and a sarcoid-derived fibroblast cell line further suggests that oncosuppressor silencing may be also involved in BPV-induced equine tumours. Abnormal DNA methylation seems to be one of the possible molecular mechanisms involved in the alteration of MGMT expression. Further studies are required to address other basic molecular mechanisms involved in reduced MGMT expression. This study underlines the possible role of DNA methylation in oncosuppressor inactivation in equine sarcoids.
Assuntos
Doenças dos Cavalos/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Neoplasias Cutâneas/veterinária , Animais , Papillomavirus Bovino 1 , Linhagem Celular Tumoral , Ilhas de CpG , Metilação de DNA , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/fisiologia , Cavalos , O(6)-Metilguanina-DNA Metiltransferase/genética , Regiões Promotoras Genéticas , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/virologiaRESUMO
BACKGROUND: Sarcoids are peculiar equine benign tumours. Their onset is associated with Bovine Papillomavirus type -1 or -2 (BPV-1/2) infection. Little is known about the molecular interplay between viral infection and neoplastic transformation. The data regarding papillomavirus infections in human species show the inactivation of a number of tumour suppressor genes as basic mechanism of transformation. In this study the putative role of the tumour suppressor gene Fragile Histidine Triad (FHIT) in sarcoid tumour was investigated in different experimental models. The expression of the oncosuppressor protein was assessed in normal and sarcoid cells and tissue. RESULTS: Nine paraffin embedded sarcoids and sarcoid derived cell lines were analysed for the expression of FHIT protein by immunohistochemistry, immunofluorescence techniques and western blotting. These analyses revealed the absence of signal in seven out of nine sarcoids. The two sarcoid derived cell lines too showed a reduced signal of the protein. To investigate the causes of the altered protein expression, the samples were analysed for the DNA methylation profile of the CpG island associated with the FHIT promoter. The analysis of the 32 CpGs encompassing the region of interest showed no significative differential methylation profile between pathological tissues and cell lines and their normal counterparts. CONCLUSION: This study represent a further evidence of the role of a tumour suppressor gene in equine sarcoids and approaches the epigenetic regulation in this well known equine neoplasm. The data obtained in sarcoid tissues and sarcoid derived cell lines suggest that also in horse, as in humans, there is a possible involvement of the tumour suppressor FHIT gene in BPV induced tumours. DNA methylation seems not to be involved in the gene expression alteration. Further studies are needed to understand the basic molecular mechanisms involved in reduced FHIT expression.
Assuntos
Hidrolases Anidrido Ácido/genética , Papillomavirus Bovino 1/genética , Epigenômica/normas , Doenças dos Cavalos/genética , Proteínas de Neoplasias/genética , Infecções por Papillomavirus/veterinária , Neoplasias Cutâneas/veterinária , Hidrolases Anidrido Ácido/metabolismo , Fatores Etários , Animais , Papillomavirus Bovino 1/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Doenças dos Cavalos/metabolismo , Doenças dos Cavalos/virologia , Cavalos , Imuno-Histoquímica/veterinária , Proteínas de Neoplasias/metabolismo , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , RNA Neoplásico/química , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/virologiaRESUMO
In this study we investigated the transcriptome and epigenome dynamics of the tomato fruit during post-harvest in a landrace belonging to a group of tomatoes (Solanum lycopersicum L.) collectively known as "Piennolo del Vesuvio", all characterized by a long shelf-life. Expression of protein-coding genes and microRNAs as well as DNA methylation patterns and histone modifications were analysed in distinct post-harvest phases. Multi-omics data integration contributed to the elucidation of the molecular mechanisms underlying processes leading to long shelf-life. We unveiled global changes in transcriptome and epigenome. DNA methylation increased and the repressive histone mark H3K27me3 was lost as the fruit progressed from red ripe to 150 days post-harvest. Thousands of genes were differentially expressed, about half of which were potentially epi-regulated as they were engaged in at least one epi-mark change in addition to being microRNA targets in ~5% of cases. Down-regulation of the ripening regulator MADS-RIN and of genes involved in ethylene response and cell wall degradation was consistent with the delayed fruit softening. Large-scale epigenome reprogramming that occurred in the fruit during post-harvest likely contributed to delayed fruit senescence.
RESUMO
Oxidative damage has been reported in Rett syndrome (RTT), a pervasive developmental disorder caused in up to 95% of cases by mutations in the X-linked methyl-CpG binding protein 2 gene. Herein, we have synthesized F(2)-dihomo-isoprostanes (F(2)-dihomo-IsoPs), peroxidation products from adrenic acid (22:4 n-6), a known component of myelin, and tested the potential value of F(2)-dihomo-IsoPs as a novel disease marker and its relationship with clinical presentation and disease progression. F(2)-dihomo-IsoPs were determined by gas chromatography/negative-ion chemical ionization tandem mass spectrometry. Newly synthesized F(2)-dihomo-IsoP isomers [ent-7(RS)-F(2t)-dihomo-IsoP and 17-F(2t)-dihomo-IsoP] were used as reference standards. The measured ions were the product ions at m/z 327 derived from the [M-181](-) precursor ions (m/z 597) produced from both the derivatized ent-7(RS)-F(2t)-dihomo-IsoP and 17-F(2t)-dihomo-IsoP. Average plasma F(2)-dihomo-IsoP levels in RTT were about one order of magnitude higher than those in healthy controls, being higher in typical RTT as compared with RTT variants, with a remarkable increase of about two orders of magnitude in patients at the earliest stage of the disease followed by a steady decrease during the natural clinical progression. hese data indicate for the first time that quantification of F(2)-dihomo-IsoPs in plasma represents an early marker of the disease and may provide a better understanding of the pathogenic mechanisms behind the neurological regression in patients with RTT.
Assuntos
F2-Isoprostanos/sangue , Peroxidação de Lipídeos , Estresse Oxidativo , Síndrome de Rett/sangue , Síndrome de Rett/metabolismo , Adolescente , Adulto , Biomarcadores/sangue , Biomarcadores/química , Criança , Pré-Escolar , Progressão da Doença , Ácidos Erúcicos/metabolismo , F2-Isoprostanos/química , Ácidos Graxos Insaturados , Feminino , Humanos , Lactente , Bainha de Mielina/metabolismo , Especificidade de Órgãos , Adulto JovemRESUMO
BACKGROUND: The control of intracellular vesicle trafficking is an ideal target to weigh the role of alternative splicing in shaping genomes to make cells. Alternative splicing has been reported for several Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptors of the vesicle (v-SNAREs) or of the target membrane (t-SNARES), which are crucial to intracellular membrane fusion and protein and lipid traffic in Eukaryotes. However, splicing has not yet been investigated in Longins, i.e. the most widespread v-SNAREs. Longins are essential in Eukaryotes and prototyped by VAMP7, Sec22b and Ykt6, sharing a conserved N-terminal Longin domain which regulates membrane fusion and subcellular targeting. Human VAMP7/TI-VAMP, encoded by gene SYBL1, is involved in multiple cell pathways, including control of neurite outgrowth. RESULTS: Alternative splicing of SYBL1 by exon skipping events results in the production of a number of VAMP7 isoforms. In-frame or frameshift coding sequence modifications modulate domain architecture of VAMP7 isoforms, which can lack whole domains or domain fragments and show variant or extra domains. Intriguingly, two main types of VAMP7 isoforms either share the inhibitory Longin domain and lack the fusion-promoting SNARE motif, or vice versa. Expression analysis in different tissues and cell lines, quantitative real time RT-PCR and confocal microscopy analysis of fluorescent protein-tagged isoforms demonstrate that VAMP7 variants have different tissue specificities and subcellular localizations. Moreover, design and use of isoform-specific antibodies provided preliminary evidence for the existence of splice variants at the protein level. CONCLUSIONS: Previous evidence on VAMP7 suggests inhibitory functions for the Longin domain and fusion/growth promoting activity for the Δ-longin molecule. Thus, non-SNARE isoforms with Longin domain and non-longin SNARE isoforms might have somehow opposite regulatory functions. When considering splice variants as "natural mutants", evidence on modulation of subcellular localization by variation in domain combination can shed further light on targeting determinants. Although further work will be needed to characterize identified variants, our data might open the route to unravel novel molecular partners and mechanisms, accounting for the multiplicity of functions carried out by the different members of the Longin proteins family.
Assuntos
Processamento Alternativo , Proteínas R-SNARE/metabolismo , Proteínas SNARE/metabolismo , Linhagem Celular , Éxons , Humanos , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas R-SNARE/análise , Proteínas R-SNARE/genéticaRESUMO
Rett syndrome (RTT) is an extremely invalidating, cureless, developmental disorder, and it is considered one of the leading causes of intellectual disability in female individuals. The vast majority of RTT cases are caused by de novo mutations in the X-linked Methyl-CpG binding protein 2 (MECP2) gene, which encodes a multifunctional reader of methylated DNA. MeCP2 is a master epigenetic modulator of gene expression, with a role in the organization of global chromatin architecture. Based on its interaction with multiple molecular partners and the diverse epigenetic scenario, MeCP2 triggers several downstream mechanisms, also influencing the epigenetic context, and thus leading to transcriptional activation or repression. In this frame, it is conceivable that defects in such a multifaceted factor as MeCP2 lead to large-scale alterations of the epigenome, ranging from an unbalanced deposition of epigenetic modifications to a transcriptional alteration of both protein-coding and non-coding genes, with critical consequences on multiple downstream biological processes. In this review, we provide an overview of the current knowledge concerning the transcriptomic and epigenomic alterations found in RTT patients and animal models.
Assuntos
Epigênese Genética/genética , Síndrome de Rett/genética , Transcriptoma/genética , Cromatina , Metilação de DNA , Epigenômica/métodos , Expressão Gênica/genética , Histonas/genética , Humanos , Proteína 2 de Ligação a Metil-CpG/genética , RNA não Traduzido/genética , Síndrome de Rett/metabolismo , Síndrome de Rett/fisiopatologia , Ativação TranscricionalRESUMO
BACKGROUND: Parkinson's disease (PD) is a neurodegenerative movement disorder affecting 1-5% of the general population for which neither effective cure nor early diagnostic tools are available that could tackle the pathology in the early phase. Here we report a multi-stage procedure to identify candidate genes likely involved in the etiopathogenesis of PD. METHODS: The study includes a discovery stage based on the analysis of whole exome data from 26 dominant late onset PD families, a validation analysis performed on 1542 independent PD patients and 706 controls from different cohorts and the assessment of polygenic variants load in the Italian cohort (394 unrelated patients and 203 controls). RESULTS: Family-based approach identified 28 disrupting variants in 26 candidate genes for PD including PARK2, PINK1, DJ-1(PARK7), LRRK2, HTRA2, FBXO7, EIF4G1, DNAJC6, DNAJC13, SNCAIP, AIMP2, CHMP1A, GIPC1, HMOX2, HSPA8, IMMT, KIF21B, KIF24, MAN2C1, RHOT2, SLC25A39, SPTBN1, TMEM175, TOMM22, TVP23A and ZSCAN21. Sixteen of them have not been associated to PD before, were expressed in mesencephalon and were involved in pathways potentially deregulated in PD. Mutation analysis in independent cohorts disclosed a significant excess of highly deleterious variants in cases (p = 0.0001), supporting their role in PD. Moreover, we demonstrated that the co-inheritance of multiple rare variants (≥ 2) in the 26 genes may predict PD occurrence in about 20% of patients, both familial and sporadic cases, with high specificity (> 93%; p = 4.4 × 10- 5). Moreover, our data highlight the fact that the genetic landmarks of late onset PD does not systematically differ between sporadic and familial forms, especially in the case of small nuclear families and underline the importance of rare variants in the genetics of sporadic PD. Furthermore, patients carrying multiple rare variants showed higher risk of manifesting dyskinesia induced by levodopa treatment. CONCLUSIONS: Besides confirming the extreme genetic heterogeneity of PD, these data provide novel insights into the genetic of the disease and may be relevant for its prediction, diagnosis and treatment.
Assuntos
Sequenciamento do Exoma/métodos , Predisposição Genética para Doença/genética , Doença de Parkinson/genética , Adulto , Idade de Início , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , LinhagemRESUMO
Before the end of the Last Glacial Maximum (LGM, â¼16.5 ka ago)1 set in motion major shifts in human culture and population structure,2 a consistent change in lithic technology, material culture, settlement pattern, and adaptive strategies is recorded in Southern Europe at â¼18-17 ka ago. In this time frame, the landscape of Northeastern Italy changed considerably, and the retreat of glaciers allowed hunter-gatherers to gradually recolonize the Alps.3-6 Change within this renewed cultural frame (i.e., during the Late Epigravettian phase) is currently associated with migrations favored by warmer climate linked to the Bølling-Allerød onset (14.7 ka ago),7-11 which replaced earlier genetic lineages with ancestry found in an individual who lived â¼14 ka ago at Riparo Villabruna, Italy, and shared among different contexts (Villabruna Cluster).9 Nevertheless, these dynamics and their chronology are still far from being disentangled due to fragmentary evidence for long-distance interactions across Europe.12 Here, we generate new genomic data from a human mandible uncovered at Riparo Tagliente (Veneto, Italy), which we directly dated to 16,980-16,510 cal BP (2σ). This individual, affected by focal osseous dysplasia, is genetically affine to the Villabruna Cluster. Our results therefore backdate by at least 3 ka the diffusion in Southern Europe of a genetic component linked to Balkan/Anatolian refugia, previously believed to have spread during the later Bølling/Allerød event. In light of the new genetic evidence, this population replacement chronologically coincides with the very emergence of major cultural transitions in Southern and Western Europe.
Assuntos
Migração Humana , Camada de Gelo , Clima , Europa (Continente) , Humanos , OcupaçõesRESUMO
Pericentric heterochromatin (PCH) is a particular form of constitutive heterochromatin that is localized to both sides of centromeres and that forms silent compartments enriched in repressive marks. These genomic regions contain species-specific repetitive satellite DNA that differs in terms of nucleotide sequences and repeat lengths. In spite of this sequence diversity, PCH is involved in many biological phenomena that are conserved among species, including centromere function, the preservation of genome integrity, the suppression of spurious recombination during meiosis, and the organization of genomic silent compartments in the nucleus. PCH organization and maintenance of its repressive state is tightly regulated by a plethora of factors, including enzymes (e.g., DNA methyltransferases, histone deacetylases, and histone methyltransferases), DNA and histone methylation binding factors (e.g., MECP2 and HP1), chromatin remodeling proteins (e.g., ATRX and DAXX), and non-coding RNAs. This evidence helps us to understand how PCH organization is crucial for genome integrity. It then follows that alterations to the molecular signature of PCH might contribute to the onset of many genetic pathologies and to cancer progression. Here, we describe the most recent updates on the molecular mechanisms known to underlie PCH organization and function.
Assuntos
Centrômero/genética , Metilação de DNA/genética , Heterocromatina/genética , Histonas/genética , Animais , Montagem e Desmontagem da Cromatina/genética , Epigênese Genética/genética , Histona Desacetilases/genética , Histona Metiltransferases , Humanos , MamíferosRESUMO
Methyl-CpG binding protein 2 (MeCP2) has historically been linked to heterochromatin organization, and in mouse cells it accumulates at pericentric heterochromatin (PCH), closely following major satellite (MajSat) DNA distribution. However, little is known about the specific function of MeCP2 in these regions. We describe the first evidence of a role in neurons for MeCP2 and MajSat forward (MajSat-fw) RNA in reciprocal targeting to PCH through their physical interaction. Moreover, MeCP2 contributes to maintenance of PCH by promoting deposition of H3K9me3 and H4K20me3. We highlight that the MeCP2B isoform is required for correct higher-order PCH organization, and underline involvement of the methyl-binding and transcriptional repression domains. The T158 residue, which is commonly mutated in Rett patients, is directly involved in this process. Our findings support the hypothesis that MeCP2 and the MajSat-fw transcript are mutually dependent for PCH organization, and contribute to clarify MeCP2 function in the regulation of chromatin architecture.
Assuntos
DNA Satélite/metabolismo , Heterocromatina/metabolismo , Histonas/metabolismo , Proteína 2 de Ligação a Metil-CpG/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Animais , DNA Satélite/genética , Heterocromatina/genética , Histonas/genética , Proteína 2 de Ligação a Metil-CpG/genética , CamundongosRESUMO
BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.