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1.
Hum Mutat ; 2019 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-31230393

RESUMO

Human retrocopies, that is messenger RNA transcripts benefitting from the long interspersed element 1 machinery for retrotransposition, may have specific consequences for genomic testing. Next genetration sequencing (NGS) techniques allow the detection of such mobile elements but they may be misinterpreted as genomic duplications or be totally overlooked. We report eight observations of retrocopies detected during diagnostic NGS analyses of targeted gene panels, exome, or genome sequencing. For seven cases, while an exons-only copy number gain was called, read alignment inspection revealed a depth of coverage shift at every exon-intron junction where indels were also systematically called. Moreover, aberrant chimeric read pairs spanned entire introns or were paired with another locus for terminal exons. The 8th retrocopy was present in the reference genome and thus showed a normal NGS profile. We emphasize the existence of retrocopies and strategies to accurately detect them at a glance during genetic testing and discuss pitfalls for genetic testing.

3.
Ann Neurol ; 83(5): 926-934, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29630738

RESUMO

OBJECTIVE: Cut homeodomain transcription factor CUX2 plays an important role in dendrite branching, spine development, and synapse formation in layer II to III neurons of the cerebral cortex. We identify a recurrent de novo CUX2 p.Glu590Lys as a novel genetic cause for developmental and epileptic encephalopathy (DEE). METHODS: The de novo p.Glu590Lys variant was identified by whole-exome sequencing (n = 5) or targeted gene panel (n = 4). We performed electroclinical and imaging phenotyping on all patients. RESULTS: The cohort comprised 7 males and 2 females. Mean age at study was 13 years (0.5-21.0). Median age at seizure onset was 6 months (2 months to 9 years). Seizure types at onset were myoclonic, atypical absence with myoclonic components, and focal seizures. Epileptiform activity on electroencephalogram was seen in 8 cases: generalized polyspike-wave (6) or multifocal discharges (2). Seizures were drug resistant in 7 or controlled with valproate (2). Six patients had a DEE: myoclonic DEE (3), Lennox-Gastaut syndrome (2), and West syndrome (1). Two had a static encephalopathy and genetic generalized epilepsy, including absence epilepsy in 1. One infant had multifocal epilepsy. Eight had severe cognitive impairment, with autistic features in 6. The p.Glu590Lys variant affects a highly conserved glutamine residue in the CUT domain predicted to interfere with CUX2 binding to DNA targets during neuronal development. INTERPRETATION: Patients with CUX2 p.Glu590Lys display a distinctive phenotypic spectrum, which is predominantly generalized epilepsy, with infantile-onset myoclonic DEE at the severe end and generalized epilepsy with severe static developmental encephalopathy at the milder end of the spectrum. Ann Neurol 2018;83:926-934.

4.
Cell Rep ; 18(8): 1996-2006, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28228264

RESUMO

MyoD is a master regulator of myogenesis. Chromatin modifications required to trigger MyoD expression are still poorly described. Here, we demonstrate that the histone demethylase LSD1/KDM1a is recruited on the MyoD core enhancer upon muscle differentiation. Depletion of Lsd1 in myoblasts precludes the removal of H3K9 methylation and the recruitment of RNA polymerase II on the core enhancer, thereby preventing transcription of the non-coding enhancer RNA required for MyoD expression (CEeRNA). Consistently, Lsd1 conditional inactivation in muscle progenitor cells during embryogenesis prevented transcription of the CEeRNA and delayed MyoD expression. Our results demonstrate that LSD1 is required for the timely expression of MyoD in limb buds and identify a new biological function for LSD1 by showing that it can activate RNA polymerase II-dependent transcription of enhancers.


Assuntos
Histona Desmetilases/metabolismo , Proteína MyoD/metabolismo , Transcrição Genética/fisiologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Histonas/metabolismo , Botões de Extremidades/metabolismo , Camundongos , Desenvolvimento Muscular/fisiologia , Mioblastos/metabolismo , Mioblastos/fisiologia , RNA Polimerase II/metabolismo , Sequências Reguladoras de Ácido Nucleico/fisiologia
5.
J Biol Chem ; 290(7): 4215-24, 2015 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-25516595

RESUMO

Skeletal muscle atrophy is a severe condition of muscle mass loss. Muscle atrophy is caused by a down-regulation of protein synthesis and by an increase of protein breakdown due to the ubiquitin-proteasome system and autophagy activation. Up-regulation of specific genes, such as the muscle-specific E3 ubiquitin ligase MAFbx, by FoxO transcription factors is essential to initiate muscle protein ubiquitination and degradation during atrophy. HDAC6 is a particular HDAC, which is functionally related to the ubiquitin proteasome system via its ubiquitin binding domain. We show that HDAC6 is up-regulated during muscle atrophy. HDAC6 activation is dependent on the transcription factor FoxO3a, and the inactivation of HDAC6 in mice protects against muscle wasting. HDAC6 is able to interact with MAFbx, a key ubiquitin ligase involved in muscle atrophy. Our findings demonstrate the implication of HDAC6 in skeletal muscle wasting and identify HDAC6 as a new downstream target of FoxO3a in stress response. This work provides new insights in skeletal muscle atrophy development and opens interesting perspectives on HDAC6 as a valuable marker of muscle atrophy and a potential target for pharmacological treatments.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/patologia , Animais , Western Blotting , Células Cultivadas , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Desacetilase 6 de Histona , Histona Desacetilases/química , Histona Desacetilases/genética , Humanos , Imunoprecipitação , Integrases/metabolismo , Camundongos , Camundongos Knockout , Denervação Muscular , Músculo Esquelético/metabolismo , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
Brain ; 136(Pt 8): 2359-68, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23824486

RESUMO

Amyotrophic lateral sclerosis is a typically rapidly progressive neurodegenerative disorder affecting motor neurons leading to progressive muscle paralysis and death, usually from respiratory failure, in 3-5 years. Some patients have slow disease progression and prolonged survival, but the underlying mechanisms remain poorly understood. Riluzole, the only approved treatment, only modestly prolongs survival and has no effect on muscle function. In the early phase of the disease, motor neuron loss is initially compensated for by collateral reinnervation, but over time this compensation fails, leading to progressive muscle wasting. The crucial role of muscle histone deacetylase 4 and its regulator microRNA-206 in compensatory reinnervation and disease progression was recently suggested in a mouse model of amyotrophic lateral sclerosis (transgenic mice carrying human mutations in the superoxide dismutase gene). Here, we sought to investigate whether the microRNA-206-histone deacetylase 4 pathway plays a role in muscle compensatory reinnervation in patients with amyotrophic lateral sclerosis and thus contributes to disease outcome differences. We studied muscle reinnervation using high-resolution confocal imaging of neuromuscular junctions in muscle samples obtained from 11 patients with amyotrophic lateral sclerosis, including five long-term survivors. We showed that the proportion of reinnervated neuromuscular junctions was significantly higher in long-term survivors than in patients with rapidly progressive disease. We analysed the expression of muscle candidate genes involved in the reinnervation process and showed that histone deacetylase 4 upregulation was significantly greater in patients with rapidly progressive disease and was negatively correlated with the extent of muscle reinnervation and functional outcome. Conversely, the proposed regulator of histone deacetylase 4, microRNA-206, was upregulated in both patient groups, but did not correlate with disease progression or reinnervation. We conclude that muscle expression of histone deacetylase 4 may be a key factor for muscle reinnervation and disease progression in patients with amyotrophic lateral sclerosis. Specific histone deacetylase 4 inhibitors may then constitute a therapeutic approach to enhancing motor performance and slowing disease progression in amyotrophic lateral sclerosis.


Assuntos
Esclerose Amiotrófica Lateral/genética , Histona Desacetilases/genética , MicroRNAs/genética , Neurônios Motores/metabolismo , Músculo Esquelético/inervação , Proteínas Repressoras/genética , Adulto , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Progressão da Doença , Feminino , Histona Desacetilases/metabolismo , Humanos , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Neurônios Motores/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Junção Neuromuscular/genética , Junção Neuromuscular/metabolismo , Junção Neuromuscular/patologia , Proteínas Repressoras/metabolismo , Sobreviventes , Regulação para Cima
8.
Nat Cell Biol ; 15(7): 818-28, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23792691

RESUMO

Dysfunctional telomeres suppress tumour progression by activating cell-intrinsic programs that lead to growth arrest. Increased levels of TRF2, a key factor in telomere protection, are observed in various human malignancies and contribute to oncogenesis. We demonstrate here that a high level of TRF2 in tumour cells decreased their ability to recruit and activate natural killer (NK) cells. Conversely, a reduced dose of TRF2 enabled tumour cells to be more easily eliminated by NK cells. Consistent with these results, a progressive upregulation of TRF2 correlated with decreased NK cell density during the early development of human colon cancer. By screening for TRF2-bound genes, we found that HS3ST4--a gene encoding for the heparan sulphate (glucosamine) 3-O-sulphotransferase 4--was regulated by TRF2 and inhibited the recruitment of NK cells in an epistatic relationship with TRF2. Overall, these results reveal a TRF2-dependent pathway that is tumour-cell extrinsic and regulates NK cell immunity.


Assuntos
Neoplasias da Mama/prevenção & controle , Neoplasias do Colo/prevenção & controle , Células Matadoras Naturais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/prevenção & controle , Sulfotransferases/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Animais , Apoptose , Western Blotting , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Adesão Celular , Proliferação de Células , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Primers do DNA/química , Receptor com Domínio Discoidina 1 , Feminino , Citometria de Fluxo , Células HeLa , Humanos , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Linfócitos do Interstício Tumoral/patologia , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Camundongos , Camundongos Nus , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfotransferases/genética , Proteína 2 de Ligação a Repetições Teloméricas/antagonistas & inibidores , Proteína 2 de Ligação a Repetições Teloméricas/genética , Células Tumorais Cultivadas
9.
EMBO Rep ; 14(4): 356-63, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23429341

RESUMO

The DNA-binding protein TRF2 is essential for telomere protection and chromosome stability in mammals. We show here that TRF2 expression is activated by the Wnt/ß-catenin signalling pathway in human cancer and normal cells as well as in mouse intestinal tissues. Furthermore, ß-catenin binds to TRF2 gene regulatory regions that are functional in a luciferase transactivating assay. Reduced ß-catenin expression in cancer cells triggers a marked increase in telomere dysfunction, which can be reversed by TRF2 overexpression. We conclude that the Wnt/ß-catenin signalling pathway maintains a level of TRF2 critical for telomere protection. This is expected to have an important role during development, adult stem cell function and oncogenesis.


Assuntos
Regulação da Expressão Gênica , Homeostase do Telômero , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Via de Sinalização Wnt , Animais , Sítios de Ligação , Feminino , Expressão Gênica , Células HCT116 , Humanos , Masculino , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/genética , Transcriptoma , beta Catenina/metabolismo
10.
Proc Natl Acad Sci U S A ; 108(20): 8305-10, 2011 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-21527717

RESUMO

Methylation of histone H3 lysine 4 (H3K4me), a mark associated with gene activation, is mediated by SET1 and the related mixed lineage leukemia (MLL) histone methyltransferases (HMTs) across species. Mammals contain seven H3K4 HMTs, Set1A, Set1B, and MLL1-MLL5. The activity of SET1 and MLL proteins relies on protein-protein interactions within large multisubunit complexes that include three core components: RbBP5, Ash2L, and WDR5. It remains unclear how the composition and specificity of these complexes varies between cell types and during development. Caenorhabditis elegans contains one SET1 protein, SET-2, one MLL-like protein, SET-16, and single homologs of RbBP5, Ash2L, and WDR5. Here we show that SET-2 is responsible for the majority of bulk H3K4 methylation at all developmental stages. However, SET-2 and absent, small, or homeotic discs 2 (ASH-2) are differentially required for tri- and dimethylation of H3K4 (H3K4me3 and -me2) in embryos and adult germ cells. In embryos, whereas efficient H3K4me3 requires both SET-2 and ASH-2, H3K4me2 relies mostly on ASH-2. In adult germ cells by contrast, SET-2 serves a major role whereas ASH-2 is dispensable for H3K4me3 and most H3K4me2. Loss of SET-2 results in progressive sterility over several generations, suggesting an important function in the maintenance of a functional germ line. This study demonstrates that individual subunits of SET1-related complexes can show tissue specificity and developmental regulation and establishes C. elegans as a model to study SET1-related complexes in a multicellular organism.


Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Embrião não Mamífero/metabolismo , Células Germinativas/metabolismo , Histona-Lisina N-Metiltransferase/fisiologia , Histonas/metabolismo , Proteínas Nucleares/fisiologia , Animais , Lisina/metabolismo , Metilação , Proteínas de Saccharomyces cerevisiae/fisiologia
11.
Cell Res ; 21(7): 1028-38, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21423270

RESUMO

The study of the proteins that bind to telomeric DNA in mammals has provided a deep understanding of the mechanisms involved in chromosome-end protection. However, very little is known on the binding of these proteins to nontelomeric DNA sequences. The TTAGGG DNA repeat proteins 1 and 2 (TRF1 and TRF2) bind to mammalian telomeres as part of the shelterin complex and are essential for maintaining chromosome end stability. In this study, we combined chromatin immunoprecipitation with high-throughput sequencing to map at high sensitivity and resolution the human chromosomal sites to which TRF1 and TRF2 bind. While most of the identified sequences correspond to telomeric regions, we showed that these two proteins also bind to extratelomeric sites. The vast majority of these extratelomeric sites contains interstitial telomeric sequences (or ITSs). However, we also identified non-ITS sites, which correspond to centromeric and pericentromeric satellite DNA. Interestingly, the TRF-binding sites are often located in the proximity of genes or within introns. We propose that TRF1 and TRF2 couple the functional state of telomeres to the long-range organization of chromosomes and gene regulation networks by binding to extratelomeric sequences.


Assuntos
DNA/metabolismo , Telômero , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Sequência de Bases , Sítios de Ligação , Imunoprecipitação da Cromatina , DNA/química , Genes , Humanos , Ligação Proteica
12.
Blood ; 118(5): 1316-22, 2011 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-21355086

RESUMO

Cells of B-cell chronic lymphocytic leukemia (B-CLL) are characterized by short telomeres despite a low proliferative index. Because telomere length has been reported to be a valuable prognosis criteria, there is a great interest in a deep understanding of the origin and consequences of telomere dysfunction in this pathology. Cases of chromosome fusion involving extremely short telomeres have been reported at advanced stage. In the present study, we address the question of the existence of early telomere dysfunction during the B-CLL time course. In a series restricted to 23 newly diagnosed Binet stage A CLL patients compared with 12 healthy donors, we found a significant increase in recruitment of DNA-damage factors to telomeres showing telomere dysfunction in the early stage of the disease. Remarkably, the presence of dysfunctional telomeres did not correlate with telomere shortening or chromatin marks deregulation but with a down-regulation of 2 shelterin genes: ACD (coding for TPP1; P = .0464) and TINF2 (coding for TIN2; P = .0177). We propose that telomeric deprotection in the early step of CLL is not merely the consequence of telomere shortening but also of shelterin alteration.


Assuntos
Dano ao DNA/fisiologia , Leucemia Linfocítica Crônica de Células B/genética , Proteínas de Ligação a Telômeros/genética , Telômero/patologia , Sequência de Bases , Estudos de Coortes , Progressão da Doença , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Modelos Biológicos , Dados de Sequência Molecular , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Telômero/genética , Proteínas de Ligação a Telômeros/metabolismo
13.
Dev Biol ; 298(1): 176-87, 2006 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-16905130

RESUMO

HP1 proteins are essential components of heterochromatin and contribute to the transcriptional repression of euchromatic genes. Although most species contain more than one HP1 family member which differ in their chromosomal distribution, it is not known to what extent the activity of these different family members is redundant or specific in a developmental context. C. elegans has two HP1 homologues, HPL-1 and HPL-2. While HPL-2 functions in vulval and germline development, no function has so far been attributed to HPL-1. Here we report the characterization of an hpl-1 null allele. We show that while the absence of hpl-1 alone results in no obvious phenotype, hpl-1;hpl-2 double mutants show synthetic, temperature sensitive phenotypes including larval lethality and severe defects in the development of the somatic gonad. Furthermore, we find that hpl-1 has an unexpected role in vulval development by acting redundantly with hpl-2, but not other genes previously implicated in vulval development. Localization studies show that like HPL-2, HPL-1 is a ubiquitously expressed nuclear protein. However, HPL-1 and HPL-2 localization does not completely overlap. Our results show that HPL-1 and HPL-2 play both unique and redundant functions in post-embryonic development.


Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/fisiologia , Proteínas Cromossômicas não Histona/fisiologia , Alelos , Animais , Biomarcadores/análise , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Diferenciação Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/crescimento & desenvolvimento , Gônadas/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento
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