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1.
Cell Rep ; 43(9): 114656, 2024 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-39240714

RESUMO

Cohesin is key to eukaryotic genome organization and acts throughout the cell cycle in an ATP-dependent manner. The mechanisms underlying cohesin ATPase activity are poorly understood. Here, we characterize distinct steps of the human cohesin ATPase cycle and show that the SMC1A and SMC3 ATPase domains undergo specific but concerted structural rearrangements along this cycle. Specifically, whereas the proximal coiled coil of the SMC1A ATPase domain remains conformationally stable, that of the SMC3 displays an intrinsic flexibility. The ATP-dependent formation of the heterodimeric SMC1A/SMC3 ATPase module (engaged state) favors this flexibility, which is counteracted by NIPBL and DNA binding (clamped state). Opening of the SMC3/RAD21 interface (open-engaged state) stiffens the SMC3 proximal coiled coil, thus constricting together with that of SMC1A the ATPase module DNA-binding chamber. The plasticity of the ATP-dependent interface between the SMC1A and SMC3 ATPase domains enables these structural rearrangements while keeping the ATP gate shut. VIDEO ABSTRACT.


Assuntos
Adenosina Trifosfatases , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Coesinas , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/química , Humanos , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/química , Domínios Proteicos , Trifosfato de Adenosina/metabolismo , Ligação Proteica , Proteoglicanas de Sulfatos de Condroitina
2.
J Cell Sci ; 136(11)2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-37288770

RESUMO

Sister chromatid cohesion is a multi-step process implemented throughout the cell cycle to ensure the correct transmission of chromosomes to daughter cells. Although cohesion establishment and mitotic cohesion dissolution have been extensively explored, the regulation of cohesin loading is still poorly understood. Here, we report that the methyltransferase NSD3 is essential for mitotic sister chromatid cohesion before mitosis entry. NSD3 interacts with the cohesin loader complex kollerin (composed of NIPBL and MAU2) and promotes the chromatin recruitment of MAU2 and cohesin at mitotic exit. We also show that NSD3 associates with chromatin in early anaphase, prior to the recruitment of MAU2 and RAD21, and dissociates from chromatin when prophase begins. Among the two NSD3 isoforms present in somatic cells, the long isoform is responsible for regulating kollerin and cohesin chromatin-loading, and its methyltransferase activity is required for efficient sister chromatid cohesion. Based on these observations, we propose that NSD3-dependent methylation contributes to sister chromatid cohesion by ensuring proper kollerin recruitment and thus cohesin loading.


Assuntos
Proteínas de Ciclo Celular , Cromátides , Histona Metiltransferases , Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Cromátides/metabolismo , Cromatina , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Histona Metiltransferases/metabolismo , Coesinas
3.
Nat Commun ; 12(1): 3014, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34021162

RESUMO

Members of the chromodomain-helicase-DNA binding (CHD) protein family are chromatin remodelers implicated in human pathologies, with CHD6 being one of its least studied members. We discovered a de novo CHD6 missense mutation in a patient clinically presenting the rare Hallermann-Streiff syndrome (HSS). We used genome editing to generate isogenic iPSC lines and model HSS in relevant cell types. By combining genomics with functional in vivo and in vitro assays, we show that CHD6 binds a cohort of autophagy and stress response genes across cell types. The HSS mutation affects CHD6 protein folding and impairs its ability to recruit co-remodelers in response to DNA damage or autophagy stimulation. This leads to accumulation of DNA damage burden and senescence-like phenotypes. We therefore uncovered a molecular mechanism explaining HSS onset via chromatin control of autophagic flux and genotoxic stress surveillance.


Assuntos
Autofagia/fisiologia , Dano ao DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Autofagia/genética , Cromatina , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/metabolismo , Epigenômica , Edição de Genes , Expressão Gênica , Síndrome de Hallermann/genética , Humanos , Mutação , Fenótipo
4.
J Clin Invest ; 131(1)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33393499

RESUMO

LMNA mutations in patients are responsible for a dilated cardiomyopathy. Molecular mechanisms underlying the origin and development of the pathology are unknown. Herein, using mouse pluripotent embryonic stem cells (ESCs) and a mouse model both harboring the p.H222P Lmna mutation, we found early defects in cardiac differentiation of mutated ESCs and dilatation of mutated embryonic hearts at E13.5, pointing to a developmental origin of the disease. Using mouse ESCs, we demonstrated that cardiac differentiation of LmnaH222P/+ was impaired at the mesodermal stage. Expression of Mesp1, a mesodermal cardiogenic gene involved in epithelial-to-mesenchymal transition of epiblast cells, as well as Snai1 and Twist expression, was decreased in LmnaH222P/+ cells compared with WT cells in the course of differentiation. In turn, cardiomyocyte differentiation was impaired. ChIP assay of H3K4me1 in differentiating cells revealed a specific decrease of this histone mark on regulatory regions of Mesp1 and Twist in LmnaH222P/+ cells. Downregulation or inhibition of LSD1 that specifically demethylated H3K4me1 rescued the epigenetic landscape of mesodermal LmnaH222P/+ cells and in turn contraction of cardiomyocytes. Inhibition of LSD1 in pregnant mice or neonatal mice prevented cardiomyopathy in E13.5 LmnaH222P/H222P offspring and adults, respectively. Thus, LSD1 appeared to be a therapeutic target to prevent or cure dilated cardiomyopathy associated with a laminopathy.


Assuntos
Cardiomiopatias/enzimologia , Cardiomiopatias/prevenção & controle , Histona Desmetilases/metabolismo , Laminopatias/complicações , Laminopatias/enzimologia , Miócitos Cardíacos/enzimologia , Substituição de Aminoácidos , Animais , Cardiomiopatias/genética , Diferenciação Celular , Modelos Animais de Doenças , Histona Desmetilases/genética , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Laminopatias/genética , Camundongos , Camundongos Mutantes , Células-Tronco Embrionárias Murinas/enzimologia , Células-Tronco Embrionárias Murinas/patologia , Mutação de Sentido Incorreto , Miócitos Cardíacos/patologia
5.
Brain ; 143(7): 2027-2038, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32542401

RESUMO

Synonymous single nucleotide variants (sSNVs) have been implicated in various genetic disorders through alterations of pre-mRNA splicing, mRNA structure and miRNA regulation. However, their impact on synonymous codon usage and protein translation remains to be elucidated in clinical context. Here, we explore the functional impact of sSNVs in the Sonic Hedgehog (SHH) gene, identified in patients affected by holoprosencephaly, a congenital brain defect resulting from incomplete forebrain cleavage. We identified eight sSNVs in SHH, selectively enriched in holoprosencephaly patients as compared to healthy individuals, and systematically assessed their effect at both transcriptional and translational levels using a series of in silico and in vitro approaches. Although no evidence of impact of these sSNVs on splicing, mRNA structure or miRNA regulation was found, five sSNVs introduced significant changes in codon usage and were predicted to impact protein translation. Cell assays demonstrated that these five sSNVs are associated with a significantly reduced amount of the resulting protein, ranging from 5% to 23%. Inhibition of the proteasome rescued the protein levels for four out of five sSNVs, confirming their impact on protein stability and folding. Remarkably, we found a significant correlation between experimental values of protein reduction and computational measures of codon usage, indicating the relevance of in silico models in predicting the impact of sSNVs on translation. Considering the critical role of SHH in brain development, our findings highlight the clinical relevance of sSNVs in holoprosencephaly and underline the importance of investigating their impact on translation in human pathologies.


Assuntos
Uso do Códon/genética , Proteínas Hedgehog/genética , Holoprosencefalia/genética , Biossíntese de Proteínas/genética , Humanos , Polimorfismo de Nucleotídeo Único
6.
Cell Rep ; 31(7): 107647, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32433956

RESUMO

The NIPBL/MAU2 heterodimer loads cohesin onto chromatin. Mutations in NIPBL account for most cases of the rare developmental disorder Cornelia de Lange syndrome (CdLS). Here we report a MAU2 variant causing CdLS, a deletion of seven amino acids that impairs the interaction between MAU2 and the NIPBL N terminus. Investigating this interaction, we discovered that MAU2 and the NIPBL N terminus are largely dispensable for normal cohesin and NIPBL function in cells with a NIPBL early truncating mutation. Despite a predicted fatal outcome of an out-of-frame single nucleotide duplication in NIPBL, engineered in two different cell lines, alternative translation initiation yields a form of NIPBL missing N-terminal residues. This form cannot interact with MAU2, but binds DNA and mediates cohesin loading. Altogether, our work reveals that cohesin loading can occur independently of functional NIPBL/MAU2 complexes and highlights a novel mechanism protective against out-of-frame mutations that is potentially relevant for other genetic conditions.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Síndrome de Cornélia de Lange/genética , Variação Genética/genética , Humanos , Coesinas
7.
Biochim Biophys Acta Mol Cell Res ; 1867(4): 118650, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31953060

RESUMO

Transfection is a powerful tool that enables introducing foreign nucleic acids into living cells in order to study the function of a gene product. Ever since the discovery of transfection many side effects or artifacts caused by transfection reagents have been reported. Here, we show that the transfection reagent, JetPRIME alters the localization of the splicing protein SC35 widely used as a nuclear speckle marker. We demonstrate that transfection of plasmids with JetPRIME leads to enlarged SC35 speckles and SC35 cytoplasmic granules. By contrast, transfection of the same plasmid with Lipofectamine 3000 does not have any effect on SC35 localization. The formation of SC35 cytoplasmic granules by JetPRIME-mediated transfection is independent of exogenous expression by plasmid and although similar in morphology they are distinct from P-bodies and stress granules. This method of transfection affected only SC35 and phosphorylated SR proteins but not the nuclear speckles. The JetPRIME-mediated transfection also showed compromised transcription in cells with enlarged SC35 speckles. Our work indicates that the use of JetPRIME alters SC35 localization and can affect gene expression and alternative splicing. Therefore, caution should be exercised when interpreting results after the use of a transient transfection system, particularly when the subject of the study is the function of a protein in the control of gene expression or mRNA splicing.


Assuntos
Artefatos , Fatores de Processamento de Serina-Arginina/análise , Transfecção , Linhagem Celular Tumoral , Estruturas do Núcleo Celular/química , Grânulos Citoplasmáticos/química , Células HeLa , Humanos , Indicadores e Reagentes , Splicing de RNA , Transcrição Gênica
8.
Hum Genet ; 138(4): 363-374, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30838450

RESUMO

Neural tube defect disorders are developmental diseases that originate from an incomplete closure of the neural tube during embryogenesis. Despite high prevalence-1 out of 3000 live births-their etiology is not yet established and both environmental and genetic factors have been proposed, with a heritability rate of about 60%. Studies in mouse models as well as in human have further suggested a multifactorial pattern of inheritance for neural tube defect disorders. Here, we report results obtained from clinical diagnosis and NGS analysis of a cohort composed of 52 patients. Using a candidate gene panel approach, we identified variants in known genes of planar cell polarity (PCP) pathway, although with higher prevalence than previously reported. Our study also reveals variants in novel genes such as FREM2 and DISP1. Altogether, these results confirm the implication of the PCP genes and involve the FRAS/FREM2 complex and Sonic Hedgehog signaling as novel components in the appearance of NTDs.


Assuntos
Polaridade Celular/genética , Estudos de Associação Genética/métodos , Defeitos do Tubo Neural/genética , Análise de Sequência de DNA/métodos , Adulto , Animais , Criança , Estudos de Coortes , Análise Mutacional de DNA/métodos , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Defeitos do Tubo Neural/patologia , Gravidez , Transdução de Sinais/genética , Transcriptoma
9.
Brain ; 142(1): 35-49, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30508070

RESUMO

Holoprosencephaly is a pathology of forebrain development characterized by high phenotypic heterogeneity. The disease presents with various clinical manifestations at the cerebral or facial levels. Several genes have been implicated in holoprosencephaly but its genetic basis remains unclear: different transmission patterns have been described including autosomal dominant, recessive and digenic inheritance. Conventional molecular testing approaches result in a very low diagnostic yield and most cases remain unsolved. In our study, we address the possibility that genetically unsolved cases of holoprosencephaly present an oligogenic origin and result from combined inherited mutations in several genes. Twenty-six unrelated families, for whom no genetic cause of holoprosencephaly could be identified in clinical settings [whole exome sequencing and comparative genomic hybridization (CGH)-array analyses], were reanalysed under the hypothesis of oligogenic inheritance. Standard variant analysis was improved with a gene prioritization strategy based on clinical ontologies and gene co-expression networks. Clinical phenotyping and exploration of cross-species similarities were further performed on a family-by-family basis. Statistical validation was performed on 248 ancestrally similar control trios provided by the Genome of the Netherlands project and on 574 ancestrally matched controls provided by the French Exome Project. Variants of clinical interest were identified in 180 genes significantly associated with key pathways of forebrain development including sonic hedgehog (SHH) and primary cilia. Oligogenic events were observed in 10 families and involved both known and novel holoprosencephaly genes including recurrently mutated FAT1, NDST1, COL2A1 and SCUBE2. The incidence of oligogenic combinations was significantly higher in holoprosencephaly patients compared to two control populations (P < 10-9). We also show that depending on the affected genes, patients present with particular clinical features. This study reports novel disease genes and supports oligogenicity as clinically relevant model in holoprosencephaly. It also highlights key roles of SHH signalling and primary cilia in forebrain development. We hypothesize that distinction between different clinical manifestations of holoprosencephaly lies in the degree of overall functional impact on SHH signalling. Finally, we underline that integrating clinical phenotyping in genetic studies is a powerful tool to specify the clinical relevance of certain mutations.


Assuntos
Holoprosencefalia/genética , Herança Multifatorial/genética , Doenças Raras/genética , Estudos de Casos e Controles , Hibridização Genômica Comparativa , Exoma/genética , Feminino , Humanos , Masculino , Mutação , Linhagem , Fenótipo
10.
Am J Med Genet C Semin Med Genet ; 178(2): 258-269, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29785796

RESUMO

Holoprosencephaly (HPE) is a complex genetic disorder of the developing forebrain characterized by high phenotypic and genetic heterogeneity. HPE was initially defined as an autosomal dominant disease, but recent research has shown that its mode of transmission is more complex. The past decade has witnessed rapid development of novel genetic technologies and significant progresses in clinical studies of HPE. In this review, we recapitulate genetic epidemiological studies of the largest European HPE cohort and summarize the novel genetic discoveries of HPE based on recently developed diagnostic methods. Our main purpose is to present different inheritance patterns that exist for HPE with a particular emphasis on oligogenic inheritance and its implications in genetic counseling.


Assuntos
Encéfalo/diagnóstico por imagem , Holoprosencefalia/genética , Encéfalo/anormalidades , Encéfalo/embriologia , Aberrações Cromossômicas , Feminino , Genes Recessivos , Aconselhamento Genético , Testes Genéticos/métodos , Proteínas Hedgehog/genética , Holoprosencefalia/etiologia , Humanos , Padrões de Herança , Masculino , Linhagem , Gravidez , Diagnóstico Pré-Natal
11.
J Cell Sci ; 131(7)2018 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-29555820

RESUMO

During the prometaphase stage of mitosis, the cell builds a bipolar spindle of microtubules that mechanically segregates sister chromatids between two daughter cells in anaphase. The spindle assembly checkpoint (SAC) is a quality control mechanism that monitors proper attachment of microtubules to chromosome kinetochores during prometaphase. Segregation occurs only when each chromosome is bi-oriented with each kinetochore pair attached to microtubules emanating from opposite spindle poles. Overexpression of the protein kinase Aurora A is a feature of various cancers and is thought to enable tumour cells to bypass the SAC, leading to aneuploidy. Here, we took advantage of a chemical and chemical-genetic approach to specifically inhibit Aurora A kinase activity in late prometaphase. We observed that a loss of Aurora A activity directly affects SAC function, that Aurora A is essential for maintaining the checkpoint protein Mad2 on unattached kinetochores and that inhibition of Aurora A leads to loss of the SAC, even in the presence of nocodazole or Taxol. This is a new finding that should affect the way Aurora A inhibitors are used in cancer treatments.This article has an associated First Person interview with the first authors of the paper.


Assuntos
Aurora Quinase A/genética , Pontos de Checagem da Fase M do Ciclo Celular/genética , Proteínas Mad2/genética , Prometáfase/genética , Anáfase/genética , Aurora Quinase A/antagonistas & inibidores , Azepinas/farmacologia , Linhagem Celular Tumoral , Cromátides/genética , Segregação de Cromossomos/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Cinetocoros/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Mitose/efeitos dos fármacos , Mitose/genética , Nocodazol/farmacologia , Paclitaxel/farmacologia , Prometáfase/efeitos dos fármacos , Pirimidinas/farmacologia , Fuso Acromático/genética
12.
Eur J Med Genet ; 61(11): 680-684, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29155047

RESUMO

Cornelia de Lange syndrome (CdLS) is a dominantly inherited developmental disorder caused by mutations in genes that encode for either structural (SMC1A, SMC3, RAD21) or regulatory (NIPBL, HDAC8) subunits of the cohesin complex. NIPBL represents the major gene of the syndrome and heterozygous mutations can be identified in more than 65% of patients. Interestingly, large portions of these variants were described as somatic mosaicism and often escape standard molecular diagnostics using lymphocyte DNA. Here we discuss the role of somatic mosaicism in CdLS and describe two additional patients with NIPBL mosaicism detected by targeted gene panel or exome sequencing. In order to verify the next generation sequencing data, Sanger sequencing or pyrosequencing on DNA extracted from different tissues were applied. None of the pathogenic variants was originally detected by Sanger sequencing on blood DNA. Patient 1 displays an unusual combination of clinical features: he is cognitively only mildly affected, but shows severe limb reduction defects. Patient 2 presents with a moderate phenotype. Interestingly, Sanger sequencing analysis on fibroblast DNA of this patient did not detect the disease-causing variant previously observed on the same DNA sample by exome sequencing. Subsequent analyses could confirm the variants by Sanger sequencing on buccal mucosa DNA. Notably, this is the first report of a higher mutational load in buccal mucosa than in fibroblast cells of a CdLS patient. Detection of low-level mosaicism is of utmost importance for an accurate molecular diagnosis and a proper genetic counseling of patients with a clinical diagnosis of CdLS. Next-generation sequencing technologies greatly facilitate the detection of low-level mosaicism, which might otherwise remain undetected by conventional sequencing approaches.


Assuntos
Síndrome de Cornélia de Lange/genética , Deficiências do Desenvolvimento/genética , Deformidades Congênitas dos Membros/genética , Proteínas/genética , Adulto , Proteínas de Ciclo Celular , Síndrome de Cornélia de Lange/fisiopatologia , Deficiências do Desenvolvimento/fisiopatologia , Feminino , Aconselhamento Genético , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Deformidades Congênitas dos Membros/fisiopatologia , Linfócitos/patologia , Masculino , Mosaicismo , Mucosa Bucal , Mutação
13.
PLoS Genet ; 13(12): e1007137, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29261648

RESUMO

Cohesin is crucial for genome stability, cell division, transcription and chromatin organization. Its functions critically depend on NIPBL, the cohesin-loader protein that is found to be mutated in >60% of the cases of Cornelia de Lange syndrome (CdLS). Other mutations are described in the cohesin subunits SMC1A, RAD21, SMC3 and the HDAC8 protein. In 25-30% of CdLS cases no mutation in the known CdLS genes is detected. Until now, functional elements in the noncoding genome were not characterized in the molecular etiology of CdLS and therefore are excluded from mutation screening, although the impact of such mutations has now been recognized for a wide range of diseases. We have identified different elements of the noncoding genome involved in regulation of the NIPBL gene. NIPBL-AS1 is a long non-coding RNA transcribed upstream and antisense to NIPBL. By knockdown and transcription blocking experiments, we could show that not the NIPBL-AS1 gene product, but its actual transcription is important to regulate NIPBL expression levels. This reveals a possibility to boost the transcriptional activity of the NIPBL gene by interfering with the NIPBL-AS1 lncRNA. Further, we have identified a novel distal enhancer regulating both NIPBL and NIPBL-AS1. Deletion of the enhancer using CRISPR genome editing in HEK293T cells reduces expression of NIPBL, NIPBL-AS1 as well as genes found to be dysregulated in CdLS.


Assuntos
Elementos Facilitadores Genéticos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Síndrome de Cornélia de Lange/genética , Regulação da Expressão Gênica , Genoma Humano , Células HEK293 , Humanos , Mutação , Fenótipo , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Análise de Sequência de DNA , Coesinas
14.
Hum Genet ; 136(3): 307-320, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28120103

RESUMO

The coordinated tissue-specific regulation of gene expression is essential for the proper development of all organisms. Mutations in multiple transcriptional regulators cause a group of neurodevelopmental disorders termed "transcriptomopathies" that share core phenotypical features including growth retardation, developmental delay, intellectual disability and facial dysmorphism. Cornelia de Lange syndrome (CdLS) belongs to this class of disorders and is caused by mutations in different subunits or regulators of the cohesin complex. Herein, we report on the clinical and molecular characterization of seven patients with features overlapping with CdLS who were found to carry mutations in chromatin regulators previously associated to other neurodevelopmental disorders that are frequently considered in the differential diagnosis of CdLS. The identified mutations affect the methyltransferase-encoding genes KMT2A and SETD5 and different subunits of the SWI/SNF chromatin-remodeling complex. Complementary to this, a patient with Coffin-Siris syndrome was found to carry a missense substitution in NIPBL. Our findings indicate that mutations in a variety of chromatin-associated factors result in overlapping clinical phenotypes, underscoring the genetic heterogeneity that should be considered when assessing the clinical and molecular diagnosis of neurodevelopmental syndromes. It is clear that emerging molecular mechanisms of chromatin dysregulation are central to understanding the pathogenesis of these clinically overlapping genetic disorders.


Assuntos
Cromatina/fisiologia , Síndrome de Cornélia de Lange/genética , Mutação , Fenótipo , Adolescente , Adulto , Criança , Pré-Escolar , Fácies , Feminino , Humanos , Masculino , Adulto Jovem
15.
Curr Opin Genet Dev ; 37: 59-66, 2016 04.
Artigo em Inglês | MEDLINE | ID: mdl-26821365

RESUMO

Consistent with the diverse roles of the cohesin complex in chromosome biology, mutations in genes encoding cohesin and its regulators are found in different types of cancer and in developmental disorders such as Cornelia de Lange Syndrome. It is so far considered that the defects caused by these mutations result from altered function of cohesin in regulating gene expression during development. Chromatin conformation analyses have established the importance of cohesin for the architecture of developmental gene clusters and in vivo studies in mouse and zebrafish demonstrated how cohesin defects lead to gene misregulation and to malformations similar to the related human syndromes. Here we present our current knowledge on cohesin's involvement in gene expression, highlighting molecular and mechanistic consequences of pathogenic mutations in the Cornelia de Lange syndrome.


Assuntos
Proteínas de Ciclo Celular/genética , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Síndrome de Cornélia de Lange/genética , Regulação da Expressão Gênica/genética , Animais , Cromatina/química , Humanos , Camundongos , Família Multigênica/genética , Mutação , Peixe-Zebra/genética , Coesinas
16.
Hum Mutat ; 36(1): 26-9, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25196272

RESUMO

Cornelia de Lange syndrome (CdLS) is a well-characterized developmental disorder. The genetic cause of CdLS is a mutation in one of five associated genes (NIPBL, SMC1A, SMC3, RAD21, and HDAC8) accounting for about 70% of cases. To improve our current molecular diagnostic and to analyze some of CdLS candidate genes, we developed and established a gene panel approach. Because recent data indicate a high frequency of mosaic NIPBL mutations that were not detected by conventional sequencing approaches of blood DNA, we started to collect buccal mucosa (BM) samples of our patients that were negative for mutations in the known CdLS genes. Here, we report the identification of three mosaic NIPBL mutations by our high-coverage gene panel sequencing approach that were undetected by classical Sanger sequencing analysis of BM DNA. All mutations were confirmed by the use of highly sensitive SNaPshot fragment analysis using DNA from BM, urine, and fibroblast samples. In blood samples, we could not detect the respective mutation. Finally, in fibroblast samples from all three patients, Sanger sequencing could identify all the mutations. Thus, our study highlights the need for highly sensitive technologies in molecular diagnostic of CdLS to improve genetic diagnosis and counseling of patients and their families.


Assuntos
Síndrome de Cornélia de Lange/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Proteínas/genética , Análise de Sequência de DNA/métodos , Proteínas de Ciclo Celular , Criança , Pré-Escolar , Síndrome de Cornélia de Lange/genética , Feminino , Predisposição Genética para Doença , Humanos , Adulto Jovem
17.
EMBO Rep ; 15(9): 948-55, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25092791

RESUMO

Sister chromatid cohesion, which depends on cohesin, is essential for the faithful segregation of replicated chromosomes. Here, we report that splicing complex Prp19 is essential for cohesion in both G2 and mitosis, and consequently for the proper progression of the cell through mitosis. Inactivation of splicing factors SF3a120 and U2AF65 induces similar cohesion defects to Prp19 complex inactivation. Our data indicate that these splicing factors are all required for the accumulation of cohesion factor Sororin, by facilitating the proper splicing of its pre-mRNA. Finally, we show that ectopic expression of Sororin corrects defective cohesion caused by Prp19 complex inactivation. We propose that the Prp19 complex and the splicing machinery contribute to the establishment of cohesion by promoting Sororin accumulation during S phase, and are, therefore, essential to the maintenance of genome stability.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Enzimas Reparadoras do DNA/genética , Proteínas Nucleares/genética , Precursores de RNA/genética , Splicing de RNA/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Segregação de Cromossomos/genética , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/biossíntese , Regulação da Expressão Gênica , Instabilidade Genômica , Células HeLa , Humanos , Mitose/genética , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/biossíntese , Fatores de Processamento de RNA , Ribonucleoproteína Nuclear Pequena U2/antagonistas & inibidores , Ribonucleoproteínas/antagonistas & inibidores , Fator de Processamento U2AF
18.
Mol Cell Biol ; 34(13): 2418-36, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24752895

RESUMO

Estradiol signaling is ideally suited for analyzing the molecular and functional linkages between the different layers of information directing transcriptional regulations: the DNA sequence, chromatin modifications, and the spatial organization of the genome. Hence, the estrogen receptor (ER) can bind at a distance from its target genes and engages timely and spatially coordinated processes to regulate their expression. In the context of the coordinated regulation of colinear genes, identifying which ER binding sites (ERBSs) regulate a given gene still remains a challenge. Here, we investigated the coordination of such regulatory events at a 2-Mb genomic locus containing the estrogen-sensitive trefoil factor (TFF) cluster of genes in breast cancer cells. We demonstrate that this locus exhibits a hormone- and cohesin-dependent reduction in the plasticity of its three-dimensional organization that allows multiple ERBSs to be dynamically brought to the vicinity of estrogen-sensitive genes. Additionally, by using triplex-forming oligonucleotides, we could precisely document the functional links between ER engagement at given ERBSs and the regulation of particular genes. Hence, our data provide evidence of a formerly suggested cooperation of enhancers toward gene regulation and also show that redundancy between ERBSs can occur.


Assuntos
Estrogênios/farmacologia , Regulação da Expressão Gênica , Peptídeos/genética , Receptores de Estrogênio/genética , Ativação Transcricional/efeitos dos fármacos , Sítios de Ligação/genética , Neoplasias da Mama/genética , Fator de Ligação a CCCTC , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA , Feminino , Humanos , Hibridização in Situ Fluorescente , Células MCF-7 , Reação em Cadeia da Polimerase Multiplex , Proteínas Nucleares/genética , Oligonucleotídeos/genética , Fosfoproteínas/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Interferência de RNA , RNA Interferente Pequeno , Sequências Reguladoras de Ácido Nucleico , Proteínas Repressoras/genética , Transcrição Gênica/efeitos dos fármacos , Fator Trefoil-2 , Coesinas
19.
Nature ; 489(7415): 313-7, 2012 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-22885700

RESUMO

Cornelia de Lange syndrome (CdLS) is a dominantly inherited congenital malformation disorder, caused by mutations in the cohesin-loading protein NIPBL for nearly 60% of individuals with classical CdLS, and by mutations in the core cohesin components SMC1A (~5%) and SMC3 (<1%) for a smaller fraction of probands. In humans, the multisubunit complex cohesin is made up of SMC1, SMC3, RAD21 and a STAG protein. These form a ring structure that is proposed to encircle sister chromatids to mediate sister chromatid cohesion and also has key roles in gene regulation. SMC3 is acetylated during S-phase to establish cohesiveness of chromatin-loaded cohesin, and in yeast, the class I histone deacetylase Hos1 deacetylates SMC3 during anaphase. Here we identify HDAC8 as the vertebrate SMC3 deacetylase, as well as loss-of-function HDAC8 mutations in six CdLS probands. Loss of HDAC8 activity results in increased SMC3 acetylation and inefficient dissolution of the 'used' cohesin complex released from chromatin in both prophase and anaphase. SMC3 with retained acetylation is loaded onto chromatin, and chromatin immunoprecipitation sequencing analysis demonstrates decreased occupancy of cohesin localization sites that results in a consistent pattern of altered transcription seen in CdLS cell lines with either NIPBL or HDAC8 mutations.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Síndrome de Cornélia de Lange/genética , Síndrome de Cornélia de Lange/metabolismo , Histona Desacetilases/genética , Mutação/genética , Proteínas Repressoras/genética , Acetilação , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Anáfase , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteoglicanas de Sulfatos de Condroitina/química , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Cromatina/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/química , Cristalografia por Raios X , Proteínas de Ligação a DNA , Feminino , Fibroblastos , Células HeLa , Histona Desacetilases/química , Histona Desacetilases/deficiência , Histona Desacetilases/metabolismo , Humanos , Masculino , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Prófase , Conformação Proteica , Proteínas/genética , Proteínas Repressoras/química , Proteínas Repressoras/deficiência , Proteínas Repressoras/metabolismo , Transcrição Gênica , Coesinas
20.
EMBO J ; 28(17): 2625-35, 2009 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-19629043

RESUMO

Cohesin complexes mediate sister chromatid cohesion. Cohesin also becomes enriched at DNA double-strand break sites and facilitates recombinational DNA repair. Here, we report that cohesin is essential for the DNA damage-induced G2/M checkpoint. In contrast to cohesin's role in DNA repair, the checkpoint function of cohesin is independent of its ability to mediate cohesion. After RNAi-mediated depletion of cohesin, cells fail to properly activate the checkpoint kinase Chk2 and have defects in recruiting the mediator protein 53BP1 to DNA damage sites. Earlier work has shown that phosphorylation of the cohesin subunits Smc1 and Smc3 is required for the intra-S checkpoint, but Smc1/Smc3 are also subunits of a distinct recombination complex, RC-1. It was, therefore, unknown whether Smc1/Smc3 function in the intra-S checkpoint as part of cohesin. We show that Smc1/Smc3 are phosphorylated as part of cohesin and that cohesin is required for the intra-S checkpoint. We propose that accumulation of cohesin at DNA break sites is not only needed to mediate DNA repair, but also facilitates the recruitment of checkpoint proteins, which activate the intra-S and G2/M checkpoints.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Proteínas Cromossômicas não Histona/metabolismo , Dano ao DNA/fisiologia , Fase G2 , Genes cdc , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Troca de Cromátide Irmã , Coesinas
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