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1.
Trends Genet ; 33(4): 233-243, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28222895

RESUMO

It has very recently become clear that the epigenetic modifier SMCHD1 has a role in two distinct disorders: facioscapulohumoral muscular dystrophy (FSHD) and Bosma arhinia and micropthalmia (BAMS). In the former there are heterozygous loss-of-function mutations, while both gain- and loss-of-function mutations have been proposed to underlie the latter. These findings have led to much interest in SMCHD1 and how it works at the molecular level. We summarise here current understanding of the mechanism of action of SMCHD1, its role in these diseases, and what has been learnt from study of mouse models null for Smchd1 in the decade since the discovery of SMCHD1.


Assuntos
Atresia das Cóanas/genética , Proteínas Cromossômicas não Histona/genética , Epigênese Genética , Microftalmia/genética , Distrofia Muscular Facioescapuloumeral/genética , Nariz/anormalidades , Animais , Metilação de DNA/genética , Heterozigoto , Humanos , Camundongos , Mutação
2.
J Biol Chem ; 293(25): 9841-9853, 2018 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-29748383

RESUMO

Structural maintenance of chromosomes flexible hinge domain-containing 1 (Smchd1) plays important roles in epigenetic silencing and normal mammalian development. Recently, heterozygous mutations in SMCHD1 have been reported in two disparate disorders: facioscapulohumeral muscular dystrophy type 2 (FSHD2) and Bosma arhinia microphthalmia syndrome (BAMS). FSHD2-associated mutations lead to loss of function; however, whether BAMS is associated with loss- or gain-of-function mutations in SMCHD1 is unclear. Here, we have assessed the effect of SMCHD1 missense mutations from FSHD2 and BAMS patients on ATP hydrolysis activity and protein conformation and the effect of BAMS mutations on craniofacial development in a Xenopus model. These data demonstrated that FSHD2 mutations only result in decreased ATP hydrolysis, whereas many BAMS mutations can result in elevated ATPase activity and decreased eye size in Xenopus Interestingly, a mutation reported in both an FSHD2 patient and a BAMS patient results in increased ATPase activity and a smaller Xenopus eye size. Mutations in the extended ATPase domain increased catalytic activity, suggesting critical regulatory intramolecular interactions and the possibility of targeting this region therapeutically to boost SMCHD1's activity to counter FSHD.


Assuntos
Trifosfato de Adenosina/metabolismo , Atresia das Cóanas/genética , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Oftalmopatias/patologia , Microftalmia/genética , Distrofia Muscular Facioescapuloumeral/genética , Mutação de Sentido Incorreto , Nariz/anormalidades , Adenosina Trifosfatases , Sequência de Aminoácidos , Animais , Atresia das Cóanas/patologia , Proteínas Cromossômicas não Histona/genética , Cristalografia por Raios X , Oftalmopatias/genética , Oftalmopatias/metabolismo , Humanos , Camundongos , Microftalmia/patologia , Distrofia Muscular Facioescapuloumeral/patologia , Nariz/patologia , Conformação Proteica , Domínios Proteicos , Homologia de Sequência , Xenopus laevis
3.
Proc Natl Acad Sci U S A ; 112(27): E3535-44, 2015 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-26091879

RESUMO

Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic repressor with described roles in X inactivation and genomic imprinting, but Smchd1 is also critically involved in the pathogenesis of facioscapulohumeral dystrophy. The underlying molecular mechanism by which Smchd1 functions in these instances remains unknown. Our genome-wide transcriptional and epigenetic analyses show that Smchd1 binds cis-regulatory elements, many of which coincide with CCCTC-binding factor (Ctcf) binding sites, for example, the clustered protocadherin (Pcdh) genes, where we show Smchd1 and Ctcf act in opposing ways. We provide biochemical and biophysical evidence that Smchd1-chromatin interactions are established through the homodimeric hinge domain of Smchd1 and, intriguingly, that the hinge domain also has the capacity to bind DNA and RNA. Our results suggest Smchd1 imparts epigenetic regulation via physical association with chromatin, which may antagonize Ctcf-facilitated chromatin interactions, resulting in coordinated transcriptional control.


Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Epigênese Genética , Genoma , Animais , Sítios de Ligação/genética , Western Blotting , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/metabolismo , Fator de Ligação a CCCTC , Células Cultivadas , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Impressão Genômica , Histonas/metabolismo , Masculino , Metilação , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/metabolismo , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcriptoma/genética
4.
Blood ; 125(12): 1890-900, 2015 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-25645357

RESUMO

Polycomb repressive complex 2 (PRC2) plays a key role in hematopoietic stem and progenitor cell (HSPC) function. Analyses of mouse mutants harboring deletions of core components have implicated PRC2 in fine-tuning multiple pathways that instruct HSPC behavior, yet how PRC2 is targeted to specific genomic loci within HSPCs remains unknown. Here we use short hairpin RNA-mediated knockdown to survey the function of PRC2 accessory factors that were defined in embryonic stem cells (ESCs) by testing the competitive reconstitution capacity of transduced murine HSPCs. We find that, similar to the phenotype observed upon depletion of core subunit Suz12, depleting Jarid2 enhances the competitive transplantation capacity of both fetal and adult mouse HSPCs. Furthermore, we demonstrate that depletion of JARID2 enhances the in vitro expansion and in vivo reconstitution capacity of human HSPCs. Gene expression profiling revealed common Suz12 and Jarid2 target genes that are enriched for the H3K27me3 mark established by PRC2. These data implicate Jarid2 as an important component of PRC2 that has a central role in coordinating HSPC function.


Assuntos
Regulação Neoplásica da Expressão Gênica , Complexo Repressor Polycomb 2/metabolismo , Animais , Antígenos CD34/metabolismo , Linhagem da Célula , Perfilação da Expressão Gênica , Hematopoese , Células-Tronco Hematopoéticas/citologia , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Fígado/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Fenótipo , RNA Interferente Pequeno/metabolismo , Células-Tronco/citologia
5.
Biochem J ; 473(6): 733-42, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26733688

RESUMO

The structural maintenance of chromosomes (SMC) proteins are fundamental to chromosome organization. They share a characteristic domain structure, featuring a central SMC hinge domain that is critical for forming SMC dimers and interacting with nucleic acids. The structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is a non-canonical member of the SMC family. Although it has been well established that Smchd1 serves crucial roles in epigenetic silencing events implicated in development and disease, much less is known about the structure and function of the Smchd1 protein. Recently, we demonstrated that the C-terminal hinge domain of Smchd1 forms a nucleic acid-binding homodimer; however, it is unclear how the protomers are assembled within the hinge homodimer and how the full-length Smchd1 protein is organized with respect to the hinge region. In the present study, by employing SAXS we demonstrate that the hinge domain of Smchd1 probably adopts an unconventional homodimeric arrangement augmented by an intermolecular coiled coil formed between the two monomers. Such a dimeric structure differs markedly from that of archetypical SMC proteins, raising the possibility that Smchd1 binds chromatin in an unconventional manner.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Repressão Epigenética/fisiologia , Regulação da Expressão Gênica/fisiologia , Animais , Proteínas Cromossômicas não Histona/genética , Células HEK293 , Humanos , Imunoprecipitação , Camundongos , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
6.
Biochem J ; 473(12): 1733-44, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27059856

RESUMO

Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic regulator that plays critical roles in gene regulation during development. Mutations in SMCHD1 were recently implicated in the pathogenesis of facioscapulohumeral muscular dystrophy (FSHD), although the mechanistic basis remains of outstanding interest. We have previously shown that Smchd1 associates with chromatin via its homodimeric C-terminal hinge domain, yet little is known about the function of the putative GHKL (gyrase, Hsp90, histidine kinase, MutL)-type ATPase domain at its N-terminus. To formally assess the structure and function of Smchd1's ATPase domain, we have generated recombinant proteins encompassing the predicted ATPase domain and the adjacent region. Here, we show that the Smchd1 N-terminal region exists as a monomer and adopts a conformation resembling that of monomeric full-length heat shock protein 90 (Hsp90) protein in solution, even though the two proteins share only ∼8% overall sequence identity. Despite being monomeric, the N-terminal region of Smchd1 exhibits ATPase activity, which can be antagonized by the reaction product, ADP, or the Hsp90 inhibitor, radicicol, at a nanomolar concentration. Interestingly, introduction of an analogous mutation to that identified in SMCHD1 of an FSHD patient compromised protein stability, suggesting a possible molecular basis for loss of protein function and pathogenesis. Together, these results reveal important structure-function characteristics of Smchd1 that may underpin its mechanistic action at the chromatin level.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Macrolídeos/farmacologia , Camundongos , Dados de Sequência Molecular , Domínios Proteicos/genética , Domínios Proteicos/fisiologia , Alinhamento de Sequência
7.
Nucleic Acids Res ; 43(15): e97, 2015 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-25925576

RESUMO

Variations in sample quality are frequently encountered in small RNA-sequencing experiments, and pose a major challenge in a differential expression analysis. Removal of high variation samples reduces noise, but at a cost of reducing power, thus limiting our ability to detect biologically meaningful changes. Similarly, retaining these samples in the analysis may not reveal any statistically significant changes due to the higher noise level. A compromise is to use all available data, but to down-weight the observations from more variable samples. We describe a statistical approach that facilitates this by modelling heterogeneity at both the sample and observational levels as part of the differential expression analysis. At the sample level this is achieved by fitting a log-linear variance model that includes common sample-specific or group-specific parameters that are shared between genes. The estimated sample variance factors are then converted to weights and combined with observational level weights obtained from the mean-variance relationship of the log-counts-per-million using 'voom'. A comprehensive analysis involving both simulations and experimental RNA-sequencing data demonstrates that this strategy leads to a universally more powerful analysis and fewer false discoveries when compared to conventional approaches. This methodology has wide application and is implemented in the open-source 'limma' package.


Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Animais , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Humanos , Modelos Lineares , Camundongos , Reprodutibilidade dos Testes
8.
Biochem J ; 457(2): 323-34, 2014 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-24107129

RESUMO

Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains.


Assuntos
Janus Quinase 2/química , Janus Quinase 2/classificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Receptor ErbB-3/química , Receptor ErbB-3/classificação , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Insetos , Janus Quinase 2/genética , Dados de Sequência Molecular , Ligação Proteica/fisiologia , Receptor ErbB-3/genética
9.
Nat Commun ; 14(1): 5466, 2023 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-37749075

RESUMO

The interplay between 3D chromatin architecture and gene silencing is incompletely understood. Here, we report a novel point mutation in the non-canonical SMC protein SMCHD1 that enhances its silencing capacity at endogenous developmental targets. Moreover, it also results in enhanced silencing at the facioscapulohumeral muscular dystrophy associated macrosatellite-array, D4Z4, resulting in enhanced repression of DUX4 encoded by this repeat. Heightened SMCHD1 silencing perturbs developmental Hox gene activation, causing a homeotic transformation in mice. Paradoxically, the mutant SMCHD1 appears to enhance insulation against other epigenetic regulators, including PRC2 and CTCF, while depleting long range chromatin interactions akin to what is observed in the absence of SMCHD1. These data suggest that SMCHD1's role in long range chromatin interactions is not directly linked to gene silencing or insulating the chromatin, refining the model for how the different levels of SMCHD1-mediated chromatin regulation interact to bring about gene silencing in normal development and disease.


Assuntos
Cromatina , Proteínas Cromossômicas não Histona , Distrofia Muscular Facioescapuloumeral , Animais , Camundongos , Cromatina/genética , Epigenômica , Inativação Gênica , Genes Homeobox , Distrofia Muscular Facioescapuloumeral/genética , Proteínas Cromossômicas não Histona/genética
10.
Sci Signal ; 13(636)2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32546545

RESUMO

Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) is an epigenetic regulator in which polymorphisms cause the human developmental disorder, Bosma arhinia micropthalmia syndrome, and the degenerative disease, facioscapulohumeral muscular dystrophy. SMCHD1 is considered a noncanonical SMC family member because its hinge domain is C-terminal, because it homodimerizes rather than heterodimerizes, and because SMCHD1 contains a GHKL-type, rather than an ABC-type ATPase domain at its N terminus. The hinge domain has been previously implicated in chromatin association; however, the underlying mechanism involved and the basis for SMCHD1 homodimerization are unclear. Here, we used x-ray crystallography to solve the three-dimensional structure of the Smchd1 hinge domain. Together with structure-guided mutagenesis, we defined structural features of the hinge domain that participated in homodimerization and nucleic acid binding, and we identified a functional hotspot required for chromatin localization in cells. This structure provides a template for interpreting the mechanism by which patient polymorphisms within the SMCHD1 hinge domain could compromise function and lead to facioscapulohumeral muscular dystrophy.


Assuntos
Proteínas Cromossômicas não Histona/química , Multimerização Proteica , Animais , Proteínas Cromossômicas não Histona/genética , Cristalografia por Raios X , Camundongos , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Domínios Proteicos , Estrutura Quaternária de Proteína , Irmãos
11.
Nat Genet ; 49(2): 249-255, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28067911

RESUMO

Bosma arhinia microphthalmia syndrome (BAMS) is an extremely rare and striking condition characterized by complete absence of the nose with or without ocular defects. We report here that missense mutations in the epigenetic regulator SMCHD1 mapping to the extended ATPase domain of the encoded protein cause BAMS in all 14 cases studied. All mutations were de novo where parental DNA was available. Biochemical tests and in vivo assays in Xenopus laevis embryos suggest that these mutations may behave as gain-of-function alleles. This finding is in contrast to the loss-of-function mutations in SMCHD1 that have been associated with facioscapulohumeral muscular dystrophy (FSHD) type 2. Our results establish SMCHD1 as a key player in nasal development and provide biochemical insight into its enzymatic function that may be exploited for development of therapeutics for FSHD.


Assuntos
Atresia das Cóanas/genética , Proteínas Cromossômicas não Histona/genética , Microftalmia/genética , Mutação de Sentido Incorreto/genética , Nariz/anormalidades , Animais , Linhagem Celular , Pré-Escolar , Epigênese Genética/genética , Feminino , Predisposição Genética para Doença/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Distrofia Muscular Facioescapuloumeral/genética , Xenopus laevis/genética
12.
Genom Data ; 7: 144-7, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26981392

RESUMO

Smchd1 is an epigenetic repressor with important functions in healthy cellular processes and disease. To elucidate its role in transcriptional regulation, we performed two independent genome-wide RNA-sequencing studies comparing wild-type and Smchd1 null samples in neural stem cells and lymphoma cell lines. Using an R-based analysis pipeline that accommodates observational and sample-specific weights in the linear modeling, we identify key genes dysregulated by Smchd1 deletion such as clustered protocadherins in the neural stem cells and imprinted genes in both experiments. Here we provide a detailed description of this analysis, from quality control to read mapping and differential expression analysis. These data sets are publicly available from the Gene Expression Omnibus database (accession numbers GSE64099 and GSE65747).

13.
Artigo em Inglês | MEDLINE | ID: mdl-27195021

RESUMO

BACKGROUND: The presence of histone 3 lysine 9 (H3K9) methylation on the mouse inactive X chromosome has been controversial over the last 15 years, and the functional role of H3K9 methylation in X chromosome inactivation in any species has remained largely unexplored. RESULTS: Here we report the first genomic analysis of H3K9 di- and tri-methylation on the inactive X: we find they are enriched at the intergenic, gene poor regions of the inactive X, interspersed between H3K27 tri-methylation domains found in the gene dense regions. Although H3K9 methylation is predominantly non-genic, we find that depletion of H3K9 methylation via depletion of H3K9 methyltransferase Set domain bifurcated 1 (Setdb1) during the establishment of X inactivation, results in failure of silencing for around 150 genes on the inactive X. By contrast, we find a very minor role for Setdb1-mediated H3K9 methylation once X inactivation is fully established. In addition to failed gene silencing, we observed a specific failure to silence X-linked long-terminal repeat class repetitive elements. CONCLUSIONS: Here we have shown that H3K9 methylation clearly marks the murine inactive X chromosome. The role of this mark is most apparent during the establishment phase of gene silencing, with a more muted effect on maintenance of the silent state. Based on our data, we hypothesise that Setdb1-mediated H3K9 methylation plays a role in epigenetic silencing of the inactive X via silencing of the repeats, which itself facilitates gene silencing through alterations to the conformation of the whole inactive X chromosome.

14.
Cancer Res ; 73(5): 1591-9, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23269277

RESUMO

SMCHD1 is an epigenetic modifier of gene expression that is critical to maintain X chromosome inactivation. Here, we show in mouse that genetic inactivation of Smchd1 accelerates tumorigenesis in male mice. Loss of Smchd1 in transformed mouse embryonic fibroblasts increased tumor growth upon transplantation into immunodeficient nude mice. In addition, loss of Smchd1 in Eµ-Myc transgenic mice that undergo lymphomagenesis reduced disease latency by 50% relative to control animals. In premalignant Eµ-Myc transgenic mice deficient in Smchd1, there was an increase in the number of pre-B cells in the periphery, likely accounting for the accelerated disease in these animals. Global gene expression profiling suggested that Smchd1 normally represses genes activated by MLL chimeric fusion proteins in leukemia, implying that Smchd1 loss may work through the same pathways as overexpressed MLL fusion proteins do in leukemia and lymphoma. Notably, we found that SMCHD1 is underexpressed in many types of human hematopoietic malignancy. Together, our observations collectively highlight a hitherto uncharacterized role for SMCHD1 as a candidate tumor suppressor gene in hematopoietic cancers.


Assuntos
Proteínas Cromossômicas não Histona/genética , Epigênese Genética , Genes Supressores de Tumor , Linfoma de Células B/genética , Animais , Transformação Celular Neoplásica , Regulação para Baixo , Fibroblastos , Técnicas de Inativação de Genes , Humanos , Masculino , Camundongos , Camundongos Nus , Camundongos Transgênicos
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