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1.
Genet Med ; 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31388190

RESUMO

PURPOSE: Sifrim-Hitz-Weiss syndrome (SIHIWES) is a recently described multisystemic neurodevelopmental disorder caused by de novo variants in CHD4. In this study, we investigated the clinical spectrum of the disorder, genotype-phenotype correlations, and the effect of different missense variants on CHD4 function. METHODS: We collected clinical and molecular data from 32 individuals with mostly de novo variants in CHD4, identified through next-generation sequencing. We performed adenosine triphosphate (ATP) hydrolysis and nucleosome remodeling assays on variants from five different CHD4 domains. RESULTS: The majority of participants had global developmental delay, mild to moderate intellectual disability, brain anomalies, congenital heart defects, and dysmorphic features. Macrocephaly was a frequent but not universal finding. Additional common abnormalities included hypogonadism in males, skeletal and limb anomalies, hearing impairment, and ophthalmic abnormalities. The majority of variants were nontruncating and affected the SNF2-like region of the protein. We did not identify genotype-phenotype correlations based on the type or location of variants. Alterations in ATP hydrolysis and chromatin remodeling activities were observed in variants from different domains. CONCLUSION: The CHD4-related syndrome is a multisystemic neurodevelopmental disorder. Missense substitutions in different protein domains alter CHD4 function in a variant-specific manner, but result in a similar phenotype in humans.

4.
Nat Commun ; 10(1): 305, 2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30659182

RESUMO

DNA methylation is an essential epigenetic process in mammals, intimately involved in gene regulation. Here we address the extent to which genetics, sex, and pregnancy influence genomic DNA methylation by intercrossing 2 inbred mouse strains, C57BL/6N and C3H/HeN, and analyzing DNA methylation in parents and offspring using whole-genome bisulfite sequencing. Differential methylation across genotype is detected at thousands of loci and is preserved on parental alleles in offspring. In comparison of autosomal DNA methylation patterns across sex, hundreds of differentially methylated regions are detected. Comparison of animals with different histories of pregnancy within our study reveals a CpG methylation pattern that is restricted to female animals that had borne offspring. Collectively, our results demonstrate the stability of CpG methylation across generations, clarify the interplay of epigenetics with genetics and sex, and suggest that CpG methylation may serve as an epigenetic record of life events in somatic tissues at loci whose expression is linked to the relevant biology.


Assuntos
Metilação de DNA/genética , Epigênese Genética , Prenhez/genética , Animais , Ilhas de CpG , Metilação de DNA/fisiologia , Feminino , Masculino , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Gravidez , Prenhez/fisiologia , Fatores Sexuais , Especificidade da Espécie , Sequenciamento Completo do Genoma
5.
Nat Commun ; 9(1): 4619, 2018 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-30397230

RESUMO

Chromatin remodeling is of crucial importance during brain development. Pathogenic alterations of several chromatin remodeling ATPases have been implicated in neurodevelopmental disorders. We describe an index case with a de novo missense mutation in CHD3, identified during whole genome sequencing of a cohort of children with rare speech disorders. To gain a comprehensive view of features associated with disruption of this gene, we use a genotype-driven approach, collecting and characterizing 35 individuals with de novo CHD3 mutations and overlapping phenotypes. Most mutations cluster within the ATPase/helicase domain of the encoded protein. Modeling their impact on the three-dimensional structure demonstrates disturbance of critical binding and interaction motifs. Experimental assays with six of the identified mutations show that a subset directly affects ATPase activity, and all but one yield alterations in chromatin remodeling. We implicate de novo CHD3 mutations in a syndrome characterized by intellectual disability, macrocephaly, and impaired speech and language.

6.
Nat Commun ; 9(1): 1059, 2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29535312

RESUMO

GATA3 is frequently mutated in breast cancer; these mutations are widely presumed to be loss-of function despite a dearth of information regarding their effect on disease course or their mechanistic impact on the breast cancer transcriptional network. Here, we address molecular and clinical features associated with GATA3 mutations. A novel classification scheme defines distinct clinical features for patients bearing breast tumors with mutations in the second GATA3 zinc-finger (ZnFn2). An engineered ZnFn2 mutant cell line by CRISPR-Cas9 reveals that mutation of one allele of the GATA3 second zinc finger (ZnFn2) leads to loss of binding and decreased expression at a subset of genes, including Progesterone Receptor. At other loci, associated with epithelial to mesenchymal transition, gain of binding correlates with increased gene expression. These results demonstrate that not all GATA3 mutations are equivalent and that ZnFn2 mutations impact breast cancer through gain and loss-of function.

7.
Nature ; 551(7678): 105-109, 2017 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-29072299

RESUMO

T helper 17 (TH17) cells are critically involved in host defence, inflammation, and autoimmunity. Transforming growth factor ß (TGFß) is instrumental in TH17 cell differentiation by cooperating with interleukin-6 (refs 6, 7). Yet, the mechanism by which TGFß enables TH17 cell differentiation remains elusive. Here we reveal that TGFß enables TH17 cell differentiation by reversing SKI-SMAD4-mediated suppression of the expression of the retinoic acid receptor (RAR)-related orphan receptor γt (RORγt). We found that, unlike wild-type T cells, SMAD4-deficient T cells differentiate into TH17 cells in the absence of TGFß signalling in a RORγt-dependent manner. Ectopic SMAD4 expression suppresses RORγt expression and TH17 cell differentiation of SMAD4-deficient T cells. However, TGFß neutralizes SMAD4-mediated suppression without affecting SMAD4 binding to the Rorc locus. Proteomic analysis revealed that SMAD4 interacts with SKI, a transcriptional repressor that is degraded upon TGFß stimulation. SKI controls histone acetylation and deacetylation of the Rorc locus and TH17 cell differentiation via SMAD4: ectopic SKI expression inhibits H3K9 acetylation of the Rorc locus, Rorc expression, and TH17 cell differentiation in a SMAD4-dependent manner. Therefore, TGFß-induced disruption of SKI reverses SKI-SMAD4-mediated suppression of RORγt to enable TH17 cell differentiation. This study reveals a critical mechanism by which TGFß controls TH17 cell differentiation and uncovers the SKI-SMAD4 axis as a potential therapeutic target for treating TH17-related diseases.


Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Proteína Smad4/metabolismo , Células Th17/citologia , Células Th17/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Diferenciação Celular/genética , Feminino , Deleção de Genes , Humanos , Interleucina-6/metabolismo , Masculino , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/deficiência , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Proteína Smad4/deficiência , Proteína Smad4/genética
8.
Nucleic Acids Res ; 45(22): 12723-12738, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-29040764

RESUMO

Transposable elements, including endogenous retroviruses (ERVs), constitute a large fraction of the mammalian genome. They are transcriptionally silenced during early development to protect genome integrity and aberrant transcription. However, the mechanisms that control their repression are not fully understood. To systematically study ERV repression, we carried out an RNAi screen in mouse embryonic stem cells (ESCs) and identified a list of novel regulators. Among them, Rif1 displays the strongest effect. Rif1 depletion by RNAi or gene deletion led to increased transcription and increased chromatin accessibility at ERV regions and their neighboring genes. This transcriptional de-repression becomes more severe when DNA methylation is lost. On the mechanistic level, Rif1 directly occupies ERVs and is required for repressive histone mark H3K9me3 and H3K27me3 assembly and DNA methylation. It interacts with histone methyltransferases and facilitates their recruitment to ERV regions. Importantly, Rif1 represses ERVs in human ESCs as well, and the evolutionally-conserved HEAT-like domain is essential for its function. Finally, Rif1 acts as a barrier during somatic cell reprogramming, and its depletion significantly enhances reprogramming efficiency. Together, our study uncovered many previously uncharacterized repressors of ERVs, and defined an essential role of Rif1 in the epigenetic defense against ERV activation.


Assuntos
Cromatina/genética , Retrovirus Endógenos/genética , Proteínas de Ligação a Telômeros/genética , Ativação Viral , Animais , Linhagem Celular , Células Cultivadas , Cromatina/metabolismo , Metilação de DNA , Células-Tronco Embrionárias/metabolismo , Retrovirus Endógenos/fisiologia , Células HEK293 , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Metilação , Camundongos , Interferência de RNA , Proteínas de Ligação a Telômeros/metabolismo
9.
Sci Rep ; 6: 24228, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-27052786

RESUMO

In eukaryotes, genomic DNA is compacted as chromatin, in which histones and DNA form the nucleosome as the basic unit. DMC1 and RAD51 are essential eukaryotic recombinases that mediate homologous chromosome pairing during homologous recombination. However, the means by which these two recombinases distinctly function in chromatin have remained elusive. Here we found that, in chromatin, the human DMC1-single-stranded DNA complex bypasses binding to the nucleosome, and preferentially promotes homologous pairing at the nucleosome-depleted regions. Consistently, DMC1 forms ternary complex recombination intermediates with the nucleosome-free DNA or the nucleosome-depleted DNA region. Surprisingly, removal of the histone tails improperly enhances the nucleosome binding by DMC1. In contrast, RAD51 does not specifically target the nucleosome-depleted region in chromatin. These are the first demonstrations that the chromatin architecture specifies the sites to promote the homologous recombination reaction by DMC1, but not by RAD51.


Assuntos
Proteínas de Ciclo Celular/genética , Cromatina/genética , Pareamento Cromossômico/genética , Proteínas de Ligação a DNA/genética , Recombinação Homóloga/genética , Rad51 Recombinase/genética , Sequência de Bases , Sítios de Ligação/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , DNA/genética , DNA/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Histonas/metabolismo , Humanos , Modelos Genéticos , Nucleossomos/genética , Nucleossomos/metabolismo , Ligação Proteica , Rad51 Recombinase/metabolismo
10.
Genom Data ; 7: 173-4, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26981404

RESUMO

Chromatin accessibility is tightly regulated by multiple factors/mechanisms to establish different cell type-specific gene expression programs from a single genome. Dysregulation of this process can lead to diseases including cancer. The Mi-2/nucleosome remodeling and deacetylase (NuRD) complex is thought to orchestrate chromatin structure using its intrinsic nucleosome remodeling and histone deacetylase activities. However, the detailed mechanisms by which the NuRD complex regulates chromatin structure in vivo are not yet known. To explore the regulatory mechanisms of the NuRD complex, we mapped genome-wide localization of MBD3, a structural component of NuRD, in a human breast cancer cell line (MDA-MB-231) using a modified ChIP-seq protocol. Our data showed high quality localization information (i.e., high mapping efficiency and low PCR duplication rate) and excellent consistency between biological replicates. The data are deposited in the Gene Expression Omnibus (GSE76116).

11.
Genome Biol ; 17: 36, 2016 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-26922637

RESUMO

BACKGROUND: Transcription factor-dependent cellular reprogramming is integral to normal development and is central to production of induced pluripotent stem cells. This process typically requires pioneer transcription factors (TFs) to induce de novo formation of enhancers at previously closed chromatin. Mechanistic information on this process is currently sparse. RESULTS: Here we explore the mechanistic basis by which GATA3 functions as a pioneer TF in a cellular reprogramming event relevant to breast cancer, the mesenchymal to epithelial transition (MET). In some instances, GATA3 binds previously inaccessible chromatin, characterized by stable, positioned nucleosomes where it induces nucleosome eviction, alters local histone modifications, and remodels local chromatin architecture. At other loci, GATA3 binding induces nucleosome sliding without concomitant generation of accessible chromatin. Deletion of the transactivation domain retains the chromatin binding ability of GATA3 but cripples chromatin reprogramming ability, resulting in failure to induce MET. CONCLUSIONS: These data provide mechanistic insights into GATA3-mediated chromatin reprogramming during MET, and suggest unexpected complexity to TF pioneering. Successful reprogramming requires stable binding to a nucleosomal site; activation domain-dependent recruitment of co-factors including BRG1, the ATPase subunit of the SWI/SNF chromatin remodeling complex; and appropriate genomic context. The resulting model provides a new conceptual framework for de novo enhancer establishment by a pioneer TF.


Assuntos
Neoplasias da Mama/genética , Reprogramação Celular/genética , Montagem e Desmontagem da Cromatina/genética , Fator de Transcrição GATA3/genética , Neoplasias da Mama/patologia , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Nucleossomos/genética , Fatores de Transcrição/genética
12.
Biochemistry ; 55(4): 637-46, 2016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26757249

RESUMO

Linker histones bind to nucleosomes and compact polynucleosomes into a higher-order chromatin configuration. Somatic and germ cell-specific linker histone subtypes have been identified and may have distinct functions. In this study, we reconstituted polynucleosomes containing human histones H1.2 and H1T, as representative somatic and germ cell-specific linker histones, respectively, and found that H1T forms less compacted chromatin, as compared to H1.2. An in vitro homologous pairing assay revealed that H1T weakly inhibited RAD51/RAD54-mediated homologous pairing in chromatin, although the somatic H1 subtypes, H1.0, H1.1, H1.2, H1.3, H1.4, and H1.5, substantially suppressed it. An in vivo recombination assay revealed that H1T overproduction minimally affected the recombination frequency, but significant suppression was observed when H1.2 was overproduced in human cells. These results suggested that the testis-specific linker histone, H1T, possesses a specific function to produce the chromatin architecture required for proper chromosome regulation, such as homologous recombination.


Assuntos
DNA Helicases/química , Histonas/química , Proteínas Nucleares/química , Nucleossomos/química , Rad51 Recombinase/química , Recombinação Genética , Linhagem Celular , DNA Helicases/genética , DNA Helicases/metabolismo , Histonas/genética , Histonas/imunologia , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo
13.
Gene Expr ; 16(4): 163-8, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26637396

RESUMO

GATA3 is a highly conserved, essential transcription factor expressed in a number of tissues, including the mammary gland. GATA3 expression is required for normal development of the mammary gland where it is estimated to be the most abundant transcription factor in luminal epithelial cells. In breast cancer, GATA3 expression is highly correlated with the luminal transcriptional program. Recent genomic analysis of human breast cancers has revealed high-frequency mutation in GATA3 in luminal tumors, suggesting "driver" function(s). Here we discuss mutation of GATA3 in breast cancer and the potential mechanism(s) by which mutation may lead to a growth advantage in cancer.


Assuntos
Neoplasias da Mama/genética , Fator de Transcrição GATA3/fisiologia , Genes Supressores de Tumor , Oncogenes , Feminino , Fator de Transcrição GATA3/genética , Humanos
14.
Sci Rep ; 4: 4863, 2014 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-24798879

RESUMO

Homologous recombination plays essential roles in mitotic DNA double strand break (DSB) repair and meiotic genetic recombination. In eukaryotes, RAD51 promotes the central homologous-pairing step during homologous recombination, but is not sufficient to overcome the reaction barrier imposed by nucleosomes. RAD54, a member of the ATP-dependent nucleosome remodeling factor family, is required to promote the RAD51-mediated homologous pairing in nucleosomal DNA. In higher eukaryotes, most nucleosomes form higher-ordered chromatin containing the linker histone H1. However, the mechanism by which RAD51/RAD54-mediated homologous pairing occurs in higher-ordered chromatin has not been elucidated. In this study, we found that a histone chaperone, Nap1, accumulates on DSB sites in human cells, and DSB repair is substantially decreased in Nap1-knockdown cells. We determined that Nap1 binds to RAD54, enhances the RAD54-mediated nucleosome remodeling by evicting histone H1, and eventually stimulates the RAD51-mediated homologous pairing in higher-ordered chromatin containing histone H1.


Assuntos
Cromatina/metabolismo , DNA Helicases/metabolismo , Histonas/metabolismo , Recombinação Homóloga/genética , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Rad51 Recombinase/metabolismo , Adenosina Trifosfatases/metabolismo , Linhagem Celular , Cromatina/genética , DNA Helicases/genética , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Histonas/genética , Humanos , Proteínas Nucleares/genética , Nucleossomos/genética , Nucleossomos/metabolismo , Rad51 Recombinase/genética
15.
BMC Cancer ; 14: 278, 2014 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-24758297

RESUMO

BACKGROUND: The transcription factor GATA3 is a favorable prognostic indicator in estrogen receptor-α (ERα)-positive breast tumors in which it participates with ERα and FOXA1 in a complex transcriptional regulatory program driving tumor growth. GATA3 mutations are frequent in breast cancer and have been classified as driver mutations. To elucidate the contribution(s) of GATA3 alterations to cancer, we studied two breast cancer cell lines, MCF7, which carries a heterozygous frameshift mutation in the second zinc finger of GATA3, and T47D, wild-type at this locus. METHODS: Immunofluorescence staining and subcellular fractionation were employed to verify cellular localization of GATA3 in T47D and MCF7 cells. To test protein stability, cells were treated with translation inhibitor, cycloheximide or proteasome inhibitor, MG132, and GATA3 abundance was measured over time using immunoblot. GATA3 turn-over in response to hormone was determined by treating the cells with estradiol or ERα agonist, ICI 182,780. DNA binding ability of recombinant GATA3 was evaluated using electrophoretic mobility shift assay and heparin chromatography. Genomic location of GATA3 in MCF7 and T47D cells was assessed by chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq). RESULTS: GATA3 localized in the nucleus in T47D and MCF7 cells, regardless of the mutation status. The truncated protein in MCF7 had impaired interaction with chromatin and was easily released from the nucleus. Recombinant mutant GATA3 was able to bind DNA to a lesser degree than the wild-type protein. Heterozygosity for the truncating mutation conferred protection from regulated turnover of GATA3, ERα and FOXA1 following estrogen stimulation in MCF7 cells. Thus, mutant GATA3 uncoupled protein-level regulation of master regulatory transcription factors from hormone action. Consistent with increased protein stability, ChIP-seq profiling identified greater genome-wide accumulation of GATA3 in MCF7 cells bearing the mutation, albeit with a similar distribution across the genome, comparing to T47D cells. CONCLUSIONS: We propose that this specific, cancer-derived mutation in GATA3 deregulates physiologic protein turnover, stabilizes GATA3 binding across the genome and modulates the response of breast cancer cells to estrogen signaling.


Assuntos
Neoplasias da Mama/genética , Estrogênios/farmacologia , Fator de Transcrição GATA3/genética , Transcrição Genética/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , Feminino , Fator de Transcrição GATA3/metabolismo , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Células MCF-7 , Mutação , Estabilidade Proteica/efeitos dos fármacos
16.
J Cell Sci ; 126(Pt 6): 1323-32, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23378026

RESUMO

In eukaryotes, transcription occurs in the chromatin context with the assistance of histone-binding proteins, such as chromatin/nucleosome remodeling factors and histone chaperones. However, it is unclear how each remodeling factor or histone chaperone functions in transcription. Here, we identify a novel histone-binding protein, Spt2, in higher eukaryotes. Recombinant human Spt2 binds to histones and DNA, and promotes nucleosome assembly in vitro. Spt2 accumulates in nucleoli and interacts with RNA polymerase I in chicken DT40 cells, suggesting its involvement in ribosomal RNA transcription. Consistently, Spt2-deficient chicken DT40 cells are sensitive to RNA polymerase I inhibitors and exhibit decreased transcription activity, as shown by a transcription run-on assay. Domain analyses of Spt2 revealed that the C-terminal region, containing the region homologous to yeast Spt2, is responsible for histone binding, while the central region is essential for nucleolar localization and DNA binding. Based on these results, we conclude that vertebrate Spt2 is a novel histone chaperone with a separate DNA-binding domain that facilitates ribosomal DNA transcription through chromatin remodeling during transcription.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Chaperonas de Histonas/metabolismo , Animais , Linhagem Celular , Galinhas , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Chaperonas de Histonas/genética , Chaperonas de Histonas/isolamento & purificação , Histonas/metabolismo , Humanos , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Transporte Proteico/genética , RNA Polimerase I/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Deleção de Sequência/genética , Transcrição Genética , Leveduras/genética
17.
Nucleic Acids Res ; 40(7): 3031-41, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22156371

RESUMO

PSF is considered to have multiple functions in RNA processing, transcription and DNA repair by mitotic recombination. In the present study, we found that PSF is produced in spermatogonia, spermatocytes and spermatids, suggesting that PSF may also function in meiotic recombination. We tested the effect of PSF on homologous pairing by the meiosis-specific recombinase DMC1, and found that human PSF robustly stimulated it. PSF synergistically enhanced the formation of a synaptic complex containing DMC1, ssDNA and dsDNA during homologous pairing. The PSF-mediated DMC1 stimulation may be promoted by its DNA aggregation activity, which increases the local concentrations of ssDNA and dsDNA for homologous pairing by DMC1. These results suggested that PSF may function as an activator for the meiosis-specific recombinase DMC1 in higher eukaryotes.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Recombinação Homóloga , Proteínas de Ligação a RNA/metabolismo , Animais , DNA de Cadeia Simples/metabolismo , Humanos , Masculino , Camundongos , Proteínas Nucleares/metabolismo , Fator de Processamento Associado a PTB , Proteínas de Ligação a RNA/biossíntese , Rad51 Recombinase/metabolismo , Espermatozoides/metabolismo
18.
Biochemistry ; 50(32): 6797-805, 2011 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-21732698

RESUMO

A deficiency in the SMN gene product causes the motor neuron degenerative disease spinal muscular atrophy. GEMIN2 was identified as an SMN-interacting protein, and the SMN-GEMIN2 complex constitutes part of the large SMN complex, which promotes the assembly of the spliceosomal small nuclear ribonucleoprotein (snRNP). In addition to its splicing function, we previously found that GEMIN2 alone stimulates RAD51-mediated recombination in vitro, and functions in DNA double-strand-break (DSB) repair through homologous recombination in vivo. However, the function of SMN in homologous recombination has not been reported. In the present study, we successfully purified the SMN-GEMIN2 complex as a fusion protein. The SMN-GEMIN2 fusion protein complemented the growth-defective phenotype of GEMIN2-knockout cells. The purified SMN-GEMIN2 fusion protein enhanced the RAD51-mediated homologous pairing much more efficiently than GEMIN2 alone. SMN-GEMIN2 possessed DNA-binding activity, which was not observed with the GEMIN2 protein, and significantly stimulated the secondary duplex DNA capture by the RAD51-single-stranded DNA complex during homologous pairing. These results provide the first evidence that the SMN-GEMIN2 complex plays a role in homologous recombination, in addition to spliceosomal snRNP assembly.


Assuntos
DNA/genética , Proteínas do Tecido Nervoso/isolamento & purificação , Proteínas de Ligação a RNA/isolamento & purificação , Rad51 Recombinase/isolamento & purificação , Recombinação Genética/fisiologia , Proteína 1 de Sobrevivência do Neurônio Motor/isolamento & purificação , Animais , Sequência de Bases , Galinhas , Primers do DNA , Reparo do DNA , Técnicas de Inativação de Genes , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Rad51 Recombinase/fisiologia , Proteína 1 de Sobrevivência do Neurônio Motor/fisiologia
19.
J Biochem ; 149(6): 721-9, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21398369

RESUMO

MENA, VASP and EVL are members of the ENA/VASP family of proteins and are involved in cytoplasmic actin remodeling. Previously, we found that EVL directly interacts with RAD51, an essential protein in the homologous recombinational repair of double-strand breaks (DSBs) and stimulates the RAD51-mediated recombination reactions in vitro. The EVL-knockdown MCF7 cells exhibited a clear reduction in RAD51-foci formation, suggesting that EVL may function in the DSB repair pathway through RAD51-mediated homologous recombination. However, the DSB repair defects were less significant in the EVL-knockdown cells, implying that two EVL paralogues, MENA and VASP, may complement the EVL function in human cells. Therefore, in the present study, we purified human MENA, VASP and EVL as recombinant proteins, and compared their biochemical activities in vitro. We found that all three proteins commonly exhibited the RAD51 binding, DNA binding and DNA-annealing activities. Stimulation of the RAD51-mediated homologous pairing was also observed with all three proteins. In addition, surface plasmon resonance analyses revealed that MENA, VASP and EVL mutually interacted. These results support the ideas that the ENA/VASP-family proteins are functionally redundant in homologous recombination, and that all three may be involved in the DSB repair pathway in humans.


Assuntos
Moléculas de Adesão Celular/metabolismo , Recombinação Homóloga , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Moléculas de Adesão Celular/genética , DNA/química , DNA/metabolismo , Humanos , Proteínas dos Microfilamentos/genética , Fosfoproteínas/genética , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície
20.
Genes Cells ; 16(4): 427-36, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21375680

RESUMO

Mutations and single-nucleotide polymorphisms affecting RAD51 gene function have been identified in several tumors, suggesting that the inappropriate expression of RAD51 activity may cause tumorigenesis. RAD51 is an essential enzyme for the homologous recombinational repair (HRR) of DNA double-strand breaks. In the HRR pathway, RAD51 catalyzes the homologous pairing between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), which is the central step of the HRR pathway. To identify a chemical compound that regulates the homologous-pairing activity of RAD51, in the present study, we screened crude extract fractions from marine sponges by the RAD51-mediated homologous-pairing assay. Halenaquinone was identified as an inhibitor of the RAD51 homologous-pairing activity. A surface plasmon resonance analysis indicated that halenaquinone directly bound to RAD51. Intriguingly, halenaquinone specifically inhibited dsDNA binding by RAD51 alone or the RAD51-ssDNA complex, but only weakly affected the RAD51-ssDNA binding. In vivo, halenaquinone significantly inhibited the retention of RAD51 at double-strand break sites. Therefore, halenaquinone is a novel type of RAD51 inhibitor that specifically inhibits the RAD51-dsDNA binding.


Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Poríferos/enzimologia , Quinonas/farmacologia , Rad51 Recombinase/antagonistas & inibidores , Animais , Misturas Complexas , Quebras de DNA de Cadeia Dupla , Humanos , Técnicas In Vitro , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Homologia de Sequência do Ácido Nucleico
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