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1.
J Exp Med ; 214(7): 1937-1947, 2017 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-28600438

RESUMO

The treatment of chronic mucocutaneous ulceration is challenging, and only some patients respond selectively to inhibitors of tumor necrosis factor-α (TNF). TNF activates opposing pathways leading to caspase-8-mediated apoptosis as well as nuclear factor κB (NF-κB)-dependent cell survival. We investigated the etiology of autosomal-dominant, mucocutaneous ulceration in a family whose proband was dependent on anti-TNF therapy for sustained remission. A heterozygous mutation in RELA, encoding the NF-κB subunit RelA, segregated with the disease phenotype and resulted in RelA haploinsufficiency. The patients' fibroblasts exhibited increased apoptosis in response to TNF, impaired NF-κB activation, and defective expression of NF-κB-dependent antiapoptotic genes. Rela+/- mice have similarly impaired NF-κB activation, develop cutaneous ulceration from TNF exposure, and exhibit severe dextran sodium sulfate-induced colitis, ameliorated by TNF inhibition. These findings demonstrate an essential contribution of biallelic RELA expression in protecting stromal cells from TNF-mediated cell death, thus delineating the mechanisms driving the effectiveness of TNF inhibition in this disease.


Assuntos
Genes Dominantes/genética , Haploinsuficiência , Úlceras Orais/genética , Úlcera Cutânea/genética , Fator de Transcrição RelA/genética , Animais , Sequência de Bases , Células Cultivadas , Doença Crônica , Colite/induzido quimicamente , Colite/genética , Sulfato de Dextrana , Exoma/genética , Feminino , Fibroblastos/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Camundongos Knockout , NF-kappa B/metabolismo , Linhagem , Análise de Sequência de DNA/métodos , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
2.
J Clin Invest ; 126(11): 4219-4236, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27760045

RESUMO

Alterations in the apoptosis of immune cells have been associated with autoimmunity. Here, we have identified a homozygous missense mutation in the gene encoding the base excision repair enzyme Nei endonuclease VIII-like 3 (NEIL3) that abolished enzymatic activity in 3 siblings from a consanguineous family. The NEIL3 mutation was associated with fatal recurrent infections, severe autoimmunity, hypogammaglobulinemia, and impaired B cell function in these individuals. The same homozygous NEIL3 mutation was also identified in an asymptomatic individual who exhibited elevated levels of serum autoantibodies and defective peripheral B cell tolerance, but normal B cell function. Further analysis of the patients revealed an absence of LPS-responsive beige-like anchor (LRBA) protein expression, a known cause of immunodeficiency. We next examined the contribution of NEIL3 to the maintenance of self-tolerance in Neil3-/- mice. Although Neil3-/- mice displayed normal B cell function, they exhibited elevated serum levels of autoantibodies and developed nephritis following treatment with poly(I:C) to mimic microbial stimulation. In Neil3-/- mice, splenic T and B cells as well as germinal center B cells from Peyer's patches showed marked increases in apoptosis and cell death, indicating the potential release of self-antigens that favor autoimmunity. These findings demonstrate that deficiency in NEIL3 is associated with increased lymphocyte apoptosis, autoantibodies, and predisposition to autoimmunity.


Assuntos
Doenças Autoimunes , Linfócitos B/imunologia , Endodesoxirribonucleases/deficiência , Predisposição Genética para Doença , N-Glicosil Hidrolases/deficiência , Linfócitos T/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/imunologia , Autoanticorpos/imunologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Linfócitos B/patologia , Endodesoxirribonucleases/imunologia , Feminino , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Knockout , N-Glicosil Hidrolases/imunologia , Poli I-C/farmacologia , Linfócitos T/patologia
3.
Nat Genet ; 48(1): 74-8, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26642240

RESUMO

Patients with a combined immunodeficiency characterized by normal numbers but impaired function of T and B cells had a homozygous p.Tyr20His substitution in transferrin receptor 1 (TfR1), encoded by TFRC. The substitution disrupts the TfR1 internalization motif, resulting in defective receptor endocytosis and markedly increased TfR1 expression on the cell surface. Iron citrate rescued the lymphocyte defects, and expression of wild-type but not mutant TfR1 rescued impaired transferrin uptake in patient-derived fibroblasts. Tfrc(Y20H/Y20H) mice recapitulated the immunological defects of patients. Despite the critical role of TfR1 in erythrocyte development and function, patients had only mild anemia and only slightly increased TfR1 expression in erythroid precursors. We show that STEAP3, a metalloreductase expressed in erythroblasts, associates with TfR1 and partially rescues transferrin uptake in patient-derived fibroblasts, suggesting that STEAP3 may provide an accessory TfR1 endocytosis signal that spares patients from severe anemia. These findings demonstrate the importance of TfR1 in adaptive immunity.


Assuntos
Antígenos CD/genética , Antígenos CD/imunologia , Síndromes de Imunodeficiência/genética , Mutação de Sentido Incorreto , Receptores da Transferrina/genética , Receptores da Transferrina/imunologia , Imunidade Adaptativa/genética , Anemia/genética , Animais , Antígenos CD/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Endocitose , Feminino , Fibroblastos/fisiologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Linhagem , Receptores da Transferrina/metabolismo
4.
J Allergy Clin Immunol ; 137(3): 879-88.e2, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26476480

RESUMO

BACKGROUND: Coronin-1A (CORO1A) is a regulator of actin dynamics important for T-cell homeostasis. CORO1A deficiency causes T(-)B(+) natural killer-positive severe combined immunodeficiency or T-cell lymphopenia with severe viral infections. However, because all known human mutations in CORO1A abrogate protein expression, the role of the protein's functional domains in host immunity is unknown. OBJECTIVE: We sought to identify the cause of the primary immunodeficiency in 2 young adult siblings with a history of disseminated varicella, cutaneous warts, and CD4(+) T-cell lymphopenia. METHODS: We performed immunologic, genetic, and biochemical studies in the patients, family members, and healthy control subjects. RESULTS: Both patients had CD4(+) T-cell lymphopenia and decreased lymphocyte proliferation to mitogens. IgG, IgM, IgA, and specific antibody responses were normal. Whole-genome sequencing identified a homozygous frameshift mutation in CORO1A disrupting the last 2 C-terminal domains by replacing 61 amino acids with a novel 91-amino-acid sequence. The CORO1A(S401fs) mutant was expressed in the patients' lymphocytes at a level comparable with that of wild-type CORO1A in normal lymphocytes but did not oligomerize and had impaired cytoskeletal association. CORO1A(S401fs) was associated with increased filamentous actin accumulation in T cells, severely defective thymic output, and impaired T-cell survival but normal calcium flux and cytotoxicity, demonstrating the importance of CORO1A oligomerization and subcellular localization in T-cell homeostasis. CONCLUSIONS: We describe a truncating mutation in CORO1A that permits protein expression and survival into young adulthood. Our studies demonstrate the importance of intact CORO1A C-terminal domains in thymic egress and T-cell survival, as well as in defense against viral pathogens.


Assuntos
Citoesqueleto/metabolismo , Homozigoto , Proteínas dos Microfilamentos/genética , Mutação , Multimerização Proteica , Viroses/etiologia , Viroses/metabolismo , Actinas/química , Actinas/metabolismo , Adolescente , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Degranulação Celular/genética , Degranulação Celular/imunologia , Sobrevivência Celular/genética , Análise Mutacional de DNA , Feminino , Mutação da Fase de Leitura , Humanos , Imunoglobulinas/sangue , Imunoglobulinas/imunologia , Contagem de Linfócitos , Linfopenia , Masculino , Camundongos , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Linhagem , Fenótipo , Multimerização Proteica/genética , Transporte Proteico , Irmãos , Transdução de Sinais , Dermatopatias/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Viroses/diagnóstico , Verrugas/patologia
5.
N Engl J Med ; 372(25): 2409-22, 2015 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-26083206

RESUMO

Background Combined immunodeficiencies are marked by inborn errors of T-cell immunity in which the T cells that are present are quantitatively or functionally deficient. Impaired humoral immunity is also common. Patients have severe infections, autoimmunity, or both. The specific molecular, cellular, and clinical features of many types of combined immunodeficiencies remain unknown. Methods We performed genetic and cellular immunologic studies involving five unrelated children with early-onset invasive bacterial and viral infections, lymphopenia, and defective T-cell, B-cell, and natural killer (NK)-cell responses. Two patients died early in childhood; after allogeneic hematopoietic stem-cell transplantation, the other three had normalization of T-cell function and clinical improvement. Results We identified biallelic mutations in the dedicator of cytokinesis 2 gene (DOCK2) in these five patients. RAC1 activation was impaired in the T cells. Chemokine-induced migration and actin polymerization were defective in the T cells, B cells, and NK cells. NK-cell degranulation was also affected. Interferon-α and interferon-λ production by peripheral-blood mononuclear cells was diminished after viral infection. Moreover, in DOCK2-deficient fibroblasts, viral replication was increased and virus-induced cell death was enhanced; these conditions were normalized by treatment with interferon alfa-2b or after expression of wild-type DOCK2. Conclusions Autosomal recessive DOCK2 deficiency is a new mendelian disorder with pleiotropic defects of hematopoietic and nonhematopoietic immunity. Children with clinical features of combined immunodeficiencies, especially with early-onset, invasive infections, may have this condition. (Supported by the National Institutes of Health and others.).


Assuntos
Doenças Genéticas Inatas/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Síndromes de Imunodeficiência/genética , Mutação , Linfócitos T/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Pré-Escolar , Evolução Fatal , Feminino , Genes Recessivos , Doenças Genéticas Inatas/terapia , Fatores de Troca do Nucleotídeo Guanina/deficiência , Transplante de Células-Tronco Hematopoéticas , Humanos , Síndromes de Imunodeficiência/terapia , Lactente , Células Matadoras Naturais/imunologia , Masculino , Linhagem , Linfócitos T/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
7.
J Allergy Clin Immunol ; 135(1): 217-27, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25468195

RESUMO

BACKGROUND: A number of heritable immune dysregulatory diseases result from defects affecting regulatory T (Treg) cell development, function, or both. They include immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, which is caused by mutations in forkhead box P3 (FOXP3), and IPEX-like disorders caused by mutations in IL-2 receptor α (IL2RA), signal transducer and activator of transcription 5b (STAT5b), and signal transducer and activator of transcription 1 (STAT1). However, the genetic defects underlying many cases of IPEX-like disorders remain unknown. OBJECTIVE: We sought to identify the genetic abnormalities in patients with idiopathic IPEX-like disorders. METHODS: We performed whole-exome and targeted gene sequencing and phenotypic and functional analyses of Treg cells. RESULTS: A child who presented with an IPEX-like syndrome and severe Treg cell deficiency was found to harbor a nonsense mutation in the gene encoding LPS-responsive beige-like anchor (LRBA), which was previously implicated as a cause of common variable immunodeficiency with autoimmunity. Analysis of subjects with LRBA deficiency revealed marked Treg cell depletion; profoundly decreased expression of canonical Treg cell markers, including FOXP3, CD25, Helios, and cytotoxic T lymphocyte-associated antigen 4; and impaired Treg cell-mediated suppression. There was skewing in favor of memory T cells and intense autoantibody production, with marked expansion of T follicular helper and contraction of T follicular regulatory cells. Whereas the frequency of recent thymic emigrants and the differentiation of induced Treg cells were normal, LRBA-deficient T cells exhibited increased apoptosis and reduced activities of the metabolic sensors mammalian target of rapamycin complexes 1 and 2. CONCLUSION: LRBA deficiency is a novel cause of IPEX-like syndrome and Treg cell deficiency associated with metabolic dysfunction and increased apoptosis of Treg cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Linfócitos T Reguladores/imunologia , Adolescente , Autoanticorpos/imunologia , Pré-Escolar , Diabetes Mellitus Tipo 1/congênito , Diarreia , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Humanos , Doenças do Sistema Imunitário/congênito , Interleucina-10/imunologia , Masculino , Mutação
9.
Curr Biol ; 24(8): 839-44, 2014 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-24684932

RESUMO

More than 2,000 C. elegans genes are targeted for RNA silencing by the mutator complex, a specialized small interfering RNA (siRNA) amplification module which is nucleated by the Q/N-rich protein MUT-16. The mutator complex localizes to Mutator foci adjacent to P granules at the nuclear periphery in germ cells. Here, we show that the DEAD box RNA helicase smut-1 functions redundantly in the mutator pathway with its paralog mut-14 during RNAi. Mutations in both smut-1 and mut-14 also cause widespread loss of endogenous siRNAs. The targets of mut-14 and smut-1 largely overlap with the targets of other mutator class genes; however, the mut-14 smut-1 double mutant and the mut-16 mutant display the most dramatic depletion of siRNAs, suggesting that they act at a similarly early step in siRNA formation. mut-14 and smut-1 are predominantly expressed in the germline and, unlike other mutator class genes, are specifically required for RNAi targeting germline genes. A catalytically inactive, dominant-negative missense mutant of MUT-14 is RNAi defective in vivo; however, mutator complexes containing the mutant protein retain the ability to synthesize siRNAs in vitro. The results point to a role for mut-14 and smut-1 in initiating siRNA amplification in germ cell Mutator foci, possibly through the recruitment or retention of target mRNAs.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , RNA Helicases DEAD-box/metabolismo , Células Germinativas/enzimologia , Interferência de RNA/fisiologia , RNA Interferente Pequeno/biossíntese , Animais , Sequência de Bases , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Fluorimunoensaio , Células Germinativas/fisiologia , Imunoprecipitação , Dados de Sequência Molecular , Reação em Cadeia da Polimerase em Tempo Real , Saccharomyces cerevisiae , Alinhamento de Sequência , Análise de Sequência de DNA
10.
Nat Neurosci ; 16(12): 1896-905, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24162652

RESUMO

Microglia, the principal neuroimmune sentinels of the brain, continuously sense changes in their environment and respond to invading pathogens, toxins and cellular debris. Microglia exhibit plasticity and can assume neurotoxic or neuroprotective priming states that determine their responses to danger. We used direct RNA sequencing, without amplification or cDNA synthesis, to determine the quantitative transcriptomes of microglia of healthy adult and aged mice. We validated our findings using fluorescence dual in situ hybridization, unbiased proteomic analysis and quantitative PCR. We found that microglia have a distinct transcriptomic signature and express a unique cluster of transcripts encoding proteins for sensing endogenous ligands and microbes that we refer to as the sensome. With aging, sensome transcripts for endogenous ligand recognition were downregulated, whereas those involved in microbe recognition and host defense were upregulated. In addition, aging was associated with an overall increase in the expression of microglial genes involved in neuroprotection.


Assuntos
Microglia/metabolismo , Análise de Sequência de RNA/métodos , Transcriptoma/fisiologia , Fatores Etários , Animais , Antígeno CD11b/metabolismo , Biologia Computacional , Citometria de Fluxo , Antígenos Comuns de Leucócito/metabolismo , Ligantes , Macrófagos Peritoneais , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Transcriptoma/genética
11.
Cell Stem Cell ; 12(6): 699-712, 2013 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-23665121

RESUMO

The chromatin state of pluripotency genes has been studied extensively in embryonic stem cells (ESCs) and differentiated cells, but their potential interactions with other parts of the genome remain largely unexplored. Here, we identified a genome-wide, pluripotency-specific interaction network around the Nanog promoter by adapting circular chromosome conformation capture sequencing. This network was rearranged during differentiation and restored in induced pluripotent stem cells. A large fraction of Nanog-interacting loci were bound by Mediator or cohesin in pluripotent cells. Depletion of these proteins from ESCs resulted in a disruption of contacts and the acquisition of a differentiation-specific interaction pattern prior to obvious transcriptional and phenotypic changes. Similarly, the establishment of Nanog interactions during reprogramming often preceded transcriptional upregulation of associated genes, suggesting a causative link. Our results document a complex, pluripotency-specific chromatin "interactome" for Nanog and suggest a functional role for long-range genomic interactions in the maintenance and induction of pluripotency.


Assuntos
Reprogramação Celular/genética , Cromatina/genética , Cromatina/metabolismo , Genoma/genética , Proteínas de Homeodomínio/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Proteína Homeobox Nanog
12.
BMC Cancer ; 13: 93, 2013 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-23442791

RESUMO

BACKGROUND: The development of resistance to chemotherapies represents a significant barrier to successful cancer treatment. Resistance mechanisms are complex, can involve diverse and often unexpected cellular processes, and can vary with both the underlying genetic lesion and the origin or type of tumor. For these reasons developing experimental strategies that could be used to understand, identify and predict mechanisms of resistance in different malignant cells would be a major advance. METHODS: Here we describe a gain-of-function forward genetic approach for identifying mechanisms of resistance. This approach uses a modified piggyBac transposon to generate libraries of mutagenized cells, each containing transposon insertions that randomly activate nearby gene expression. Genes of interest are identified using next-gen high-throughput sequencing and barcode multiplexing is used to reduce experimental cost. RESULTS: Using this approach we successfully identify genes involved in paclitaxel resistance in a variety of cancer cell lines, including the multidrug transporter ABCB1, a previously identified major paclitaxel resistance gene. Analysis of co-occurring transposons integration sites in single cell clone allows for the identification of genes that might act cooperatively to produce drug resistance a level of information not accessible using RNAi or ORF expression screening approaches. CONCLUSION: We have developed a powerful pipeline to systematically discover drug resistance in mammalian cells in vitro. This cost-effective approach can be readily applied to different cell lines, to identify canonical or context specific resistance mechanisms. Its ability to probe complex genetic context and non-coding genomic elements as well as cooperative resistance events makes it a good complement to RNAi or ORF expression based screens.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Análise Mutacional de DNA/métodos , Elementos de DNA Transponíveis/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias/tratamento farmacológico , Paclitaxel/farmacologia , Western Blotting , Linhagem Celular Tumoral , Código de Barras de DNA Taxonômico/métodos , Humanos , Mutagênese Insercional/métodos , Neoplasias/genética , RNA Mensageiro/análise
13.
Curr Opin Allergy Clin Immunol ; 12(6): 623-8, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23095910

RESUMO

PURPOSE OF REVIEW: This review discusses the strengths and challenges of using whole genome sequencing (WGS)/whole exome sequencing (WES) for identifying novel genetic causes of primary immunodeficiencies. RECENT FINDINGS: WGS permits comprehensive sequencing of introns and exons, whereas WES allows deeper sequencing of exonic regions at a lower cost. Due to the large number of genetic variants found in each genome, it is necessary to use filtering approaches to distinguish deleterious from benign variants. WES has been used successfully to identify novel genetic causes of primary immunodeficiency. Complex structural variations and non-Mendelian disorders remain challenges for WGS/WES. SUMMARY: WGS/WES is a powerful screening tool with great potential to identify genetic causes of primary immunodeficiencies for research and clinical applications. To use WGS/WES effectively, it is necessary to understand how to filter the sequencing data and to realize its limitations as well as its strengths.


Assuntos
Exoma , Genoma Humano , Síndromes de Imunodeficiência/genética , Animais , Sequência de Bases , Variação Genética , Humanos , Análise de Sequência de DNA/métodos
14.
Genome Res ; 22(10): 1864-76, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22948768

RESUMO

X chromosome inactivation (XCI) achieves dosage balance in mammals by repressing one of two X chromosomes in females. During XCI, the long noncoding Xist RNA and Polycomb proteins spread along the inactive X (Xi) to initiate chromosome-wide silencing. Although inactivation is known to commence at the X-inactivation center (Xic), how it propagates remains unknown. Here, we examine allele-specific binding of Polycomb repressive complex 2 (PRC2) and chromatin composition during XCI and generate a chromosome-wide profile of Xi and Xa (active X) at nucleosome-resolution. Initially, Polycomb proteins are localized to ∼150 strong sites along the X and concentrated predominantly within bivalent domains coinciding with CpG islands ("canonical sites"). As XCI proceeds, ∼4000 noncanonical sites are recruited, most of which are intergenic, nonbivalent, and lack CpG islands. Polycomb sites are depleted of LINE repeats but enriched for SINEs and simple repeats. Noncanonical sites cluster around the ∼150 strong sites, and their H3K27me3 levels reflect a graded concentration originating from strong sites. This suggests that PRC2 and H3K27 methylation spread along a gradient unique to XCI. We propose that XCI is governed by a hierarchy of defined Polycomb stations that spread H3K27 methylation in cis.


Assuntos
Proteínas do Grupo Polycomb/metabolismo , Inativação do Cromossomo X , Alelos , Animais , Sítios de Ligação , Linhagem Celular , Imunoprecipitação da Cromatina , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Complexo Repressor Polycomb 2/metabolismo , Proteínas do Grupo Polycomb/química , Domínios e Motivos de Interação entre Proteínas , Sequências Repetitivas de Ácido Nucleico , Cromossomo X
15.
Bioinformatics ; 28(19): 2412-6, 2012 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-22815363

RESUMO

SUMMARY: We developed MolBioLib to address the need for adaptable next-generation sequencing analysis tools. The result is a compact, portable and extensively tested C++11 software framework and set of applications tailored to the demands of next-generation sequencing data and applicable to many other applications. MolBioLib is designed to work with common file formats and data types used both in genomic analysis and general data analysis. A central relational-database-like Table class is a flexible and powerful object to intuitively represent and work with a wide variety of tabular datasets, ranging from alignment data to annotations. MolBioLib has been used to identify causative single-nucleotide polymorphisms in whole genome sequencing, detect balanced chromosomal rearrangements and compute enrichment of messenger RNAs (mRNAs) on microtubules, typically requiring applications of under 200 lines of code. MolBioLib includes programs to perform a wide variety of analysis tasks, such as computing read coverage, annotating genomic intervals and novel peak calling with a wavelet algorithm. Although MolBioLib was designed primarily for bioinformatics purposes, much of its functionality is applicable to a wide range of problems. Complete documentation and an extensive automated test suite are provided. AVAILABILITY: MolBioLib is available for download at: http://sourceforge.net/projects/molbiolib CONTACT: ohsumit@molbio.mgh.harvard.edu.


Assuntos
Biologia Computacional/métodos , Análise de Sequência/métodos , Software , Algoritmos , Bases de Dados Factuais , Genômica , Linguagens de Programação
16.
Cell Rep ; 1(2): 83-90, 2012 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-22720264

RESUMO

The preferential in vitro interaction of the PHD finger of RAG2, a subunit of the V(D)J recombinase, with histone H3 tails simultaneously trimethylated at lysine 4 and symmetrically dimethylated at arginine 2 (H3R2me2sK4me3) predicted the existence of the previously unknown histone modification H3R2me2s. Here, we report the in vivo identification of H3R2me2s . Consistent with the binding specificity of the RAG2 PHD finger, high levels of H3R2me2sK4me3 are found at antigen receptor gene segments ready for rearrangement. However, this double modification is much more general; it is conserved throughout eukaryotic evolution. In mouse, H3R2me2s is tightly correlated with H3K4me3 at active promoters throughout the genome. Mutational analysis in S. cerevisiae reveals that deposition of H3R2me2s requires the same Set1 complex that deposits H3K4me3. Our work suggests that H3R2me2sK4me3, not simply H3K4me3 alone, is the mark of active promoters and that factors that recognize H3K4me3 will have their binding modulated by their preference for H3R2me2s.


Assuntos
Arginina/metabolismo , Eucariotos/genética , Eucariotos/metabolismo , Genoma/genética , Histonas/metabolismo , Lisina/metabolismo , Animais , Sequência Conservada/genética , Evolução Molecular , Loci Gênicos/genética , Histona-Lisina N-Metiltransferase/metabolismo , Metilação , Camundongos , RNA Interferente Pequeno/metabolismo , Receptores de Antígenos/imunologia , Recombinação Genética/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
17.
Cell ; 149(3): 525-37, 2012 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-22521361

RESUMO

Balanced chromosomal abnormalities (BCAs) represent a relatively untapped reservoir of single-gene disruptions in neurodevelopmental disorders (NDDs). We sequenced BCAs in patients with autism or related NDDs, revealing disruption of 33 loci in four general categories: (1) genes previously associated with abnormal neurodevelopment (e.g., AUTS2, FOXP1, and CDKL5), (2) single-gene contributors to microdeletion syndromes (MBD5, SATB2, EHMT1, and SNURF-SNRPN), (3) novel risk loci (e.g., CHD8, KIRREL3, and ZNF507), and (4) genes associated with later-onset psychiatric disorders (e.g., TCF4, ZNF804A, PDE10A, GRIN2B, and ANK3). We also discovered among neurodevelopmental cases a profoundly increased burden of copy-number variants from these 33 loci and a significant enrichment of polygenic risk alleles from genome-wide association studies of autism and schizophrenia. Our findings suggest a polygenic risk model of autism and reveal that some neurodevelopmental genes are sensitive to perturbation by multiple mutational mechanisms, leading to variable phenotypic outcomes that manifest at different life stages.


Assuntos
Transtornos Globais do Desenvolvimento Infantil/genética , Aberrações Cromossômicas , Transtorno Autístico/diagnóstico , Transtorno Autístico/genética , Criança , Transtornos Globais do Desenvolvimento Infantil/diagnóstico , Quebra Cromossômica , Deleção Cromossômica , Variações do Número de Cópias de DNA , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Sistema Nervoso/crescimento & desenvolvimento , Esquizofrenia/genética , Análise de Sequência de DNA , Transdução de Sinais
18.
Nat Genet ; 44(4): 390-7, S1, 2012 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-22388000

RESUMO

We defined the genetic landscape of balanced chromosomal rearrangements at nucleotide resolution by sequencing 141 breakpoints from cytogenetically interpreted translocations and inversions. We confirm that the recently described phenomenon of 'chromothripsis' (massive chromosomal shattering and reorganization) is not unique to cancer cells but also occurs in the germline, where it can resolve to a relatively balanced state with frequent inversions. We detected a high incidence of complex rearrangements (19.2%) and substantially less reliance on microhomology (31%) than previously observed in benign copy-number variants (CNVs). We compared these results to experimentally generated DNA breakage-repair by sequencing seven transgenic animals, revealing extensive rearrangement of the transgene and host genome with similar complexity to human germline alterations. Inversion was the most common rearrangement, suggesting that a combined mechanism involving template switching and non-homologous repair mediates the formation of balanced complex rearrangements that are viable, stably replicated and transmitted unaltered to subsequent generations.


Assuntos
Quebra Cromossômica , Reparo do DNA por Junção de Extremidades/genética , Rearranjo Gênico , Mutação em Linhagem Germinativa , Animais , Animais Geneticamente Modificados , Inversão Cromossômica , Humanos , Dados de Sequência Molecular , Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de DNA , Translocação Genética
19.
Mol Biol Cell ; 22(22): 4312-23, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21937723

RESUMO

RNA localization is an important mechanism for achieving precise control of posttranscriptional gene expression. Previously, we demonstrated that a subset of cellular mRNAs copurify with mitotic microtubules in egg extracts of Xenopus laevis. Due to limited genomic sequence information available for X. laevis, we used RNA-seq to comprehensively identify the microtubule-interacting transcriptome of the related frog Xenopus tropicalis. We identified ~450 mRNAs that showed significant enrichment on microtubules (MT-RNAs). In addition, we demonstrated that the MT-RNAs incenp, xrhamm, and tpx2 associate with spindle microtubules in vivo. MT-RNAs are enriched with transcripts associated with cell division, spindle formation, and chromosome function, demonstrating an overrepresentation of genes involved in mitotic regulation. To test whether uncharacterized MT-RNAs have a functional role in mitosis, we performed RNA interference and discovered that several MT-RNAs are required for normal spindle pole organization and γ-tubulin distribution. Together, these data demonstrate that microtubule association is one mechanism for compartmentalizing functionally related mRNAs within the nucleocytoplasmic space of mitotic cells and suggest that MT-RNAs are likely to contribute to spindle-localized mitotic translation.


Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fuso Acromático/metabolismo , Transcriptoma , Animais , Sequência de Bases , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Divisão Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Mitose , Proteínas Nucleares/genética , Fosfoproteínas/genética , Interferência de RNA , RNA Interferente Pequeno , Alinhamento de Sequência , Análise de Sequência de RNA , Fuso Acromático/genética , Tubulina (Proteína) , Xenopus , Proteínas de Xenopus/genética
20.
Am J Hum Genet ; 88(4): 469-81, 2011 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-21473983

RESUMO

The contribution of balanced chromosomal rearrangements to complex disorders remains unclear because they are not detected routinely by genome-wide microarrays and clinical localization is imprecise. Failure to consider these events bypasses a potentially powerful complement to single nucleotide polymorphism and copy-number association approaches to complex disorders, where much of the heritability remains unexplained. To capitalize on this genetic resource, we have applied optimized sequencing and analysis strategies to test whether these potentially high-impact variants can be mapped at reasonable cost and throughput. By using a whole-genome multiplexing strategy, rearrangement breakpoints could be delineated at a fraction of the cost of standard sequencing. For rearrangements already mapped regionally by karyotyping and fluorescence in situ hybridization, a targeted approach enabled capture and sequencing of multiple breakpoints simultaneously. Importantly, this strategy permitted capture and unique alignment of up to 97% of repeat-masked sequences in the targeted regions. Genome-wide analyses estimate that only 3.7% of bases should be routinely omitted from genomic DNA capture experiments. Illustrating the power of these approaches, the rearrangement breakpoints were rapidly defined to base pair resolution and revealed unexpected sequence complexity, such as co-occurrence of inversion and translocation as an underlying feature of karyotypically balanced alterations. These findings have implications ranging from genome annotation to de novo assemblies and could enable sequencing screens for structural variations at a cost comparable to that of microarrays in standard clinical practice.


Assuntos
Cromossomos Humanos/genética , Rearranjo Gênico , Análise de Sequência de DNA/métodos , Inversão Cromossômica , Biologia Computacional , Código de Barras de DNA Taxonômico , Quebras de DNA , Feminino , Biblioteca Gênica , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Sequências Repetitivas Dispersas , Cariotipagem , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Translocação Genética
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