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1.
Cell ; 172(4): 784-796.e18, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29358051

RESUMO

Mammalian barrier surfaces are constitutively colonized by numerous microorganisms. We explored how the microbiota was sensed by the immune system and the defining properties of such responses. Here, we show that a skin commensal can induce T cell responses in a manner that is restricted to non-classical MHC class I molecules. These responses are uncoupled from inflammation and highly distinct from pathogen-induced cells. Commensal-specific T cells express a defined gene signature that is characterized by expression of effector genes together with immunoregulatory and tissue-repair signatures. As such, non-classical MHCI-restricted commensal-specific immune responses not only promoted protection to pathogens, but also accelerated skin wound closure. Thus, the microbiota can induce a highly physiological and pleiotropic form of adaptive immunity that couples antimicrobial function with tissue repair. Our work also reveals that non-classical MHC class I molecules, an evolutionarily ancient arm of the immune system, can promote homeostatic immunity to the microbiota.


Assuntos
Imunidade Adaptativa , Bactérias/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Microbiota/imunologia , Pele/imunologia , Linfócitos T/imunologia , Animais , Regulação da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Camundongos , Camundongos Transgênicos
2.
Mol Carcinog ; 59(2): 237-245, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31898340

RESUMO

In humans, bone marrow (BM) failure syndromes, both constitutional and acquired, predispose to myeloid malignancies. We have modeled acquired immune aplastic anemia, the paradigmatic disease of these syndromes, in the mouse by infusing lymph node cells from specific pathogen-free (SPF) CD45.1 congenic C57BL/6 (B6) donors into hybrid CByB6F1 recipients housed either in conventional (CVB) or SPF facilities. The severity of BM damage was reduced in CVB recipients; they also had reduced levels of CD44+ CD62L- effector memory T cells, reduced numbers of donor-type CD44+ T cells, and reduced expansion of donor-type CD8 T cells carrying T-cell receptor ß-variable regions 07, 11, and 17. Analyses of fecal samples through 16S ribosomal RNA amplicon sequencing revealed greater gut microbial alpha diversity in CVB mice relative to that of SPF mice. Thus, the presence of a broader spectrum of gut microorganisms in CVB-housed CByB6F1 could have primed recipient animal's immune system leading to suppression of allogeneic donor T-cell activation and expansion and attenuation of host BM destruction. These results suggest the potential benefit of diverse gut microbiota in patients receiving BM transplants.


Assuntos
Anemia Aplástica/terapia , Transplante de Medula Óssea/métodos , Medula Óssea/imunologia , Microbioma Gastrointestinal/imunologia , Linfócitos T/imunologia , Anemia Aplástica/imunologia , Anemia Aplástica/patologia , Animais , Medula Óssea/patologia , Fezes/microbiologia , Receptores de Hialuronatos/imunologia , Receptores de Hialuronatos/metabolismo , Memória Imunológica/imunologia , Ativação Linfocitária/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Organismos Livres de Patógenos Específicos , Linfócitos T/metabolismo , Linfócitos T/transplante , Imunologia de Transplantes , Transplante Homólogo
3.
J Med Genet ; 56(7): 444-452, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30842225

RESUMO

BACKGROUND: A single variant in NAA10 (c.471+2T>A), the gene encoding N-acetyltransferase 10, has been associated with Lenz microphthalmia syndrome. In this study, we aimed to identify causative variants in families with syndromic X-linked microphthalmia. METHODS: Three families, including 15 affected individuals with syndromic X-linked microphthalmia, underwent analyses including linkage analysis, exome sequencing and targeted gene sequencing. The consequences of two identified variants in NAA10 were evaluated using quantitative PCR and RNAseq. RESULTS: Genetic linkage analysis in family 1 supported a candidate region on Xq27-q28, which included NAA10. Exome sequencing identified a hemizygous NAA10 polyadenylation signal (PAS) variant, chrX:153,195,397T>C, c.*43A>G, which segregated with the disease. Targeted sequencing of affected males from families 2 and 3 identified distinct NAA10 PAS variants, chrX:g.153,195,401T>C, c.*39A>G and chrX:g.153,195,400T>C, c.*40A>G. All three variants were absent from gnomAD. Quantitative PCR and RNAseq showed reduced NAA10 mRNA levels and abnormal 3' UTRs in affected individuals. Targeted sequencing of NAA10 in 376 additional affected individuals failed to identify variants in the PAS. CONCLUSION: These data show that PAS variants are the most common variant type in NAA10-associated syndromic microphthalmia, suggesting reduced RNA is the molecular mechanism by which these alterations cause microphthalmia/anophthalmia. We reviewed recognised variants in PAS associated with Mendelian disorders and identified only 23 others, indicating that NAA10 harbours more than 10% of all known PAS variants. We hypothesise that PAS in other genes harbour unrecognised pathogenic variants associated with Mendelian disorders. The systematic interrogation of PAS could improve genetic testing yields.


Assuntos
Regiões 3' não Traduzidas , Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética , Acetiltransferase N-Terminal A/genética , Acetiltransferase N-Terminal E/genética , Poli A , Alelos , Anoftalmia , Feminino , Genes Ligados ao Cromossomo X , Genótipo , Humanos , Escore Lod , Masculino , Microftalmia , Linhagem , Análise de Sequência de DNA , Inativação do Cromossomo X
4.
Int J Mol Sci ; 21(17)2020 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-32858886

RESUMO

Specific-pathogen-free (SPF) mice have improved hematopoietic characteristics relative to germ-free mice, however, it is not clear whether improvements in hematopoietic traits will continue when the level of microorganism exposure is further increased. We co-housed SPF C57BL/6 mice in a conventional facility (CVT) and found a significant increase in gut microbiota diversity along with increased levels of myeloid cells and T cells, especially effector memory T cells. Through single cell RNA sequencing of sorted KL (c-Kit+Lin-) cells, we imputed a decline in long-term hematopoietic stem cells and an increase in granulocyte-monocyte progenitors in CVT mice with up-regulation of genes associated with cell survival. Bone marrow transplantation through competitive repopulation revealed a significant increase in KSL (c-Kit+Sca-1+Lin-) cell reconstitution in recipients of CVT donor cells which occurred when donors were co-housed for both one and twelve months. However, there was minimal to no gain in mature blood cell engraftment in recipients of CVT donor cells relative to those receiving SPF donor cells. We conclude that co-housing SPF mice with mice born in a conventional facility increased gut microbiota diversity, augmented myeloid cell production and T cell activation, stimulated KSL cell reconstitution, and altered hematopoietic gene expression.


Assuntos
Bactérias/classificação , Perfilação da Expressão Gênica/métodos , Hematopoese , Células Mieloides/metabolismo , Análise de Sequência de RNA/métodos , Linfócitos T/metabolismo , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Transplante de Medula Óssea , Microbioma Gastrointestinal , Regulação da Expressão Gênica , Abrigo para Animais , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Filogenia , Análise de Célula Única , Organismos Livres de Patógenos Específicos
5.
Am J Hum Genet ; 95(1): 66-76, 2014 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-24975946

RESUMO

Coronary artery calcification (CAC) is a heritable and definitive morphologic marker of atherosclerosis that strongly predicts risk for future cardiovascular events. To search for genes involved in CAC, we used an integrative transcriptomic, genomic, and protein expression strategy by using next-generation DNA sequencing in the discovery phase with follow-up studies using traditional molecular biology and histopathology techniques. RNA sequencing of peripheral blood from a discovery set of CAC cases and controls was used to identify dysregulated genes, which were validated by ClinSeq and Framingham Heart Study data. Only a single gene, TREML4, was upregulated in CAC cases in both studies. Further examination showed that rs2803496 was a TREML4 cis-eQTL and that the minor allele at this locus conferred up to a 6.5-fold increased relative risk of CAC. We characterized human TREML4 and demonstrated by immunohistochemical techniques that it is localized in macrophages surrounding the necrotic core of coronary plaques complicated by calcification (but not in arteries with less advanced disease). Finally, we determined by von Kossa staining that TREML4 colocalizes with areas of microcalcification within coronary plaques. Overall, we present integrative RNA, DNA, and protein evidence implicating TREML4 in coronary artery calcification. Our findings connect multimodal genomics data with a commonly used clinical marker of cardiovascular disease.


Assuntos
Calcinose , Vasos Coronários/patologia , DNA/metabolismo , Proteínas/metabolismo , RNA/metabolismo , Receptores Imunológicos/fisiologia , Sequência de Bases , Primers do DNA , Células HEK293 , Humanos , Locos de Características Quantitativas , Receptores Imunológicos/genética
6.
Am J Physiol Endocrinol Metab ; 309(6): E534-45, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26173457

RESUMO

Pendrin (Slc26a4) is a Cl(-)/HCO3 (-) exchanger expressed in renal intercalated cells and mediates renal Cl(-) absorption. With pendrin gene ablation, blood pressure and vascular volume fall, which increases plasma renin concentration. However, serum aldosterone does not significantly increase in pendrin-null mice, suggesting that pendrin regulates adrenal zona glomerulosa aldosterone production. Therefore, we examined pendrin expression in the adrenal gland using PCR, immunoblots, and immunohistochemistry. Pendrin protein was detected in adrenal lysates from wild-type but not pendrin-null mice. However, immunohistochemistry and qPCR of microdissected adrenal zones showed that pendrin was expressed in the adrenal medulla, rather than in cortex. Within the adrenal medulla, pendrin localizes to both epinephrine- and norepinephrine-producing chromaffin cells. Therefore, we examined plasma catecholamine concentration and blood pressure in wild-type and pendrin-null mice under basal conditions and then after 5 and 20 min of immobilization stress. Under basal conditions, blood pressure was lower in the mutant than in the wild-type mice, although epinephrine and norepinephrine concentrations were similar. Catecholamine concentration and blood pressure increased markedly in both groups with stress. With 20 min of immobilization stress, epinephrine and norepinephrine concentrations increased more in pendrin-null than in wild-type mice, although stress produced a similar increase in blood pressure in both groups. We conclude that pendrin is expressed in the adrenal medulla, where it blunts stress-induced catecholamine release.


Assuntos
Medula Suprarrenal/metabolismo , Proteínas de Transporte de Ânions/genética , Antiportadores de Cloreto-Bicarbonato/genética , Epinefrina/metabolismo , Norepinefrina/metabolismo , RNA Mensageiro/metabolismo , Estresse Psicológico/metabolismo , Glândulas Suprarrenais/metabolismo , Animais , Proteínas de Transporte de Ânions/metabolismo , Pressão Sanguínea , Antiportadores de Cloreto-Bicarbonato/metabolismo , Perfilação da Expressão Gênica , Immunoblotting , Imuno-Histoquímica , Rim/metabolismo , Camundongos , Camundongos Knockout , Ratos , Restrição Física , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transportadores de Sulfato
7.
Blood ; 121(22): e138-48, 2013 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-23613520

RESUMO

Current methods for detecting mutations in Fanconi anemia (FA)-suspected patients are inefficient and often miss mutations. We have applied recent advances in DNA sequencing and genomic capture to the diagnosis of FA. Specifically, we used custom molecular inversion probes or TruSeq-enrichment oligos to capture and sequence FA and related genes, including introns, from 27 samples from the International Fanconi Anemia Registry at The Rockefeller University. DNA sequencing was complemented with custom array comparative genomic hybridization (aCGH) and RNA sequencing (RNA-seq) analysis. aCGH identified deletions/duplications in 4 different FA genes. RNA-seq analysis revealed lack of allele specific expression associated with a deletion and splicing defects caused by missense, synonymous, and deep-in-intron variants. The combination of TruSeq-targeted capture, aCGH, and RNA-seq enabled us to identify the complementation group and biallelic germline mutations in all 27 families: FANCA (7), FANCB (3), FANCC (3), FANCD1 (1), FANCD2 (3), FANCF (2), FANCG (2), FANCI (1), FANCJ (2), and FANCL (3). FANCC mutations are often the cause of FA in patients of Ashkenazi Jewish (AJ) ancestry, and we identified 2 novel FANCC mutations in 2 patients of AJ ancestry. We describe here a strategy for efficient molecular diagnosis of FA.


Assuntos
Hibridização Genômica Comparativa/métodos , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/genética , Judeus/genética , Análise de Sequência de RNA/métodos , Fatores de Transcrição de Zíper de Leucina Básica/genética , Saúde da Família , Anemia de Fanconi/etnologia , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Proteína do Grupo de Complementação C da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Deleção de Genes , Duplicação Gênica , Humanos , Mutação
8.
BMC Genomics ; 15: 198, 2014 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-24628908

RESUMO

BACKGROUND: Massively-parallel cDNA sequencing (RNA-Seq) is a new technique that holds great promise for cardiovascular genomics. Here, we used RNA-Seq to study the transcriptomes of matched coronary artery disease cases and controls in the ClinSeq® study, using cell lines as tissue surrogates. RESULTS: Lymphoblastoid cell lines (LCLs) from 16 cases and controls representing phenotypic extremes for coronary calcification were cultured and analyzed using RNA-Seq. All cell lines were then independently re-cultured and along with another set of 16 independent cases and controls, were profiled with Affymetrix microarrays to perform a technical validation of the RNA-Seq results. Statistically significant changes (p < 0.05) were detected in 186 transcripts, many of which are expressed at extremely low levels (5-10 copies/cell), which we confirmed through a separate spike-in control RNA-Seq experiment. Next, by fitting a linear model to exon-level RNA-Seq read counts, we detected signals of alternative splicing in 18 transcripts. Finally, we used the RNA-Seq data to identify differential expression (p < 0.0001) in eight previously unannotated regions that may represent novel transcripts. Overall, differentially expressed genes showed strong enrichment (p = 0.0002) for prior association with cardiovascular disease. At the network level, we found evidence for perturbation in pathways involving both cardiovascular system development and function as well as lipid metabolism. CONCLUSIONS: We present a pilot study for transcriptome involvement in coronary artery calcification and demonstrate how RNA-Seq analyses using LCLs as a tissue surrogate may yield fruitful results in a clinical sequencing project. In addition to canonical gene expression, we present candidate variants from alternative splicing and novel transcript detection, which have been unexplored in the context of this disease.


Assuntos
Vasos Coronários/metabolismo , Vasos Coronários/patologia , Perfilação da Expressão Gênica , Transcriptoma , Calcificação Vascular/genética , Processamento Alternativo , Estudos de Casos e Controles , Linhagem Celular , Biologia Computacional/métodos , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Pessoa de Meia-Idade , Anotação de Sequência Molecular , Reprodutibilidade dos Testes
9.
Arterioscler Thromb Vasc Biol ; 33(6): 1418-26, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23539218

RESUMO

OBJECTIVE: To identify transcriptomic biomarkers of coronary heart disease (CHD) in 188 cases with CHD and 188 age- and sex-matched controls who were participants in the Framingham Heart Study. APPROACH AND RESULTS: A total of 35 genes were differentially expressed in cases with CHD versus controls at false discovery rate<0.5, including GZMB, TMEM56, and GUK1. Cluster analysis revealed 3 gene clusters associated with CHD, 2 linked to increased erythrocyte production and a third to reduced natural killer and T cell activity in cases with CHD. Exon-level results corroborated and extended the gene-level results. Alternative splicing analysis suggested that GUK1 and 38 other genes were differentially spliced in cases with CHD versus controls. Gene Ontology analysis linked ubiquitination and T-cell-related pathways with CHD. CONCLUSIONS: Two bioinformatically defined groups of genes show consistent associations with CHD. Our findings are consistent with the hypotheses that hematopoesis is upregulated in CHD, possibly reflecting a compensatory mechanism, and that innate immune activity is disrupted in CHD or altered by its treatment. Transcriptomic signatures may be useful in identifying pathways associated with CHD and point toward novel therapeutic targets for its treatment and prevention.


Assuntos
Doença das Coronárias/epidemiologia , Doença das Coronárias/genética , DNA Recombinante/genética , Predisposição Genética para Doença/epidemiologia , Transcriptoma/genética , Distribuição por Idade , Idoso , Estudos de Casos e Controles , Análise por Conglomerados , Éxons/genética , Feminino , Granzimas/genética , Humanos , Incidência , Masculino , Proteínas de Membrana , Proteínas dos Microfilamentos , Pessoa de Meia-Idade , Neurofibromina 2/genética , Reação em Cadeia da Polimerase em Tempo Real , Valores de Referência , Reprodutibilidade dos Testes , Fatores de Risco , Distribuição por Sexo
10.
Cell Genom ; 4(1): 100466, 2024 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-38190108

RESUMO

The data-intensive fields of genomics and machine learning (ML) are in an early stage of convergence. Genomics researchers increasingly seek to harness the power of ML methods to extract knowledge from their data; conversely, ML scientists recognize that genomics offers a wealth of large, complex, and well-annotated datasets that can be used as a substrate for developing biologically relevant algorithms and applications. The National Human Genome Research Institute (NHGRI) inquired with researchers working in these two fields to identify common challenges and receive recommendations to better support genomic research efforts using ML approaches. Those included increasing the amount and variety of training datasets by integrating genomic with multiomics, context-specific (e.g., by cell type), and social determinants of health datasets; reducing the inherent biases of training datasets; prioritizing transparency and interpretability of ML methods; and developing privacy-preserving technologies for research participants' data.


Assuntos
Bioética , Genômica , Humanos , Algoritmos , Privacidade , Aprendizado de Máquina
11.
Genome Res ; 19(9): 1516-26, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19439515

RESUMO

Structural variants (SVs) are common in the human genome. Because approximately half of the human genome consists of repetitive, transposable DNA sequences, it is plausible that these elements play an important role in generating SVs in humans. Sequencing of the diploid genome of one individual human (HuRef) affords us the opportunity to assess, for the first time, the impact of mobile elements on SVs in an individual in a thorough and unbiased fashion. In this study, we systematically evaluated more than 8000 SVs to identify mobile element-associated SVs as small as 100 bp and specific to the HuRef genome. Combining computational and experimental analyses, we identified and validated 706 mobile element insertion events (including Alu, L1, SVA elements, and nonclassical insertions), which added more than 305 kb of new DNA sequence to the HuRef genome compared with the Human Genome Project (HGP) reference sequence (hg18). We also identified 140 mobile element-associated deletions, which removed approximately 126 kb of sequence from the HuRef genome. Overall, approximately 10% of the HuRef-specific indels larger than 100 bp are caused by mobile element-associated events. More than one-third of the insertion/deletion events occurred in genic regions, and new Alu insertions occurred in exons of three human genes. Based on the number of insertions and the estimated time to the most recent common ancestor of HuRef and the HGP reference genome, we estimated the Alu, L1, and SVA retrotransposition rates to be one in 21 births, 212 births, and 916 births, respectively. This study presents the first comprehensive analysis of mobile element-related structural variants in the complete DNA sequence of an individual and demonstrates that mobile elements play an important role in generating inter-individual structural variation.


Assuntos
Biologia Computacional/métodos , Elementos de DNA Transponíveis/genética , Variação Genética , Genoma Humano , Elementos Alu , Deleção de Genes , Humanos , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA
12.
PLoS Genet ; 5(5): e1000469, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19424419

RESUMO

DNA double-strand breaks (DSBs) are a common form of cellular damage that can lead to cell death if not repaired promptly. Experimental systems have shown that DSB repair in eukaryotic cells is often imperfect and may result in the insertion of extra chromosomal DNA or the duplication of existing DNA at the breakpoint. These events are thought to be a source of genomic instability and human diseases, but it is unclear whether they have contributed significantly to genome evolution. Here we developed an innovative computational pipeline that takes advantage of the repetitive structure of genomes to detect repair-mediated duplication events (RDs) that occurred in the germline and created insertions of at least 50 bp of genomic DNA. Using this pipeline we identified over 1,000 probable RDs in the human genome. Of these, 824 were intra-chromosomal, closely linked duplications of up to 619 bp bearing the hallmarks of the synthesis-dependent strand-annealing repair pathway. This mechanism has duplicated hundreds of sequences predicted to be functional in the human genome, including exons, UTRs, intron splice sites and transcription factor binding sites. Dating of the duplication events using comparative genomics and experimental validation revealed that the mechanism has operated continuously but with decreasing intensity throughout primate evolution. The mechanism has produced species-specific duplications in all primate species surveyed and is contributing to genomic variation among humans. Finally, we show that RDs have also occurred, albeit at a lower frequency, in non-primate mammals and other vertebrates, indicating that this mechanism has been an important force shaping vertebrate genome evolution.


Assuntos
Reparo do DNA/genética , Evolução Molecular , Duplicação Gênica , Vertebrados/genética , Animais , Sequência de Bases , Biometria , DNA/genética , Quebras de DNA de Cadeia Dupla , Técnicas Genéticas , Variação Genética , Genoma Humano , Humanos , Modelos Genéticos , Polimorfismo Genético , Primatas/genética , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Fatores de Tempo
13.
PLoS Genet ; 3(10): 1939-49, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17953488

RESUMO

With more than 1.2 million copies, Alu elements are one of the most important sources of structural variation in primate genomes. Here, we compare the chimpanzee and human genomes to determine the extent of Alu recombination-mediated deletion (ARMD) in the chimpanzee genome since the divergence of the chimpanzee and human lineages ( approximately 6 million y ago). Combining computational data analysis and experimental verification, we have identified 663 chimpanzee lineage-specific deletions (involving a total of approximately 771 kb of genomic sequence) attributable to this process. The ARMD events essentially counteract the genomic expansion caused by chimpanzee-specific Alu inserts. The RefSeq databases indicate that 13 exons in six genes, annotated as either demonstrably or putatively functional in the human genome, and 299 intronic regions have been deleted through ARMDs in the chimpanzee lineage. Therefore, our data suggest that this process may contribute to the genomic and phenotypic diversity between chimpanzees and humans. In addition, we found four independent ARMD events at orthologous loci in the gorilla or orangutan genomes. This suggests that human orthologs of loci at which ARMD events have already occurred in other nonhuman primate genomes may be "at-risk" motifs for future deletions, which may subsequently contribute to human lineage-specific genetic rearrangements and disorders.


Assuntos
Elementos Alu , Genoma , Recombinação Genética , Animais , Linhagem da Célula , Éxons , Deleção de Genes , Variação Genética , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Pan troglodytes , Retroelementos/genética , Software
14.
Genomics ; 93(3): 205-12, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18951971

RESUMO

The Alu family is a highly successful group of non-LTR retrotransposons ubiquitously found in primate genomes. Similar to the L1 retrotransposon family, Alu elements integrate primarily through an endonuclease-dependent mechanism termed target site-primed reverse transcription (TPRT). Recent studies have suggested that, in addition to TPRT, L1 elements occasionally utilize an alternative endonuclease-independent pathway for genomic integration. To determine whether an analogous mechanism exists for Alu elements, we have analyzed three publicly available primate genomes (human, chimpanzee and rhesus macaque) for endonuclease-independent recently integrated or lineage specific Alu insertions. We recovered twenty-three examples of such insertions and show that these insertions are recognizably different from classical TPRT-mediated Alu element integration. We suggest a role for this process in DNA double-strand break repair and present evidence to suggest its association with intra-chromosomal translocations, in-vitro RNA recombination (IVRR), and synthesis-dependent strand annealing (SDSA).


Assuntos
Elementos Alu/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Retroelementos/genética , Animais , Biologia Computacional , Genoma Humano , Humanos , Macaca mulatta , Dados de Sequência Molecular , Pan troglodytes , Homologia de Sequência do Ácido Nucleico
15.
Front Immunol ; 11: 397, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32292401

RESUMO

The Triggering Receptor Expressed on Myeloid cells-like 4 (TREML4) is a member of the TREM receptor family, known modulators of inflammatory responses. We have previously found that TREML4 expression positively correlates with human coronary arterial calcification (CAC). However, the role of TREML4 in the pathogenesis of cardiovascular disease remains incompletely defined. Since macrophages play a key role in inflammatory conditions, we investigated if activated macrophages selectively expressed TREML4 and found that carriage of either one of the eQTL SNP's previously associated with increased TREML4 expression conferred higher expression in human inflammatory macrophages (M1) compared to alternatively activated macrophages (M2). Furthermore, we found that TREML4 expression in human M1 dysregulated several inflammatory pathways related to leukocyte activation, apoptosis and extracellular matrix degradation. Similarly, murine M1 expressed substantial levels of Treml4, as did oxLDL treated macrophages. Transcriptome analysis confirmed that murine Treml4 controls the expression of genes related to inflammation and lipid regulation pathways, suggesting a possible role in atherosclerosis. Analysis of Apoe-/-/Treml4-/- mice showed reduced plaque burden and lesion complexity as indicated by decreased stage scores, macrophage content and collagen deposition. Finally, transcriptome analysis of oxLDL-loaded murine macrophages showed that Treml4 represses a specific set of genes related to carbohydrate, ion and amino acid membrane transport. Metabolomic analysis confirmed that Treml4 deficiency may promote a beneficial relationship between iron homeostasis and glucose metabolism. Together, our results suggest that Treml4 plays a role in the development of cardiovascular disease, as indicated by Treml4-dependent dysregulation of macrophage inflammatory pathways, macrophage metabolism and promotion of vulnerability features in advanced lesions.


Assuntos
Aterosclerose/patologia , Doenças Cardiovasculares/patologia , Macrófagos/metabolismo , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Animais , Apolipoproteínas E/deficiência , Aterosclerose/imunologia , Aterosclerose/metabolismo , Doenças Cardiovasculares/imunologia , Doenças Cardiovasculares/metabolismo , Regulação da Expressão Gênica/imunologia , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Macrófagos/imunologia
16.
Nucleic Acids Res ; 35(11): 3741-51, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17517773

RESUMO

LINE-1 elements (L1s) are a family of highly successful retrotransposons comprising approximately 17% of the human genome, the majority of which have inserted through an endonuclease-dependent mechanism termed target-primed reverse transcription. Recent in vitro analyses suggest that in the absence of non-homologous end joining proteins, L1 elements may utilize an alternative, endonuclease-independent pathway for insertion. However, it remains unknown whether this pathway operates in vivo or in cell lines where all DNA repair mechanisms are functional. Here, we have analyzed the human genome to demonstrate that this alternative pathway for L1 insertion has been active in recent human evolution and characterized 21 loci where L1 elements have integrated without signs of endonuclease-related activity. The structural features of these loci suggest a role for this process in DNA double-strand break repair. We show that endonuclease-independent L1 insertions are structurally distinguishable from classical L1 insertion loci, and that they are associated with inter-chromosomal translocations and deletions of target genomic DNA.


Assuntos
Genoma Humano , Elementos Nucleotídeos Longos e Dispersos , Sequência de Bases , Biologia Computacional , Sequência Consenso , Reparo do DNA , Endodesoxirribonucleases/metabolismo , Genômica , Humanos , Modelos Genéticos , Homologia de Sequência do Ácido Nucleico
17.
Science ; 363(6422)2019 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-30523076

RESUMO

Barrier tissues are primary targets of environmental stressors and are home to the largest number of antigen-experienced lymphocytes in the body, including commensal-specific T cells. We found that skin-resident commensal-specific T cells harbor a paradoxical program characterized by a type 17 program associated with a poised type 2 state. Thus, in the context of injury and exposure to inflammatory mediators such as interleukin-18, these cells rapidly release type 2 cytokines, thereby acquiring contextual functions. Such acquisition of a type 2 effector program promotes tissue repair. Aberrant type 2 responses can also be unleashed in the context of local defects in immunoregulation. Thus, commensal-specific T cells co-opt tissue residency and cell-intrinsic flexibility as a means to promote both local immunity and tissue adaptation to injury.


Assuntos
Plasticidade Celular , Pele/lesões , Pele/microbiologia , Simbiose , Células Th17/imunologia , Células Th17/microbiologia , Ferimentos e Lesões/imunologia , Alarminas/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/microbiologia , Candida albicans , Feminino , Fator de Transcrição GATA3/metabolismo , Interleucinas/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Microscopia de Fluorescência , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Análise de Sequência de RNA , Staphylococcus epidermidis , Transcriptoma
18.
Science ; 365(6452)2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31371577

RESUMO

Laboratory mouse studies are paramount for understanding basic biological phenomena but also have limitations. These include conflicting results caused by divergent microbiota and limited translational research value. To address both shortcomings, we transferred C57BL/6 embryos into wild mice, creating "wildlings." These mice have a natural microbiota and pathogens at all body sites and the tractable genetics of C57BL/6 mice. The bacterial microbiome, mycobiome, and virome of wildlings affect the immune landscape of multiple organs. Their gut microbiota outcompete laboratory microbiota and demonstrate resilience to environmental challenges. Wildlings, but not conventional laboratory mice, phenocopied human immune responses in two preclinical studies. A combined natural microbiota- and pathogen-based model may enhance the reproducibility of biomedical studies and increase the bench-to-bedside safety and success of immunological studies.


Assuntos
Animais Selvagens/microbiologia , Microbioma Gastrointestinal , Interações entre Hospedeiro e Microrganismos/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Pesquisa Translacional Biomédica/normas
19.
Science ; 360(6391)2018 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-29798856

RESUMO

Primary liver tumors and liver metastasis currently represent the leading cause of cancer-related death. Commensal bacteria are important regulators of antitumor immunity, and although the liver is exposed to gut bacteria, their role in antitumor surveillance of liver tumors is poorly understood. We found that altering commensal gut bacteria in mice induced a liver-selective antitumor effect, with an increase of hepatic CXCR6+ natural killer T (NKT) cells and heightened interferon-γ production upon antigen stimulation. In vivo functional studies showed that NKT cells mediated liver-selective tumor inhibition. NKT cell accumulation was regulated by CXCL16 expression of liver sinusoidal endothelial cells, which was controlled by gut microbiome-mediated primary-to-secondary bile acid conversion. Our study suggests a link between gut bacteria-controlled bile acid metabolism and liver antitumor immunosurveillance.


Assuntos
Ácidos e Sais Biliares/metabolismo , Microbioma Gastrointestinal/imunologia , Vigilância Imunológica , Neoplasias Hepáticas/imunologia , Fígado/metabolismo , Células T Matadoras Naturais/imunologia , Animais , Quimiocina CXCL16/metabolismo , Clostridium/metabolismo , Humanos , Fígado/imunologia , Fígado/patologia , Neoplasias Hepáticas/patologia , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Receptores CXCR6/metabolismo
20.
Nucleic Acids Res ; 33(13): 4040-52, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16034026

RESUMO

Long INterspersed Elements (LINE-1s or L1s) are abundant non-LTR retrotransposons in mammalian genomes that are capable of insertional mutagenesis. They have been associated with target site deletions upon insertion in cell culture studies of retrotransposition. Here, we report 50 deletion events in the human and chimpanzee genomes directly linked to the insertion of L1 elements, resulting in the loss of approximately 18 kb of sequence from the human genome and approximately 15 kb from the chimpanzee genome. Our data suggest that during the primate radiation, L1 insertions may have deleted up to 7.5 Mb of target genomic sequences. While the results of our in vivo analysis differ from those of previous cell culture assays of L1 insertion-mediated deletions in terms of the size and rate of sequence deletion, evolutionary factors can reconcile the differences. We report a pattern of genomic deletion sizes similar to those created during the retrotransposition of Alu elements. Our study provides support for the existence of different mechanisms for small and large L1-mediated deletions, and we present a model for the correlation of L1 element size and the corresponding deletion size. In addition, we show that internal rearrangements can modify L1 structure during retrotransposition events associated with large deletions.


Assuntos
Genoma Humano , Elementos Nucleotídeos Longos e Dispersos , Mutagênese Insercional , Pan troglodytes/genética , Deleção de Sequência , Animais , Sequência de Bases , Evolução Molecular , Genômica , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Polimorfismo Genético , Recombinação Genética
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