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1.
PLoS One ; 15(1): e0225289, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31961910

RESUMO

TYK2 belongs to the JAK protein tyrosine kinase family and mediates signaling of numerous antiviral and immunoregulatory cytokines (type I and type III IFNs, IL-10, IL-12, IL-22, IL-23) in immune and non-immune cells. After many years of genetic association studies, TYK2 is recognized as a susceptibility gene for some inflammatory and autoimmune diseases (AID). Seven TYK2 variants have been associated with AIDs in Europeans, and establishing their causality remains challenging. Previous work showed that a protective variant (P1104A) is hypomorphic and also a risk allele for mycobacterial infection. Here, we have studied two AID-associated common TYK2 variants: rs12720270 located in intron 7 and rs2304256, a non-synonymous variant in exon 8 that causes a valine to phenylalanine substitution (c.1084 G > T, Val362Phe). We found that this amino acid substitution does not alter TYK2 expression, catalytic activity or ability to relay signaling in EBV-B cell lines or in reconstituted TYK2-null cells. Based on in silico predictions that these variants may impact splicing of exon 8, we: i) analyzed TYK2 transcripts in genotyped EBV-B cells and in CRISPR/Cas9-edited cells, ii) measured splicing using minigene assays, and iii) performed eQTL (expression quantitative trait locus) analysis of TYK2 transcripts in primary monocytes and whole blood cells. Our results reveal that the two variants promote the inclusion of exon 8, which, we demonstrate, is essential for TYK2 binding to cognate receptors. In addition and in line with GTEx (Genetic Tissue Expression) data, our eQTL results show that rs2304256 mildly enhances TYK2 expression in whole blood. In all, these findings suggest that these TYK2 variants are not neutral but instead have a potential impact in AID.


Assuntos
Doenças Autoimunes/genética , Predisposição Genética para Doença , Inflamação/genética , TYK2 Quinase/genética , Alelos , Substituição de Aminoácidos/genética , Doenças Autoimunes/sangue , Doenças Autoimunes/patologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Citocinas/química , Citocinas/genética , Regulação da Expressão Gênica/genética , Estudos de Associação Genética , Genótipo , Humanos , Inflamação/sangue , Inflamação/patologia , Fenilalanina/genética , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética , TYK2 Quinase/sangue
4.
Nat Commun ; 10(1): 3616, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399586

RESUMO

Cardiac fibrosis is a final common pathology in inherited and acquired heart diseases that causes cardiac electrical and pump failure. Here, we use systems genetics to identify a pro-fibrotic gene network in the diseased heart and show that this network is regulated by the E3 ubiquitin ligase WWP2, specifically by the WWP2-N terminal isoform. Importantly, the WWP2-regulated pro-fibrotic gene network is conserved across different cardiac diseases characterized by fibrosis: human and murine dilated cardiomyopathy and repaired tetralogy of Fallot. Transgenic mice lacking the N-terminal region of the WWP2 protein show improved cardiac function and reduced myocardial fibrosis in response to pressure overload or myocardial infarction. In primary cardiac fibroblasts, WWP2 positively regulates the expression of pro-fibrotic markers and extracellular matrix genes. TGFß1 stimulation promotes nuclear translocation of the WWP2 isoforms containing the N-terminal region and their interaction with SMAD2. WWP2 mediates the TGFß1-induced nucleocytoplasmic shuttling and transcriptional activity of SMAD2.


Assuntos
Fibrose/metabolismo , Redes Reguladoras de Genes , Predisposição Genética para Doença , Proteína Smad2/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adolescente , Adulto , Idoso , Animais , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibrose/genética , Regulação da Expressão Gênica , Predisposição Genética para Doença/genética , Cardiopatias/genética , Cardiopatias/metabolismo , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Isoformas de Proteínas , Proteína Smad2/genética , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases/genética , Adulto Jovem
5.
Am J Hum Genet ; 104(6): 1241-1250, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31155285

RESUMO

Archaic admixture is increasingly recognized as an important source of diversity in modern humans, and Neanderthal haplotypes cover 1%-3% of the genome of present-day Eurasians. Recent work has shown that archaic introgression has contributed to human phenotypic diversity, mostly through the regulation of gene expression. Yet the mechanisms through which archaic variants alter gene expression and the forces driving the introgression landscape at regulatory regions remain elusive. Here, we explored the impact of archaic introgression on transcriptional and post-transcriptional regulation. We focused on promoters and enhancers across 127 different tissues as well as on microRNA (miRNA)-mediated regulation. Although miRNAs themselves harbor few archaic variants, we found that some of these variants may have a strong impact on miRNA-mediated gene regulation. Enhancers were by far the regulatory elements most affected by archaic introgression: up to one-third of the tissues we tested presented significant enrichments. Specifically, we found strong enrichments of archaic variants in adipose-related tissues and primary T cells, even after accounting for various genomic and evolutionary confounders such as recombination rate and background selection. Interestingly, we identified signatures of adaptive introgression at enhancers of some key regulators of adipogenesis, raising the interesting hypothesis of a possible adaptation of early Eurasians to colder climates. Collectively, this study sheds new light on the mechanisms through which archaic admixture has impacted gene regulation in Eurasians and, more generally, increases our understanding of the contribution of Neanderthals to the regulation of acquired immunity and adipose homeostasis in modern humans.

6.
HLA ; 94(3): 275-284, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31115186

RESUMO

Over the last 10 years, genome-wide association studies (GWAS) have identified hundreds of susceptibility loci for autoimmune diseases. However, despite increasing power for the detection of both common and rare coding variants affecting disease susceptibility, a large fraction of disease heritability has remained unexplained. In addition, a majority of the identified loci are located in noncoding regions, and translation of disease-associated loci into new biological insights on the etiology of immune disorders has been lagging. This highlights the need for a better understanding of noncoding variation and new strategies to identify causal genes at disease loci. In this review, I will first detail the molecular basis of gene expression and review the various mechanisms that contribute to alter gene activity at the transcriptional and post-transcriptional level. I will then review the findings from 10 years of functional genomics studies regarding the genetics on gene expression, in particular in the context of infection. Finally, I will discuss the extent to which genetic variants that modulate gene expression at transcriptional and post-transcriptional level contribute to disease susceptibility and present strategies to leverage this information for the identification of causal mechanisms at disease loci in the era of whole genome sequencing.

7.
Nat Commun ; 10(1): 1671, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30975994

RESUMO

Host and environmental factors contribute to variation in human immune responses, yet the genetic and evolutionary drivers of alternative splicing in response to infection remain largely uncharacterised. Leveraging 970 RNA-sequencing profiles of resting and stimulated monocytes from 200 individuals of African- and European-descent, we show that immune activation elicits a marked remodelling of the isoform repertoire, while increasing the levels of erroneous splicing. We identify 1,464 loci associated with variation in isoform usage (sQTLs), 9% of them being stimulation-specific, which are enriched in disease-related loci. Furthermore, we detect a longstanding increased plasticity of immune gene splicing, and show that positive selection and Neanderthal introgression have both contributed to diversify the splicing landscape of human populations. Together, these findings suggest that differential isoform usage has been an important substrate of innovation in the long-term evolution of immune responses and a more recent vehicle of population local adaptation.


Assuntos
Processamento Alternativo/imunologia , Imunidade/genética , Seleção Genética/imunologia , Transcriptoma/imunologia , Grupo com Ancestrais do Continente Africano/genética , Animais , Evolução Biológica , Grupo com Ancestrais do Continente Europeu/genética , Variação Genética/imunologia , Voluntários Saudáveis , Humanos , Masculino , Homem de Neandertal/genética , Polimorfismo de Nucleotídeo Único , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Locos de Características Quantitativas/imunologia , Análise de Sequência de RNA , Sequenciamento Completo do Exoma
8.
J Clin Endocrinol Metab ; 104(2): 465-486, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30137523

RESUMO

Context: Insulin resistance (IR) and obesity differ among ethnic groups in Singapore, with the Malays more obese yet less IR than Asian-Indians. However, the molecular basis underlying these differences is not clear. Objective: As the skeletal muscle (SM) is metabolically relevant to IR, we investigated molecular pathways in SM that are associated with ethnic differences in IR, obesity, and related traits. Design, Setting, and Main Outcome Measures: We integrated transcriptomic, genomic, and phenotypic analyses in 156 healthy subjects representing three major ethnicities in the Singapore Adult Metabolism Study. Patients: This study contains Chinese (n = 63), Malay (n = 51), and Asian-Indian (n = 42) men, aged 21 to 40 years, without systemic diseases. Results: We found remarkable diversity in the SM transcriptome among the three ethnicities, with >8000 differentially expressed genes (40% of all genes expressed in SM). Comparison with blood transcriptome from a separate Singaporean cohort showed that >95% of SM expression differences among ethnicities were unique to SM. We identified a network of 46 genes that were specifically downregulated in Malays, suggesting dysregulation of components of cellular respiration in SM of Malay individuals. We also report 28 differentially expressed gene clusters, four of which were also enriched for genes that were found in genome-wide association studies of metabolic traits and disease and correlated with variation in IR, obesity, and related traits. Conclusion: We identified extensive gene-expression changes in SM among the three Singaporean ethnicities and report specific genes and molecular pathways that might underpin and explain the differences in IR among these ethnic groups.


Assuntos
Grupos Étnicos/genética , Resistência à Insulina/genética , Músculo Esquelético/metabolismo , Transcriptoma , Adulto , Índice de Massa Corporal , Estudos de Coortes , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Resistência à Insulina/etnologia , Masculino , Transdução de Sinais/genética , Singapura , Adulto Jovem
9.
Genome Biol ; 19(1): 222, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30563547

RESUMO

BACKGROUND: DNA methylation is influenced by both environmental and genetic factors and is increasingly thought to affect variation in complex traits and diseases. Yet, the extent of ancestry-related differences in DNA methylation, their genetic determinants, and their respective causal impact on immune gene regulation remain elusive. RESULTS: We report extensive population differences in DNA methylation between 156 individuals of African and European descent, detected in primary monocytes that are used as a model of a major innate immunity cell type. Most of these differences (~ 70%) are driven by DNA sequence variants nearby CpG sites, which account for ~ 60% of the variance in DNA methylation. We also identify several master regulators of DNA methylation variation in trans, including a regulatory hub nearby the transcription factor-encoding CTCF gene, which contributes markedly to ancestry-related differences in DNA methylation. Furthermore, we establish that variation in DNA methylation is associated with varying gene expression levels following mostly, but not exclusively, a canonical model of negative associations, particularly in enhancer regions. Specifically, we find that DNA methylation highly correlates with transcriptional activity of 811 and 230 genes, at the basal state and upon immune stimulation, respectively. Finally, using a Bayesian approach, we estimate causal mediation effects of DNA methylation on gene expression in ~ 20% of the studied cases, indicating that DNA methylation can play an active role in immune gene regulation. CONCLUSION: Using a system-level approach, our study reveals substantial ancestry-related differences in DNA methylation and provides evidence for their causal impact on immune gene regulation.


Assuntos
Grupo com Ancestrais do Continente Africano/genética , Metilação de DNA , Grupo com Ancestrais do Continente Europeu/genética , Regulação da Expressão Gênica , Imunidade Inata , Adulto , Epigênese Genética , Humanos , Masculino , Monócitos , Locos de Características Quantitativas
10.
Genetics ; 206(2): 1139-1151, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28450461

RESUMO

Crescentic glomerulonephritis (Crgn) is a complex disorder where macrophage activity and infiltration are significant effector causes. In previous linkage studies using the uniquely susceptible Wistar Kyoto (WKY) rat strain, we have identified multiple crescentic glomerulonephritis QTL (Crgn) and positionally cloned genes underlying Crgn1 and Crgn2, which accounted for 40% of total variance in glomerular inflammation. Here, we have generated a backcross (BC) population (n = 166) where Crgn1 and Crgn2 were genetically fixed and found significant linkage to glomerular crescents on chromosome 2 (Crgn8, LOD = 3.8). Fine mapping analysis by integration with genome-wide expression QTLs (eQTLs) from the same BC population identified ceruloplasmin (Cp) as a positional eQTL in macrophages but not in serum. Liquid chromatography-tandem mass spectrometry confirmed Cp as a protein QTL in rat macrophages. WKY macrophages overexpress Cp and its downregulation by RNA interference decreases markers of glomerular proinflammatory macrophage activation. Similarly, short incubation with Cp results in a strain-dependent macrophage polarization in the rat. These results suggest that genetically determined Cp levels can alter susceptibility to Crgn through macrophage function and propose a new role for Cp in early macrophage activation.


Assuntos
Ceruloplasmina/genética , Predisposição Genética para Doença , Glomerulonefrite/genética , Animais , Ceruloplasmina/biossíntese , Mapeamento Cromossômico , Regulação da Expressão Gênica , Ligação Genética , Glomerulonefrite/patologia , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , Ratos , Ratos Endogâmicos WKY
11.
Genome Res ; 27(3): 440-450, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28250018

RESUMO

The recoding of genetic information through RNA editing contributes to proteomic diversity, but the extent and significance of RNA editing in disease is poorly understood. In particular, few studies have investigated the relationship between RNA editing and disease at a genome-wide level. Here, we developed a framework for the genome-wide detection of RNA sites that are differentially edited in disease. Using RNA-sequencing data from 100 hippocampi from mice with epilepsy (pilocarpine-temporal lobe epilepsy model) and 100 healthy control hippocampi, we identified 256 RNA sites (overlapping with 87 genes) that were significantly differentially edited between epileptic cases and controls. The degree of differential RNA editing in epileptic mice correlated with frequency of seizures, and the set of genes differentially RNA-edited between case and control mice were enriched for functional terms highly relevant to epilepsy, including "neuron projection" and "seizures." Genes with differential RNA editing were preferentially enriched for genes with a genetic association to epilepsy. Indeed, we found that they are significantly enriched for genes that harbor nonsynonymous de novo mutations in patients with epileptic encephalopathy and for common susceptibility variants associated with generalized epilepsy. These analyses reveal a functional convergence between genes that are differentially RNA-edited in acquired symptomatic epilepsy and those that contribute risk for genetic epilepsy. Taken together, our results suggest a potential role for RNA editing in the epileptic hippocampus in the occurrence and severity of epileptic seizures.


Assuntos
Epilepsia/genética , Edição de RNA , Animais , Estudo de Associação Genômica Ampla , Hipocampo/metabolismo , Masculino , Camundongos , Transcriptoma
12.
Cell ; 167(3): 643-656.e17, 2016 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-27768888

RESUMO

Humans differ in the outcome that follows exposure to life-threatening pathogens, yet the extent of population differences in immune responses and their genetic and evolutionary determinants remain undefined. Here, we characterized, using RNA sequencing, the transcriptional response of primary monocytes from Africans and Europeans to bacterial and viral stimuli-ligands activating Toll-like receptor pathways (TLR1/2, TLR4, and TLR7/8) and influenza virus-and mapped expression quantitative trait loci (eQTLs). We identify numerous cis-eQTLs that contribute to the marked differences in immune responses detected within and between populations and a strong trans-eQTL hotspot at TLR1 that decreases expression of pro-inflammatory genes in Europeans only. We find that immune-responsive regulatory variants are enriched in population-specific signals of natural selection and show that admixture with Neandertals introduced regulatory variants into European genomes, affecting preferentially responses to viral challenges. Together, our study uncovers evolutionarily important determinants of differences in host immune responsiveness between human populations.


Assuntos
Adaptação Fisiológica/genética , Adaptação Fisiológica/imunologia , Imunidade Adaptativa , Homem de Neandertal/genética , Homem de Neandertal/imunologia , Imunidade Adaptativa/genética , Grupo com Ancestrais do Continente Africano/genética , Alelos , Animais , Infecções Bacterianas/genética , Infecções Bacterianas/imunologia , Sequência de Bases , Evolução Biológica , Grupo com Ancestrais do Continente Europeu/genética , Regulação da Expressão Gênica , Variação Genética , Humanos , Sistema Imunitário , Locos de Características Quantitativas , RNA/genética , Seleção Genética , Análise de Sequência de RNA , Receptores Toll-Like/genética , Transcrição Genética , Viroses/genética , Viroses/imunologia
13.
Nat Commun ; 7: 12061, 2016 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-27389904

RESUMO

Coronary flow (CF) measured ex vivo is largely determined by capillary density that reflects angiogenic vessel formation in the heart in vivo. Here we exploit this relationship and show that CF in the rat is influenced by a locus on rat chromosome 2 that is also associated with cardiac capillary density. Mitochondrial tryptophanyl-tRNA synthetase (Wars2), encoding an L53F protein variant within the ATP-binding motif, is prioritized as the candidate at the locus by integrating genomic data sets. WARS2(L53F) has low enzyme activity and inhibition of WARS2 in endothelial cells reduces angiogenesis. In the zebrafish, inhibition of wars2 results in trunk vessel deficiencies, disordered endocardial-myocardial contact and impaired heart function. Inhibition of Wars2 in the rat causes cardiac angiogenesis defects and diminished cardiac capillary density. Our data demonstrate a pro-angiogenic function for Wars2 both within and outside the heart that may have translational relevance given the association of WARS2 with common human diseases.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Genoma , Células Endoteliais da Veia Umbilical Humana/enzimologia , Mitocôndrias/genética , Neovascularização Fisiológica/genética , Triptofano-tRNA Ligase/genética , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Cromossomos de Mamíferos/química , Embrião não Mamífero , Loci Gênicos , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Mitocôndrias/metabolismo , Miocárdio/citologia , Miocárdio/enzimologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Triptofano-tRNA Ligase/antagonistas & inibidores , Triptofano-tRNA Ligase/metabolismo , Peixe-Zebra
14.
Nat Neurosci ; 19(2): 223-32, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26691832

RESUMO

Genetic determinants of cognition are poorly characterized, and their relationship to genes that confer risk for neurodevelopmental disease is unclear. Here we performed a systems-level analysis of genome-wide gene expression data to infer gene-regulatory networks conserved across species and brain regions. Two of these networks, M1 and M3, showed replicable enrichment for common genetic variants underlying healthy human cognitive abilities, including memory. Using exome sequence data from 6,871 trios, we found that M3 genes were also enriched for mutations ascertained from patients with neurodevelopmental disease generally, and intellectual disability and epileptic encephalopathy in particular. M3 consists of 150 genes whose expression is tightly developmentally regulated, but which are collectively poorly annotated for known functional pathways. These results illustrate how systems-level analyses can reveal previously unappreciated relationships between neurodevelopmental disease-associated genes in the developed human brain, and provide empirical support for a convergent gene-regulatory network influencing cognition and neurodevelopmental disease.


Assuntos
Cognição , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/fisiopatologia , Redes Reguladoras de Genes/genética , Sistema Nervoso/crescimento & desenvolvimento , Animais , Química Encefálica/genética , Deficiências do Desenvolvimento/psicologia , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/cirurgia , Expressão Gênica , Variação Genética , Estudo de Associação Genômica Ampla , Hipocampo/cirurgia , Humanos , Sistema Nervoso/fisiopatologia , Sinapses/genética
15.
Nat Commun ; 6: 10047, 2015 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-26616214

RESUMO

The genetic history of African populations is increasingly well documented, yet their patterns of epigenomic variation remain uncharacterized. Moreover, the relative impacts of DNA sequence variation and temporal changes in lifestyle and habitat on the human epigenome remain unknown. Here we generate genome-wide genotype and DNA methylation profiles for 362 rainforest hunter-gatherers and sedentary farmers. We find that the current habitat and historical lifestyle of a population have similarly critical impacts on the methylome, but the biological functions affected strongly differ. Specifically, methylation variation associated with recent changes in habitat mostly concerns immune and cellular functions, whereas that associated with historical lifestyle affects developmental processes. Furthermore, methylation variation--particularly that correlated with historical lifestyle--shows strong associations with nearby genetic variants that, moreover, are enriched in signals of natural selection. Our work provides new insight into the genetic and environmental factors affecting the epigenomic landscape of human populations over time.


Assuntos
Grupo com Ancestrais do Continente Africano/genética , Genética Populacional , Metilação de DNA , Ecossistema , Epigenômica , Fazendeiros , Feminino , Variação Genética , Humanos , Masculino , Floresta Úmida
16.
Nat Commun ; 6: 6031, 2015 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-25615886

RESUMO

Gene-regulatory network analysis is a powerful approach to elucidate the molecular processes and pathways underlying complex disease. Here we employ systems genetics approaches to characterize the genetic regulation of pathophysiological pathways in human temporal lobe epilepsy (TLE). Using surgically acquired hippocampi from 129 TLE patients, we identify a gene-regulatory network genetically associated with epilepsy that contains a specialized, highly expressed transcriptional module encoding proconvulsive cytokines and Toll-like receptor signalling genes. RNA sequencing analysis in a mouse model of TLE using 100 epileptic and 100 control hippocampi shows the proconvulsive module is preserved across-species, specific to the epileptic hippocampus and upregulated in chronic epilepsy. In the TLE patients, we map the trans-acting genetic control of this proconvulsive module to Sestrin 3 (SESN3), and demonstrate that SESN3 positively regulates the module in macrophages, microglia and neurons. Morpholino-mediated Sesn3 knockdown in zebrafish confirms the regulation of the transcriptional module, and attenuates chemically induced behavioural seizures in vivo.


Assuntos
Epilepsia do Lobo Temporal/genética , Redes Reguladoras de Genes , Proteínas de Choque Térmico/genética , Hipocampo/patologia , Convulsões/genética , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Epilepsia do Lobo Temporal/fisiopatologia , Feminino , Proteínas de Choque Térmico/metabolismo , Hipocampo/fisiopatologia , Humanos , Lactente , Inflamação/genética , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Microglia/metabolismo , Microglia/patologia , Pessoa de Meia-Idade , Atividade Motora , Neurônios/metabolismo , Neurônios/patologia , Pentilenotetrazol , Convulsões/fisiopatologia , Adulto Jovem , Peixe-Zebra
17.
Nucleic Acids Res ; 43(3): 1418-32, 2015 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-25605797

RESUMO

The large number of chemical modifications that are found on the histone proteins of eukaryotic cells form multiple complex combinations, which can act as recognition signals for reader proteins. We have used peptide capture in conjunction with super-SILAC quantification to carry out an unbiased high-throughput analysis of the composition of protein complexes that bind to histone H3K9/S10 and H3K27/S28 methyl-phospho modifications. The accurate quantification allowed us to perform Weighted correlation network analysis (WGCNA) to obtain a systems-level view of the histone H3 histone tail interactome. The analysis reveals the underlying modularity of the histone reader network with members of nuclear complexes exhibiting very similar binding signatures, which suggests that many proteins bind to histones as part of pre-organized complexes. Our results identify a novel complex that binds to the double H3K9me3/S10ph modification, which includes Atrx, Daxx and members of the FACT complex. The super-SILAC approach allows comparison of binding to multiple peptides with different combinations of modifications and the resolution of the WGCNA analysis is enhanced by maximizing the number of combinations that are compared. This makes it a useful approach for assessing the effects of changes in histone modification combinations on the composition and function of bound complexes.


Assuntos
Histonas/metabolismo , Proteínas/metabolismo , Animais , Linhagem Celular Tumoral , Técnicas de Química Combinatória , Espectrometria de Massas , Camundongos
18.
Mol Cell Proteomics ; 14(3): 484-98, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25532521

RESUMO

Macrophage multinucleation (MM) is essential for various biological processes such as osteoclast-mediated bone resorption and multinucleated giant cell-associated inflammatory reactions. Here we study the molecular pathways underlying multinucleation in the rat through an integrative approach combining MS-based quantitative phosphoproteomics (LC-MS/MS) and transcriptome (high-throughput RNA-sequencing) to identify new regulators of MM. We show that a strong metabolic shift toward HIF1-mediated glycolysis occurs at transcriptomic level during MM, together with modifications in phosphorylation of over 50 proteins including several ARF GTPase activators and polyphosphate inositol phosphatases. We use shortest-path analysis to link differential phosphorylation with the transcriptomic reprogramming of macrophages and identify LRRFIP1, SMARCA4, and DNMT1 as novel regulators of MM. We experimentally validate these predictions by showing that knock-down of these latter reduce macrophage multinucleation. These results provide a new framework for the combined analysis of transcriptional and post-translational changes during macrophage multinucleation, prioritizing essential genes, and revealing the sequential events leading to the multinucleation of macrophages.


Assuntos
Núcleo Celular/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Helicases/metabolismo , Perfilação da Expressão Gênica/métodos , Macrófagos/metabolismo , Proteínas Nucleares/metabolismo , Proteoma/análise , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA Helicases/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteínas Nucleares/genética , Fosforilação , Proteínas de Ligação a RNA/genética , Ratos , Ratos Endogâmicos Lew , Ratos Endogâmicos WKY , Análise de Sequência de RNA/métodos , Fatores de Transcrição/genética
19.
PLoS Genet ; 10(12): e1004813, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25474312

RESUMO

Epigenetic marks such as cytosine methylation are important determinants of cellular and whole-body phenotypes. However, the extent of, and reasons for inter-individual differences in cytosine methylation, and their association with phenotypic variation are poorly characterised. Here we present the first genome-wide study of cytosine methylation at single-nucleotide resolution in an animal model of human disease. We used whole-genome bisulfite sequencing in the spontaneously hypertensive rat (SHR), a model of cardiovascular disease, and the Brown Norway (BN) control strain, to define the genetic architecture of cytosine methylation in the mammalian heart and to test for association between methylation and pathophysiological phenotypes. Analysis of 10.6 million CpG dinucleotides identified 77,088 CpGs that were differentially methylated between the strains. In F1 hybrids we found 38,152 CpGs showing allele-specific methylation and 145 regions with parent-of-origin effects on methylation. Cis-linkage explained almost 60% of inter-strain variation in methylation at a subset of loci tested for linkage in a panel of recombinant inbred (RI) strains. Methylation analysis in isolated cardiomyocytes showed that in the majority of cases methylation differences in cardiomyocytes and non-cardiomyocytes were strain-dependent, confirming a strong genetic component for cytosine methylation. We observed preferential nucleotide usage associated with increased and decreased methylation that is remarkably conserved across species, suggesting a common mechanism for germline control of inter-individual variation in CpG methylation. In the RI strain panel, we found significant correlation of CpG methylation and levels of serum chromogranin B (CgB), a proposed biomarker of heart failure, which is evidence for a link between germline DNA sequence variation, CpG methylation differences and pathophysiological phenotypes in the SHR strain. Together, these results will stimulate further investigation of the molecular basis of locally regulated variation in CpG methylation and provide a starting point for understanding the relationship between the genetic control of CpG methylation and disease phenotypes.


Assuntos
Doenças Cardiovasculares/genética , Metilação de DNA , Genoma , Miocárdio/metabolismo , Animais , Sequência de Bases , Doenças Cardiovasculares/patologia , Células Cultivadas , Modelos Animais de Doenças , Humanos , Masculino , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Polimorfismo de Nucleotídeo Único , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos SHR , Análise de Sequência de DNA/métodos
20.
Genetics ; 198(3): 1277-90, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25189874

RESUMO

Interindividual differences in many behaviors are partly due to genetic differences, but the identification of the genes and variants that influence behavior remains challenging. Here, we studied an F2 intercross of two outbred lines of rats selected for tame and aggressive behavior toward humans for >64 generations. By using a mapping approach that is able to identify genetic loci segregating within the lines, we identified four times more loci influencing tameness and aggression than by an approach that assumes fixation of causative alleles, suggesting that many causative loci were not driven to fixation by the selection. We used RNA sequencing in 150 F2 animals to identify hundreds of loci that influence brain gene expression. Several of these loci colocalize with tameness loci and may reflect the same genetic variants. Through analyses of correlations between allele effects on behavior and gene expression, differential expression between the tame and aggressive rat selection lines, and correlations between gene expression and tameness in F2 animals, we identify the genes Gltscr2, Lgi4, Zfp40, and Slc17a7 as candidate contributors to the strikingly different behavior of the tame and aggressive animals.


Assuntos
Agressão , Comportamento Animal , Encéfalo/metabolismo , Regulação da Expressão Gênica , Alelos , Animais , Cruzamentos Genéticos , Feminino , Perfilação da Expressão Gênica , Genoma , Humanos , Masculino , Locos de Características Quantitativas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Análise de Sequência de RNA
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