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1.
BMC Genomics ; 22(1): 726, 2021 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-34620074

RESUMO

BACKGROUND: The golden lion tamarin (Leontopithecus rosalia) is an endangered Platyrrhine primate endemic to the Atlantic coastal forests of Brazil. Despite ongoing conservation efforts, genetic data on this species remains scarce. Complicating factors include limitations on sample collection and a lack of high-quality reference sequences. Here, we used nanopore adaptive sampling to resequence the L. rosalia mitogenome from feces, a sample which can be collected non-invasively. RESULTS: Adaptive sampling doubled the fraction of both host-derived and mitochondrial sequences compared to sequencing without enrichment. 258x coverage of the L. rosalia mitogenome was achieved in a single flow cell by targeting the unfinished genome of the distantly related emperor tamarin (Saguinus imperator) and the mitogenome of the closely related black lion tamarin (Leontopithecus chrysopygus). The L. rosalia mitogenome has a length of 16,597 bp, sharing 99.68% sequence identity with the L. chrysopygus mitogenome. A total of 38 SNPs between them were identified, with the majority being found in the non-coding D-loop region. DNA methylation and hydroxymethylation were directly detected using a neural network model applied to the raw signal from the MinION sequencer. In contrast to prior reports, DNA methylation was negligible in mitochondria in both CpG and non-CpG contexts. Surprisingly, a quarter of the 642 CpG sites exhibited DNA hydroxymethylation greater than 1% and 44 sites were above 5%, with concentration in the 3' side of several coding regions. CONCLUSIONS: Overall, we report a robust new mitogenome assembly for L. rosalia and direct detection of cytosine base modifications in all contexts.


Assuntos
Genoma Mitocondrial , Leontopithecus , Nanoporos , Animais , DNA , Epigenoma , Fezes
3.
FASEB J ; 35(9): e21864, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34423880

RESUMO

Resistance training (RT) dynamically alters the skeletal muscle nuclear DNA methylome. However, no study has examined if RT affects the mitochondrial DNA (mtDNA) methylome. Herein, ten older, Caucasian untrained males (65 ± 7 y.o.) performed six weeks of full-body RT (twice weekly). Body composition and knee extensor torque were assessed prior to and 72 h following the last RT session. Vastus lateralis (VL) biopsies were also obtained. VL DNA was subjected to reduced representation bisulfite sequencing providing excellent coverage across the ~16-kilobase mtDNA methylome (254 CpG sites). Biochemical assays were also performed, and older male data were compared to younger trained males (22 ± 2 y.o., n = 7, n = 6 Caucasian & n = 1 African American). RT increased whole-body lean tissue mass (p = .017), VL thickness (p = .012), and knee extensor torque (p = .029) in older males. RT also affected the mtDNA methylome, as 63% (159/254) of the CpG sites demonstrated reduced methylation (p < .05). Several mtDNA sites presented a more "youthful" signature in older males after RT in comparison to younger males. The 1.12 kilobase mtDNA D-loop/control region, which regulates replication and transcription, possessed enriched hypomethylation in older males following RT. Enhanced expression of mitochondrial H- and L-strand genes and complex III/IV protein levels were also observed (p < .05). While limited to a shorter-term intervention, this is the first evidence showing that RT alters the mtDNA methylome in skeletal muscle. Observed methylome alterations may enhance mitochondrial transcription, and RT evokes mitochondrial methylome profiles to mimic younger men. The significance of these findings relative to broader RT-induced epigenetic changes needs to be elucidated.


Assuntos
Envelhecimento , Metilação de DNA , DNA Mitocondrial/metabolismo , Epigenoma , Regulação da Expressão Gênica , Genes Mitocondriais/genética , Músculo Esquelético/metabolismo , Treinamento de Força , Idoso , Envelhecimento/genética , Envelhecimento/metabolismo , DNA Mitocondrial/genética , Humanos , Masculino , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/citologia , RNA Mensageiro/análise , RNA Mensageiro/genética , Adulto Jovem
4.
Ecotoxicol Environ Saf ; 223: 112612, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34371455

RESUMO

Excessive fluoride exposure and epigenetic change can induce numerous adverse health outcomes, but the role of epigenetics underneath the harmful health effects induced by fluoride exposure is unclear. In such gap, we evaluated the associations between fluoride exposure and genome-wide DNA methylation, and identified that novel candidate genes associated with fluoride exposure. A total of 931 school-age children (8-12 years) in Tongxu County of Henan Province (China) were recruited in 2017. Urinary fluoride (UF) concentrations were measured using the national standardized ion selective electrode method. Participants were divided into a high fluoride-exposure group (HFG) and control group (CG) according to the UF concentrations. Candidate differentially methylated regions (DMRs) were screened by Infinium-Methylation EPIC BeadChip of DNA samples collected from 16 participants (eight each from each group). Differentially methylated genes (DMGs) containing DMRs associated with skeletal and neuronal development influenced by fluoride exposure were confirmed using MethylTarget™ technology from 100 participants (fifty each from each group). DMGs were verified by quantitative methylation specific PCR from 815 participants. Serum levels of hormones were measured by auto biochemical analyzer. The mediation analysis of methylation in the effect of fluoride exposure on hormone levels was also performed. A total of 237 differentially methylated sites (DMSs) and 212 DMRs were found in different fluoride-exposure groups in the epigenome-wide phase. Methylation of the target sequences of neuronatin (NNAT), calcitonin-related polypeptide alpha (CALCA) and methylenetetrahydrofolate dehydrogenase 1 showed significant difference between the HFG and CG. Each 0.06% (95% CI: -0.11%, -0.01%) decreased in NNAT methylation status correlated with each increase of 1.0 mg/L in UF concentration in 815 school-age children using QMSP. Also, each 1.88% (95% CI: 0.04%, 3.72%) increase in CALCA methylation status correlated with each increase of 1.0 mg/L in UF concentration. The mediating effect of NNAT methylation was found in alterations of ACTH levels influenced by fluoride exposure, with a ß value of 11.7% (95% CI: 3.4%, 33.4%). In conclusion, long-term fluoride exposure affected the methylation pattern of genomic DNA. NNAT and CALCA as DMGs might be susceptible to fluoride exposure in school-age children.


Assuntos
Metilação de DNA , Fluoretos , Criança , Epigênese Genética , Epigenoma , Fluoretos/toxicidade , Humanos , Instituições Acadêmicas
5.
Genes (Basel) ; 12(8)2021 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-34440372

RESUMO

DNA methylation (DNAm) patterns over time at 1146 CpGs on coronavirus-related genes were assessed to understand whether the varying differences in susceptibility, symptoms, and the outcomes of the SARS-CoV-2 infection in children and young adults could be explained through epigenetic alterations in a host cell's transcriptional apparatus to coronaviruses. DNAm data from the Isle of Wight birth cohort (IOWBC) at birth, 10, 18, and 26 years of age were included. Linear mixed models with repeated measurements stratified by sex were used to examine temporal patterns, and cluster analysis was performed to identify CpGs following similar patterns. CpGs on autosomes and sex chromosomes were analyzed separately. The association of identified CpGs and expression of their genes were evaluated. Pathway enrichment analyses of the genes was conducted at FDR = 0.05. DNAm at 635 of the 1146 CpGs on autosomes showed statistically significant time effects (FDR = 0.05). The 635 CpGs were classified into five clusters with each representing a unique temporal pattern of DNAm. Of the 29 CpGs on sex chromosomes, DNAm at seven CpGs in males and eight CpGs in females showed time effects (FDR = 0.05). Sex-specific and non-specific associations of DNAm with gene expression were found at 24 and 93 CpGs, respectively. Genes which mapped the 643 CpGs represent 460 biological processes. We suggest that the observed variability in DNAm with advancing age may partially explain differing susceptibility, disease severity, and mortality of coronavirus infections among different age groups.


Assuntos
COVID-19/genética , Metilação de DNA , Adolescente , Adulto , Criança , Ilhas de CpG , Epigenoma , Feminino , Humanos , Masculino
6.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445317

RESUMO

The 22q11.2 deletion syndrome (22q11.2DS) is the most common genomic disorder in humans and is the result of a recurrent 1.5 to 2.5 Mb deletion, encompassing approximately 20-40 genes, respectively. The clinical presentation of the typical deletion includes: Velocardiofacial, Di George, Opitz G/BBB and Conotruncalanomaly face syndromes. Atypical deletions (proximal, distal or nested) are rare and characterized mainly by normal phenotype or mild intellectual disability and variable clinical features. The pathogenetic mechanisms underlying this disorder are not completely understood. Because the 22q11.2 region harbours genes coding for transcriptional factors and chromatin remodelers, in this study, we performed analysis of genome-wide DNA methylation of peripheral blood from 49 patients with 22q11.2DS using the Illumina Infinium Methylation EPIC bead chip arrays. This cohort comprises 43 typical, 2 proximal and 4 distal deletions. We demonstrated the evidence of a unique and highly specific episignature in all typical and proximal 22q11.2DS. The sensitivity and specificity of this signature was further confirmed by comparing it to over 1500 patients with other neurodevelopmental disorders with known episignatures. Mapping the 22q11.2DS DNA methylation episignature provides both novel insights into the molecular pathogenesis of this disorder and an effective tool in the molecular diagnosis of 22q11.2DS.


Assuntos
Metilação de DNA , Síndrome de DiGeorge/genética , Epigenoma , Feminino , Humanos , Lactente , Masculino
7.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445323

RESUMO

In plants, the gene expression and associated phenotypes can be modulated by dynamic changes in DNA methylation, occasionally being fixed in certain genomic loci and inherited stably as epialleles. Epiallelic variations in a population can occur as methylation changes at an individual cytosine position, methylation changes within a stretch of genomic regions, and chromatin changes in certain loci. Here, we focus on methylated regions, since it is unclear whether variations at individual methylated cytosines can serve any regulatory function, and the evidence for heritable chromatin changes independent of genetic changes is limited. While DNA methylation is known to affect and regulate wide arrays of plant phenotypes, most epialleles in the form of methylated regions have not been assigned any biological function. Here, we review how epialleles can be established in plants, serve a regulatory function, and are involved in adaptive processes. Recent studies suggest that most epialleles occur as byproducts of genetic variations, mainly from structural variants and Transposable Element (TE) activation. Nevertheless, epialleles that occur spontaneously independent of any genetic variations have also been described across different plant species. Here, we discuss how epialleles that are dependent and independent of genetic architecture are stabilized in the plant genome and how methylation can regulate a transcription relative to its genomic location.


Assuntos
Epigenoma , Variação Genética , Plantas/genética , Metilação de DNA , Elementos de DNA Transponíveis , Regulação da Expressão Gênica de Plantas
8.
Nat Commun ; 12(1): 4706, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34349112

RESUMO

During mammalian pregnancy, immune cells are vertically transferred from mother to fetus. The functional role of these maternal microchimeric cells (MMc) in the offspring is mostly unknown. Here we show a mouse model in which MMc numbers are either normal or low, which enables functional assessment of MMc. We report a functional role of MMc in promoting fetal immune development. MMc induces preferential differentiation of hematopoietic stem cells in fetal bone marrow towards monocytes within the myeloid compartment. Neonatal mice with higher numbers of MMc and monocytes show enhanced resilience against cytomegalovirus infection. Similarly, higher numbers of MMc in human cord blood are linked to a lower number of respiratory infections during the first year of life. Our data highlight the importance of MMc in promoting fetal immune development, potentially averting the threats caused by early life exposure to pathogens.


Assuntos
Quimerismo , Feto/imunologia , Imunidade Materno-Adquirida/imunologia , Infecções/imunologia , Animais , Medula Óssea/metabolismo , Epigenoma , Feminino , Sangue Fetal/citologia , Hematopoese , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Lactente , Camundongos , Monócitos/citologia , Gravidez , Linfócitos T/citologia
9.
Science ; 373(6558)2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34446580

RESUMO

The immune system has evolved in the face of microbial exposure. How maternal infection experienced at distinct developmental stages shapes the offspring immune system remains poorly understood. Here, we show that during pregnancy, maternally restricted infection can have permanent and tissue-specific impacts on offspring immunity. Mechanistically, maternal interleukin-6 produced in response to infection can directly impose epigenetic changes on fetal intestinal epithelial stem cells, leading to long-lasting impacts on intestinal immune homeostasis. As a result, offspring of previously infected dams develop enhanced protective immunity to gut infection and increased inflammation in the context of colitis. Thus, maternal infection can be coopted by the fetus to promote long-term, tissue-specific fitness, a phenomenon that may come at the cost of predisposition to inflammatory disorders.


Assuntos
Colite/imunologia , Imunidade , Interleucina-6/imunologia , Intestinos/imunologia , Complicações Infecciosas na Gravidez/imunologia , Células Th17/imunologia , Infecções por Yersinia pseudotuberculosis/imunologia , Animais , Candidíase/imunologia , Cromatina/metabolismo , Epigênese Genética , Epigenoma , Feminino , Desenvolvimento Fetal , Microbioma Gastrointestinal/imunologia , Microbioma Gastrointestinal/fisiologia , Interleucina-6/sangue , Interleucina-6/farmacologia , Mucosa Intestinal/citologia , Mucosa Intestinal/embriologia , Mucosa Intestinal/imunologia , Intestinos/embriologia , Intestinos/microbiologia , Camundongos , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Salmonelose Animal/imunologia , Células-Tronco/imunologia , Células-Tronco/fisiologia , Subpopulações de Linfócitos T/imunologia
10.
Nat Methods ; 18(8): 965-974, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34341582

RESUMO

CRISPR-Cas9 technologies have dramatically increased the ease of targeting DNA sequences in the genomes of living systems. The fusion of chromatin-modifying domains to nuclease-deactivated Cas9 (dCas9) has enabled targeted epigenome editing in both cultured cells and animal models. However, delivering large dCas9 fusion proteins to target cells and tissues is an obstacle to the widespread adoption of these tools for in vivo studies. Here, we describe the generation and characterization of two conditional transgenic mouse lines for epigenome editing, Rosa26:LSL-dCas9-p300 for gene activation and Rosa26:LSL-dCas9-KRAB for gene repression. By targeting the guide RNAs to transcriptional start sites or distal enhancer elements, we demonstrate regulation of target genes and corresponding changes to epigenetic states and downstream phenotypes in the brain and liver in vivo, and in T cells and fibroblasts ex vivo. These mouse lines are convenient and valuable tools for facile, temporally controlled, and tissue-restricted epigenome editing and manipulation of gene expression in vivo.


Assuntos
Sistemas CRISPR-Cas , Epigênese Genética , Epigenoma , Edição de Genes/métodos , Regulação da Expressão Gênica , Animais , Encéfalo/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Linfócitos T/metabolismo
11.
Nat Genet ; 53(8): 1221-1232, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34294917

RESUMO

Driver mutations in genes encoding histone H3 proteins resulting in p.Lys27Met substitutions (H3-K27M) are frequent in pediatric midline brain tumors. However, the precise mechanisms by which H3-K27M causes tumor initiation remain unclear. Here, we use human hindbrain neural stem cells to model the consequences of H3.3-K27M on the epigenomic landscape in a relevant developmental context. Genome-wide mapping of epitope-tagged histone H3.3 revealed that both the wild type and the K27M mutant incorporate abundantly at pre-existing active enhancers and promoters, and to a lesser extent at Polycomb repressive complex 2 (PRC2)-bound regions. At active enhancers, H3.3-K27M leads to focal H3K27ac loss, decreased chromatin accessibility and reduced transcriptional expression of nearby neurodevelopmental genes. In addition, H3.3-K27M deposition at a subset of PRC2 target genes leads to increased PRC2 and PRC1 binding and augmented transcriptional repression that can be partially reversed by PRC2 inhibitors. Our work suggests that, rather than imposing de novo transcriptional circuits, H3.3-K27M drives tumorigenesis by locking initiating cells in their pre-existing, immature epigenomic state, via disruption of PRC2 and enhancer functions.


Assuntos
Elementos Facilitadores Genéticos , Histonas/metabolismo , Células-Tronco Neurais/fisiologia , Complexo Repressor Polycomb 2/genética , Rombencéfalo/citologia , Animais , Neoplasias Encefálicas/genética , Diferenciação Celular/genética , Linhagem Celular , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Epigenoma , Regulação da Expressão Gênica no Desenvolvimento , Glioma/genética , Histonas/genética , Humanos , Lisina/metabolismo , Masculino , Camundongos Endogâmicos , Mutação , Células-Tronco Neurais/transplante , Oncogenes , Complexo Repressor Polycomb 2/antagonistas & inibidores , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas , Rombencéfalo/fisiologia
12.
Artigo em Inglês | MEDLINE | ID: mdl-34299878

RESUMO

Epigenetics is known to be involved in regulatory pathways through which greenness exposure influences child development and health. We aimed to investigate the associations between residential surrounding greenness and DNA methylation changes in children, and further assessed the association between DNA methylation and children's intelligence quotient (IQ) in a prospective cohort study. We identified cytosine-guanine dinucleotide sites (CpGs) associated with cognitive abilities from epigenome- and genome-wide association studies through a systematic literature review for candidate gene analysis. We estimated the residential surrounding greenness at age 2 using a geographic information system. DNA methylation was analyzed from whole blood using the HumanMethylationEPIC array in 59 children at age 2. We analyzed the association between greenness exposure and DNA methylation at age 2 at the selected CpGs using multivariable linear regression. We further investigated the relationship between DNA methylation and children's IQ. We identified 8743 CpGs associated with cognitive ability based on the literature review. Among these CpGs, we found that 25 CpGs were significantly associated with greenness exposure at age 2, including cg26269038 (Bonferroni-corrected p ≤ 0.05) located in the body of SLC6A3, which encodes a dopamine transporter. DNA methylation at cg26269038 at age 2 was significantly associated with children's performance IQ at age 6. Exposure to surrounding greenness was associated with cognitive ability-related DNA methylation changes, which was also associated with children's IQ. Further studies are warranted to clarify the epigenetic pathways linking greenness exposure and neurocognitive function.


Assuntos
Metilação de DNA , Estudo de Associação Genômica Ampla , Criança , Pré-Escolar , Proteínas da Membrana Plasmática de Transporte de Dopamina , Epigênese Genética , Epigenoma , Humanos , Estudos Prospectivos
13.
Nat Rev Immunol ; 21(8): 472, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34239105
14.
Nutrients ; 13(6)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207686

RESUMO

Here, we performed a genome-wide search for methylation sites that contribute to the risk of obesity. We integrated methylation quantitative trait locus (mQTL) data with BMI GWAS information through a SNP-based multiomics approach to identify genomic regions where mQTLs for a methylation site co-localize with obesity risk SNPs. We then tested whether the identified site contributed to BMI through Mendelian randomization. We identified multiple methylation sites causally contributing to the risk of obesity. We validated these findings through a replication stage. By integrating expression quantitative trait locus (eQTL) data, we noted that lower methylation at cg21178254 site upstream of CCNL1 contributes to obesity by increasing the expression of this gene. Higher methylation at cg02814054 increases the risk of obesity by lowering the expression of MAST3, whereas lower methylation at cg06028605 contributes to obesity by decreasing the expression of SLC5A11. Finally, we noted that rare variants within 2p23.3 impact obesity by making the cg01884057 site more susceptible to methylation, which consequently lowers the expression of POMC, ADCY3 and DNAJC27. In this study, we identify methylation sites associated with the risk of obesity and reveal the mechanism whereby a number of these sites exert their effects. This study provides a framework to perform an omics-wide association study for a phenotype and to understand the mechanism whereby a rare variant causes a disease.


Assuntos
Metilação de DNA/genética , Epigenoma/genética , Predisposição Genética para Doença/genética , Obesidade/genética , Adenilil Ciclases/genética , Índice de Massa Corporal , Ciclinas/genética , Epigenômica/métodos , Estudo de Associação Genômica Ampla , Proteínas de Choque Térmico HSP40/genética , Humanos , Análise da Randomização Mendeliana , Proteínas Associadas aos Microtúbulos/genética , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Pró-Opiomelanocortina/genética , Proteínas Serina-Treonina Quinases/genética , Locos de Características Quantitativas , Fatores de Risco , Proteínas de Transporte de Sódio-Glucose/genética , Proteínas rab de Ligação ao GTP/genética
15.
Int J Mol Sci ; 22(12)2021 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-34204821

RESUMO

Hematologic malignancies comprise a heterogeneous group of neoplasms arising from hematopoietic cells or their precursors and most commonly presenting as leukemias, lymphomas, and myelomas. Genetic analyses have uncovered recurrent mutations which initiate or accumulate in the course of malignant transformation, as they provide selective growth advantage to the cell. These include mutations in genes encoding transcription factors and epigenetic regulators of metabolic genes, as well as genes encoding key metabolic enzymes. The resulting alterations contribute to the extensive metabolic reprogramming characterizing the transformed cell, supporting its increased biosynthetic needs and allowing it to withstand the metabolic stress that arises as a consequence of increased metabolic rates and changes in its microenvironment. Interestingly, this cross-talk is bidirectional, as metabolites also signal back to the nucleus and, via their widespread effects on modulating epigenetic modifications, shape the chromatin landscape and the transcriptional programs of the cell. In this article, we provide an overview of the main metabolic changes and relevant genetic alterations that characterize malignant hematopoiesis and discuss how, in turn, metabolites regulate epigenetic events during this process. The aim is to illustrate the intricate interrelationship between the genome (and epigenome) and metabolism and its relevance to hematologic malignancy.


Assuntos
Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Animais , Epigenoma , Hematopoese/genética , Humanos , Redes e Vias Metabólicas/genética , Modelos Biológicos , Mutação/genética
16.
Transl Psychiatry ; 11(1): 398, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34282125

RESUMO

Epigenetic changes are currently invoked as explanations for both the chronicity and tenacity of post-traumatic stress disorder (PTSD), a heterogeneous condition showing varying, sometimes idiosyncratic responses to treatment. This study evaluated epigenetic markers in the context of a randomized clinical trial of PTSD patients undergoing prolonged-exposure psychotherapy with and without a hydrocortisone augmentation prior to each session. The purpose of the longitudinal epigenome-wide analyses was to identify predictors of recovery (from pretreatment data) or markers associated with symptom change (based on differences between pre- and post-therapy epigenetic changes). The results of these analyses identified the CREB-BDNF signaling pathway, previously linked to startle reaction and fear learning and memory processes, as a convergent marker predicting both symptom change and severity. Several previous-reported resilience markers were also identified (FKBP5, NR3C1, SDK1, and MAD1L1) to associate with PTSD recovery in this study. Especially, the methylation levels of FKBP5 in the gene body region decreased significantly as CAPS score decreased in responders, while no changes occurred in nonresponders. These biomarkers may have future utility in understanding clinical recovery in PTSD and potential applications in predicting treatment effects.


Assuntos
Transtornos de Estresse Pós-Traumáticos , Metilação de DNA , Epigênese Genética , Epigenoma , Humanos , Hidrocortisona , Transtornos de Estresse Pós-Traumáticos/tratamento farmacológico , Transtornos de Estresse Pós-Traumáticos/genética
17.
Nat Protoc ; 16(8): 4004-4030, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34244697

RESUMO

The integration of DNA methylation and transcriptional state within single cells is of broad interest. Several single-cell dual- and multi-omics approaches have been reported that enable further investigation into cellular heterogeneity, including the discovery and in-depth study of rare cell populations. Such analyses will continue to provide important mechanistic insights into the regulatory consequences of epigenetic modifications. We recently reported a new method for profiling the DNA methylome and transcriptome from the same single cells in a cancer research study. Here, we present details of the protocol and provide guidance on its utility. Our Smart-RRBS (reduced representation bisulfite sequencing) protocol combines Smart-seq2 and RRBS and entails physically separating mRNA from the genomic DNA. It generates paired epigenetic promoter and RNA-expression measurements for ~24% of protein-coding genes in a typical single cell. It also works for micro-dissected tissue samples comprising hundreds of cells. The protocol, excluding flow sorting of cells and sequencing, takes ~3 d to process up to 192 samples manually. It requires basic molecular biology expertise and laboratory equipment, including a PCR workstation with UV sterilization, a DNA fluorometer and a microfluidic electrophoresis system.


Assuntos
DNA/metabolismo , Análise de Célula Única , Sequência de Aminoácidos , Antibacterianos/farmacologia , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Doxiciclina/farmacologia , Epigenoma , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma
18.
Int J Mol Sci ; 22(14)2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34299232

RESUMO

The genetic architecture of complex traits is multifactorial. Genome-wide association studies (GWASs) have identified risk loci for complex traits and diseases that are disproportionately located at the non-coding regions of the genome. On the other hand, we have just begun to understand the regulatory roles of the non-coding genome, making it challenging to precisely interpret the functions of non-coding variants associated with complex diseases. Additionally, the epigenome plays an active role in mediating cellular responses to fluctuations of sensory or environmental stimuli. However, it remains unclear how exactly non-coding elements associate with epigenetic modifications to regulate gene expression changes and mediate phenotypic outcomes. Therefore, finer interrogations of the human epigenomic landscape in associating with non-coding variants are warranted. Recently, chromatin-profiling techniques have vastly improved our understanding of the numerous functions mediated by the epigenome and DNA structure. Here, we review various chromatin-profiling techniques, such as assays of chromatin accessibility, nucleosome distribution, histone modifications, and chromatin topology, and discuss their applications in unraveling the brain epigenome and etiology of complex traits at tissue homogenate and single-cell resolution. These techniques have elucidated compositional and structural organizing principles of the chromatin environment. Taken together, we believe that high-resolution epigenomic and DNA structure profiling will be one of the best ways to elucidate how non-coding genetic variations impact complex diseases, ultimately allowing us to pinpoint cell-type targets with therapeutic potential.


Assuntos
Cromatina/genética , Cromatina/metabolismo , Cromatina/fisiologia , Sítios de Ligação/genética , Imunoprecipitação da Cromatina/métodos , Epigênese Genética/genética , Epigenoma/genética , Epigenômica/métodos , Regulação da Expressão Gênica/genética , Genoma , Estudo de Associação Genômica Ampla/métodos , Código das Histonas/genética , Humanos , Herança Multifatorial/genética , Nucleossomos/metabolismo , Nucleossomos/fisiologia , Polimorfismo de Nucleotídeo Único/genética , RNA não Traduzido/genética , RNA não Traduzido/metabolismo
19.
Nat Commun ; 12(1): 3987, 2021 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-34183656

RESUMO

Here we examine the association between DNA methylation in circulating leukocytes and blood lipids in a multi-ethnic sample of 16,265 subjects. We identify 148, 35, and 4 novel associations among Europeans, African Americans, and Hispanics, respectively, and an additional 186 novel associations through a trans-ethnic meta-analysis. We observe a high concordance in the direction of effects across racial/ethnic groups, a high correlation of effect sizes between high-density lipoprotein and triglycerides, a modest overlap of associations with epigenome-wide association studies of other cardio-metabolic traits, and a largely non-overlap with lipid loci identified to date through genome-wide association studies. Thirty CpGs reached significance in at least 2 racial/ethnic groups including 7 that showed association with the expression of an annotated gene. CpGs annotated to CPT1A showed evidence of being influenced by triglycerides levels. DNA methylation levels of circulating leukocytes show robust and consistent association with blood lipid levels across multiple racial/ethnic groups.


Assuntos
Metilação de DNA/genética , Leucócitos/citologia , Lipídeos/sangue , Lipoproteínas HDL/sangue , Adulto , Afro-Americanos , Idoso , Carnitina O-Palmitoiltransferase/genética , Ilhas de CpG/genética , Epigênese Genética , Epigenoma/genética , Epigenômica , Grupo com Ancestrais do Continente Europeu , Feminino , Hispano-Americanos , Humanos , Masculino , Pessoa de Meia-Idade , Locos de Características Quantitativas/genética
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