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1.
Nat Commun ; 12(1): 5242, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34475398

RESUMO

Genome-wide association studies (GWAS) have linked single nucleotide polymorphisms (SNPs) at >250 loci in the human genome to type 2 diabetes (T2D) risk. For each locus, identifying the functional variant(s) among multiple SNPs in high linkage disequilibrium is critical to understand molecular mechanisms underlying T2D genetic risk. Using massively parallel reporter assays (MPRA), we test the cis-regulatory effects of SNPs associated with T2D and altered in vivo islet chromatin accessibility in MIN6 ß cells under steady state and pathophysiologic endoplasmic reticulum (ER) stress conditions. We identify 1,982/6,621 (29.9%) SNP-containing elements that activate transcription in MIN6 and 879 SNP alleles that modulate MPRA activity. Multiple T2D-associated SNPs alter the activity of short interspersed nuclear element (SINE)-containing elements that are strongly induced by ER stress. We identify 220 functional variants at 104 T2D association signals, narrowing 54 signals to a single candidate SNP. Together, this study identifies elements driving ß cell steady state and ER stress-responsive transcriptional activation, nominates causal T2D SNPs, and uncovers potential roles for repetitive elements in ß cell transcriptional stress response and T2D genetics.


Assuntos
Diabetes Mellitus Tipo 2/genética , Estresse do Retículo Endoplasmático/genética , Células Secretoras de Insulina/patologia , Polimorfismo de Nucleotídeo Único , Ativação Transcricional/genética , Alelos , Animais , Linhagem Celular , Cromatina/metabolismo , Diabetes Mellitus Tipo 2/patologia , Estudo de Associação Genômica Ampla , Humanos , Camundongos , Locos de Características Quantitativas , Elementos Nucleotídeos Curtos e Dispersos/genética
3.
Nat Genet ; 53(8): 1166-1176, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34326544

RESUMO

Effective interpretation of genome function and genetic variation requires a shift from epigenetic mapping of cis-regulatory elements (CREs) to characterization of endogenous function. We developed hybridization chain reaction fluorescence in situ hybridization coupled with flow cytometry (HCR-FlowFISH), a broadly applicable approach to characterize CRISPR-perturbed CREs via accurate quantification of native transcripts, alongside CRISPR activity screen analysis (CASA), a hierarchical Bayesian model to quantify CRE activity. Across >325,000 perturbations, we provide evidence that CREs can regulate multiple genes, skip over the nearest gene and display activating and/or silencing effects. At the cholesterol-level-associated FADS locus, we combine endogenous screens with reporter assays to exhaustively characterize multiple genome-wide association signals, functionally nominate causal variants and, importantly, identify their target genes.


Assuntos
Hibridização in Situ Fluorescente/métodos , Sequências Reguladoras de Ácido Nucleico , Proteínas Adaptadoras de Transdução de Sinal/genética , Teorema de Bayes , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Ácidos Graxos Dessaturases/genética , Citometria de Fluxo , Fator de Transcrição GATA1/genética , Humanos , Células K562 , Proteínas com Domínio LIM/genética , Modelos Genéticos , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas/genética , Locos de Características Quantitativas , RNA Guia
4.
Chem Biol ; 20(2): 272-84, 2013 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-23438756

RESUMO

Innovative strategies are needed to combat drug resistance associated with methicillin-resistant Staphylococcus aureus (MRSA). Here, we investigate the potential of wall teichoic acid (WTA) biosynthesis inhibitors as combination agents to restore ß-lactam efficacy against MRSA. Performing a whole-cell pathway-based screen, we identified a series of WTA inhibitors (WTAIs) targeting the WTA transporter protein, TarG. Whole-genome sequencing of WTAI-resistant isolates across two methicillin-resistant Staphylococci spp. revealed TarG as their common target, as well as a broad assortment of drug-resistant bypass mutants mapping to earlier steps of WTA biosynthesis. Extensive in vitro microbiological analysis and animal infection studies provide strong genetic and pharmacological evidence of the potential effectiveness of WTAIs as anti-MRSA ß-lactam combination agents. This work also highlights the emerging role of whole-genome sequencing in antibiotic mode-of-action and resistance studies.


Assuntos
Antibacterianos/farmacologia , Parede Celular/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Ácidos Teicoicos/biossíntese , beta-Lactamas/metabolismo , Substituição de Aminoácidos , Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Avaliação Pré-Clínica de Medicamentos , Farmacorresistência Bacteriana , Genoma Bacteriano , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Concentração Osmolar , Fenótipo , Análise de Sequência de DNA , Ácidos Teicoicos/química , Temperatura , beta-Lactamas/química
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