RESUMEN
A nonsynonymous single-nucleotide polymorphism at codon 47 in TP53 exists in African-descent populations (P47S, rs1800371; referred to here as S47). Here we report that, in human cell lines and a mouse model, the S47 variant exhibits a modest decrease in apoptosis in response to most genotoxic stresses compared with wild-type p53 but exhibits a significant defect in cell death induced by cisplatin. We show that, compared with wild-type p53, S47 has nearly indistinguishable transcriptional function but shows impaired ability to transactivate a subset of p53 target genes, including two involved in metabolism:Gls2(glutaminase 2) and Sco2 We also show that human and mouse cells expressing the S47 variant are markedly resistant to cell death by agents that induce ferroptosis (iron-mediated nonapoptotic cell death). We show that mice expressing S47 in homozygous or heterozygous form are susceptible to spontaneous cancers of diverse histological types. Our data suggest that the S47 variant may contribute to increased cancer risk in individuals of African descent, and our findings highlight the need to assess the contribution of this variant to cancer risk in these populations. These data also confirm the potential relevance of metabolism and ferroptosis to tumor suppression by p53.
Asunto(s)
Genes p53/genética , Polimorfismo de Nucleótido Simple , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Población Negra/genética , Carcinoma Hepatocelular/genética , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Línea Celular , Cisplatino/farmacología , Codón/química , Codón/genética , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Neoplasias/genética , Unión Proteica/genética , Factores de Riesgo , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genéticaRESUMEN
Cationic antimicrobial peptides (CAMPs) are important elements of innate immunity in higher organisms, representing an ancient defense mechanism against pathogenic bacteria. These peptides exhibit broad-spectrum antimicrobial activities, utilizing mechanisms that involve targeting bacterial membranes. Recently, a 34-residue CAMP (NA-CATH) was identified in cDNA from the venom gland of the Chinese cobra (Naja atra). A semi-conserved 11-residue pattern observed in the NA-CATH sequence provided the basis for generating an 11-residue truncated peptide, ATRA-1A, and its corresponding D-peptide isomer. While the antimicrobial and biophysical properties of the ATRA-1A stereoisomers have been investigated, their modes of action remain unclear. More broadly, mechanistic differences that can arise when investigating minimal antimicrobial units within larger naturally occurring CAMPs have not been rigorously explored. Therefore, the studies reported here are focused on this question and the interactions of full-length NA-CATH and the truncated ATRA-1A isomers with bacterial membranes. The results of these studies indicate that in engineering the ATRA-1A isomers, the associated change in peptide length and charge dramatically impacts not only their antimicrobial effectiveness, but also the mechanism of action they employ relative to that of the full-length parent peptide NA-CATH. These insights are relevant to future efforts to develop shorter versions of larger naturally occurring CAMPs for potential therapeutic applications.
Asunto(s)
Antibacterianos/farmacología , Bacillus cereus/efectos de los fármacos , Catelicidinas/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Oligopéptidos/farmacología , Fragmentos de Péptidos/farmacología , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Bacillus cereus/metabolismo , Bacillus cereus/ultraestructura , Catelicidinas/química , Catelicidinas/aislamiento & purificación , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Relación Dosis-Respuesta a Droga , Venenos Elapídicos/química , Escherichia coli/metabolismo , Escherichia coli/ultraestructura , Cinética , Viabilidad Microbiana/efectos de los fármacos , Oligopéptidos/química , Fragmentos de Péptidos/química , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
Mouse embryonic stem cells (mESCs) cultured in the presence of LIF occupy a ground state with highly active pluripotency-associated transcriptional and epigenetic circuitry. However, ground state pluripotency in some inbred strain backgrounds is unstable in the absence of ERK1/2 and GSK3 inhibition. Using an unbiased genetic approach, we dissect the basis of this divergent response to extracellular cues by profiling gene expression and chromatin accessibility in 170 genetically heterogeneous mESCs. We map thousands of loci affecting chromatin accessibility and/or transcript abundance, including 10 QTL hotspots where genetic variation at a single locus coordinates the regulation of genes throughout the genome. For one hotspot, we identify a single enhancer variant â¼10 kb upstream of Lifr associated with chromatin accessibility and mediating a cascade of molecular events affecting pluripotency. We validate causation through reciprocal allele swaps, demonstrating the functional consequences of noncoding variation in gene regulatory networks that stabilize pluripotent states in vitro.
Asunto(s)
Cromatina , Células Madre Pluripotentes , Animales , Diferenciación Celular , Cromatina/genética , Expresión Génica , Variación Genética , Glucógeno Sintasa Quinasa 3 , RatonesRESUMEN
BACKGROUND: Cigarette smoke is a causal factor in cancers and cardiovascular disease. Smoking-associated differentially methylated regions (SM-DMRs) have been observed in disease studies, but the causal link between altered DNA methylation and transcriptional change is obscure. OBJECTIVE: Our objectives were to finely resolve SM-DMRs and to interrogate the mechanistic link between SM-DMRs and altered transcription of enhancer noncoding RNA (eRNA) and mRNA in human circulating monocytes. METHOD: We integrated SM-DMRs identified by reduced representation bisulfite sequencing (RRBS) of circulating CD14+ monocyte DNA collected from two independent human studies [n=38 from Clinical Research Unit (CRU) and n=55 from the Multi-Ethnic Study of Atherosclerosis (MESA), about half of whom were active smokers] with gene expression for protein-coding genes and noncoding RNAs measured by RT-PCR or RNA sequencing. Candidate SM-DMRs were compared with RRBS of purified CD4+ T cells, CD8+ T cells, CD15+ granulocytes, CD19+ B cells, and CD56+ NK cells (n=19 females, CRU). DMRs were validated using pyrosequencing or bisulfite amplicon sequencing in up to 85 CRU volunteers, who also provided saliva DNA. RESULTS: RRBS identified monocyte SM-DMRs frequently located in putative gene regulatory regions. The most significant monocyte DMR occurred at a poised enhancer in the aryl-hydrocarbon receptor repressor gene (AHRR) and it was also detected in both granulocytes and saliva DNA. To our knowledge, we identify for the first time that SM-DMRs in or near AHRR, C5orf55-EXOC-AS, and SASH1 were associated with increased noncoding eRNA as well as mRNA in monocytes. Functionally, the AHRR SM-DMR appeared to up-regulate AHRR mRNA through activating the AHRR enhancer, as suggested by increased eRNA in the monocytes, but not granulocytes, from smokers compared with nonsmokers. CONCLUSIONS: Our findings suggest that AHRR SM-DMR up-regulates AHRR mRNA in a monocyte-specific manner by activating the AHRR enhancer. Cell type-specific activation of enhancers at SM-DMRs may represent a mechanism driving smoking-related disease. https://doi.org/10.1289/EHP2395.
Asunto(s)
Metilación de ADN/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , ARN no Traducido/efectos de los fármacos , Fumar/efectos adversos , Sulfitos/efectos adversos , Transcripción Genética/efectos de los fármacos , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismo , ARN no Traducido/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/efectos de los fármacos , Secuencias Reguladoras de Ácidos Nucleicos/genéticaRESUMEN
ARID1A, a subunit of the SWI/SNF chromatin remodeling complex, is frequently mutated in cancer. Deficiency in its homolog ARID1B is synthetically lethal with ARID1A mutation. However, the functional relationship between these homologs has not been explored. Here, we use ATAC-seq, genome-wide histone modification mapping, and expression analysis to examine colorectal cancer cells lacking one or both ARID proteins. We find that ARID1A has a dominant role in maintaining chromatin accessibility at enhancers, while the contribution of ARID1B is evident only in the context of ARID1A mutation. Changes in accessibility are predictive of changes in expression and correlate with loss of H3K4me and H3K27ac marks, nucleosome spacing, and transcription factor binding, particularly at growth pathway genes including MET. We find that ARID1B knockdown in ARID1A mutant ovarian cancer cells causes similar loss of enhancer architecture, suggesting that this is a conserved function underlying the synthetic lethality between ARID1A and ARID1B.
Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Neoplasias/patología , Proteínas Nucleares/metabolismo , Mutaciones Letales Sintéticas , Factores de Transcripción/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Histonas/análisis , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Nucleares/genética , Procesamiento Proteico-Postraduccional , Factores de Transcripción/genéticaRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0166486.].
RESUMEN
Tobacco smoke exposure dramatically alters DNA methylation in blood cells and may mediate smoking-associated complex diseases through effects on immune cell function. However, knowledge of smoking effects in specific leukocyte subtypes is limited. To better characterize smoking-associated methylation changes in whole blood and leukocyte subtypes, we used Illumina 450K arrays and Reduced Representation Bisulfite Sequencing (RRBS) to assess genome-wide DNA methylation. Differential methylation analysis in whole blood DNA from 172 smokers and 81 nonsmokers revealed 738 CpGs, including 616 previously unreported CpGs, genome-wide significantly associated with current smoking (p <1.2x10-7, Bonferroni correction). Several CpGs (MTSS1, NKX6-2, BTG2) were associated with smoking duration among heavy smokers (>22 cigarettes/day, n = 86) which might relate to long-term heavy-smoking pathology. In purified leukocyte subtypes from an independent group of 20 smokers and 14 nonsmokers we further examined methylation and gene expression for selected genes among CD14+ monocytes, CD15+ granulocytes, CD19+ B cells, and CD2+ T cells. In 10 smokers and 10 nonsmokers we used RRBS to fine map differential methylation in CD4+ T cells, CD8+ T cells, CD14+, CD15+, CD19+, and CD56+ natural killer cells. Distinct cell-type differences in smoking-associated methylation and gene expression were identified. AHRR (cg05575921), ALPPL2 (cg21566642), GFI1 (cg09935388), IER3 (cg06126421) and F2RL3 (cg03636183) showed a distinct pattern of significant smoking-associated methylation differences across cell types: granulocytes> monocytes>> B cells. In contrast GPR15 (cg19859270) was highly significant in T and B cells and ITGAL (cg09099830) significant only in T cells. Numerous other CpGs displayed distinctive cell-type responses to tobacco smoke exposure that were not apparent in whole blood DNA. Assessing the overlap between these CpG sites and differential methylated regions (DMRs) with RRBS in 6 cell types, we confirmed cell-type specificity in the context of DMRs. We identified new CpGs associated with current smoking, pack-years, duration, and revealed unique profiles of smoking-associated DNA methylation and gene expression among immune cell types, providing potential clues to hematopoietic lineage-specific effects in disease etiology.
Asunto(s)
Metilación de ADN , Epigenómica/métodos , Leucocitos/metabolismo , Fumar , Adulto , Fosfatasa Alcalina/genética , Proteínas Reguladoras de la Apoptosis/genética , Linfocitos B/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Islas de CpG/genética , Proteínas de Unión al ADN/genética , Epigénesis Genética , Femenino , Proteínas Ligadas a GPI/genética , Expresión Génica , Estudio de Asociación del Genoma Completo/métodos , Granulocitos/metabolismo , Humanos , Leucocitos/clasificación , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Monocitos/metabolismo , Receptores de Trombina/genética , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN/métodos , Linfocitos T/metabolismo , Factores de Transcripción/genética , Adulto JovenRESUMEN
BACKGROUND: Tobacco smoke contains numerous agonists of the aryl hydrocarbon receptor (AhR) pathway, and activation of the AhR pathway was shown to promote atherosclerosis in mice. Intriguingly, cigarette smoking is most strongly and robustly associated with DNA modifications to an AhR pathway gene, the AhR repressor (AHRR). We hypothesized that altered AHRR methylation in monocytes, a cell type sensitive to cigarette smoking and involved in atherogenesis, may be a part of the biological link between cigarette smoking and atherosclerosis. METHODS AND RESULTS: DNA methylation profiles of AHRR in monocytes (542 CpG sites ± 150 kb of AHRR, using Illumina 450K array) were integrated with smoking habits and ultrasound-measured carotid plaque scores from 1256 participants of the Multi-Ethnic Study of Atherosclerosis (MESA). Methylation of cg05575921 significantly associated (P=6.1 × 10(-134)) with smoking status (current versus never). Novel associations between cg05575921 methylation and carotid plaque scores (P=3.1 × 10(-10)) were identified, which remained significant in current and former smokers even after adjusting for self-reported smoking habits, urinary cotinine, and well-known cardiovascular disease risk factors. This association replicated in an independent cohort using hepatic DNA (n=141). Functionally, cg05575921 was located in a predicted gene expression regulatory element (enhancer) and had methylation correlated with AHRR mRNA profiles (P=1.4 × 10(-17)) obtained from RNA sequencing conducted on a subset (n=373) of the samples. CONCLUSIONS: These findings suggest that AHRR methylation may be functionally related to AHRR expression in monocytes and represents a potential biomarker of subclinical atherosclerosis in smokers.